【摘要】：目的:探討過表達GATA-4小鼠骨髓間充質(zhì)干細胞(BMSCs)通過分泌外排體(exosome)修復(fù)心肌損傷的機制。方法:構(gòu)建過表達GATA-4的小鼠BMSCs并提取其分泌的exosome。而后將GATA-4-BMSCs-exosome與BMSCs共同培養(yǎng),空載體-BMSCs-exosome與BMSCs共同培養(yǎng),BMSCs-exosome與BMSCs共同培養(yǎng)、BMSCs單獨培養(yǎng)及小鼠心肌細胞單獨培養(yǎng)48h,采用Q-PCR定量檢測心肌肌鈣蛋白T(cTnT)、α肌動蛋白(α-actin)、連接蛋白43(connexin 43)、結(jié)蛋白(Desmin)心肌特異性抗原表達量。再將過表達GATA-4BMSCs分泌的exosome與心肌細胞共培養(yǎng),空載體-BMSCs分泌的exosome與心肌細胞共培養(yǎng),BMSCs分泌的exosome與心肌細胞共培養(yǎng)及心肌細胞單獨用于低氧培養(yǎng)無血清培養(yǎng)誘導(dǎo)細胞凋亡作為陽性對照培養(yǎng)48h誘導(dǎo)細胞凋亡。采用流式細胞技術(shù)檢測各組細胞的凋亡率。進一步采用TMT標記定量蛋白質(zhì)組學(xué)分析過表達GATA-4小鼠BMSCs分泌exosome內(nèi)有關(guān)細胞分化及抗凋亡的蛋白。結(jié)果:Q-PCR顯示:過表達GATA-4-BMSCs分泌的exosome可以有效促進BMSCs向心肌細胞轉(zhuǎn)化并具有極強的抗凋亡能力。蛋白質(zhì)譜提示有8個蛋白有關(guān)細胞分化。有6個蛋白有關(guān)細胞調(diào)亡。其中Slit homolog 2protein及Connective tissue growth factor蛋白既涉及細胞凋亡又涉及細胞分化。結(jié)論:過表達GATA-4-BMSCs分泌的exosome可以促進BMSCs向心肌細胞分化并具有抗凋亡能力。Slit homolog 2protein及Connective tissue growth factor蛋白既涉及細胞凋亡又涉及細胞分化。
[Abstract]:Aim: to investigate the mechanism of over-expression of GATA-4 mouse bone marrow mesenchymal stem cells (BMSCs) in repairing myocardial injury by secreting efflux (exosome). Methods: the mouse BMSCs expressing GATA-4 was constructed and the exosome. secreted by it was extracted. Then GATA-4-BMSCs-exosome was co-cultured with BMSCs, empty vector-BMSCs-exosome was co-cultured with BMSCs, BMSCs was cultured alone and mouse cardiomyocytes were cultured for 48 h. The expression levels of cardiac troponin T (cTnT), 偽 actin 43 (connexin 43) and desmin (Desmin) myocardial specific antigen were detected by Q-PCR. The exosome secreted by GATA-4BMSCs was co-cultured with cardiomyocytes, and the exosome secreted by empty vector-BMSCs was co-cultured with cardiomyocytes. Exosome secreted by BMSCs was co-cultured with cardiomyocytes and cardiomyocytes were used alone in hypoxia culture to induce apoptosis in serum-free culture as a positive control culture for 48 hours. The apoptosis rate of each group was detected by flow cytometry. Furthermore, TMT labeled quantitative proteomics was used to analyze the proteins related to cell differentiation and anti-apoptosis in exosome secreted by over-expressed BMSCs in GATA-4 mice. Results: Q-PCR showed that over-expression of exosome secreted by GATA-4-BMSCs could effectively promote the transformation of BMSCs into cardiomyocytes and had strong anti-apoptosis ability. The protein profiles suggest that there are 8 proteins related to cell differentiation. There are six proteins involved in cell apoptosis. Among them, Slit homolog 2protein and Connective tissue growth factor proteins are involved in both apoptosis and differentiation. Conclusion: overexpression of exosome secreted by GATA-4-BMSCs can promote the differentiation of BMSCs into cardiomyocytes and has the ability of anti-apoptosis. Slit homolog 2protein and Connective tissue growth factor proteins are involved in both apoptosis and differentiation of cardiomyocytes.
【作者單位】： 云南省第一人民醫(yī)院心臟大血管外科;
【基金】：國家自然科學(xué)基金(No:81460073,31460298) 云南省科技廳-昆明醫(yī)科大學(xué)應(yīng)用基礎(chǔ)研究聯(lián)合專項(No:2014FB089) 云南省教育廳科學(xué)研究基金(No:2015Z051) 中國博士后科學(xué)基金(No:2015M582764XB) 成都醫(yī)學(xué)院2015年度科研項目(No:CYZ15-18) 云南省醫(yī)學(xué)后備人才(No:H-201607)
【分類號】：R542.2

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