肝脏靶向治疗载体系统的基础研究

发布时间：2018-04-20 14:02

  本文选题：腺相关病毒2 + 靶向基因治疗　； 参考：《华中科技大学》2010年博士论文

【摘要】： 目的 1.通过分子生物学技术对腺相关病毒(Adeno-associated virus, AAV)的改构以获得既能够特异性结合乙肝表面抗原(Hepatitis B surface antigen, HBsAg)又携带shRNA的重组病毒,为开发治疗HBV的靶向性试剂和药物奠定基础。 2.研究肝脏去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)大亚基异构体的生物学功能,为进一步以其为靶标的肝脏靶向治疗奠定基础。 方法 1.运用重组PCR技术将本室筛选得到的针对乙肝表面抗原的特异性多肽插入到2型腺相关病毒(AAV2)核衣壳蛋白的587位氨基酸处,同时将针对HBsAg的shRNA插入到AAV2的基因组。构建既携带shRNA又靶向HBsAg的重组AAV2病毒。 2.运用磷酸钙共转染法包装重组的病毒,病毒经过纯化后,Real-Time PCR测定滴度。重组病毒按照一定的滴度感染HepG2、HepG2.215细胞,流式细胞仪检测感染效率。 3.肝素封闭实验和HBsAb封闭实验验证重组病毒感染HepG2.215细胞的特异性,ELISA检测病毒对HBsAg、HBeAg的抑制。 4.从正常人肝组织中克隆出ASGPR大亚基Hla、Hlb和小亚基H2c,构建EGFP融合表达质粒,荧光显微镜下观察大亚基Hla、Hlb和小亚基H2c的细胞定位。 5.建立稳定表达ASGPRH1a、ASGPRH2c的功能性细胞系来研究ASGPR大亚基异构体H1b在ASGPR分子结合配体功能中的作用,同时研究sASGPR的生物学功能。 结果 1.成功构建了同时携带靶向HBsAg多肽和携带shRNA的重组AAV2,命名为rAAVssyU6-shRNA-hrGFP.纯化的病毒滴度在109v.g/ml以上。 2. rAAVssyU6-shRNA-hrGFP对HepG2、HepG2.215细胞的感染率较AAV2明显升高,并且对HepG2.215细胞最为明显。并且可以抑制HepG2.215细胞HBsAg、HBeAg的表达。 3.肝素封闭实验可以促进rAAVssyU6-shRNA-hrGFP病毒感染HepG2.215细胞,同时HBsAb封闭实验明显降低rAAVssyU6-shRNA-hrGFP病毒感染HepG2.215细胞。 4.成功构建了EGFP融合表达质粒,EGFP-H1a主要在细胞膜表达；EGFP-H1b主要在细胞质内形成块状或颗粒状的聚集物；EGFP-H2c在细胞膜和细胞质内均匀分布。 5.成功建立了表达ASGPR分子的功能性细胞系。并且ASGPR大亚基异构体H1b不影响ASGPR分子结合配体分子ASOR, sASGPR下调ASGPR分子结合配体的功能；sASGPR可以非特异性地和细胞结合,ASOR可以特异性上调sASGPR与细胞的结合。 结论 1. rAAVssyU6-shRNA-hrGFP对HepG2.215细胞的感染率明显升高,并且可以抑制HepG2.215细胞HBsAg、HBeAg的表达。 2.rAAVssyU6-shRNA-hrGFP同时保留天然的趋向性和对HBsAg的靶向性。 3.单独的ASGPR大亚基异构体H1b不影响ASGPR分子结合配体分子ASOR。 4. sASGPR可以和ASOR分子形成复合物。sASGPR在维持体内糖基配体复合物代谢中发挥保护作用。当大量有害的糖基配体在外周血中出现时,sASGPR结合配体使其转运至肝脏代谢。本研究的创新点及意义 1.首次构建了rAAVssyU6-shRNA-hrGFP病毒,并且该病毒在体外具有一定的靶向性和效应性。为靶向HBV的基因治疗提供了实验基础。 2.首次探索了ASGPR大亚基异构体H1b及sASGPR在ASGPR结合配体功能中的作用。为系统了解ASGPR的生物学特性奠定了基础。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

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