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Previous issue date: 2016-07-29 / CNPQ / FACEPE / A regulação do volume celular é importante em vários processos fisiológicos. A diminuição regulatória do volume (RVD) é o processo pelo qual as células recuperam o seu volume após terem sido submetidas a um choque hipoosmótico. Este processo se deve principalmente à liberação de K⁺ e Cl⁻ através de canais e transportadores iônicos. O objetivo deste trabalho foi estudar a participação dos canais de potássio e canais de cloreto envolvidos na RVD e na proliferação das células-tronco mesenquimais da geléia de Wharton do cordão umbilical humano (hWJ-MSCs). As hWJ-MSCs foram isoladas de acordo com a técnica de migração espontânea do explante. As células foram cultivadas em meio DMEM suplementado com 15 % soro fetal bovino, 10 % F-12, 100 U/ml de penicilina e 100 μg/ml de estreptomicina e mantidas em atmosfera de 5% de CO₂ a 37 °C. Nos experimentos da RVD, as hWJ-MSCs foram depositadas em uma cubeta acoplada a um microscópio invertido com um sistema de vídeo imagem, sendo submetidas inicialmente a um choque hipoosmótico (300 mOsm→200 mOsm) por perfusão. A dinâmica de variação do volume foi monitorada por 30 min e as imagens antes (300 mOsm) e durante o choque hipoosmótico (200 mOsm) foram obtidas a cada minuto e analisadas usando o software ImageJ. As hWJ-MSCs foram submetidas aos seguintes inibidores de canais: tetraetilamônio (TEA) 10 mM, (canal de Kv); glibenclamida (GB) 100 µm, (canal de Kir6.x); 4-aminopiridina (4-AP) 5 mM, (canal de Kv1, KCNA), Iberiotoxina (IBTX) 10 nM (canal BKca), (ácido 5-nitro-2-(3-fenilpropilamino) benzóico (NPPB), (canal de Cl⁻ ), ácido disulfônico di-isocianatoestilbeno (DIDS) 100 µm e Tamoxifeno (TAM) 10 µm. Posteriormente, as células foram submetidas aos ensaios de MTT, curva de crescimento, quantificação do conteúdo de DNA e RT-PCR. A técnica de patch clamp na configuração Whole-cell foi empregada para caracterização das correntes iônicas. Os dados foram analisados usando o teste t Student’s ou ANOVA, com pós-teste de Tukey ou de Bonferroni. Um valor de p ≤ 0,05 foi considerado estatisticamente significativo. A RVD presente nas hWJ-MSCs foi suprimida na presença dos inibidores de canais de potássio e de canais de cloreto. As hWJ-MSCs apresentaram três perfis de corrente iônica: Maxi K (BK ou Kca); corrente de potássio transiente de saída (Ito) e uma corrente de retificação tardia (Kᴅʀ). As hWJ-MSCs na presença dos inibidores de canais iônicos reduziram a proliferação permanecendo na fase G0/G1 do ciclo celular. As hWJ-MSCs expressaram os canais de potássio (MaxiK, KCND2, KCND3, KCNS1, KCNH2 e KCNH1) e os canais de cloreto (pl-VDAC e CLCN3). Deste modo, conclui-se que os canais de potássio dependentes de voltagem (Kv), canais de potássio dependentes de ATP, ativados por cálcio de alta condutância (Kca) e canais de cloreto participam no mecanismo da RVD e consequentemente interferem na proliferação das hWJ-MSCs. / Cell volume regulation is one of the most fundamental homeostatic mechanisms and essential for normal cellular function. The phenomenon of shrinkage and / or cell swelling caused by osmotic changes are called regulatory volume increase (RVI) and regulatory volume decrease (RVD), respectively. The RVD is the process by which cells recovery its volume after being subjected to hypoosmotic environment. This process occurs due to release K⁺ and Cl⁻ through ion channels and transporters. The aim of this study was to investigate the involvement of ionic channels involved in RVD and proliferation of mesenchymal stem cells from Wharton's Jelly of the human umbilical cord (hWJ-MSCs). The hWJ-MSCs were isolated according to the spontaneous migration of the explant technique. Cells were grown in DMEM supplemented with 15% fetal bovine serum, 10 % F-12, 100 U / ml penicillin and 100 ug / ml streptomycin and maintained in an atmosphere of 5 % CO₂ at 37 °C. In the experiments the RVD, the hWJ-MSCs were placed in a chamber coupled to an inverted microscope with a video image system and initially subjected to shock hypo-osmotic (300 mOsm → 200 mOsm) perfusion. The dynamics of volume change was monitored for 30 minutes before (300 mOsm) and during hypo-osmotic shock (200 mOsm) and the images (every min) analyzed using the ImageJ software. Specific cation channel inhibitors such as tetraethylammonium (TEA) 10 mM (Kv channel); glibenclamide (GB) 100 μm (Kir6.x channel); 4-aminopyridine (4-AP) 5 mM (Kv1 channel KCNA), iberotoxin (IBTX) 10 nM (BKCa channel) and cellular anion inhibitors (5-Nitro-2- (3-phenylpropylamino) benzoic acid (NPPB) (Cl⁻channel), disulfonic acid di-isocianatoestilben- (DIDS) 100 μm (Cl⁻ channel) and tamoxifen (TAM) 10 μm (Cl- channel) were used as molecular tools to evaluate the influence of ion channels in regulate mechanism RVD and cell proliferation. Subsequently, the cells were subjected to MTT assay, growth curve, flow cytometer to quantify the DNA content and RT-PCR. The patch clamp technique in Whole-cell configuration was employed to characterize the ionic currents. For statistical analysis, one-way ANOVA followed Tukey's or Bonferroni post hoc test were performed. A p-value less than 0.05 were considered statistically significant. The RVD in hWJ-MSCs was abolished in the presence of ionic channels inhibitors (potassium and chloride). The electrophysiological recording presents, at least, three different profiles compatible with: noisy current as a Maxi K current (BK or Kca), transient outward K+ current (Ito) and a slower activating delayed rectifier (Kᴅʀ). Also showed that hWJ- MSCs in the presence of the ionic channel inhibitors reduced the cell proliferation and arrest cells in G0/G1 cell cycle stage. As hWJ-MSCs expressed of the potassium channels (MaxiK, KCND2, KCND3, KCNS1, KCNH2 and KCNH1) and chloride channels (pl-VDAC and CLCN3). The results showed that there are a relationship of cell cycle progression and membrane permeability. Thus, we concluded that there is participation of KATP, BKCa Kv and Cl⁻ channels in the mechanism of RVD and proliferation of hWJ-MSCs.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufpe.br:123456789/25053 |
| Date | 29 July 2016 |
| Creators | ALBERTIM, Gisely Juliane Barbosa de |
| Contributors | http://lattes.cnpq.br/0681322721384641, RODRIGUES, Claudio Gabriel, SILVA, Marcia Bezerra da |
| Publisher | Universidade Federal de Pernambuco, Programa de Pos Graduacao em Inovacao Terapeutica, UFPE, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFPE, instname:Universidade Federal de Pernambuco, instacron:UFPE |
| Rights | Attribution-NonCommercial-NoDerivs 3.0 Brazil, http://creativecommons.org/licenses/by-nc-nd/3.0/br/, info:eu-repo/semantics/openAccess |
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