發(fā)布時間：2018-05-07 02:08

  本文選題：BMSCs + NT-3��； 參考：《中國醫(yī)科大學》2010年碩士論文

【摘要】： 目的 本實驗擬觀察陽離子脂質(zhì)體介導的含人神經(jīng)營養(yǎng)素-3(NT-3)和綠色熒光蛋白(EGFP)基因共表達真核載體轉(zhuǎn)染骨髓基質(zhì)細胞(BMSCs)后的表達及其是否促進BMSCs向神經(jīng)元樣細胞方向分化。 材料與方法 1、骨髓基質(zhì)細胞(BMSCs)的分離及培養(yǎng) 選用體重100-150g的雄性Wistar大鼠,麻醉后在無菌條件下取得雙側(cè)股骨,取其骨髓用全骨髓貼壁法進行細胞培養(yǎng),通過傳代使BMSCs不斷純化,計數(shù)后備用。 2、BMSCs的鑒定 通過用流式細胞儀方法檢測大鼠BMSCs的CD34及CD90的表達情況,并誘導BMSCs向脂肪細胞及成骨細胞方向分化的方法進行綜合鑒定。 3、陽離子脂質(zhì)體介導的NT-3基因轉(zhuǎn)染BMSCs 選取第3代BMSCs,接種于6孔培養(yǎng)板中,用脂質(zhì)體介導的方法進行轉(zhuǎn)染,分別設(shè)未轉(zhuǎn)染組(A組)、空載體轉(zhuǎn)染組(B組)和轉(zhuǎn)染組(C組)。48h后檢測瞬時表達。 4、檢測指標 (1)倒置熒光顯微鏡下觀察各組細胞GFP的表達情況并記錄。 (2)利用反轉(zhuǎn)錄-PCR (RT-PCR)方法檢測各組細胞轉(zhuǎn)染48h時NT-3、NSE mRNA的表達情況。 (3)利用Western-blot方法檢測各組細胞轉(zhuǎn)染48h時NT-3蛋白的表達情況。 結(jié)果 1、大鼠BMSCs的形態(tài)學觀察及鑒定 原代細胞接種后3天鏡下可見大量懸浮的死亡細胞,圓形,多為血細胞,換液后可見呈集落樣生長的貼壁細胞群,分布不均勻,呈梭形、三角形,約8-10天細胞達到90%以上融合。隨著傳代BMSCs純度越來越高,大量長梭形的細胞均勻貼壁生長,成簇排列,傳代24h即進入對數(shù)生長期,約傳到P3代時細胞均勻的呈漩渦狀排列生長,呈長梭形,形態(tài)基本均一。 流式細胞儀檢測大鼠BMSCs的CD34呈陰性表達(表達率0.43%)；CD90呈陽性表達(表達率96.77%),BMSCs成骨誘導后,堿性磷酸酶呈強陽性。BMSCs成脂誘導后,油紅O染色可見胞漿中大小不等的紅色脂滴。 2、GFP的表達 各組細胞在倒置熒光顯微鏡下觀察,除A組(未轉(zhuǎn)染組)外,B組和C組均可見到發(fā)綠色熒光的細胞, 3、RT-PCR結(jié)果 C組NT-3的mRNA表達明顯高于A組和B組,A、B兩組幾乎未見NT-3mRNA的表達,C組NSE的mRNA表達明顯高于A組, 4、Western-blot結(jié)果 C組NT-3蛋白的表達明顯高于A組和B組,A、B兩組表達較弱 結(jié)論 1、通過全骨髓貼壁培養(yǎng)法成功分離培養(yǎng)BMSCs,利用其貼壁特性,經(jīng)過傳代可以使其不斷純化而達到實驗要求。 2、脂質(zhì)體Lipofectamin2000可以順利將真核共表達質(zhì)粒pIRES2-EGFP-NT-3轉(zhuǎn)染大鼠BMSCs。 3、轉(zhuǎn)染后的BMSCs細胞生長狀態(tài)良好,目的基因NT-3表達良好,示蹤基因GFP表達良好。 4、NT-3轉(zhuǎn)入大鼠BMSCs后可以促進其向神經(jīng)元樣細胞方向分化。
[Abstract]:Purpose The aim of this study was to investigate the expression of human neurotrophin-3 (NT-3) and green fluorescent protein (EGFP) gene co-expressed in bone marrow stromal cells (BMSCs) transfected with cationic liposome and whether it could promote the differentiation of BMSCs into neuron-like cells. Materials and methods 1. Isolation and culture of bone marrow stromal cells (BMSCs) Male Wistar rats weighing 100-150 g were used to obtain bilateral femurs under anaesthetized conditions. Bone marrow was harvested and cultured by whole bone marrow adherent method. BMSCs was purified continuously by passage and then counted and set aside. Identification of BMSCs The expression of CD34 and CD90 in rat BMSCs was detected by flow cytometry, and the differentiation of BMSCs into adipocytes and osteoblasts was identified. Cationic liposome mediated NT-3 gene transfection into BMSCs The third generation BMSCs were inoculated in 6-well culture plate and transfected with liposome. The transient expression was detected after 48 hours in the untransfected group (group A) and empty vector transfection group (group B) and the transfection group (group C). 4, test index The expression of GFP was observed and recorded under inverted fluorescence microscope. The expression of NT-3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) at 48h after transfection. Western-blot method was used to detect the expression of NT-3 protein at 48 h after transfection. Result 1. Morphological observation and identification of rat BMSCs Three days after primary cell inoculation, a large number of suspended dead cells, round and mostly blood cells, could be seen in colony like adherent cells, distributed inhomogeneously, fusiform, triangular, and fused more than 90% in 8-10 days. With the purity of passage BMSCs becoming higher and higher, a large number of long fusiform cells grew evenly and clustered in clusters. After 24 hours of passage, they entered the logarithmic growth period. At about P3 generation, the cells grew in a whirlpool shape, long fusiform and almost uniform in shape. Flow cytometry was used to detect the negative expression of CD34 in rat BMSCs (the positive expression rate was 0.43% and 0.43%). (the expression rate was 96.77% and 96.77%; after osteogenesis induction, alkaline phosphatase was strongly positive. After lipogenesis, oil red O staining showed red lipid droplets of different sizes in the cytoplasm. Expression of GFP The cells in each group were observed under inverted fluorescence microscope. Except group A (untransfected group), green fluorescent cells were found in group B and C, respectively. 3 RT-PCR results The expression of NT-3 mRNA in group C was significantly higher than that in group A and group B. The expression of NSE mRNA in group C was significantly higher than that in group A, and the expression of NSE in group C was significantly higher than that in group A. 4 Western-blot results The expression of NT-3 protein in group C was significantly higher than that in group A and group B. Conclusion 1. BMSCs were isolated and cultured successfully by whole bone marrow adherent culture method. By using the adherent characteristics of BMSCs, the BMSCs could be purified continuously by subculture to meet the experimental requirements. 2. Liposome Lipofectamin2000 could successfully transfect eukaryotic co-expression plasmid pIRES2-EGFP-NT-3 into rat BMSCs. 3. After transfection, the BMSCs cells grew well, the target gene NT-3 expressed well and the tracer gene GFP expressed well. 4 NT-3 can promote the differentiation of rat BMSCs into neuron-like cells.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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