特异性抑制Tim-1表达的siRNA表达质粒的构建和Tim家族分子在小鼠肝炎模型中表达的初步研究
发布时间：2018-01-18 14:06
本文关键词：特异性抑制Tim-1表达的siRNA表达质粒的构建和Tim家族分子在小鼠肝炎模型中表达的初步研究 出处：《山东大学》2006年硕士论文 论文类型：学位论文
更多相关文章： Tim-1 Tim-2 Tim-3 基因表达 RNA干扰 抑制作用 ConA 急性肝炎模型
【摘要】：目的： 首先构建带HA标签的Tim-1真核表达载体和针对Tim-1的具有良好抑制作用的siRNA表达载体。其次通过real-time PCR的方法初步研究Tim家族分子在ConA诱导的小鼠急性肝炎模型和HBV全基因转基因鼠中的表达水平。该实验不仅为进一步研究Tim家族分子在免疫系统中的作用提供新的实验工具，而且为下一步研究Tim家族分子在肝脏炎症疾病中的作用机制奠定基础。 方法： 1．Tim-1HA融合蛋白在肝癌细胞系HepG2中的表达 以小鼠脾细胞cDNA为模板，设计引物，PCR扩增Tim-1HA融合蛋白基因片段，T-A克隆入真核表达载体pTARGE-T，构建重组表达载体pTARGETim-1HA。重组子经酶切、PCR及测序鉴定。将重组子以脂质体法转染肝癌细胞系HepG2，RT-PCR和Western blot的方法检测Tim-1HA融合蛋白表达。 2．针对Tim-1的siRNA表达载体的构建及其对Tim-1HA的抑制效果 参考siRNA的设计策略，通过基因Blast，设计并合成4对针对Tim-1 cDNA序列的寡核苷酸，退火后克隆入含有U6启动子的pAVU6+27载体，构建针对Tim-1的siRNA表达载体，重组子经酶切及测序鉴定。将真核表达载体pTARGETim-1HA与4个siRNA表达载体分别共转染肝癌细胞系HepG2细胞。转染72小时后，利用RT-PCR和Western blot的方法检测siRNA对Tim-1的抑制效果。 3．制备ConA诱导的小鼠急性肝炎模型 小鼠尾静脉注射ConA 25mg／kg体重制备急性肝炎模型，，以尾静脉注射生理盐水小鼠为对照鼠，6～8小时后处死小鼠，测定小鼠血清ALT水平，取部分肝组织切片进行HE染色。 4．实时定量PCR测定Tim分子在模型中的表达
[Abstract]:Objective: Firstly, the eukaryotic expression vector of Tim-1 with HA tag and the expression vector of siRNA with good inhibitory effect on Tim-1 were constructed. Secondly, the expression vector of siRNA was constructed by real-time. The PCR method was used to study the expression level of Tim family molecules in ConA induced mouse acute hepatitis model and HBV transgenic mice. This study is not only for the further study of Tim family molecules in mice. The role of the immune system provides new experimental tools. It also lays a foundation for the further study of the role of Tim family in liver inflammatory diseases. Methods: 1. Expression of Tim-1 HA fusion protein in hepatocellular carcinoma cell line HepG2 Using mouse spleen cell cDNA as template, primers were designed to amplify the Tim-1HA fusion protein gene fragment, T-A, and cloned into eukaryotic expression vector pTARGE-T. The recombinant expression vector pTARGETim-1 HAwas constructed. The recombinant plasmid was identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was transfected into hepatoma cell line HepG2 by liposome method. The expression of Tim-1HA fusion protein was detected by RT-PCR and Western blot. 2. Construction of siRNA expression vector for Tim-1 and its inhibitory effect on Tim-1HA According to the design strategy of siRNA, four pairs of oligonucleotides targeting Tim-1 cDNA sequence were designed and synthesized by gene Blast. After annealing, the pAVU6 27 vector containing U6 promoter was cloned and the siRNA expression vector for Tim-1 was constructed. The eukaryotic expression vector pTARGETim-1HA and four siRNA expression vectors were co-transfected into the hepatoma cell line HepG2 cells after 72 hours of transfection. The inhibition effect of siRNA on Tim-1 was detected by RT-PCR and Western blot. 3. The mouse model of acute hepatitis induced by ConA was established. Acute hepatitis model was established by injecting ConA 25 mg / kg body weight into tail vein of mice. The mice were killed after 6 hours of injection of normal saline into caudal vein. The serum ALT level was measured and some liver sections were taken for HE staining. 4. The expression of Tim molecules in the model was determined by real-time quantitative PCR.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
【共引文献】
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