不同浓度Wnt-11诱导骨髓间充质干细胞向心肌样细胞定向分化的实验研究

发布时间：2018-11-28 20:42

【摘要】：目的探讨Wnt-11体外诱导骨髓间充质干细胞(BMMSCs)分化为心肌样细胞的最适浓度。方法采用全骨髓贴壁法分离培养SD大鼠BMMSCs,取第2代BMMSCs培养48h后进行定向诱导,根据Wnt-11终浓度的不同分为A组(100ng/mL),B组(200ng/mL),C组(400ng/mL)和D组(空白对照组),诱导72h后更换为完全培养液继续培养4周,D组仅用完全培养液培养。倒置相差显微镜下观察培养细胞的形态学变化。运用流式细胞仪检测技术对BMMSCs表面联合标记物进行鉴定。各组细胞培养至第4周时,运用免疫细胞化学染色技术检测结蛋白(Desmin)、间隙连接蛋白43(Connexin43)及心肌肌钙蛋白I(cTnI)的表达情况。运用透射电镜观察分化细胞的超微结构变化。实时荧光定量多聚核苷酸链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测各组细胞在诱导后培养第1、2、4周时心肌早期转录因子GATA-4、Nkx2.5及α-MHC的表达。结果 1原代BMMSCs于第2周形成集落,多呈梭形、星形,少数形状不规则。传代后细胞体积变大,诱导后细胞多为长梭形,呈一致性生长。2流式细胞仪检测技术对BMMSCs表面联合标记物的鉴定结果显示CD29、CD45、CD90的阳性表达率分别为97.9%、0.4%和99.5%。3免疫细胞化学染色技术检测结果显示:诱导后常规培养至第4周,A组、B组、C组细胞胞质均呈阳性表达Desmin、Connexin43和cTnI,而D组细胞呈弱阳性或阴性表达,B组细胞的阳性表达率均最高,差异具有统计学意义(P0.05)。4透射电镜结果显示:各诱导组细胞于诱导后常规培养至第4周,胞质内可见平行排列的肌丝及亚细胞结构如线粒体、粗面内质网和核糖体。5RT-qPCR检测结果显示:BMMSCs经诱导后,常规培养第1周时各诱导组细胞均表达GATA-4及Nkx2.5基因,在第2周时表达减弱,在第4周时表达增强,就基因表达而言,三个诱导组相比较,B组明显优于其他两组(P0.05)。α-MHC基因在诱导后常规培养期间均不表达。对照组细胞在常规培养期间GATA-4及Nkx2.5基因的表达量为1,而不表达α-MHC基因。结论 Wnt-11可在体外诱导SD大鼠BMMSCs定向分化为心肌样细胞,其诱导分化最适浓度为200ng/mL。
[Abstract]:Objective to investigate the optimal concentration of Wnt-11 in inducing (BMMSCs) differentiation of bone marrow mesenchymal stem cells into cardiomyoid cells. Methods BMMSCs, of SD rats were isolated and cultured by whole bone marrow adherent method for 48h and then cultured for 48h. According to the final concentration of Wnt-11, they were divided into group A (100ng/mL), B group) and group A (200ng/mL). Group C (400ng/mL) and group D (blank control group) were treated with complete culture medium for 4 weeks after 72 h induction. The morphological changes of cultured cells were observed under inverted phase contrast microscope. BMMSCs surface markers were identified by flow cytometry. The expression of desmin (Desmin), gap junction protein 43 (Connexin43) and cardiac troponin I (cTnI) were detected by immunocytochemical staining at the 4th week. The ultrastructural changes of differentiated cells were observed by transmission electron microscope (TEM). Real-time quantitative polynucleotide chain reaction (real-time quantitative polymerase chain reaction,RT-qPCR) was used to detect the expression of early transcription factors (GATA-4,Nkx2.5 and 伪-MHC) in myocardium of each group at 4 weeks after induction. Results 1 the primary BMMSCs formed colony at the 2nd week, mostly fusiform, starlike, and a few irregular shape. After passage, the cells became larger, and most of the cells were spindle shape after induction. 2 the results of identification of BMMSCs surface markers by flow cytometry showed that the positive expression rates of CD29,CD45,CD90 were 97.9%, respectively. The results of immunocytochemical staining of 0.4% and 99.5.3% showed that the cytoplasm of the cells in groups A, B and C were positive for Desmin,Connexin43 and cTnI, in the 4th week after induction. Group D showed weak positive or negative expression, and group B had the highest positive rate (P0.05). 4 the results of transmission electron microscope showed that the cells of each induction group were cultured to 4 weeks after induction. In the cytoplasm, parallel arrangement of myofilaments and subcellular structures such as mitochondria, rough endoplasmic reticulum and ribosome were observed. The results of 5RT-qPCR detection showed that GATA-4 and Nkx2.5 genes were expressed in the cells of each group at the first week of conventional culture after induction of BMMSCs. At the second week, the expression of 伪-MHC was decreased and increased at the 4th week. Compared with the other two groups, group B was significantly better than the other two groups in gene expression (P0.05). 伪-MHC gene was not expressed in the normal culture period after induction. In the control group, the expression of GATA-4 and Nkx2.5 genes was 1 during conventional culture, but 伪-MHC gene was not expressed. Conclusion Wnt-11 can induce SD rat BMMSCs to differentiate into cardiomyocyte-like cells in vitro, and the optimal concentration is 200ng / mL.
【作者单位】：河北北方学院组织学与胚胎学教研室;河北北方学院人体解剖学教研室;
【基金】：河北省自然科学基金(No.H2015405017);河北省自然科学基金(No.H2014405005) 河北北方学院创新培育项目(No.CXRC1315) 河北省教育厅重大项目(No.ZH2012001)~~
【分类号】：R54

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