本文選題：乙型肝炎病毒　切入點(diǎn)：RNA干擾　出處：《華中科技大學(xué)》2009年碩士論文

【摘要】：【目的】 設(shè)計(jì)與構(gòu)建針對(duì)乙型肝炎病毒（HBV）基因的不同序列特異性siRNA載體，探討各siRNA對(duì)HepG2.2.15細(xì)胞HBV復(fù)制和表達(dá)的作用。篩選獲得具有高效沉默HBV的載體，以去唾液酸受體為靶點(diǎn)將其靶向HepG2.2.15細(xì)胞，觀察靶向與沉默效應(yīng)、以及對(duì)細(xì)胞活性的影響，為進(jìn)一步體內(nèi)靶向研究奠定基礎(chǔ)。 【方法】 1.特異性siRNA真核載體的構(gòu)建：設(shè)計(jì)并合成6對(duì)針對(duì)HBV基因的不同序列siRNA寡核苷酸，，經(jīng)退火形成雙鏈，然后經(jīng)T4連接酶連接，克隆入表達(dá)EGFP的真核載體，命名為pGenesil-siHBV1～6，并設(shè)立與HBV序列無(wú)關(guān)的pGenesil-siHBV-HK作為陰性對(duì)照。 2.重組體的生物活性鑒定：將重組質(zhì)粒pGenesil-siHBV1～6和pGenesilsiHBV-HK用脂質(zhì)體分別轉(zhuǎn)染HepG2.2.15細(xì)胞。采用實(shí)時(shí)熒光定量PCR檢測(cè)轉(zhuǎn)染后2d、3d、4d靶基因的mRNA和細(xì)胞培養(yǎng)上清中cccDNA的水平；ELISA檢測(cè)轉(zhuǎn)染后2d、3d、5d、7d細(xì)胞培養(yǎng)上清中HBsAg、HBeAg的表達(dá)。 3.去唾液酸受體的靶向作用：將具有高效沉默HBV的siRNA載體利用jetPEI-Hepatocyte靶向轉(zhuǎn)染HepG2.2.15細(xì)胞，倒置相差熒光顯微鏡和流式細(xì)胞術(shù)（FCM）檢測(cè)細(xì)胞內(nèi)EGFP的表達(dá)，檢測(cè)轉(zhuǎn)染效率；FCM檢測(cè)細(xì)胞內(nèi)HBcAg的表達(dá)以及轉(zhuǎn)染后3d細(xì)胞的凋亡；ELISA檢測(cè)細(xì)胞培養(yǎng)上清中HBsAg、HBeAg的表達(dá)，細(xì)胞免疫化學(xué)酶標(biāo)技術(shù)觀察細(xì)胞內(nèi)HBsAg的表達(dá)。 【結(jié)果】 1. HBV特異性siRNA載體的構(gòu)建：PCR、酶切及測(cè)序鑒定結(jié)果表明，針對(duì)HBV基因特異性的siRNA真核載體構(gòu)建成功。 2. HBV特異性siRNA載體的生物活性：Realtime-PCR、ELISA分別從mRNA、蛋白質(zhì)和cccDNA水平檢測(cè)，結(jié)果表明pGenesil-siHBV1～6均可不同程度的抑制HBV的復(fù)制和抗原的表達(dá)，其中pGenesil-siHBV1的抑制效果最強(qiáng)。 3. ASGPR靶向pGenesil-siHBV1對(duì)HBV的沉默效應(yīng)：將pGenesil-siHBV1利用jetPEI-Hepatocyte靶向轉(zhuǎn)染HepG2.2.15細(xì)胞，倒置相差熒光顯微鏡下可見(jiàn)綠色熒光，而無(wú)jetPEI-Hepatocyte轉(zhuǎn)染對(duì)照組未見(jiàn)綠色熒光，F(xiàn)CM檢測(cè)綠色熒光陽(yáng)性細(xì)胞約為45％；FCM檢測(cè)細(xì)胞內(nèi)HBcAg的表達(dá)以及ELISA檢測(cè)靶向?qū)嶒?yàn)組細(xì)胞培養(yǎng)上清中HBsAg、HBeAg的表達(dá)低于LipofectamineTM2000轉(zhuǎn)染對(duì)照組，且二者均顯著低于無(wú)關(guān)序列對(duì)照組；細(xì)胞免疫化學(xué)酶標(biāo)技術(shù)觀察細(xì)胞內(nèi)HBsAg的表達(dá)與無(wú)關(guān)序列對(duì)照組比較顯著降低；FCM檢測(cè)轉(zhuǎn)染后3d的細(xì)胞凋亡無(wú)明顯變化。 【結(jié)論】 結(jié)果表明針對(duì)HBV的不同siRNA的沉默效應(yīng)存在差異；ASGPR介導(dǎo)的靶向遞送siRNA載體能更好的發(fā)揮沉默HBV基因復(fù)制與表達(dá)的作用，為下一步的體內(nèi)實(shí)驗(yàn)奠定了良好的基礎(chǔ)。
[Abstract]:[purpose]To design and construct different sequence specific siRNA vectors for hepatitis B virus (HBV) gene, and to explore the role of siRNA in HBV replication and expression in HepG2.2.15 cells.The vector with high efficiency silencing HBV was obtained. The sialic acid receptor was used as the target to target HepG2.2.15 cells. The effects of targeting and silencing and the effect on cell activity were observed, which laid a foundation for further research on target in vivo.[methods]1.Construction of specific siRNA eukaryotic vector: six pairs of siRNA oligodeoxynucleotides targeting HBV gene were designed and synthesized. After annealing to form double strand, and then ligated with T4 ligase, they were cloned into eukaryotic vector expressing EGFP.It was named pGenesil-siHBV1 / 6, and pGenesil-siHBV-HK unrelated to HBV sequence was established as negative control.2.Identification of the bioactivity of the recombinant plasmid: the recombinant plasmid pGenesil-siHBV1~6 and pGenesilsiHBV-HK were transfected into HepG2.2.15 cells by liposome respectively.Real-time fluorescence quantitative PCR was used to detect the expression of HBeAg in the cell culture supernatant of 2 days and 3 days after transfection and the level of cccDNA in the supernatant of cell culture by Elisa. The expression of HBeAg was detected in the supernatant of 2 days, 3 days, 5 days and 7 days after transfection.3.The targeting effect of sialic acid receptor: the siRNA vector with high efficiency silencing HBV was transfected into HepG2.2.15 cells by jetPEI-Hepatocyte, and the expression of EGFP was detected by inverted phase contrast fluorescence microscopy and flow cytometry.The expression of HBcAg in cells was detected by FCM and the expression of HBsAg HBeAg in the supernatant of cells was detected by Elisa on the 3rd day after transfection. The expression of HBsAg in cells was observed by immunocytochemical enzyme labeling.[results]1.Construction of HBV specific siRNA vector, restriction endonuclease digestion and sequencing analysis showed that the siRNA eukaryotic vector specific for HBV gene was successfully constructed.2.The biological activity of HBV specific siRNA vector was detected by Elisa from the levels of mRNAs, proteins and cccDNA, respectively. The results showed that pGenesil-siHBV1~6 could inhibit the replication of HBV and the expression of HBV antigens to varying degrees, and pGenesil-siHBV1 had the strongest inhibitory effect.3.The silencing effect of ASGPR targeted pGenesil-siHBV1 on HBV: pGenesil-siHBV1 was transfected into HepG2.2.15 cells by jetPEI-Hepatocyte, and green fluorescence was observed under inverted phase contrast fluorescence microscope.In the control group without jetPEI-Hepatocyte transfection, the expression of HBcAg and the expression of HBeAg in the culture supernatant of targeted experimental group were detected by FCM and the expression of HBeAg in the culture supernatant of targeted experimental group was lower than that in LipofectamineTM2000 transfected control group.The expression of HBsAg in the cells was significantly lower than that in the unrelated sequence control group, and the expression of HBsAg in the cells was significantly lower than that in the unrelated sequence control group on the 3rd day after transfection.[conclusion]The results showed that the silencing effect of different siRNA against HBV was different. ASGPR-mediated targeted delivery siRNA vector could play a better role in silencing the replication and expression of HBV gene, which laid a good foundation for further in vivo experiments.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R346

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本文編號(hào)：1721441