發(fā)布時(shí)間：2018-04-20 14:02

  本文選題：腺相關(guān)病毒2 + 靶向基因治療��； 參考：《華中科技大學(xué)》2010年博士論文

【摘要】： 目的 1.通過分子生物學(xué)技術(shù)對(duì)腺相關(guān)病毒(Adeno-associated virus, AAV)的改構(gòu)以獲得既能夠特異性結(jié)合乙肝表面抗原(Hepatitis B surface antigen, HBsAg)又?jǐn)y帶shRNA的重組病毒,為開發(fā)治療HBV的靶向性試劑和藥物奠定基礎(chǔ)。 2.研究肝臟去唾液酸糖蛋白受體(asialoglycoprotein receptor, ASGPR)大亞基異構(gòu)體的生物學(xué)功能,為進(jìn)一步以其為靶標(biāo)的肝臟靶向治療奠定基礎(chǔ)。 方法 1.運(yùn)用重組PCR技術(shù)將本室篩選得到的針對(duì)乙肝表面抗原的特異性多肽插入到2型腺相關(guān)病毒(AAV2)核衣殼蛋白的587位氨基酸處,同時(shí)將針對(duì)HBsAg的shRNA插入到AAV2的基因組。構(gòu)建既攜帶shRNA又靶向HBsAg的重組AAV2病毒。 2.運(yùn)用磷酸鈣共轉(zhuǎn)染法包裝重組的病毒,病毒經(jīng)過純化后,Real-Time PCR測定滴度。重組病毒按照一定的滴度感染HepG2、HepG2.215細(xì)胞,流式細(xì)胞儀檢測感染效率。 3.肝素封閉實(shí)驗(yàn)和HBsAb封閉實(shí)驗(yàn)驗(yàn)證重組病毒感染HepG2.215細(xì)胞的特異性,ELISA檢測病毒對(duì)HBsAg、HBeAg的抑制。 4.從正常人肝組織中克隆出ASGPR大亞基Hla、Hlb和小亞基H2c,構(gòu)建EGFP融合表達(dá)質(zhì)粒,熒光顯微鏡下觀察大亞基Hla、Hlb和小亞基H2c的細(xì)胞定位。 5.建立穩(wěn)定表達(dá)ASGPRH1a、ASGPRH2c的功能性細(xì)胞系來研究ASGPR大亞基異構(gòu)體H1b在ASGPR分子結(jié)合配體功能中的作用,同時(shí)研究sASGPR的生物學(xué)功能。 結(jié)果 1.成功構(gòu)建了同時(shí)攜帶靶向HBsAg多肽和攜帶shRNA的重組AAV2,命名為rAAVssyU6-shRNA-hrGFP.純化的病毒滴度在109v.g/ml以上。 2. rAAVssyU6-shRNA-hrGFP對(duì)HepG2、HepG2.215細(xì)胞的感染率較AAV2明顯升高,并且對(duì)HepG2.215細(xì)胞最為明顯。并且可以抑制HepG2.215細(xì)胞HBsAg、HBeAg的表達(dá)。 3.肝素封閉實(shí)驗(yàn)可以促進(jìn)rAAVssyU6-shRNA-hrGFP病毒感染HepG2.215細(xì)胞,同時(shí)HBsAb封閉實(shí)驗(yàn)明顯降低rAAVssyU6-shRNA-hrGFP病毒感染HepG2.215細(xì)胞。 4.成功構(gòu)建了EGFP融合表達(dá)質(zhì)粒,EGFP-H1a主要在細(xì)胞膜表達(dá)；EGFP-H1b主要在細(xì)胞質(zhì)內(nèi)形成塊狀或顆粒狀的聚集物；EGFP-H2c在細(xì)胞膜和細(xì)胞質(zhì)內(nèi)均勻分布。 5.成功建立了表達(dá)ASGPR分子的功能性細(xì)胞系。并且ASGPR大亞基異構(gòu)體H1b不影響ASGPR分子結(jié)合配體分子ASOR, sASGPR下調(diào)ASGPR分子結(jié)合配體的功能；sASGPR可以非特異性地和細(xì)胞結(jié)合,ASOR可以特異性上調(diào)sASGPR與細(xì)胞的結(jié)合。 結(jié)論 1. rAAVssyU6-shRNA-hrGFP對(duì)HepG2.215細(xì)胞的感染率明顯升高,并且可以抑制HepG2.215細(xì)胞HBsAg、HBeAg的表達(dá)。 2.rAAVssyU6-shRNA-hrGFP同時(shí)保留天然的趨向性和對(duì)HBsAg的靶向性。 3.單獨(dú)的ASGPR大亞基異構(gòu)體H1b不影響ASGPR分子結(jié)合配體分子ASOR。 4. sASGPR可以和ASOR分子形成復(fù)合物。sASGPR在維持體內(nèi)糖基配體復(fù)合物代謝中發(fā)揮保護(hù)作用。當(dāng)大量有害的糖基配體在外周血中出現(xiàn)時(shí),sASGPR結(jié)合配體使其轉(zhuǎn)運(yùn)至肝臟代謝。本研究的創(chuàng)新點(diǎn)及意義 1.首次構(gòu)建了rAAVssyU6-shRNA-hrGFP病毒,并且該病毒在體外具有一定的靶向性和效應(yīng)性。為靶向HBV的基因治療提供了實(shí)驗(yàn)基礎(chǔ)。 2.首次探索了ASGPR大亞基異構(gòu)體H1b及sASGPR在ASGPR結(jié)合配體功能中的作用。為系統(tǒng)了解ASGPR的生物學(xué)特性奠定了基礎(chǔ)。
[Abstract]:objective
1. through the modification of Adeno-associated virus (AAV) by molecular biology technology, the recombinant virus which can specifically bind the hepatitis B surface antigen (Hepatitis B surface antigen, HBsAg) and carry shRNA is obtained, which lays the foundation for the development of targeted reagents and drugs for the treatment of HBV.
2. study the biological functions of the asialoglycoprotein receptor (ASGPR) large subunit isomer of the liver and lay the foundation for further targeting treatment of liver targeting.
Method
1. the recombinant PCR technique was used to insert the specific polypeptide against HBsAg to the 587 bit amino acid of the AAV2 nucleocapsid protein, and the shRNA of HBsAg was inserted into the genome of AAV2. The recombinant AAV2 virus, which carried both shRNA and target to HBsAg, was constructed.
2. the recombinant virus was packaged by calcium phosphate co transfection. After the virus was purified, the titer was measured by Real-Time PCR. The recombinant virus was infected with HepG2, HepG2.215 cells, and flow cytometry to detect the infection efficiency.
3. heparin blocking test and HBsAb blocking test confirmed the specificity of recombinant virus infected HepG2.215 cells, and ELISA detected virus inhibition on HBsAg and HBeAg.
4. ASGPR large subunit Hla, Hlb and small subunit H2c were cloned from normal human liver tissue, and EGFP fusion expression plasmid was constructed. The cell localization of large subunit Hla, Hlb and small subunit H2c was observed under the fluorescence microscope.
5. a functional cell line expressing ASGPRH1a and ASGPRH2c is established to study the role of ASGPR large subunit isomer H1b in the function of ASGPR molecular binding ligand, and to study the biological function of sASGPR.
Result
1. successfully constructed a recombinant AAV2 carrying HBsAg peptide and carrying shRNA at the same time, named rAAVssyU6-shRNA-hrGFP., and the virus titer was above 109v.g/ml.
The infection rate of 2. rAAVssyU6-shRNA-hrGFP to HepG2, HepG2.215 cells was significantly higher than that of AAV2, and was most obvious to HepG2.215 cells, and it could inhibit the expression of HBsAg and HBeAg in HepG2.215 cells.
3. heparin sealing experiment can promote rAAVssyU6-shRNA-hrGFP virus infection of HepG2.215 cells, while HBsAb blocking experiment obviously reduces the rAAVssyU6-shRNA-hrGFP virus infection of HepG2.215 cells.
4. the EGFP fusion expression plasmid was successfully constructed, and EGFP-H1a was mainly expressed in the cell membrane; EGFP-H1b mainly formed lump or granular aggregates in the cytoplasm; EGFP-H2c was distributed evenly in the cell membrane and cytoplasm.
5. the functional cell lines expressing the ASGPR molecule have been successfully established. And the ASGPR large subunit isomer H1b does not affect the function of the ASGPR binding ligand molecule ASOR, sASGPR downregulation the function of the ASGPR binding ligand; sASGPR can bind nonspecifically to the cell, and ASOR can specifically regulate the binding of sASGPR to the cell.
conclusion
1. rAAVssyU6-shRNA-hrGFP infection rate of HepG2.215 cells increased significantly, and could inhibit the expression of HBsAg and HBeAg in HepG2.215 cells.
2.rAAVssyU6-shRNA-hrGFP also retained natural tendency and targeted HBsAg.
3. the independent ASGPR large subunit isomer H1b does not affect the ASGPR binding ligand molecule ASOR..
4. sASGPR can form complex.SASGPR with ASOR molecules to protect the metabolism of glycosyl ligand complexes in the body. When a large number of harmful glycosyl ligands appear in peripheral blood, sASGPR binding ligands are transported to the liver metabolism. The innovation and significance of this study
1. the rAAVssyU6-shRNA-hrGFP virus has been constructed for the first time, and the virus has a certain targeting and effect in vitro. It provides an experimental basis for gene therapy targeting HBV.
2. for the first time, we explored the role of ASGPR subunit H1b and sASGPR in the function of ASGPR binding ligand for the first time, and laid a foundation for understanding the biological characteristics of ASGPR.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

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本文編號(hào)：1778070