脂质体介导的pIRES2-EGFP-NT-3转染骨髓基质细胞的实验研究

发布时间：2018-05-07 02:08

本文选题：BMSCs + NT-3　；参考：《中国医科大学》2010年硕士论文

【摘要】： 目的本实验拟观察阳离子脂质体介导的含人神经营养素-3(NT-3)和绿色荧光蛋白(EGFP)基因共表达真核载体转染骨髓基质细胞(BMSCs)后的表达及其是否促进BMSCs向神经元样细胞方向分化。材料与方法 1、骨髓基质细胞(BMSCs)的分离及培养选用体重100-150g的雄性Wistar大鼠,麻醉后在无菌条件下取得双侧股骨,取其骨髓用全骨髓贴壁法进行细胞培养,通过传代使BMSCs不断纯化,计数后备用。 2、BMSCs的鉴定通过用流式细胞仪方法检测大鼠BMSCs的CD34及CD90的表达情况,并诱导BMSCs向脂肪细胞及成骨细胞方向分化的方法进行综合鉴定。 3、阳离子脂质体介导的NT-3基因转染BMSCs 选取第3代BMSCs,接种于6孔培养板中,用脂质体介导的方法进行转染,分别设未转染组(A组)、空载体转染组(B组)和转染组(C组)。48h后检测瞬时表达。 4、检测指标 (1)倒置荧光显微镜下观察各组细胞GFP的表达情况并记录。 (2)利用反转录-PCR (RT-PCR)方法检测各组细胞转染48h时NT-3、NSE mRNA的表达情况。 (3)利用Western-blot方法检测各组细胞转染48h时NT-3蛋白的表达情况。结果 1、大鼠BMSCs的形态学观察及鉴定原代细胞接种后3天镜下可见大量悬浮的死亡细胞,圆形,多为血细胞,换液后可见呈集落样生长的贴壁细胞群,分布不均匀,呈梭形、三角形,约8-10天细胞达到90%以上融合。随着传代BMSCs纯度越来越高,大量长梭形的细胞均匀贴壁生长,成簇排列,传代24h即进入对数生长期,约传到P3代时细胞均匀的呈漩涡状排列生长,呈长梭形,形态基本均一。流式细胞仪检测大鼠BMSCs的CD34呈阴性表达(表达率0.43%)；CD90呈阳性表达(表达率96.77%),BMSCs成骨诱导后,碱性磷酸酶呈强阳性。BMSCs成脂诱导后,油红O染色可见胞浆中大小不等的红色脂滴。 2、GFP的表达各组细胞在倒置荧光显微镜下观察,除A组(未转染组)外,B组和C组均可见到发绿色荧光的细胞, 3、RT-PCR结果 C组NT-3的mRNA表达明显高于A组和B组,A、B两组几乎未见NT-3mRNA的表达,C组NSE的mRNA表达明显高于A组, 4、Western-blot结果 C组NT-3蛋白的表达明显高于A组和B组,A、B两组表达较弱结论 1、通过全骨髓贴壁培养法成功分离培养BMSCs,利用其贴壁特性,经过传代可以使其不断纯化而达到实验要求。 2、脂质体Lipofectamin2000可以顺利将真核共表达质粒pIRES2-EGFP-NT-3转染大鼠BMSCs。 3、转染后的BMSCs细胞生长状态良好,目的基因NT-3表达良好,示踪基因GFP表达良好。 4、NT-3转入大鼠BMSCs后可以促进其向神经元样细胞方向分化。
[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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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