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

【摘要】：【目的】 設(shè)計與構(gòu)建針對乙型肝炎病毒（HBV）基因的不同序列特異性siRNA載體，探討各siRNA對HepG2.2.15細胞HBV復制和表達的作用。篩選獲得具有高效沉默HBV的載體，以去唾液酸受體為靶點將其靶向HepG2.2.15細胞，觀察靶向與沉默效應、以及對細胞活性的影響，為進一步體內(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)計并合成6對針對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EGFP的真核載體，命名為pGenesil-siHBV1～6，并設(shè)立與HBV序列無關(guān)的pGenesil-siHBV-HK作為陰性對照。 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細胞。采用實時熒光定量PCR檢測轉(zhuǎn)染后2d、3d、4d靶基因的mRNA和細胞培養(yǎng)上清中cccDNA的水平；ELISA檢測轉(zhuǎn)染后2d、3d、5d、7d細胞培養(yǎng)上清中HBsAg、HBeAg的表達。 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細胞，倒置相差熒光顯微鏡和流式細胞術(shù)（FCM）檢測細胞內(nèi)EGFP的表達，檢測轉(zhuǎn)染效率；FCM檢測細胞內(nèi)HBcAg的表達以及轉(zhuǎn)染后3d細胞的凋亡；ELISA檢測細胞培養(yǎng)上清中HBsAg、HBeAg的表達，細胞免疫化學酶標技術(shù)觀察細胞內(nèi)HBsAg的表達。 【結(jié)果】 1. HBV特異性siRNA載體的構(gòu)建：PCR、酶切及測序鑒定結(jié)果表明，針對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，結(jié)果表明pGenesil-siHBV1～6均可不同程度的抑制HBV的復制和抗原的表達，其中pGenesil-siHBV1的抑制效果最強。 3. ASGPR靶向pGenesil-siHBV1對HBV的沉默效應：將pGenesil-siHBV1利用jetPEI-Hepatocyte靶向轉(zhuǎn)染HepG2.2.15細胞，倒置相差熒光顯微鏡下可見綠色熒光，而無jetPEI-Hepatocyte轉(zhuǎn)染對照組未見綠色熒光，F(xiàn)CM檢測綠色熒光陽性細胞約為45％；FCM檢測細胞內(nèi)HBcAg的表達以及ELISA檢測靶向?qū)嶒灲M細胞培養(yǎng)上清中HBsAg、HBeAg的表達低于LipofectamineTM2000轉(zhuǎn)染對照組，且二者均顯著低于無關(guān)序列對照組；細胞免疫化學酶標技術(shù)觀察細胞內(nèi)HBsAg的表達與無關(guān)序列對照組比較顯著降低；FCM檢測轉(zhuǎn)染后3d的細胞凋亡無明顯變化。 【結(jié)論】 結(jié)果表明針對HBV的不同siRNA的沉默效應存在差異；ASGPR介導的靶向遞送siRNA載體能更好的發(fā)揮沉默HBV基因復制與表達的作用，為下一步的體內(nèi)實驗奠定了良好的基礎(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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346

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本文編號：1721441