發(fā)布時(shí)間：2018-07-27 19:38

【摘要】： 目的:探討體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞(human bone marrow-derived mesenchymal stem cells, hBMSCs)定向誘導(dǎo)分化為心肌樣細(xì)胞的實(shí)驗(yàn)條件。 方法:取成人棄骨骨髓,通過全骨髓貼壁法分離培養(yǎng)hBMSCs。在傳至第三代時(shí),篩選具有心肌特異性分化潛能的c-kit~+的hBMSCs細(xì)胞克隆,用含20%胎牛血清的低糖DMEM培養(yǎng)液繼續(xù)克隆培養(yǎng)。當(dāng)細(xì)胞達(dá)到70%融合時(shí),分別用10μmol/l5-氮胞苷(5-azacytidine crystalline,5-aza),100mg/l參麥注射液,10μmol/l5-氮胞苷和100mg/l參麥注射液3組誘導(dǎo)劑誘導(dǎo)hBMSCs。24小時(shí)后,更換為含10%胎牛血清的低糖DMEM培養(yǎng)液繼續(xù)培養(yǎng)。在誘導(dǎo)3周后,通過顯微鏡下觀察其形態(tài)變化。用免疫細(xì)胞化學(xué)方法檢測(cè)心肌特異性標(biāo)記抗原Desmin和肌鈣蛋白T的表達(dá);用RT-PCR方法檢測(cè)心肌特異基因心房利尿肽(atrial natriuretic peptide,ANP)和腦鈉肽(brain natriuretic peptide,BNP)表達(dá)。 結(jié)果:骨髓中含有多種hBMSCs細(xì)胞克隆,其中成纖維細(xì)胞樣克隆表達(dá)c-kit~+。分別用3組誘導(dǎo)劑誘導(dǎo)c-kit~+細(xì)胞15天后,部分細(xì)胞胞體明顯增粗,可見有些細(xì)胞間有融合樣現(xiàn)象發(fā)生。誘導(dǎo)3周后,3組都有類似肌管樣細(xì)胞出現(xiàn),細(xì)胞短粗,呈不規(guī)則的圓柱形,有分支,互相連成網(wǎng)。蘇木精-伊紅(hematoxylin-eosin HE)染色后,3組細(xì)胞都排列規(guī)則,類似于心肌細(xì)胞的形態(tài),可見部分細(xì)胞有排列規(guī)則的橫紋樣結(jié)構(gòu)。免疫細(xì)胞化學(xué)顯示誘導(dǎo)3周后的hBMSCs,Desmin陽性細(xì)胞數(shù)目分別是:5-aza組(65.3±4.7%),參麥注射液組(62.3±3.9%),5-aza和參麥注射液組(68.5±2.1%),3組Desmin陽性細(xì)胞表達(dá)率無顯著差異; cTnT陽性細(xì)胞數(shù)目分別是:5-aza組(62.2±4.1%),參麥注射液組(63.8±3.4%), 5-aza和參麥注射液組(66.0±6.2%),3組Desmin陽性細(xì)胞表達(dá)率無顯著差異。陰性對(duì)照組和未分化的hBMSCs均無Desmin和cTnT的表達(dá)。RT-PCR顯示3種不同的誘導(dǎo)劑誘導(dǎo)hBMSCs3周后都有ANP,BNP的表達(dá),未分化的hBMSCs無ANP,BNP的表達(dá)。 結(jié)論:5-aza和中藥可以誘導(dǎo)c-kit~+的hBMSCs細(xì)胞克隆分化為心肌樣細(xì)胞。體外誘導(dǎo)成的心肌樣細(xì)胞表現(xiàn)為未成熟心肌細(xì)胞的結(jié)構(gòu)和功能特征。證明骨髓中含有多種干細(xì)胞克隆,c-kit可作為篩選hBMSCs向心肌樣細(xì)胞分化的一項(xiàng)檢測(cè)指標(biāo)。篩選具有心肌特異性分化潛能的hMSCs可提高誘導(dǎo)分化率。
[Abstract]:Objective: To investigate the experimental conditions of induced differentiation of human bone marrow mesenchymal stem cells (human bone marrow-derived mesenchymal stem cells, hBMSCs) into cardiomyocytes in vitro.
Methods: the bone marrow of adult discarded bone was taken from the whole bone marrow adherent method to isolate and culture hBMSCs. in the third generation, and the c-kit~+ hBMSCs cell clones with the specific differentiation potential of the myocardium were screened. The low sugar DMEM culture medium containing 20% fetal bovine serum was continued to be cloned and cultured. When the cells reached 70% fusion, 10 micron mol/l5- azacytidine (5-azacytidine) was used respectively. Crystalline, 5-aza), 100mg/l Shenmai injection, 10 mol/l5- azacytidine and 100mg/l Shenmai injection 3 inducers were induced for hBMSCs.24 hours, and changed into low sugar DMEM culture solution containing 10% fetal bovine serum. After 3 weeks of induction, the morphological changes were observed under the microscope. The specific markers of cardiac muscle were detected by immunocytochemical method. The expression of antigen Desmin and troponin T; the expression of cardiac specific gene atrial natriuretic peptide (ANP) and brain natriuretic peptide (brain natriuretic peptide, BNP) by RT-PCR method.
Results: there were a variety of hBMSCs cell clones in the bone marrow, in which the fibroblast like clone and expression c-kit~+. induced c-kit~+ cells with 3 groups of inducers for 15 days, and some of the cell bodies were obviously thickened, and some of the cells had the phenomenon of fusion. After 3 weeks of induction, the 3 groups had the appearance of myotube like cells, and the cells were rough and irregular. A cylindrical, branched, interconnected network. After hematoxylin eosin (hematoxylin-eosin HE) staining, the 3 groups of cells were arranged regularly, similar to the morphology of the cardiac myocytes, and some cells were arranged in a regular pattern. The immunocytochemistry showed that the number of hBMSCs in the 3 weeks after induction was: the number of Desmin positive cells was (65.3 + 4.7%) in the 5-aza group, respectively. In the group of Mai Injection (62.3 + 3.9%), 5-aza and Shenmai injection group (68.5 + 2.1%), there was no significant difference in the expression rate of Desmin positive cells in the 3 groups, and the number of cTnT positive cells were in group 5-aza (62.2 + 4.1%), Shenmai injection group (63.8 + 3.4%), 5-aza and Shenmai injection group (66 + 6.2%), and there was no significant difference in the expression rate of Desmin positive cells in the 3 group. No Desmin and cTnT expression.RT-PCR in both group and undifferentiated hBMSCs showed that 3 different inducers were induced to have ANP, BNP expression, and undifferentiated hBMSCs without ANP and BNP expression after hBMSCs3 weeks.
Conclusion: 5-aza and traditional Chinese medicine can induce the hBMSCs cell clone of c-kit~+ to differentiate into myocardial like cells. The induced cardiomyocytes in vitro are characterized by the structure and function of immature cardiomyocytes. It is proved that there are a variety of stem cell clones in the bone marrow, and c-kit can be used as a detection index for screening the differentiation of hBMSCs centripetal muscle like cells. HMSCs with cardiac specific differentiation potential can increase the differentiation rate.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

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本文編號(hào)：2148925