建立组特异性过表达血栓调蛋白的小鼠模型

发布时间：2018-01-26 07:15

本文关键词： 转基因小鼠血栓调节蛋白活化蛋白C 组织特异性表达 Tie2-Cre小鼠　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：目的 1.构建血栓调节蛋白(TM)表达质粒并分析其在细胞中的生物学活性; 2.建立组织特异性过表达TM的小鼠模型并分析其在动物体内的生物学活性。方法 1. RT-PCR获取野生型小鼠肺组织中TM基因片段; 2. TOPO表达载体连接TM基因片段,相应质粒转染SVEC细胞后,检测细胞增殖和凋亡,以及蛋白C活化试验; 3. PCCALL2表达载体连接TM基因片段,相应质粒PTMHL与Cre质粒共转染SVEC细胞后,检测TM的表达,以及蛋白C活化试验; 4.显微注射法将构建成功的PTMHL质粒注入供体C57BL/6小鼠受精卵细胞后,采用转基因小鼠的个体组织(鼠尾)中提取基因组DNA进行PCR分析,鉴定基因型; 5.新生PTMHL转基因小鼠与Tie2-Cre小鼠杂交,采用转基因小鼠的个体组织(鼠尾)中提取基因组DNA进行PCR分析,对其TM基因型和Cre基因型鉴定,Western blot和免疫组化法检测组织中TM抗体的表达,用蛋白C活性分析法检测TM转基因小鼠对蛋白C活化的影响。结果 1.成功构建真核表达TM的质粒,转染SVEC细胞后,细胞的增殖和凋亡得到抑制,细胞中的TM生物活性增强; 2.成功构建组织特异性表达TM的质粒PTMHL,用此质粒和表达Cre质粒共转染SVEC细胞后只有在表达Cre的情况下TM才会过表达,此外细胞中的TM生物活性增强; 3.成功构建组织特异性过表达TM的小鼠模型,同Tie2-Cre小鼠杂交后,新生小鼠的心脏,肾脏,肝脏,肺等组织中内皮细胞的TM表达明显增强,转基因小鼠体内TM的生物活性增加。
[Abstract]:Purpose 1. The expression plasmid of thrombomodulin (TMN) was constructed and its biological activity in cells was analyzed. 2. To establish a mouse model of tissue specific overexpression of TM and to analyze its biological activity in vivo. Method 1. The TM gene fragment in the lung tissue of wild-type mice was obtained by RT-PCR. 2. The TM gene fragment was ligated into the TOPO expression vector. After transfection of the corresponding plasmid into SVEC cells, cell proliferation and apoptosis, as well as protein C activation test were detected. 3. The TM gene fragment was ligated into the PCCALL2 expression vector. After the corresponding plasmid PTMHL and Cre plasmid were co-transfected into SVEC cells, the expression of TM and protein C activation test were detected. 4. The constructed PTMHL plasmid was injected into donor C57BL / 6 mouse fertilized egg cells by microinjection. Genomic DNA was extracted from the individual tissues of transgenic mice (mouse tail) for PCR analysis and genotypes were identified. 5. PTMHL transgenic mice were hybridized with Tie2-Cre mice. Genomic DNA was extracted from the individual tissues of transgenic mice (mouse tail) for PCR analysis. The expression of TM antibody was detected by Western blot and immunohistochemistry. The effect of TM transgenic mice on the activation of protein C was detected by protein C activity analysis. Results 1. The eukaryotic plasmids expressing TM were successfully constructed. After transfection into SVEC cells, the proliferation and apoptosis of the cells were inhibited, and the biological activity of TM in the cells was enhanced. 2. The tissue specific expression plasmid PTMHL1 was successfully constructed, which was co-transfected into SVEC cells by using this plasmid and the expressed Cre plasmid. Only in the case of Cre expression could TM be overexpressed. In addition, the biological activity of TM in cells was enhanced. 3.The mouse model of tissue specific overexpression of TM was successfully constructed. After hybridization with Tie2-Cre mice, the heart, kidney and liver of newborn mice were obtained. The expression of TM in endothelial cells was significantly increased in lung tissues, and the biological activity of TM in transgenic mice was increased.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R-332

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