發(fā)布時(shí)間：2018-01-18 14:06

  本文關(guān)鍵詞：特異性抑制Tim-1表達(dá)的siRNA表達(dá)質(zhì)粒的構(gòu)建和Tim家族分子在小鼠肝炎模型中表達(dá)的初步研究　出處：《山東大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Tim-1 Tim-2 Tim-3 基因表達(dá) RNA干擾 抑制作用 ConA 急性肝炎模型

【摘要】：目的： 首先構(gòu)建帶HA標(biāo)簽的Tim-1真核表達(dá)載體和針對(duì)Tim-1的具有良好抑制作用的siRNA表達(dá)載體。其次通過real-time PCR的方法初步研究Tim家族分子在ConA誘導(dǎo)的小鼠急性肝炎模型和HBV全基因轉(zhuǎn)基因鼠中的表達(dá)水平。該實(shí)驗(yàn)不僅為進(jìn)一步研究Tim家族分子在免疫系統(tǒng)中的作用提供新的實(shí)驗(yàn)工具，而且為下一步研究Tim家族分子在肝臟炎癥疾病中的作用機(jī)制奠定基礎(chǔ)。 方法： 1．Tim-1HA融合蛋白在肝癌細(xì)胞系HepG2中的表達(dá) 以小鼠脾細(xì)胞cDNA為模板，設(shè)計(jì)引物，PCR擴(kuò)增Tim-1HA融合蛋白基因片段，T-A克隆入真核表達(dá)載體pTARGE-T，構(gòu)建重組表達(dá)載體pTARGETim-1HA。重組子經(jīng)酶切、PCR及測(cè)序鑒定。將重組子以脂質(zhì)體法轉(zhuǎn)染肝癌細(xì)胞系HepG2，RT-PCR和Western blot的方法檢測(cè)Tim-1HA融合蛋白表達(dá)。 2．針對(duì)Tim-1的siRNA表達(dá)載體的構(gòu)建及其對(duì)Tim-1HA的抑制效果 參考siRNA的設(shè)計(jì)策略，通過基因Blast，設(shè)計(jì)并合成4對(duì)針對(duì)Tim-1 cDNA序列的寡核苷酸，退火后克隆入含有U6啟動(dòng)子的pAVU6+27載體，構(gòu)建針對(duì)Tim-1的siRNA表達(dá)載體，重組子經(jīng)酶切及測(cè)序鑒定。將真核表達(dá)載體pTARGETim-1HA與4個(gè)siRNA表達(dá)載體分別共轉(zhuǎn)染肝癌細(xì)胞系HepG2細(xì)胞。轉(zhuǎn)染72小時(shí)后，利用RT-PCR和Western blot的方法檢測(cè)siRNA對(duì)Tim-1的抑制效果。 3．制備ConA誘導(dǎo)的小鼠急性肝炎模型 小鼠尾靜脈注射ConA 25mg／kg體重制備急性肝炎模型，，以尾靜脈注射生理鹽水小鼠為對(duì)照鼠，6～8小時(shí)后處死小鼠，測(cè)定小鼠血清ALT水平，取部分肝組織切片進(jìn)行HE染色。 4．實(shí)時(shí)定量PCR測(cè)定Tim分子在模型中的表達(dá)
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張志宏;曾永秋;稅青林;田強(qiáng);黃燕;趙小平;;hTERT-siRNA表達(dá)載體的構(gòu)建及對(duì)MCF-7細(xì)胞生長(zhǎng)、端粒酶活性的抑制作用[J];解放軍醫(yī)學(xué)雜志;2007年06期

2 文維群,章廉,胡章勇,張明霞,肖紅,駱抗先;IL-18表達(dá)水平與HBV感染相關(guān)性的臨床研究[J];臨床肝膽病雜志;2001年04期

3 李寧,范學(xué)工,黃燕,劉征波,全俊,湯參娥,朱才;CpG-ODN對(duì)慢性乙型肝炎患者外周血單個(gè)核細(xì)胞T-bet表達(dá)的影響[J];免疫學(xué)雜志;2005年03期

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5 劉海英,單瑞芹,李曉梅,孔北華,王成香;乙型肝炎病毒母嬰垂直傳播與Th1/Th2型細(xì)胞因子相關(guān)性研究[J];實(shí)用婦產(chǎn)科雜志;2003年04期

6 段國(guó)榮;丙型肝炎發(fā)病機(jī)制研究進(jìn)展[J];陜西醫(yī)學(xué)雜志;2004年03期

7 馬玲娣;張彥;文世宏;何於娟;劉小珊;康格非;蔣紀(jì)愷;;小鼠TIM2基因真核表達(dá)載體的構(gòu)建及鑒定[J];細(xì)胞與分子免疫學(xué)雜志;2006年06期

8 劉海英,馬玉燕,崔保霞,戴e

本文編號(hào)：1441243