体外可控性Neurod2基因载体的构建及对MSCs定向分化的研究

发布时间：2018-04-21 01:00

  本文选题：Neurod2基因 + Hsp70　； 参考：《甘肃农业大学》2017年硕士论文

【摘要】：研究表明大鼠骨髓间充质干细胞(MSCs)有益于脊髓损伤(SCI)后的功能恢复,本研究利用基因编辑技术构建人热休克蛋白启动子的真核表达载体hHsp70-ND2,并探讨了不同诱导时间对小鼠间充质干细胞(MSCs)向神经元样细胞定向分化的影响。主要研究方法有,1.首先克隆了wistar大鼠Neurod2基因,对其序列进行生物信息学分析后,构建pIRES2-AcGFP1-ND2真核表达载体,分对照组(pIRES2-AcGFP1转染组)与实验组(pIRES2-AcGFP1-ND2转染组)转染小鼠MSCs后,观察AcGFP1绿色荧光蛋白表达,采用real-time PCR、免疫荧光染色和western blotting技术分别检测小鼠MSCs中Neurod2基因及蛋白的表达水平。2.替换原载体pIRES2-AcGFP1中启动子CMV为hHsp70启动子,经双酶切及转染验证其生物活性后与Neurod2基因连接获得重组质粒hHsp70-ND2,将其提取纯化后转染小鼠MSCs,转染实验分对照组(诱导0 h)与实验组(0.5 h、1h、2 h、3 h),在42℃分别进行热诱导后24h观察Ac GFP1绿色荧光蛋白的表达,利用real-time PCR、western blotting和免疫荧光染色技术,检测小鼠MSCs中Neurod2基因及蛋白表达水平。3.分析转染后细胞形态的变化和神经分化相关的髓磷脂碱蛋白(myelin basic protein,MBP)、微管相关蛋白2(microtubule associated protein 2,MAP2)、髓磷脂P0蛋白(myelin protein zero,MPZ)、表皮生长因子(epidermal growth factor,EGF)、神经胶质原纤维酸性蛋白质(glial fibrillary acidic protein,GFAP)、神经丝蛋白(neurofilament,NF)、突触囊泡蛋白(synaptophysin,Syn)、200 kD神经丝蛋白重链(200 kD neurofilament heavy,200KD NFH)等因子表达量。结果表明,1.大鼠Neurod2基因CDS区全长1 149 bp,与Norway大鼠同区域相比较,起始密码子后672、703、747、770、794位出现胞嘧啶的缺失与插入,共编码382个氨基酸,属于bHLH家族;氨基酸序列与家鼠(99%)、罗猴(98%)、人(97.7%)同源性较高,二级结构以α-螺旋和无规卷曲为主,空间结构呈现近似“螺旋-环-螺旋(HLH)”结构,主要分布于细胞核中;转染细胞后Neurod2基因的表达及蛋白水平显著高于对照组(P0.05);2.替换启动子转染后AcGFP1蛋白表达率为39%,插入Neurod2克隆基因后,AcGFP1绿色荧光蛋白表达从高到低依次分别为0.5 h、对照组、1 h、2 h、3 h。real-time PCR分析结果显示,热诱导0.5 h后Neurod2基因表达与对照组、2 h和3 h相比均有显著性差异(P0.05),western blotting分析结果显示,0.5 h热诱导后NeuroD2蛋白表达与2 h有显著性差异(P0.05)。3.细胞免疫荧光染色显示MBP、GFAP、NF蛋白表达明显上调,同样进行real-time PCR和western blotting分析得出,诱导0.5 h和1 h后NF基因及蛋白表达量最高,并与其余诱导组比较差异极显著(P0.01),诱导1 h后MBP基因表达与其余组比较差异显著(P0.05),蛋白表达于2 h和3 h诱导组差异极显著(P0.01);GFAP基因表达在诱导0.5 h和1 h后与2 h和3 h差异显著(P0.05),蛋白表达在诱导2 h后表达量显著低于其余组(P0.01);而且诱导后的细胞形态较转染前有明显变化,呈神经样的长梭状细胞数目明显增多。以上结果表明成功构建了含人热休克蛋白启动子的真核重组表达载体hHsp70-ND2,并在42℃条件下,可利用不同热激时间实现对小鼠MSCs向神经元样细胞定向分化的调控,且得出在热诱导0.5 h和1 h时,其诱导分化效率最高。
[Abstract]:The study showed that rat bone marrow mesenchymal stem cells (MSCs) was beneficial to functional recovery after spinal cord injury (SCI). This study constructed the eukaryotic expression vector hHsp70-ND2 of human heat shock protein promoter using gene editing technique, and explored the effect of different induction time on the directional differentiation of mouse mesenchymal stem cells (MSCs) to neuron like cells. The main research methods are: 1. first, the Neurod2 gene of Wistar rat was cloned. After bioinformatics analysis of the sequence, the pIRES2-AcGFP1-ND2 eukaryotic expression vector was constructed. The expression of AcGFP1 green fluorescent protein was observed in the control group (pIRES2-AcGFP1 transfection group) and the experimental group (pIRES2-AcGFP1-ND2 transfection group) was transfected to the mouse MSCs, and real-time was observed with real-time. PCR, immunofluorescence staining and Western blotting technique were used to detect the expression level of Neurod2 gene and protein in mouse MSCs,.2. replacement of original carrier pIRES2-AcGFP1 in pIRES2-AcGFP1, pIRES2-AcGFP1 promoter CMV was a hHsp70 promoter. After double enzyme digestion and transfection, the biological activity was verified by connection with Neurod2 gene to obtain a heavy group plasmid hHsp70-ND2, then it was extracted and purified. Mice MSCs was transfected with the control group (induced 0 h) and the experimental group (0.5 h, 1H, 2 h, 3 h). The expression of Ac GFP1 green fluorescent protein was observed by 24h at 42 degrees centigrade respectively. Real-time PCR, Western enrichment and immunofluorescence staining techniques were used to detect the gene and protein expression level of the mice. Myelin basic protein (MBP), microtubule related protein 2 (microtubule associated protein 2, MAP2), myelin P0 protein (myelin protein zero), epidermal growth factor, and glial fibrillary acidic protein. GFAP), neurofilament (NF), synaptic vesicle protein (synaptophysin, Syn), 200 kD neurofilament heavy chain (200 kD neurofilament heavy, 200KD NFH). The results showed that the total length of the 1. rat Neurod2 genes was 1149. Compared with the same region of the rat, the 672703747770794 place appeared after the initial codon. Cytosine deletion and insertion, cocoding 382 amino acids, belonging to the bHLH family; the amino acid sequence and the family mouse (99%), the romaceo (98%), the human (97.7%) homology, the two structure is mainly alpha helix and random curl, the spatial structure is similar to the "spiral ring spiral (HLH)" structure, mainly distributed in the nucleus; after transfection of the cell Neurod2 gene The expression and protein level were significantly higher than that of the control group (P0.05), and the expression rate of AcGFP1 protein was 39% after transfection of the 2. promoter. After the insertion of Neurod2 gene, the expression of AcGFP1 green fluorescent protein was 0.5 h respectively from high to low. The control group, 1 h, 2 h, 3 h.real-time PCR segregation results showed that the Neurod2 gene expression and control after the heat induction of 0.5 h Compared with 2 h and 3 h, there were significant differences (P0.05). Western blotting analysis showed that the expression of NeuroD2 protein was significantly different from 2 h (P0.05) after 0.5 h heat induction (P0.05).3. cell immunofluorescence staining showed MBP, GFAP, and protein expression was up to be analyzed and induced by 0.5 and 1. The expression of gene and protein was the highest (P0.01). After 1 h induction, the expression of MBP gene was significantly different from the other groups (P0.05). The expression of protein in the 2 h and 3 h induced groups was very significant (P0.01), and the expression of GFAP gene was significantly different from 2 h and 3 h after the induction of 0.5 h and 1 h, and the protein expression was induced 2. The post expression amount was significantly lower than that of the other groups (P0.01), and the induced cell morphology changed obviously before the transfection, and the number of long spindle cells in the nerve like cells increased significantly. The above results showed that the recombinant expression vector hHsp70-ND2 containing human heat shock protein promoter was successfully constructed, and the different heat shock time could be used under the condition of 42 degrees C. The regulation of directional differentiation of mouse MSCs into neuron like cells was achieved, and it was concluded that the induction efficiency was highest when the heat was induced at 0.5 h and 1 h.

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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