本文選題：骨髓間充質(zhì)干細(xì)胞 + 心肌細(xì)胞�。� 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： 目的:通過體外實(shí)驗(yàn),觀察化學(xué)誘導(dǎo)劑和模擬的生物微環(huán)境誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向心肌細(xì)胞分化。 方法:取6~8w齡SD大鼠股骨骨髓,培養(yǎng)至第二代,經(jīng)流式細(xì)胞儀檢測(cè)骨髓間充質(zhì)干細(xì)胞純度97%以上,然后分成五組:A組,單純DMEM細(xì)胞培養(yǎng)組(對(duì)照組);B組,5-氮雜胞苷誘導(dǎo)組;C組,心肌細(xì)胞裂解液組;D組,心肌細(xì)胞裂解液組+5-氮雜胞苷組;E組,細(xì)胞間接接觸組。根據(jù)各自實(shí)驗(yàn)條件誘導(dǎo)至第2w和第4w時(shí),收獲細(xì)胞。應(yīng)用免疫組化法檢測(cè)誘導(dǎo)后骨髓間充質(zhì)干細(xì)胞表達(dá)心肌特異性肌鈣蛋白T(cTnT)、連接蛋白43(CX-43),血管內(nèi)皮細(xì)胞表面抗原標(biāo)志CD31及肌性標(biāo)志α-橫紋肌肌動(dòng)蛋白(α-SCA)的表達(dá)情況。應(yīng)用RT-PCR法檢測(cè)各組早期心肌細(xì)胞特異性轉(zhuǎn)錄因子GATA4、NKX2.5、β-MHC及成熟心肌細(xì)胞特異性轉(zhuǎn)錄因子α-MHC基因的表達(dá)情況,并進(jìn)行簡(jiǎn)單量的比較。 結(jié)果:免疫組化檢測(cè):實(shí)驗(yàn)組誘導(dǎo)2w后,cTnT、CX-43表達(dá)陰性或低表達(dá),誘導(dǎo)4w后呈陽性表達(dá),實(shí)驗(yàn)組較對(duì)照組有統(tǒng)計(jì)學(xué)差異(P0.05),實(shí)驗(yàn)組之間無顯著差異(P0.05);對(duì)照組及B組無CD31表達(dá),C, D, E組表達(dá)呈弱陽性,組間無統(tǒng)計(jì)學(xué)差異(P0.05);α-SCA誘導(dǎo)前骨髓間充質(zhì)細(xì)胞中既有低度表達(dá),誘導(dǎo)后濃度明顯增高,實(shí)驗(yàn)組間無差異(P0.05)。 RT-PCR表達(dá)情況:實(shí)驗(yàn)組誘導(dǎo)2w后條帶不明顯;誘導(dǎo)4w后均表達(dá)GATA4, NKX2.5,β-MHC,無α-MHC表達(dá),從條帶密度和亮度分析,實(shí)驗(yàn)組較對(duì)照組有顯著差異(P0.05),實(shí)驗(yàn)組間無顯著差異(P0.05)。 結(jié)論: 1. 5-氮雜胞苷可以誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向心肌細(xì)胞分化。 2.骨髓間充質(zhì)干細(xì)胞因其“環(huán)境依賴性分化”特性,具有分化為心肌細(xì)胞的潛能。心肌細(xì)胞裂解液和細(xì)胞間接接觸模擬的心肌微環(huán)境也可以誘導(dǎo)骨髓間充質(zhì)干細(xì)胞分化成心肌樣細(xì)胞,且誘導(dǎo)的效果與誘導(dǎo)的時(shí)間及誘導(dǎo)劑濃度相關(guān),分化的細(xì)胞介于成熟的心肌細(xì)胞和心肌祖細(xì)胞之間的心肌細(xì)胞前體。
[Abstract]:Aim: to observe the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes induced by chemical inducers and simulated microenvironment in vitro. Methods: bone marrow of SD rats aged 6 to 8 weeks was cultured to the second generation. The purity of bone marrow mesenchymal stem cells was detected by flow cytometry. The bone marrow mesenchymal stem cells were divided into five groups: group A, group B (control group) and group C (group C) induced by 5-azacytidine. Group D was treated with cardiomyocyte lysate, group E with 5-azacytidine, group with indirect cell contact. According to the experimental conditions, the cells were harvested at the 2nd and 4th week. The expression of myocardial specific troponin T (cTnT), connexin 43 (CX-43), CD31 and 偽 -rhabdomyactin (偽 -SCA) in bone marrow mesenchymal stem cells (BMSCs) were detected by immunohistochemistry. RT-PCR was used to detect the expression of NKX2.5, 尾 -MHC and 偽 -MHC genes in the early cardiac myocyte specific transcription factor GATA4, NKX2.5, and mature cardiomyocyte specific transcription factor 偽 -MHC, and a simple comparison was made. Results: the expression of cTnTnTnTnTnTnCX-43 in the experimental group was negative or low after 2 weeks of induction, and was positive after 4 weeks of induction. There was statistical difference between the experimental group and the control group (P0.05), there was no significant difference between the experimental group and the experimental group (P0.05); in the control group and group B, the expression of CD31 was weakly positive, but there was no statistical difference between the two groups (P0.05); there was a low expression in the bone marrow mesenchymal cells before 偽 -SCA induction. After induction, the concentration of GATA4, NKX2.5, 尾 -MHC and 偽 -MHC were significantly increased (P0.05). The expression of RT-PCR: the bands were not obvious after 2 weeks of induction, but GATA4, NKX2.5, 尾 -MHCand 偽 -MHC were all expressed at 4 weeks after induction. There was significant difference between the experimental group and the control group (P0.05), but there was no significant difference between the experimental group (P0.05). Conclusion: 1. 5-azacytidine could induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes. 2. Bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into cardiomyocytes due to their environmental dependent differentiation. Myocardial cell lysate and cell contact with the simulated myocardial microenvironment could also induce bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells, and the induction effect was related to the time of induction and the concentration of inducer. Differentiated cells are precursors of cardiac myocytes between mature cardiomyocytes and cardiac progenitor cells.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 閆穎穎;兔骨髓間充質(zhì)干細(xì)胞向心肌樣細(xì)胞的體外誘導(dǎo)分化研究[D];西北農(nóng)林科技大學(xué);2010年

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本文編號(hào)：2080066