Introdução: O transplante de células-tronco hematopoiéticas (TCTH) tornou-se um procedimento terapêutico mundialmente aceito sobretudo pelo impacto positivo na sobrevida e na qualidade de vida dos pacientes com doenças onco-hematológicas. No entanto, a mortalidade ainda é alta e influenciada por fatores de natureza individual e terapêutica. Objetivo: Analisar a mortalidade relacionada ao transplante (MRT) nos pacientes submetidos ao TCTH e seus fatores associados. Método: Coorte prospectiva realizada com 60 pacientes internados na unidade de TCTH do Hospital de Clínicas da Universidade Estadual de Campinas. Os dados foram obtidos pela análise diária dos prontuários. A variável dependente foi a MRT e as variáveis independentes foram demográficas e de evolução clínicas, incluindo escore de risco pré-TCTH (EBMT) e o SAPS II. Na análise dos dados foram utilizados os testes Qui-quadrado, Exato de Fisher, o teste t de Student e Mann-Whitney. Na análise da MRT utilizou-se o método de kaplan-Meier e o Modelo de Cox. Considerou-se nível de significância igual a 5%. Resultados: A MRT foi de 15% aos cem dias do TCTH, de 18,9% no grupo de pacientes de TCTH alogênico e de 8,7% para os de TCTH autólogo. A infecção foi a principal causa de óbito. Na amostra, o tempo médio de sobrevida dos pacientes foi de 83,2 dias (DP 32,7). No grupo de pacientes não sobreviventes a maioria pertencia ao sexo masculino, com média de idade de 48,7 anos e diagnóstico principal de leucemia. Quanto à gravidade destes pacientes, o escore de risco pré-TCTH (EBMT) foi de 4,1 pontos e do SAPS II geral foi de 52,6 pontos, o que correspondeu a um risco médio de morte de de 38,4%. Os fatores associados à MRT, em cem dias, foram faixa etária (p=0,0306), presença de infecção (p=0,0216), número de infecções (p=0,0386), ocorrência de enxertia (p<0,0001), uso de ventilação mecânica (p<0,0001) e de drogas vasoativas (p<0,0001). O índice de gravidade SAPS II foi fator preditor para MRT (p=0,0001). Conclusão: O índice de gravidade SAPS II, preditor para MRT em cem dias, mostrou que o paciente submetido ao TCTH é grave e necessita de cuidado especializado e intensivo. / Hematopoietic stem cells transplantation (HSCT) has become a therapeutic procedure accepted worldwide, particularly because of its positive impact on survival and quality of life of patients with onco-hematological diseases. However, the mortality is still high and it is influenced by factors of individual and therapeutic kinds. Objective: To analyze the transplant-related mortality (TRM) on patients submitted to HSCT and its associated factors. Methodology: Prospective cohort study with 60 patients hospitalized in the HSTC unit of the Clinical Hospital of the State University of Campinas (Unicamp). Data was obtained by daily analysis of the medical records. The dependent variable was the TRM and the independent variables were demographic and clinical development, including pre-HSCT risk score (EBMT) and SAPS II. For data analysis were used the Chi-square, Fishers exact tests, Students t-test, Mann-Whitney. On TRM analysis were used Kaplan-Meier and Cox Model method. It was considered a significance level of 5%. Results: The TRM was 15% to a hundred days of HSCT, 18,9% to allogeneic HSCT patients and 8,7% to autologous HSCT. Infection was the main cause of death. In the sample, the median survival time of the patients was 83,2 days (DP 32,7). In the group of non-surviving patients the most were male, with an average age of 48,7 years and the main diagnosis was leukemia. Regarding to the severity of these patients, the pre-HSCT risk score (EBMT) was 4,1 points and general SAPS II was 52,6 points, which corresponds to an average death risk of 38,4%. The TRM associated factors on a hundred days were age (p=0,0306), presence of infection (p=0,0216), number of infections (p=0,0386), occurrence of grafting (p<0,0001), mechanical ventilation use (p<0,0001) and vasoactive drugs (p<0,0001). The severity rate SAPS II was a predictive factor for TRM (p=0,0001). Conclusion: The severity rate SAPS II was predictive for TRM on a hundred days and showed that the patient submitted to HSCT is severe and demands specialized and intensive care.

Introdução: O transplante de células-tronco hematopoiéticas (TCTH) tornou-se um procedimento terapêutico mundialmente aceito sobretudo pelo impacto positivo na sobrevida e na qualidade de vida dos pacientes com doenças onco-hematológicas. No entanto, a mortalidade ainda é alta e influenciada por fatores de natureza individual e terapêutica. Objetivo: Analisar a mortalidade relacionada ao transplante (MRT) nos pacientes submetidos ao TCTH e seus fatores associados. Método: Coorte prospectiva realizada com 60 pacientes internados na unidade de TCTH do Hospital de Clínicas da Universidade Estadual de Campinas. Os dados foram obtidos pela análise diária dos prontuários. A variável dependente foi a MRT e as variáveis independentes foram demográficas e de evolução clínicas, incluindo escore de risco pré-TCTH (EBMT) e o SAPS II. Na análise dos dados foram utilizados os testes Qui-quadrado, Exato de Fisher, o teste t de Student e Mann-Whitney. Na análise da MRT utilizou-se o método de kaplan-Meier e o Modelo de Cox. Considerou-se nível de significância igual a 5%. Resultados: A MRT foi de 15% aos cem dias do TCTH, de 18,9% no grupo de pacientes de TCTH alogênico e de 8,7% para os de TCTH autólogo. A infecção foi a principal causa de óbito. Na amostra, o tempo médio de sobrevida dos pacientes foi de 83,2 dias (DP 32,7). No grupo de pacientes não sobreviventes a maioria pertencia ao sexo masculino, com média de idade de 48,7 anos e diagnóstico principal de leucemia. Quanto à gravidade destes pacientes, o escore de risco pré-TCTH (EBMT) foi de 4,1 pontos e do SAPS II geral foi de 52,6 pontos, o que correspondeu a um risco médio de morte de de 38,4%. Os fatores associados à MRT, em cem dias, foram faixa etária (p=0,0306), presença de infecção (p=0,0216), número de infecções (p=0,0386), ocorrência de enxertia (p<0,0001), uso de ventilação mecânica (p<0,0001) e de drogas vasoativas (p<0,0001). O índice de gravidade SAPS II foi fator preditor para MRT (p=0,0001). Conclusão: O índice de gravidade SAPS II, preditor para MRT em cem dias, mostrou que o paciente submetido ao TCTH é grave e necessita de cuidado especializado e intensivo. / Hematopoietic stem cells transplantation (HSCT) has become a therapeutic procedure accepted worldwide, particularly because of its positive impact on survival and quality of life of patients with onco-hematological diseases. However, the mortality is still high and it is influenced by factors of individual and therapeutic kinds. Objective: To analyze the transplant-related mortality (TRM) on patients submitted to HSCT and its associated factors. Methodology: Prospective cohort study with 60 patients hospitalized in the HSTC unit of the Clinical Hospital of the State University of Campinas (Unicamp). Data was obtained by daily analysis of the medical records. The dependent variable was the TRM and the independent variables were demographic and clinical development, including pre-HSCT risk score (EBMT) and SAPS II. For data analysis were used the Chi-square, Fishers exact tests, Students t-test, Mann-Whitney. On TRM analysis were used Kaplan-Meier and Cox Model method. It was considered a significance level of 5%. Results: The TRM was 15% to a hundred days of HSCT, 18,9% to allogeneic HSCT patients and 8,7% to autologous HSCT. Infection was the main cause of death. In the sample, the median survival time of the patients was 83,2 days (DP 32,7). In the group of non-surviving patients the most were male, with an average age of 48,7 years and the main diagnosis was leukemia. Regarding to the severity of these patients, the pre-HSCT risk score (EBMT) was 4,1 points and general SAPS II was 52,6 points, which corresponds to an average death risk of 38,4%. The TRM associated factors on a hundred days were age (p=0,0306), presence of infection (p=0,0216), number of infections (p=0,0386), occurrence of grafting (p<0,0001), mechanical ventilation use (p<0,0001) and vasoactive drugs (p<0,0001). The severity rate SAPS II was a predictive factor for TRM (p=0,0001). Conclusion: The severity rate SAPS II was predictive for TRM on a hundred days and showed that the patient submitted to HSCT is severe and demands specialized and intensive care.

Lau, Wai Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 141-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

A mucosite bucal (MB) é uma complicação comum no tratamento do câncer e o desenvolvimento de intervenções efetivas para sua prevenção e tratamento são vistos como prioridade nos cuidados de suporte ao paciente oncológico. O objetivo do presente estudo foi investigar fatores de risco relacionados à incidência de mucosite bucal em pacientes submetidos a transplante de células progenitoras hematopoiéticas (TCPH) e em pacientes oncológicos pediátricos. Foram realizados dois estudos: o primeiro analisando a relação entre a incidência de mucosite bucal e o estado de saúde bucal, neutropenia, leucopenia e níveis de IL-1β em pacientes submetidos ao TCPH; e, o segundo, avaliando a incidência de mucosite bucal em pacientes oncopediátricos submetidos a diferentes protocolos quimioterápicos e sua relação com toxicidade hematológica, hepática e renal. Estudo 1: Foram avaliados 54 pacientes submetidos ao TCPH coletados dados demográficos e de à história médica foram coletados. Todos os pacientes foram avaliados quanto a saúde bucal através da análise do índice de placa (IP), índice gengival (IG), número de dentes cariados, perdidos e obturados (CPOD) e exame da mucosa bucal. Todos os pacientes receberam tratamento dentário e orientações de higiene bucal prévio bem como, fotobiomodulação (FBM) com laser de diodo InGaAlP como protocolo preventivo para mucosite bucal. Os pacientes foram avaliados diariamente desde o condicionamento ate o final do transplante. Avaliações de mucosite bucal, níveis de neutrófilos e leucócitos e análise de IL-1β foram realizados nos períodos de condicionamento, D+3 e D+8. Os pacientes que apresentaram gengivite severa anterior ao condicionamento para o transplante e que apresentaram neutropenia grave e leucopenia mostraram associação com o desenvolvimento OM. Os pacientes com mucosite bucal apresentaram níveis mais baixos de IL-1β. Estudo 2: Foram acompanhados 172 ciclos de quimioterapia realizados em 40 pacientes pediátricos. Dados de toxicidade hematológica (níveis de plaquetas, leucócitos, neutrófilos e hemoglobina), hepática (níveis de bilirrubina, TGO, TGP) e renal (creatinina e uréia) nos períodos D1, D5, D10 e D15 foram coletados. Avaliação do grau de mucosite bucal foi realizado diariamente a partir de D1 até D15. Os pacientes que desenvolveram mucosite receberam FBM 3 vezes por semana como tratamento. Os resultados mostraram que a mucosite bucal em pacientes oncológicos pediátricos tem relação com o tipo de protocolo quimioterápico utilizado, com a diminuição nos níveis de plaquetas, leucócitos e hemoglobina bem como, com o aumento dos níveis de bilirrubina. Os níveis de plaquetas e de bilirrubina podem ser considerados como fatores de risco para predizer o desenvolvimento de mucosite bucal. Conclui-se que ambos os trabalhos vieram a contribuir para a elucidação de fatores envolvidos no desenvolvimiento de mucosite bucal em pacientes submetidos ao TCPH e em pacientes oncopediátricos. / Oral mucositis (OM) is a common complication in cancer treatment. The development of effective interventions for prevention and treatment are seen as priority in supportive care cancer patients. The aim of this study was to investigate risk factors related to the incidence of oral mucositis in patients undergoing to hematopoietic stem cells transplantation (HSCT) and in pediatric oncology patients. Two studies were performed: the first analyzing the relationship between oral mucositis incidence with oral health status, neutropenia, leukopenia, and IL-1β levels in patients undergoing to HPCT; and the second, evaluating the incidence of oral mucositis in pediatric oncological patients undergoing to different chemotherapy protocols and their relationship with toxicity haematological, of liver and of kidney. Study 1: A total of 54 patients undergoing to HSCT were collected demographic data and medical history. All patients were evaluated for the oral health through plaque index (PI), gingival index (GI), number of decayed, missing and filled (DMF) and oral mucosa examination. All patients received prior dental and oral hygiene as well as photobiomodulation (PBM) InGaAlP diode laser as a preventive protocol for oral mucositis. Patients were evaluated daily from the conditioning until the end of transplantation. Reviews of oral mucositis, neutrophil ans leukocytes levels and IL- 1β analysis were performed in periods of conditioning, D+3 and D+8. Patients with previous severe gingivitis to conditioning for transplantation and who had severe neutropenia and leukopenia showed association with OM development. The oral mucositis patients had lower levels of IL-1β. Study 2: Wewre analyzed a total of 172 cycles of chemotherapy conducted in 40 oncological pediatric patients. Haematological toxicity data (levels of platelets, leukocytes, neutrophils, and hemoglobin), liver (bilirubin, GOT, GPT) and renal (creatinine and urea) in the periods D1, D5, D10 and D15 were collected. oral mucositis grade evaluation was performed daily from D1 to D15. Patients who developed oral mucositis received three times a week PBM as treatment. The results showed that oral mucositis in pediatric oncology patients is related to the type of chemotherapy protocol used with the decrease in the levels of platelets, leucocytes and hemoglobin as well as with the increase of the bilirubin level. The levels of platelets and bilirubin may be considered as risk factors to predict the development of oral mucositis. We conclude that both studies contributed to the elucidation of factors involved in the development of oral mucositis in patients undergoing HSCT and pediatric oncology patients.

A mucosite bucal (MB) é uma complicação comum no tratamento do câncer e o desenvolvimento de intervenções efetivas para sua prevenção e tratamento são vistos como prioridade nos cuidados de suporte ao paciente oncológico. O objetivo do presente estudo foi investigar fatores de risco relacionados à incidência de mucosite bucal em pacientes submetidos a transplante de células progenitoras hematopoiéticas (TCPH) e em pacientes oncológicos pediátricos. Foram realizados dois estudos: o primeiro analisando a relação entre a incidência de mucosite bucal e o estado de saúde bucal, neutropenia, leucopenia e níveis de IL-1β em pacientes submetidos ao TCPH; e, o segundo, avaliando a incidência de mucosite bucal em pacientes oncopediátricos submetidos a diferentes protocolos quimioterápicos e sua relação com toxicidade hematológica, hepática e renal. Estudo 1: Foram avaliados 54 pacientes submetidos ao TCPH coletados dados demográficos e de à história médica foram coletados. Todos os pacientes foram avaliados quanto a saúde bucal através da análise do índice de placa (IP), índice gengival (IG), número de dentes cariados, perdidos e obturados (CPOD) e exame da mucosa bucal. Todos os pacientes receberam tratamento dentário e orientações de higiene bucal prévio bem como, fotobiomodulação (FBM) com laser de diodo InGaAlP como protocolo preventivo para mucosite bucal. Os pacientes foram avaliados diariamente desde o condicionamento ate o final do transplante. Avaliações de mucosite bucal, níveis de neutrófilos e leucócitos e análise de IL-1β foram realizados nos períodos de condicionamento, D+3 e D+8. Os pacientes que apresentaram gengivite severa anterior ao condicionamento para o transplante e que apresentaram neutropenia grave e leucopenia mostraram associação com o desenvolvimento OM. Os pacientes com mucosite bucal apresentaram níveis mais baixos de IL-1β. Estudo 2: Foram acompanhados 172 ciclos de quimioterapia realizados em 40 pacientes pediátricos. Dados de toxicidade hematológica (níveis de plaquetas, leucócitos, neutrófilos e hemoglobina), hepática (níveis de bilirrubina, TGO, TGP) e renal (creatinina e uréia) nos períodos D1, D5, D10 e D15 foram coletados. Avaliação do grau de mucosite bucal foi realizado diariamente a partir de D1 até D15. Os pacientes que desenvolveram mucosite receberam FBM 3 vezes por semana como tratamento. Os resultados mostraram que a mucosite bucal em pacientes oncológicos pediátricos tem relação com o tipo de protocolo quimioterápico utilizado, com a diminuição nos níveis de plaquetas, leucócitos e hemoglobina bem como, com o aumento dos níveis de bilirrubina. Os níveis de plaquetas e de bilirrubina podem ser considerados como fatores de risco para predizer o desenvolvimento de mucosite bucal. Conclui-se que ambos os trabalhos vieram a contribuir para a elucidação de fatores envolvidos no desenvolvimiento de mucosite bucal em pacientes submetidos ao TCPH e em pacientes oncopediátricos. / Oral mucositis (OM) is a common complication in cancer treatment. The development of effective interventions for prevention and treatment are seen as priority in supportive care cancer patients. The aim of this study was to investigate risk factors related to the incidence of oral mucositis in patients undergoing to hematopoietic stem cells transplantation (HSCT) and in pediatric oncology patients. Two studies were performed: the first analyzing the relationship between oral mucositis incidence with oral health status, neutropenia, leukopenia, and IL-1β levels in patients undergoing to HPCT; and the second, evaluating the incidence of oral mucositis in pediatric oncological patients undergoing to different chemotherapy protocols and their relationship with toxicity haematological, of liver and of kidney. Study 1: A total of 54 patients undergoing to HSCT were collected demographic data and medical history. All patients were evaluated for the oral health through plaque index (PI), gingival index (GI), number of decayed, missing and filled (DMF) and oral mucosa examination. All patients received prior dental and oral hygiene as well as photobiomodulation (PBM) InGaAlP diode laser as a preventive protocol for oral mucositis. Patients were evaluated daily from the conditioning until the end of transplantation. Reviews of oral mucositis, neutrophil ans leukocytes levels and IL- 1β analysis were performed in periods of conditioning, D+3 and D+8. Patients with previous severe gingivitis to conditioning for transplantation and who had severe neutropenia and leukopenia showed association with OM development. The oral mucositis patients had lower levels of IL-1β. Study 2: Wewre analyzed a total of 172 cycles of chemotherapy conducted in 40 oncological pediatric patients. Haematological toxicity data (levels of platelets, leukocytes, neutrophils, and hemoglobin), liver (bilirubin, GOT, GPT) and renal (creatinine and urea) in the periods D1, D5, D10 and D15 were collected. oral mucositis grade evaluation was performed daily from D1 to D15. Patients who developed oral mucositis received three times a week PBM as treatment. The results showed that oral mucositis in pediatric oncology patients is related to the type of chemotherapy protocol used with the decrease in the levels of platelets, leucocytes and hemoglobin as well as with the increase of the bilirubin level. The levels of platelets and bilirubin may be considered as risk factors to predict the development of oral mucositis. We conclude that both studies contributed to the elucidation of factors involved in the development of oral mucositis in patients undergoing HSCT and pediatric oncology patients.

A mucosite bucal (MB) é uma complicação comum no tratamento do câncer e o desenvolvimento de intervenções efetivas para sua prevenção e tratamento são vistos como prioridade nos cuidados de suporte ao paciente oncológico. O objetivo do presente estudo foi investigar fatores de risco relacionados à incidência de mucosite bucal em pacientes submetidos a transplante de células progenitoras hematopoiéticas (TCPH) e em pacientes oncológicos pediátricos. Foram realizados dois estudos: o primeiro analisando a relação entre a incidência de mucosite bucal e o estado de saúde bucal, neutropenia, leucopenia e níveis de IL-1β em pacientes submetidos ao TCPH; e, o segundo, avaliando a incidência de mucosite bucal em pacientes oncopediátricos submetidos a diferentes protocolos quimioterápicos e sua relação com toxicidade hematológica, hepática e renal. Estudo 1: Foram avaliados 54 pacientes submetidos ao TCPH coletados dados demográficos e de à história médica foram coletados. Todos os pacientes foram avaliados quanto a saúde bucal através da análise do índice de placa (IP), índice gengival (IG), número de dentes cariados, perdidos e obturados (CPOD) e exame da mucosa bucal. Todos os pacientes receberam tratamento dentário e orientações de higiene bucal prévio bem como, fotobiomodulação (FBM) com laser de diodo InGaAlP como protocolo preventivo para mucosite bucal. Os pacientes foram avaliados diariamente desde o condicionamento ate o final do transplante. Avaliações de mucosite bucal, níveis de neutrófilos e leucócitos e análise de IL-1β foram realizados nos períodos de condicionamento, D+3 e D+8. Os pacientes que apresentaram gengivite severa anterior ao condicionamento para o transplante e que apresentaram neutropenia grave e leucopenia mostraram associação com o desenvolvimento OM. Os pacientes com mucosite bucal apresentaram níveis mais baixos de IL-1β. Estudo 2: Foram acompanhados 172 ciclos de quimioterapia realizados em 40 pacientes pediátricos. Dados de toxicidade hematológica (níveis de plaquetas, leucócitos, neutrófilos e hemoglobina), hepática (níveis de bilirrubina, TGO, TGP) e renal (creatinina e uréia) nos períodos D1, D5, D10 e D15 foram coletados. Avaliação do grau de mucosite bucal foi realizado diariamente a partir de D1 até D15. Os pacientes que desenvolveram mucosite receberam FBM 3 vezes por semana como tratamento. Os resultados mostraram que a mucosite bucal em pacientes oncológicos pediátricos tem relação com o tipo de protocolo quimioterápico utilizado, com a diminuição nos níveis de plaquetas, leucócitos e hemoglobina bem como, com o aumento dos níveis de bilirrubina. Os níveis de plaquetas e de bilirrubina podem ser considerados como fatores de risco para predizer o desenvolvimento de mucosite bucal. Conclui-se que ambos os trabalhos vieram a contribuir para a elucidação de fatores envolvidos no desenvolvimiento de mucosite bucal em pacientes submetidos ao TCPH e em pacientes oncopediátricos. / Oral mucositis (OM) is a common complication in cancer treatment. The development of effective interventions for prevention and treatment are seen as priority in supportive care cancer patients. The aim of this study was to investigate risk factors related to the incidence of oral mucositis in patients undergoing to hematopoietic stem cells transplantation (HSCT) and in pediatric oncology patients. Two studies were performed: the first analyzing the relationship between oral mucositis incidence with oral health status, neutropenia, leukopenia, and IL-1β levels in patients undergoing to HPCT; and the second, evaluating the incidence of oral mucositis in pediatric oncological patients undergoing to different chemotherapy protocols and their relationship with toxicity haematological, of liver and of kidney. Study 1: A total of 54 patients undergoing to HSCT were collected demographic data and medical history. All patients were evaluated for the oral health through plaque index (PI), gingival index (GI), number of decayed, missing and filled (DMF) and oral mucosa examination. All patients received prior dental and oral hygiene as well as photobiomodulation (PBM) InGaAlP diode laser as a preventive protocol for oral mucositis. Patients were evaluated daily from the conditioning until the end of transplantation. Reviews of oral mucositis, neutrophil ans leukocytes levels and IL- 1β analysis were performed in periods of conditioning, D+3 and D+8. Patients with previous severe gingivitis to conditioning for transplantation and who had severe neutropenia and leukopenia showed association with OM development. The oral mucositis patients had lower levels of IL-1β. Study 2: Wewre analyzed a total of 172 cycles of chemotherapy conducted in 40 oncological pediatric patients. Haematological toxicity data (levels of platelets, leukocytes, neutrophils, and hemoglobin), liver (bilirubin, GOT, GPT) and renal (creatinine and urea) in the periods D1, D5, D10 and D15 were collected. oral mucositis grade evaluation was performed daily from D1 to D15. Patients who developed oral mucositis received three times a week PBM as treatment. The results showed that oral mucositis in pediatric oncology patients is related to the type of chemotherapy protocol used with the decrease in the levels of platelets, leucocytes and hemoglobin as well as with the increase of the bilirubin level. The levels of platelets and bilirubin may be considered as risk factors to predict the development of oral mucositis. We conclude that both studies contributed to the elucidation of factors involved in the development of oral mucositis in patients undergoing HSCT and pediatric oncology patients.

幹細胞療法是近年的研究熱點之一，然而幹細胞在組織修復中的實際應用受到移植後幹細胞存活率低的制約，約80% 的幹細胞在移植至組織後不能存活。 人類臍帶血管周皮 (HUCPV) 幹細胞為多功能間充質幹細胞移植提供豐富的細胞來源。 在合適的誘導環境下，它們具有向多種間充質細胞系分化的能力。 與從骨髓或臍帶血中提取的間充質幹細胞比較，人類臍帶血管周皮幹細胞的體外增殖更為容易。 在本研究中，我們從人類臍帶血管周圍組織中分離人類臍帶血管周皮幹細胞，並採用流式細胞技術分選細胞表面標記物CD34、CD45呈陰性同時CD44 、CD90、 CD105、 CD146呈陽性的HUCPV細胞。HUCPV細胞在體外培養以及三維支架的環境下具有分化為骨和軟骨的能力。 / 在本研究中，我們主要研究腦和生殖器官表達基因（BRE）在HUCPV細胞中的功能。 BRE蛋白與其他已知蛋白的同源性均不高，目前尚未鑑定出任何功能性的結構域。 至今為止，BRE基因的已知功能大多數是通過對腫瘤模型的研究發現的。 據報導，BRE能夠提高DNA損傷的腫瘤細胞的存活率，但BRE在幹細胞中的作用仍不清楚。 我們發現，當HUCPV細胞分化後，其BRE的表達水平降低。 此外，利用BRE-siRNA降低HUCPV細胞中BRE基因的表達，能夠促進HUCPV細胞向骨和軟骨分化的進程。 因此，我們假設BRE對維持HUCPV細胞的幹細胞功能具有重要的作用。 由於經過BRE基因沉默處理的HUCPV細胞與對照組相比並無顯著的表型差別，我們採用微陣列（microarray）以及比較蛋白組學的方法研究兩者間的區別，從而找出BRE基因的功能以及可能涉及BRE的信號通路。 / 通過微陣列技術，我們深入地分析了BRE基因表達沉默後HUCPV細胞的轉錄組。 在經過BRE基因沉默處理的HUCPV細胞中，我們發現與維持幹細胞多向分化潛能有關的OCT4、 FGF5和FOXO1A等基因的表達顯著下調。 另外，BRE基因的沉默能夠影響表觀遺傳調控基因以及TGF-β 信號通路組成部件的表達，而TGF -β 信號通路是維持幹細胞自我更新的重要通路。 這些結果提示，BRE作為一個重要的調控因子，在維持HUCPV細胞的多向分化潛能的同時能夠防止細胞分化。 / 在比較蛋白組學的研究中，我們發現BRE基因的沉默能夠降低細胞骨架結合蛋白的表達，例如actin, annexin II 及 tropomyosin。 此外，我們利用免疫共沉澱的方法證明了BRE蛋白與actin及 annexin II蛋白直接結合。 細胞骨架的改變可能為HUCPV細胞的分化提供了一個有利的環境，因而BRE基因的沉默能夠促進HUCPV細胞向骨和軟骨分化。 支持這一推論的其中一個依據是Lim et al., 2000; Solursh, 1989; Zhang et al., 2006，文獻報導肌動蛋白多聚化抑製劑能夠促進軟骨形成的過程。 綜上所述，本研究為進一步研究BRE基因在HUCPV細胞中的功能以及與BRE直接作用的蛋白打下了基礎。 / Stem cells therapy has gained considerable attention in recent years. However, the practical use of stem cells for tissue repair has been hindered due to their low survival rate after grafting into tissues, for approximately 80% of the stem cells died after implantation. Human umbilical cord perivascular (HUCPV) stem cells offer a new and rich resource of multipotent mesenchymal stem cells. These cells possess the ability to differentiate into various mesenchymal cell lineages when induced. HUCPV cells can be more easily amplified in culture than mesenchymal stem cells extracted from bone marrow or umbilical cord blood. In this study, HUCPV cells were isolated from the perivascular regions of human umbilical cords. The HUCPV cells were sorted using flow cytometer for CD34⁻, CD44⁺, CD45⁻, CD90⁺, CD105⁺ and CD146⁺ surface markers. These HUCPV cells were found to be capable of differentiating into osteogenic lineage in monolayer culture and chondrogenic lineage in pellet culture. These cells were also found to be capable of differentiating into osteogenic and chondrogenic lineage in silk fibroin which acted as three-dimensional scaffolds for the cells to grow on. / The function of the Brain and Reproductive Organ-Expressed (BRE) gene in the context of HUCPV cells was investigated. The BRE protein shares no homology with any other known gene products and contains no known functional domain. To date, most of what we know about the function of this gene has been conducted in the tumor model. It has been reported that BRE can enhance the cellular survival of cancer cells following DNA damage. The role of BRE in stem cells has never been examined. We have established that BRE expression was down-regulated when HUCPV cells started to differentiate. In addition, silencing BRE expression, using BRE-siRNA, in HUCPV cells could accelerate osteogenic and chondrogenic differentiation. Hence, we hypothesized that BRE played an important role in maintaining the stemness of HUCPV cells. Because there was a lack of phenotypic difference between the BRE-silenced HUCPV cells and cells transfected with the control-siRNA, we decided to profile these cells using microarray and proteomic analyses. The aim was to elucidate the function of the BRE gene and establish whether BRE was involved in any signaling pathways. / In the microarray analysis, we examined the transcriptome of HUCPV cells in response to BRE-silencing in depth. Amongst the genes that we identified were significantly down-regulated by BRE-silencing and involved in the maintenance of pluripotency in ES cells were OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and also components of the TGF-β signaling pathway. This pathway is crucially involved in maintaining stem cell self-renewal. Therefore, we propose that BRE acts like a modulator that promotes stemness and at the same time inhibits the differentiation of HUCPV cells. / In the comparative proteomic study, BRE-silencing resulted in decreased expression patterns of cytoskeletal binding proteins such as actin, annexin II and tropomyosin. In addition, co-immunoprecipitation experiments revealed that the BRE protein can bind directly with actin and annexin II. It is possible that altering the cytoskeleton may provide a favorable environment for HUCPV cells to differentiate. This may explain why we were able to accelerate osteogenic and chondrogenic differentiation following BRE-silencing. In support of the view, it has been reported that chondrogenesis could be enhanced after cells have been treated with actin polymerization inhibitors (Lim et al., 2000; Solursh, 1989; Zhang et al., 2006). In sum, our studies provide an insight into the function of the BRE gene in HUCPV cells and the proteins that BRE can directly act on. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Elve. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 135-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / List of Figures --- p.ix / List of Tables --- p.xiii / Table of Abbreviations --- p.xiv / Contents --- p.xviii / Chapter 1 --- p.1 / Literature Review --- p.1 / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.2 --- Embryonic stem cells (ESCs) --- p.2 / Chapter 1.3 --- Epiblast-derived stem (EpiS) cells --- p.2 / Chapter 1.4 --- Somatic stem cells (SSCs) --- p.3 / Chapter 1.5 --- Induced pluripotent stem (iPS) cells --- p.5 / Chapter 1.6 --- Human umbilical cord perivascular (HUCPV) cells --- p.7 / Chapter 1.7 --- CD146 --- p.8 / Chapter 1.8 --- Stem cell senescence --- p.9 / Chapter 1.9 --- Brain and reproductive organ-expressed (BRE) protein --- p.12 / Chapter 1.10 --- Stem cell self-renewal --- p.14 / Chapter 1.11 --- Apoptosis --- p.16 / Chapter 1.12 --- Stem cell niche --- p.21 / Chapter 1.13 --- Stem cell homing --- p.22 / Chapter 1.14 --- Objective --- p.22 / Chapter 2 --- p.24 / Accelerated osteogenic and chondrogenic differentiation of HUCPV cells by modulating the expression of BRE --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Rationale --- p.27 / Chapter 2.3 --- Materials and Methods --- p.27 / Chapter 2.3.1 --- Extraction of HUCPV cells from umbilical cord --- p.27 / Chapter 2.3.2 --- Cell culture condition --- p.28 / Chapter 2.3.3 --- Flow cytometry analysis and cell sorting --- p.28 / Chapter 2.3.4 --- In vitro osteogenic differentiation --- p.29 / Chapter 2.3.5 --- In vitro chondrogenic differentiation --- p.29 / Chapter 2.3.6 --- Alcian blue staining --- p.29 / Chapter 2.3.7 --- Alizarin red S staining --- p.30 / Chapter 2.3.8 --- Immunofluorescence analysis --- p.30 / Chapter 2.3.9 --- Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) --- p.31 / Chapter 2.3.10 --- Transfection with siRNA --- p.35 / Chapter 2.3.11 --- Microarray --- p.35 / Chapter 2.3.12 --- Cell lysis and immunoprecipitation --- p.36 / Chapter 2.3.13 --- SDS-PAGE and Western blot --- p.36 / Chapter 2.3.14 --- Isoelectric focusing and 2-dimensional gel electrophoresis --- p.37 / Chapter 2.3.15 --- Migration (wound healing) assay --- p.38 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- HUCPV cells were capable to differentiate into osteoblasts and chondrocytes --- p.38 / Chapter 2.4.2 --- BRE expression is down-regulated when HUCPV cells begins to differentiate --- p.40 / Chapter 2.4.3 --- Silencing of BRE expression accelerates induction of osteogenesis and chondrogenesis --- p.40 / Chapter 2.4.4 --- Microarray analysis of BRE-silenced HUCPV cells --- p.42 / Chapter 2.4.4.1 --- Stemness factors --- p.43 / Chapter 2.4.4.2 --- Epigenetic regulation --- p.43 / Chapter 2.4.4.3 --- Signaling pathways crucial for stemness maintenance --- p.44 / Chapter 2.4.4.4 --- TGF-β signaling --- p.44 / Chapter 2.4.4.5 --- FGF signaling --- p.44 / Chapter 2.4.4.6 --- NOTCH signaling --- p.45 / Chapter 2.4.4.7 --- WNT signaling --- p.46 / Chapter 2.4.4.8 --- Homeobox transcription factors (HOX) --- p.46 / Chapter 2.4.4.9 --- Cell cycle regulation --- p.47 / Chapter 2.4.4.10 --- Chemokines and cytokines regulation --- p.48 / Chapter 2.4.4.11 --- Apoptosis --- p.49 / Chapter 2.4.5 --- BRE-silencing alters the cellular proteome of HUCPV cells --- p.50 / Chapter 2.4.5.1 --- BRE-silencing alters the cytoskeletal binding proteins of HUCPV cells --- p.51 / Chapter 2.4.5.2 --- BRE-silencing alters the expressions of stemness-related proteins in HUCPV cells --- p.52 / Chapter 2.4.5.3 --- BRE-silencing alters the expressions of apoptosis-related proteins in HUCPV cells --- p.53 / Chapter 2.5 --- Discussion --- p.86 / Chapter 2.5.1 --- Microarray study discussion --- p.87 / Chapter 2.5.2 --- Proteomic study discussion --- p.89 / Chapter 3 --- p.93 / Replicative senescence alters the transcriptome and proteome of HUCPV cells --- p.93 / Chapter 3.1 --- Introduction --- p.93 / Chapter 3.2 --- Materials and methods --- p.93 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Microarray analysis of aged HUCPV cells --- p.94 / Chapter 3.3.1.1 --- Stemness factors --- p.95 / Chapter 3.3.1.2 --- Epigenetic regulation --- p.96 / Chapter 3.3.1.3 --- Senescence associated markers --- p.96 / Chapter 3.3.1.4 --- Chemokines and cytokines regulation --- p.97 / Chapter 3.3.1.5 --- Matrix metalloproteinases regulation --- p.97 / Chapter 3.3.1.6 --- WNT signaling --- p.98 / Chapter 3.3.1.7 --- Toll-like receptor signaling pathway --- p.98 / Chapter 3.3.2 --- Proteomic profiling of aged HUCPV cells --- p.98 / Chapter 3.4 --- Discussion --- p.117 / Chapter 3.4.1 --- Aging alters the transcriptome of HUCPV cells --- p.117 / Chapter 3.4.2 --- Aging alters the proteome of HUCPV cells --- p.118 / Chapter 4 --- p.121 / Osteogenic and chondrogenic differentiation capacities of HUCPV cells in silk fibroin scaffold --- p.121 / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.2 --- Materials and methods --- p.121 / Chapter 4.2.1 --- Extraction of silk fibroin --- p.121 / Chapter 4.2.2 --- Fabrication of porous silk fibroin scaffold --- p.122 / Chapter 4.2.3 --- Scanning electron microscopy --- p.123 / Chapter 4.2.4 --- Cell culture --- p.123 / Chapter 4.3 --- Results --- p.124 / Chapter 4.4 --- Discussion --- p.132 / Chapter 5 --- p.133 / Conclusions --- p.133 / References --- p.135

皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能. / 本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。 / 本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子. / 本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。 / 結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性. / The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored. / The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks. / The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates. / The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes. / In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cai, Yijun. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-129). / Abstract also in Chinese. / Abstrac --- p.i / Table of Contents --- p.v / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Chapter Chapter I --- Introduction --- p.1 / Skin --- p.3 / Wound healing --- p.6 / Wound regeneration and repair --- p.6 / Recent history of wound treatment --- p.9 / Skin substitutes --- p.11 / Stem cells for wound treatment --- p.14 / Stem cells overview --- p.15 / Adult stem cells --- p.16 / Fetal stem cells --- p.18 / Amniotic membrane derived stem cells --- p.19 / Umbilical cord stem cells --- p.22 / Hypothesis and Specific aims --- p.24 / Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28 / Introduction --- p.28 / Materials and methods --- p.30 / Results --- p.47 / Discussion --- p.62 / Conclusion --- p.67 / Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69 / Introduction --- p.69 / Materials and methods --- p.72 / Results --- p.75 / Discussion --- p.83 / Conclusion --- p.88 / Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89 / Introduction --- p.89 / Materials and methods --- p.91 / Results --- p.94 / Discussion --- p.101 / Conclusion --- p.104 / Chapter Chapter V --- Summary and Future Plan --- p.105 / Summary --- p.105 / Future plan --- p.108 / Acknowledgements --- p.113 / References --- p.114 / Appendix --- p.130

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. Little is known about the changes in the bone marrow (BM) and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, we have previously reported that hematopoietic stem cells (HSC) undergo a significant expansion in the BM accompanied with myeloid suppression. This bone marrow response was Toll-like Receptor 4 (TLR4)-dependent. TLR4 is activated by bacterial lipopolysaccharide (LPS) and signals through two major independent downstream molecules: TRIF and MyD88. In the present study, I found that the TLR4/TRIF and the TLR4/MyD88 pathways contribute in a distinct manner to the BM response to P. aeruginosa's LPS. TRIF plays a major role in the expansion of the HSC pool, whereas MyD88 is required for myeloid suppression. Following LPS stimulation, HSCs enter in the cell cycle, expand and exhaust when transplanted in healthy mice. Loss of TRIF rescued completely the long-term engraftment and multilineage reconstitution potential of septic HSCs, but did not affect myeloid differentiation. Conversely, MyD88 deficiency prevented completely the myeloid suppression in the myeloid progenitors, but conferred limited protective effects on the HSC function. It is of great therapeutic value to identify the downstream molecules involved in TLR4/MyD88 dependent myeloid suppression. I found miR-21, a microRNA that is involved in inflammation, was up-regulated upon LPS challenge in a MyD88-dependent manner. However, deletion of miR-21 in the BM did not rescue LPS-induced bone marrow dysfunction, demonstrating that miR-21 is not a critical regulator in these processes. Further studies are warranted to determine the precise molecular mechanisms involved in the complex pathogenesis of BM response to sepsis. Taken together, my results show for the first time that the TLR4/TRIF signaling as a key mediator of HSC damage during acute LPS exposure and that activation of the TLR4/MyD88 signaling pathway play a dominant role in myeloid suppression. These results provide novel insights into our understanding of the molecular mechanisms underlying bone marrow injury during severe sepsis and may lead to the development of new therapeutic approaches in this disease.