本文選題：丙型肝炎病毒核心蛋白 + 基質(zhì)金屬蛋白酶組織抑制因子-1啟動(dòng)子　； 參考：《蘭州大學(xué)》2006年碩士論文

【摘要】：目的 肝硬化是丙型肝炎嚴(yán)重的并發(fā)癥之一,然而丙型肝炎病毒(HCV)引起纖維化、肝硬化的機(jī)制尚不清楚,鑒于大量文獻(xiàn)報(bào)道HCV核心蛋白在細(xì)胞生長(zhǎng)及肝癌發(fā)生方面具有重要調(diào)控作用,我們采用分子生物學(xué)方法研究HCV核心蛋白對(duì)TIMP-1基因表達(dá)的影響,以探討HCV的致肝纖維化機(jī)制。 方法 1.以人基因組DNA為模板,根據(jù)參考文獻(xiàn)設(shè)計(jì)引物,采用聚合酶鏈反應(yīng),克隆TIMP1p。構(gòu)建TIMP1p的氯霉素乙酰轉(zhuǎn)移酶(CAT)報(bào)告基因表達(dá)載體轉(zhuǎn)染HepG2細(xì)胞,同時(shí)平行設(shè)立Pcat3-basic陰性對(duì)照組、Pcat3-control陽(yáng)性對(duì)照組,用ELISA法檢測(cè)各組氯霉素乙酰轉(zhuǎn)移酶(CAT)的表達(dá)來鑒定TIMP1啟動(dòng)子的活性。 2.應(yīng)用瞬時(shí)轉(zhuǎn)染技術(shù),將PcDNA3.1(-)-HCVcore與Pcat3-TIMP-1p共轉(zhuǎn)染入LX-2細(xì)胞,同時(shí)平行設(shè)立Pcat3-basic陰性對(duì)照組、Pcat3-control陽(yáng)性對(duì)照組、Pcat3-TIMP-1p單獨(dú)轉(zhuǎn)染組,用ELISA法檢測(cè)各組CAT的表達(dá)來鑒定HCV核心蛋白對(duì)TIMP1表達(dá)的影響。 結(jié)果 1.成功克隆TIMP1啟動(dòng)子片斷,經(jīng)測(cè)序鑒定正確。成功構(gòu)建了TIMP1p的CAT報(bào)告基因表達(dá)載體,Pcat3-TIMP-1p轉(zhuǎn)染HepG2細(xì)胞后,EHSA法檢測(cè)結(jié)果顯示Pcat3-basic的吸光度值為0.004±0.002,Pcat3-TIMP-1p為2.329±0.685,經(jīng)方差分析各組差異有統(tǒng)計(jì)學(xué)意義,F=26.075(P0.05),LSD法兩兩比較均顯示P0.05,證明TIMP-1p能夠啟動(dòng)CAT的表達(dá)。 2.PcDNA3.1(-)-HCVcore與Pcat3-TIMP-1p共轉(zhuǎn)染LX-2細(xì)胞后,ELISA法檢測(cè)結(jié)果顯示Pcat3-TIMP-1p吸光度值為0.833±0.040,同期單獨(dú)轉(zhuǎn)染LX-2細(xì)胞的Pcat3-TIMP-1p吸光度值為0.677±0.049,方差分析各組差異有統(tǒng)計(jì)學(xué)意義,F=132.401(P0.05),LSD法兩兩比較均顯示P0.05,提示PcDNA3.1(-)-HCVcore與Pcat3-TIMP-1p共轉(zhuǎn)
[Abstract]:Objective liver cirrhosis is one of the serious complications of hepatitis C. However, the mechanism of cirrhosis is not clear because of the fibrosis caused by hepatitis C virus (HCV). As a large number of literatures have reported that HCV core protein plays an important role in the regulation of cell growth and hepatocellular carcinogenesis, we used molecular biological methods to study the effect of HCV core protein on TIMP-1 gene expression in order to explore the mechanism of hepatic fibrosis induced by HCV. Method 1. Using human genomic DNA as template, primers were designed according to references, and TIMP 1p was cloned by polymerase chain reaction (PCR). The expression vector of chloramphenicol acetyltransferase (CAT) reporter gene of TIMP1p was constructed and transfected into HepG2 cells, and the Pcat3-basic negative control group was set up in parallel with Pcat3-control positive control group. Enzyme linked immunosorbent assay (Elisa) was used to detect the expression of chloramphenicol acetyltransferase (CAT) in order to identify the activity of TIMP1 promoter. Using transient transfection technique, PcDNA3.1 ~ (-1) -Pcat3-TIMP-1p was co-transfected into LX-2 cells, and Pcat3-basic negative control group (Pcat3-control) was established in parallel. The expression of cat in each group was detected by Elisa to determine the effect of HCV core protein on the expression of TIMP _ (1) in LX-2 cells, and Pcat3-TIMP-1p was transfected into LX-2 cells in parallel with Pcat3-basic negative control group. The expression of cat in each group was detected by Elisa. Result 1. The promoter of TIMP1 was successfully cloned and confirmed by sequencing. The cat reporter gene expression vector of TIMP1p was successfully constructed. After transfected HepG2 cells with Pcat3-TIMP-1p, the results of EHSA assay showed that the absorbance of Pcat3-basic was 0.004 鹵0.002, Pcat3-TIMP-1p was 2.329 鹵0.685, and the difference of F26.075P0.05LSD between the two groups was statistically significant by ANOVA analysis of variance, which proved that TIMP-1p could be detected in both groups. 2. After PcDNA3.1 ~ + -HCVcore and Pcat3-TIMP-1p co-transfected LX-2 cells, the results of Elisa showed that the absorbance of Pcat3-TIMP-1p was 0.833 鹵0.040, and the absorbance of Pcat3-TIMP-1p was 0.677 鹵0.049 when LX-2 cells were transfected alone at the same time. There was statistical significance in the analysis of the differences among the three groups. There was significant difference between the two groups (P < 0.05), indicating that the PcDNA3.1 ~ (-1) -ti-HCV core and Pcat3-TIMP-1p were cotransferred with Pcat3-TIMP-1p.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R373.21

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