本文選題：骨髓間充質(zhì)干細胞(BMSCs) + 成神經(jīng)誘導　； 參考：《蘇州大學》2008年碩士論文

【摘要】： 目的探討抑制Rac1的表達對于骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells, BMSCs)向神經(jīng)樣細胞分化過程中的影響,為研究BMSCs向神經(jīng)樣細胞分化過程中Rac1的作用奠定基礎(chǔ),有助于闡明BMSCs的成神經(jīng)誘導分化機理,為BMSCs在臨床再生醫(yī)學上的應(yīng)用提供理論依據(jù)。 方法本實驗分三個階段研究抑制Rac1表達對BMSCs向神經(jīng)樣細胞分化的影響。(1)采用Percoll分離法在體外培養(yǎng)及擴增BMSCs。通過免疫熒光染色的方法檢測BMSCs的表面抗原以及將BMSCs進行體外成骨誘導,對BMSCs進行鑒定。(2)采用抗氧化劑誘導方案誘導BMSCs向神經(jīng)樣細胞分化,即先用濃度為10 ng/ml的bFGF預誘導24 h,加入濃度為200μM丁羥基茴香醚誘導(BHA)和2%的二甲基亞砜(DMSO)誘導5 h,再用H-DMEM培養(yǎng)基和N2維持48 h。觀察誘導中的形態(tài)變化,用免疫熒光檢測神經(jīng)細胞特異性標志物的表達。(3)通過免疫熒光染色檢測BMSCs在成神經(jīng)誘導過程中的Rac1的表達情況。設(shè)計并合成三對Rac1特異性miRNA干擾序列,并將其定向克隆至干擾載體pcDNA?6.2-GW/EmGFP-miR上,提取質(zhì)粒送公司測序。再將重組質(zhì)粒通過脂質(zhì)體轉(zhuǎn)染BMSCs。通過Western blotting檢測三對序列的干擾效果。使用BHA誘導方案將轉(zhuǎn)染干擾質(zhì)粒的BMSCs向神經(jīng)樣細胞誘導,研究抑制Rac1表達對于BMSCs向神經(jīng)樣細胞分化過程中形態(tài)學變化及神經(jīng)細胞特異性標志物的表達情況等方面的影響。 結(jié)果(1)采用Percoll淋巴細胞分離液及不連續(xù)密度梯度法,成功分離培養(yǎng)出大鼠骨髓間充質(zhì)干細胞,可穩(wěn)定傳至20代以上。表型鑒定結(jié)果為CD90、CD106、CD71、CD29陽性,CD45陰性。誘導BMSCs向成骨細胞分化28 d時,ALP和von kossa染色均為陽性。(2)用bFGF/BHA誘導BMSCs向神經(jīng)樣細胞分化,誘導過程中細胞形態(tài)學發(fā)生了改變,胞體變圓,折光率增加,并伸出有2到3或更多突起。誘導初期表達神經(jīng)前體細胞的標志物Nestin,陽性率為66.52%±0.8%,95%的細胞表達β-Ⅲ-Tubulin,隨著維持時間的延長,約有29.5%的細胞表達神經(jīng)特異性烯醇化酶NSE。GFAP在誘導過程中始終為陰性。(3) BMSCs向神經(jīng)樣細胞分化過程中,Rac1始終表達。重組質(zhì)粒測序結(jié)果顯示質(zhì)粒中包含的含有序列與設(shè)計的序列完全一致。將包含干擾序列的重組質(zhì)粒轉(zhuǎn)染至BMSCs中,轉(zhuǎn)染率達到62.6%。Western blotting結(jié)果證實三種RNA干擾序列均可抑制Rac1的表達。抑制Rac1表達后BMSCs向神經(jīng)樣細胞分化過程中未出現(xiàn)神經(jīng)元樣的形態(tài)變化,不表達神經(jīng)細胞特異性標記。轉(zhuǎn)染了亂序?qū)φ召|(zhì)粒的BMSCs在成神經(jīng)誘導過程中出現(xiàn)類似于神經(jīng)元的形態(tài),并且表達神經(jīng)細胞特異性標記Nestin和NSE。 結(jié)論抑制Rac1的表達可阻止骨髓間充質(zhì)干細胞向神經(jīng)樣細胞分化。
[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% 鹵0.8% and 95% of the cells expressed 尾-鈪，

本文編號：2087414