肝脏靶向治疗载体系统的基础研究
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。
Objective
1. To obtain HBsAg targeting recombinant adeno-associated virus (rAAV) encoding HBsAg-shRNA by molecular biological techniques, and lay a foundation for developing new reagents or drugs for the targeting treatment of HBV.
2. To study the biological function of the major subunit variants of human asialoglycoprotein receptor and provide foundation for hepatic targeting therapy through ASGPR.
Methods
1. In order to construct both expressing shRNA and HBsAg targeting recombinant AAV2 virus,a HBsAg specific peptide was inserted into the 587AA position of AAV2 capside by recombinant PCR and meanwhile a DNA sequence expressing HBsAg-shRNA was inserted into the genome of AAV2.
2. The recombinant AAV2 was paked by cotransfected with three plasmids into AAV-293 cells mediated by calcium acid phosphate. The rAAVs (rAAVssy-shHBs) infected HepG2,HepG2215 after purified and quntitated by Real-Time PCR.The efficiency of infection was tested by FACS.
3. The specificity of the rAAVssyU6-shRNA-hrGFP on HepG2.215 cell was identified by heparin and HBsAb blocking Test. The silencing effect of the virus on HBsAg, HBeAg gene expression was assessed by ELISA.
4. Two major subunit variants of ASGPRH1 (Hla and Hlb) and a minor.subunit variant H2c were cloned from normal human liver tissues. These genes were subcloned into eukaryotic expression vector to construct EGFP fusion protein in order to test their location in cell.
5. To constructed a cell line which stably expression ASGPRH1a and H2c. The function of the cell line was tested by FACS.The influence of the split variant ASGPRHlb on the ASGPR-ASOR binding ability was tested by this cell line; the biological function of sASGPR was also studied by this cell line.
Results
1. The HBsAg targeting and shRNA expressing recombinant AAV2 virus was constructed successfully, designated rAAVssyU6-shRNA-hrGFP.The titer of the purified rAAVssyU6-shRNA-hrGFP was above 1×109v.g/ml.
2. The efficiency of rAAVssyU6-shRNA-hrGFP infection on HepG2, HepG2.215 was increased, especially on HepG2.215. The HBsAg, HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAVssy-shHBs.
3. The efficiency of the rAAVssyU6-shRNA-hrGFP on HepG2.215 cell was increased by heparin and decreased by HBsAb.
4. The EGFP fusional protein vectors were constructed successfully. EGFP-Hla located on cell membrane; EGFP-Hlb expressed in cytoplasm by particle; EGFP-H2c expressed on cell membrane and in cytoplasm equably.
5. A cell line which stably expression ASGPRHla and H2c was constructed successfully. The function of the cell line was confirmed by ASOR binding assay. Single Hlb showed no effect on ASOR binding to cells.sASGPR down regulated the ASGPR-ASOR binding.sASGPR could nonspecific bind to cells and ASOR specifically increased its binding.
Conclusions
1. The efficiency of rAAVssyU6-shRNA-hrGFP infection on HepG2.215 were increased markly.The HBsAg, HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAVssyU6-shRNA-hrGFP.
2. rAAVssyU6-shRNA-hrGFP could target HBsAg but remain natural tropism.
3. Single H1b showed no effect on ASGPR-ASOR binding.
4. sASGPR could bind ASOR to form complex. SASGPR played a protective role in maintaining the balance of glycoconjugates metabolism.SASGPR recognized and bound the deleterious soluble glycoconjugates and then transported them safely to the liver, where they will be removed and degraded by the hepatic ASGPR system.
Significances of the study
1. This is the first reported rAAVssyU6-shRNA-hrGFP could targeting HBsAg and silencing HBsAg, HBeAg in vitro. This study laid a foundation for targeting gene therapy of HBV.
2. Our works provide the first evidence of the function of human hepatic ASGPR Hlb and sASGPR in ASGPR-ligand binding. The study laid a foundation for further comprehensive study of the bionomics of ASGPR.
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。
Objective
1. To obtain HBsAg targeting recombinant adeno-associated virus (rAAV) encoding HBsAg-shRNA by molecular biological techniques, and lay a foundation for developing new reagents or drugs for the targeting treatment of HBV.
2. To study the biological function of the major subunit variants of human asialoglycoprotein receptor and provide foundation for hepatic targeting therapy through ASGPR.
Methods
1. In order to construct both expressing shRNA and HBsAg targeting recombinant AAV2 virus,a HBsAg specific peptide was inserted into the 587AA position of AAV2 capside by recombinant PCR and meanwhile a DNA sequence expressing HBsAg-shRNA was inserted into the genome of AAV2.
2. The recombinant AAV2 was paked by cotransfected with three plasmids into AAV-293 cells mediated by calcium acid phosphate. The rAAVs (rAAVssy-shHBs) infected HepG2,HepG2215 after purified and quntitated by Real-Time PCR.The efficiency of infection was tested by FACS.
3. The specificity of the rAAVssyU6-shRNA-hrGFP on HepG2.215 cell was identified by heparin and HBsAb blocking Test. The silencing effect of the virus on HBsAg, HBeAg gene expression was assessed by ELISA.
4. Two major subunit variants of ASGPRH1 (Hla and Hlb) and a minor.subunit variant H2c were cloned from normal human liver tissues. These genes were subcloned into eukaryotic expression vector to construct EGFP fusion protein in order to test their location in cell.
5. To constructed a cell line which stably expression ASGPRH1a and H2c. The function of the cell line was tested by FACS.The influence of the split variant ASGPRHlb on the ASGPR-ASOR binding ability was tested by this cell line; the biological function of sASGPR was also studied by this cell line.
Results
1. The HBsAg targeting and shRNA expressing recombinant AAV2 virus was constructed successfully, designated rAAVssyU6-shRNA-hrGFP.The titer of the purified rAAVssyU6-shRNA-hrGFP was above 1×109v.g/ml.
2. The efficiency of rAAVssyU6-shRNA-hrGFP infection on HepG2, HepG2.215 was increased, especially on HepG2.215. The HBsAg, HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAVssy-shHBs.
3. The efficiency of the rAAVssyU6-shRNA-hrGFP on HepG2.215 cell was increased by heparin and decreased by HBsAb.
4. The EGFP fusional protein vectors were constructed successfully. EGFP-Hla located on cell membrane; EGFP-Hlb expressed in cytoplasm by particle; EGFP-H2c expressed on cell membrane and in cytoplasm equably.
5. A cell line which stably expression ASGPRHla and H2c was constructed successfully. The function of the cell line was confirmed by ASOR binding assay. Single Hlb showed no effect on ASOR binding to cells.sASGPR down regulated the ASGPR-ASOR binding.sASGPR could nonspecific bind to cells and ASOR specifically increased its binding.
Conclusions
1. The efficiency of rAAVssyU6-shRNA-hrGFP infection on HepG2.215 were increased markly.The HBsAg, HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAVssyU6-shRNA-hrGFP.
2. rAAVssyU6-shRNA-hrGFP could target HBsAg but remain natural tropism.
3. Single H1b showed no effect on ASGPR-ASOR binding.
4. sASGPR could bind ASOR to form complex. SASGPR played a protective role in maintaining the balance of glycoconjugates metabolism.SASGPR recognized and bound the deleterious soluble glycoconjugates and then transported them safely to the liver, where they will be removed and degraded by the hepatic ASGPR system.
Significances of the study
1. This is the first reported rAAVssyU6-shRNA-hrGFP could targeting HBsAg and silencing HBsAg, HBeAg in vitro. This study laid a foundation for targeting gene therapy of HBV.
2. Our works provide the first evidence of the function of human hepatic ASGPR Hlb and sASGPR in ASGPR-ligand binding. The study laid a foundation for further comprehensive study of the bionomics of ASGPR.
引文
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