發(fā)布時間：2018-07-17 08:09

【摘要】： 目的:探討體外分離、純化大鼠骨髓間充質干細胞(bone marrow mesenchymal stem cells BMSCs)的方法及骨髓間充質干細胞向神經元樣細胞的分化潛能,并分析其部分表型特點。動態(tài)觀察體外腦脊液對骨髓間充質干細胞的影響,為骨髓間充質干細胞的移植提供初步的實驗依據。 方法: 1.選取清潔級SD大鼠,體重150g～200g。用密度梯度離心結合貼壁培養(yǎng)法及全骨髓培養(yǎng)法分離純化大鼠骨髓間充質干細胞,并傳代擴增。進行形態(tài)學觀察,免疫組化法分析測定細胞表面抗原的表達。2.采用抗氧化劑誘導方案誘導骨髓間充質干細胞向神經元樣細胞分化,觀察誘導過程中細胞的形態(tài)變化,并用免疫熒光法檢測神經樣細胞的特異性標志物表達,即免疫組化法檢測誘導后神經元樣細胞的特異性烯醇化酶、微管相關蛋白和巢蛋白的表達。3.選擇生長良好的第3、4代細胞接種于腦脊液中,觀察細胞生長狀況,并通過細胞免疫化學染色法鑒定腦脊液對細胞表型的影響。制定細胞生長曲線。 結果: 1. BMSCs屬骨髓中單個核細胞,運用密度梯度離心結合貼壁培養(yǎng)法及全骨髓培養(yǎng)法能成功有效的分離純化大鼠BMSCs,并可穩(wěn)定傳至20代以上。表型鑒定結果為CD90、CD106、CD71、CD29陽性,CD45陰性。2.采用bFGF/BHA誘導BMSCs向神經樣細胞分化,誘導過程中細胞形態(tài)學發(fā)生了改變,bFGF誘導24h后細胞由扁平和長梭狀變?yōu)槎噙呅渭安灰?guī)則形。加入DMSO和BHA5h后,胞體變圓,折光率增加,并伸出有2到3或更多突起。誘導24h后細胞形態(tài)更像神經元,并交織呈網狀。誘導后神經細胞的標志物Nestin,β-Ⅲ-Tubulin和NSE表達陽性,并且BMSCs的表面標記CD106表達陽性。3.骨髓間充干質細胞在腦脊液培養(yǎng)基中仍可繼續(xù)生長、增殖,提示這種細胞對生長環(huán)境有高度的適應性,并不影響其特性和表型。 結論:本實驗建立了一種體外分離純化、培養(yǎng)擴增大鼠骨髓BMSCs的方法,用Percoll淋巴細胞分離液分離培養(yǎng)BMSCs可以快速擴增和純化,并能穩(wěn)定傳代培養(yǎng)。采用bFGF/BHA誘導方案,能成功誘導骨髓間充質干細胞分化為神經元樣細胞。骨髓間充質干細胞對腦脊液有高度的適應性,可作為一種新的培養(yǎng)方法。
[Abstract]:Aim: to investigate the method of isolation and purification of rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells BMSCs) in vitro and the differentiation potential of bone marrow mesenchymal stem cells into neuron-like cells. To observe the effect of cerebrospinal fluid (CSF) on bone marrow mesenchymal stem cells (BMSCs) in vitro and to provide a preliminary experimental basis for the transplantation of BMSCs. Methods: 1. SD rats of clean grade were selected, weighing 150 g or 200 g. Rat bone marrow mesenchymal stem cells were purified by density gradient centrifugation combined with adherent culture and whole bone marrow culture. Morphological observation and immunohistochemical analysis were used to detect the expression of cell surface antigen. 2. 2. Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate into neuron-like cells by antioxidant induction. The morphological changes of the cells were observed and the expression of specific markers of neuron-like cells was detected by immunofluorescence. The expression of specific enolase, microtubule-associated protein and nestin in neuron-like cells was detected by immunohistochemistry. The third generation of well growing cells were inoculated into cerebrospinal fluid (CSF) to observe the growth of the cells and to identify the effect of CSF on the phenotype of the cells by immunocytochemical staining. Draw up cell growth curve. Results: 1. BMSCs are mononuclear cells from bone marrow. Using density gradient centrifugation combined with adherent culture method and whole bone marrow culture method, BMSCs can be separated and purified effectively, and can be transferred to more than 20 generations. The results of phenotypic identification were CD90, CD106, CD71, CD29 positive and CD45 negative. 2. BMSCs were induced to differentiate into neural like cells by bFGF- / BHA. The morphology of BMSCs changed from flat and long spindle to polygonal and irregular shape after 24 hours of induction by bFGF. After the addition of DMSO and BHA for 5 h, the cell body became round, the refractive index increased, and there were 2 to 3 or more protrusions. After 24 hours of induction, the cells were more neuronal in shape and intertwined in a reticular form. After induction, Nestin, 尾-鈪，

本文編號：2129738