目的 黑色素瘤是一种高度恶性肿瘤，极易耐药。而肿瘤细胞的免疫逃逸很可能是产生耐药的原因之一。参茋扶正注射液（SQFZ）广泛应用于联合放化疗药物治疗癌症，且具有一定的临床疗效基础。通过研究参茋扶正注射液（SQFZ）对A375细胞分泌的免疫抑制因子的作用和影响和研究SQFZ是否可以提高jurkat细胞的功能，并间接对肿瘤细胞起到杀伤作用。探讨SQFZ对抑制黑色素瘤细胞A375免疫逃逸的影响和作用机制，以及SQFZ用于治疗黑色素瘤的可能性。 方法 外培养黑色素瘤细胞A375，应用不同的SQFZ干预细胞。 1. 采用MTT技术检测SQFZ对A375的直接作用； 2. 采用Real-Time PCR技术检测SQFZ对A375细胞表达免疫抑制因子IL-10、TGF-beta、VEGF的影响； 3. 用ELISA检测SQFZ对A375细胞分泌IL-10、TGF-beta、VEGF的影响； 4. 建立transwell小室模型，检测SQFZ对jurkat T细胞迁徙功能的影响； 5. 利用transwell小室，建立共培养体系, 检测SQFZ对jurkat T细胞分泌细胞因子功能的影响。 结果 1. SQFZ浓度为1280μg/mL时，对A375细胞有抑制作用，与空白组比较，差异具有统计学意义（p<0.01），但抑制率低于20%，无研究意义； 2. 不同浓度（1280μg/mL、640μg/mL)SQFZ干预A375细胞24h，48h后，可以降低IL-10、TGF-beta 在mRNA水平的表达，与对照组比较结果有统计学意义(p<0.05），并且抑制呈时间剂量依赖关系；SQFZ(l280μg/mL）干预A375細胞48h后，VEGF在mRNA水平的表达降低，与对照组比较结果有统计学 意义（p<0.05); 3. 经不同浓度SQFZ处理24h后，A375细胞IL-10在SQFZ浓度为640μg/mL表达降低，；VEGF-beta、VEGF在SQFZ浓度为1280μg/mL、640μg/mL 时，蛋白水平表达降低，以上结果均与对照组比结果有统计学意义（p<0 .05); 4. 经不同浓度SQFZ处理A375细胞48h后，jurkat T细胞相对迁徙面积和穿膜细胞数变化显著，与对照组比较结果具有统计学意义（p

No description available.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 1991.

Les cellules souches pluripotentes d’origines embryonnaires (cellules hES) ou induites à la pluripotence (cellules iPS) possèdent deux propriétés essentielles, l’autorenouvellement et la pluripotence. ces deux propriétés font de ces cellules une ressource biologique unique pour l’étude des mécanismes moléculaires et cellulaires impliqués dans le développement embryonnaire précoce. l’objectif de mes travaux a, dans un premier temps, été de mettre en place des modèles expérimentaux permettant l’engagement des cellules hES et iPS dans les deux principaux types cellulaires de l’épiderme : les kératinocytes et les mélanocytes. J’ai ainsi participé à l’élaboration de protocoles de différentiation permettant d’engager les cellules hES et iPS dans les lignages épithéliaux et mélanocytaires. Enfin sur la base de ces résultats, la dernière partie de ma thèse a eu pour but de comprendre le rôle des microarns dans le contrôle de ces mécanismes développementaux. L’ensemble de ces données démontre que les cellules souches pluripotentes représentent un modèle pertinent pour étudier les mécanismes cellulaires et moléculaires impliqués dans le contrôle du développement embryonnaire humain. Nous avons ainsi démontré que la différenciation des cellules souches pluripotentes dans le lignage épidermique respectait la chronobiologie du développement et mis en évidence le rôle régulateur de deux microarns dont les fonctions n’avaient pas été identifiées, à ce jour, dans le développement embryonnaire humain. / Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) are characterized by their two fundamental properties: pluripotency and self-renewal. Due to these unique capacities, pluripotent stem cells offer a virtually unlimited biological resource capable to differentiate into any cell type of the organism. In parallel to their great potential for regenerative medicine, these cells represent a unique in vitro model of early human development. The aim of this thesis is to investigate the molecular and cellular events occurred during epidermis formation in the Human. The first objective of this work was to set up protocols to differentiate pluripotent stem cells, (hES and iPS) into the two major cell types of epidermis: Keratinocytes and Melanocytes. In parallel to this work the last part of my thesis was to study the role of a new class of developmental regulator: The microRNAs. Taken together, all these data demonstrate that human pluripotent stem cells represent a unique model to study molecular and cellular mechanisms involved in early embryonic development in the Human. By defining experimental procedure to differentiate these cells into pure populations of keratinocytes, melanocytes and neural progenitors we have been able to demonstrate that pluripotent stem cells follow the entire chronobiology of embryonic development and highlighted the regulatory functions of two microRNAs previously described in the mouse but never identified in the Human.

published_or_final_version / Anatomy / Master / Master of Philosophy

No description available.

Yes / Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10–15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2–related factor 2 (Nrf2)–dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.

A study was undertaken to determine whether cytogenetic abnormalities can be identified in an invasive melanoma cell population that has been selected in vitro out of a larger cell population of low invasive potential. The selecting agent was a denuded human amniotic membrane situated within Mega-Membrane Invasion Culture System chambers. Invasive cells were collected, grown, and harvested for cytogenetic analysis. Metaphases of these cells were examined for chromosomal abnormalities and for evidence of gene amplification in the form of double minute chromosomes. Invasive cell lines evinced changes in their degree of aneuploidy which were not seen in parental control lines of the same passage number. Significant karyotypic abnormalities identified in invasive cell lines were an increased dosage of chromosome 7 and multiple 1q translocation marker chromosomes. Double minute chromosomes were found in up to 18% of invasive cell metaphases examined and in 3% of parental controls. The incidence of double minutes was found to decrease as a function of passage number.

Hypomelanosis, a severe reduction in pigmentation, is a widespread phenomenon which affects many different vertebrate species, including humans and chickens. The cause(s) of various forms of hypomelanosis is (are) not known. The aim of this study was to determine the cause of hypomelanosis in a breed of white chickens (White Plymouth Rock x Pile Game). It was hoped that this hypomelanotic breed may provide insight into the etiopathogenesis of certain human hypomelanotic disorders, such as vitiligo and albinism. To determine whether melanocytes are present in the hypomelanotic skin, two melanocyte-specific assays were carried out, in situ DOPA histochemistry and a sensitive radiometric assay for tyrosinase. The results show that active tyrosinase was present in 8, 9 and 10 day skins. However, unlike normal black skin, the level of tyrosinase did not increase with age, suggesting that the melanocytes either die or that they do not continue to synthesise tyrosinase. Ultrastructurally, these melanocytes appeared to be morphologically normal and they did not show signs of premature degeneration. Unlike black chick melanocytes, however, they contained very few premelanosomes and fully melanised melanosomes were never observed, suggesting that hypomelanosis results from the arrested development (melanisation) of melanosomes in vivo. Two different experiments were carried out to determine whether this blockage in melanogenesis is intrinsic in the melanocyte or whether it is caused by extrinsic environmental factors. The outcome of these studies were conflicting: 1) In culture, white chick neural crest cells produced pigment, suggesting that the melanocyte is not defective. However, ultrastructural examination of these cultured melanocytes showed that they contained a large proportion of partially melanised melanosomes. 2) Black chick neural crest cells migrated into white skin explants and contributed towards pigment in the developing feathers, suggesting that the white chick tissue environment is also not defective. The results hint that hypomelanosis in the white chicks is caused by the interaction of at least two genetic defects: an intrinsic mutation of the melanocyte, as well as an extrinsic mutation in the melanocyte environment that, in combination, exert an inhibitory influence on melanin synthesis. This study shows that, in situ, white chick melanocytes share some features with ty-pos albino melanocytes and may be representative of this pigmentary disorder. White Plymouth Rock x Pile Game chicks may also be useful as a model for the multi-faceted disorder, vitiligo.