抑制Rac1表达对骨髓间充质干细胞向神经样细胞分化的影响研究

发布时间：2018-07-01 10:02

  本文选题：骨髓间充质干细胞(BMSCs) + 成神经诱导　； 参考：《苏州大学》2008年硕士论文

【摘要】： 目的探讨抑制Rac1的表达对于骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)向神经样细胞分化过程中的影响,为研究BMSCs向神经样细胞分化过程中Rac1的作用奠定基础,有助于阐明BMSCs的成神经诱导分化机理,为BMSCs在临床再生医学上的应用提供理论依据。 方法本实验分三个阶段研究抑制Rac1表达对BMSCs向神经样细胞分化的影响。(1)采用Percoll分离法在体外培养及扩增BMSCs。通过免疫荧光染色的方法检测BMSCs的表面抗原以及将BMSCs进行体外成骨诱导,对BMSCs进行鉴定。(2)采用抗氧化剂诱导方案诱导BMSCs向神经样细胞分化,即先用浓度为10 ng/ml的bFGF预诱导24 h,加入浓度为200μM丁羟基茴香醚诱导(BHA)和2%的二甲基亚砜(DMSO)诱导5 h,再用H-DMEM培养基和N2维持48 h。观察诱导中的形态变化,用免疫荧光检测神经细胞特异性标志物的表达。(3)通过免疫荧光染色检测BMSCs在成神经诱导过程中的Rac1的表达情况。设计并合成三对Rac1特异性miRNA干扰序列,并将其定向克隆至干扰载体pcDNA?6.2-GW/EmGFP-miR上,提取质粒送公司测序。再将重组质粒通过脂质体转染BMSCs。通过Western blotting检测三对序列的干扰效果。使用BHA诱导方案将转染干扰质粒的BMSCs向神经样细胞诱导,研究抑制Rac1表达对于BMSCs向神经样细胞分化过程中形态学变化及神经细胞特异性标志物的表达情况等方面的影响。 结果(1)采用Percoll淋巴细胞分离液及不连续密度梯度法,成功分离培养出大鼠骨髓间充质干细胞,可稳定传至20代以上。表型鉴定结果为CD90、CD106、CD71、CD29阳性,CD45阴性。诱导BMSCs向成骨细胞分化28 d时,ALP和von kossa染色均为阳性。(2)用bFGF/BHA诱导BMSCs向神经样细胞分化,诱导过程中细胞形态学发生了改变,胞体变圆,折光率增加,并伸出有2到3或更多突起。诱导初期表达神经前体细胞的标志物Nestin,阳性率为66.52%±0.8%,95%的细胞表达β-Ⅲ-Tubulin,随着维持时间的延长,约有29.5%的细胞表达神经特异性烯醇化酶NSE。GFAP在诱导过程中始终为阴性。(3) BMSCs向神经样细胞分化过程中,Rac1始终表达。重组质粒测序结果显示质粒中包含的含有序列与设计的序列完全一致。将包含干扰序列的重组质粒转染至BMSCs中,转染率达到62.6%。Western blotting结果证实三种RNA干扰序列均可抑制Rac1的表达。抑制Rac1表达后BMSCs向神经样细胞分化过程中未出现神经元样的形态变化,不表达神经细胞特异性标记。转染了乱序对照质粒的BMSCs在成神经诱导过程中出现类似于神经元的形态,并且表达神经细胞特异性标记Nestin和NSE。 结论抑制Rac1的表达可阻止骨髓间充质干细胞向神经样细胞分化。
[Abstract]:Objective to investigate the effect of inhibition of Rac1 expression on the differentiation of bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) into neuron-like cells, and to establish a foundation for the study of the role of Rac1 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells. It is helpful to elucidate the mechanism of neurogenesis and differentiation of BMSCs and provide theoretical basis for the application of BMSCs in clinical regenerative medicine. Methods the effects of inhibition of Rac1 expression on the differentiation of BMSCs into neuron-like cells were studied in three stages. (1) BMSCs were cultured and amplified by Percoll method in vitro. The surface antigen of BMSCs was detected by immunofluorescence staining, and BMSCs were induced by osteogenesis in vitro. (2) differentiation of BMSCs into neuroblast-like cells was induced by antioxidant induction. It was pretreated with 10 ng/ml bFGF for 24 h, then induced with 200 渭 M butadiol anisole (BHA) and 2% dimethyl sulfoxide (DMSO) for 5 h, then maintained in H-DMEM medium and N2 for 48 h. The morphological changes were observed and the expression of specific markers of nerve cells was detected by immunofluorescence. (3) the expression of Rac1 in the induction of BMSCs was detected by immunofluorescence staining. Three pairs of Rac1 specific miRNA interference sequences were designed and synthesized and cloned into the interference vector pcDNA6.2-GW / EmGFP-miR for sequencing. The recombinant plasmid was transfected into BMSCs by liposome. The interference effect of three pairs of sequences was detected by Western blotting. BMSCs transfected with interference plasmids were induced to neuron-like cells by BHA. The effects of inhibition of Rac1 expression on morphological changes and expression of neuronal specific markers during differentiation of BMSCs into neuron-like cells were studied. Results (1) Percoll lymphocyte isolate and discontinuous density gradient method were used to isolate and culture rat bone marrow mesenchymal stem cells. The results of phenotypic identification were CD90, CD106, CD71, CD29 positive and CD45 negative. Both ALP and von kossa staining were positive at 28 days after differentiation of BMSCs into osteoblasts. (2) BMSCs were induced to differentiate into nerve like cells by bFGF / BHA. During the induction, the morphology of BMSCs was changed, the cell body became round and the refractive index increased. And protruding two to three or more protrusions. The positive rate of Nestin was 66.52% 卤0.8% and 95% of the cells expressed 尾-鈪，

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