携带HBB基因shRNA的人脐血CD34+细胞构建及其应用基础研究
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摘要
β-血红蛋白病是一组多相群的单基因疾病,重型可致死。为了探讨其基因治疗的可行性及潜在价值。本课题拟选择对β-珠蛋白基因具有靶向RNAi的特异序列,并构建慢病毒载体,采用体外筛选法获得目的序列及质粒,以期能与β-珠蛋白等位基因特异性结合抑制RNA的表达,并了解感染细胞的体内植入和增殖情况,以期在以后试验中与其它基因表达载体组合共同用于治疗β-血红蛋白病。
内容和方法
1.从GeneBank查找HBB mRNA序列,设计挑选3个RNAi靶点,每个合成两条单寡核苷酸链,退火后连入酶切的pGCL-GFP载体,将连接产物转染感受态细菌,对长出的克隆进行PCR鉴定,测序比对。对阳性克隆进行质粒抽提。
2.用PCR方法从模板钓取相应的HBB序列片段,将片段与pdsRED2-N1载体进行双酶切后定向连接,转染感受态细菌,长出的克隆进行PCR鉴定,测序和比对分析后,对阳性克隆进行质粒抽提。
3.HBB-RNAi-LV质粒和HBB过表达质粒,按低浓度组(0.5:1)和高浓度组(1:1)用Lipofectamine2000共转染293T细胞,48h后FM观察GFP表达,Western Blot检测FLAG-HBB融合蛋白表达。
4.将HBB-RNAi-LV质粒和慢病毒包装载体pHelper 1.0及pHelper 2.0载体按4:3:2比例添加,转染293T细胞,培养48小时后收集培养上清,利用Plus-20离心超滤装置对病毒上清进行离心超浓缩。
5.人脐血MACS法分离的CD34+细胞,加入细胞因子进行无血清无基质预培养,慢病毒感染,48h后FM观察感染率,Real-Time PCR检测感染2、4、5天的HBB\HBG基因表达情况,Western Blot检测感染7天的HBB蛋白表达。
6.人脐血CD34+细胞,体外用高浓度细胞因子预刺激培养过夜后感染慢病毒24小时,经皮注射入SCID新生小鼠腹腔,密切观察小鼠存活情况。
7.移植后3周,用FM和FCM检测小鼠外周血中人HbF细胞和成人红细胞,外周血有核细胞中的GFP阳性细胞;骨髓细胞中的GFP阳性细胞,并观察小鼠肝、脾、肾脏、小肠等组织中的GFP阳性细胞分布。
结果
1.构建了3个HBB基因的RNAi-LV载体(PSC1、PSC2、PSC3)及一个对照质粒PSCNC。
2.构建了含有相应HBB基因片段的过表达载体HBB- pdsRED2-N1-3Flag质粒。
3.外源筛靶试验中PSC1、PSC3对HBB融合蛋白有明显敲减作用,随着浓度增加敲减作用增强,其中PSC1靶点的敲减效果最好。
4.包装并获得高滴度的PSC1、PSC3和PSCNC慢病毒。
5.慢病毒在CD34+细胞中的感染率为15~30%,病毒感染明显激活细胞中的HBB和HBG基因表达,PSC1促进HBB珠蛋白表达,PSC3则显著抑制HBB和HBG珠蛋白表达,其抑制效果与感染率有关。
6.移植CD34+细胞后,小鼠外周血中人HbF细胞及成人红细胞为0.1~1.2%;有核细胞中GFP细胞,PSCNC组为3.2~10.2%, PSC3组为4.4~8.8%;骨髓细胞中GFP细胞PSCNC组为1.5~5.5%, PSC3组为1.3~6.2%,各组间均无统计学意义。肝、脾、肾和小肠等组织均未见明显的GFP阳性细胞。
结论
1.系统建立了完整的人脐带血CD34+细胞分离、无血清无基质培养和慢病毒感染的实验方法。
2.成功构建了3个β-珠蛋白基因shRNA的慢病毒载体,通过外源筛靶方法初筛出2个有效序列,经慢病毒包装获得了高滴度的病毒颗粒。
3.成功筛选到一条对HBB基因有抑制作用的RNAi序列。
4.人脐血CD34+细胞移植入未骨髓抑制的免疫缺陷病新生小鼠腹腔,可成功归巢、增殖,主要增殖为自身缺陷的淋巴系统,红系缺乏表达优势。
Beta-hemoglobinopathies is caused by singleβ-globin genetic variation, of which the most frequent occurrence isβ-thalassemia and sickle cell disease. The patients with severe form maybe die without any treatment. For approaching the feasibility and potential value of gene RNA interference, the objective of this study is to design special RNAi target and to construct the lentiviral vector, to screen the effective target to specific bind and restrainβ-globin allele gene expression, and to observe the homing and repopulation of human infected CB CD34+ cells, and hope to conduce gene thrapy inβ-hemoglobinopathies by combination with other expression vector in follow-up experiment.
Contents and Methods
1. Based on the HBB mRNA sequence from GeneBank, we designed and selected target sequences for RNAi to HBB gene. After designed and synthesized, two single oligonucleotide DNA chains of each target were annealed and linked to enzyme lysised pGCL-GFP vector, then transferred to competence bacterial. The positive colony was amplified and extracted, and identified by PCR and sequencing.
2. The relevantβ-globin gene fragment were copied and linked with pdsRED2-N1vector, then transferred to competence bacterial. The positive colony was amplified and extracted, and identified by PCR, sequencing and Blast analysis.
3. HBB-RNAi-LV plasmid and HBB over-expression plasmid were cotransfected into 293T cells by lipofectamine 2000 with the low concentration group(0.5:1) and high concentration(1:1) experimental group. Cells were observed under fluorescence microscopy, and the Flag and HBB fusion protein were detected.
4. Packaged lentivirus particles with HBB-RNAi-LV plasmid, pHelper 1.0 vector and pHelper 2.0 vector, the viral supernatant was then concentrated by Plus-20 ultrafiltration kit.
5. CD34+ cells were isolated from human cord blood (CB) by MACS, cultured with cell factors and stroma-free SFM medium, and infected with MOI of 10 by LV particles. The ration of GFP positive cells was detected with FM and FCM. The HBB and HBG mRNA were detected by Real-Time PCR by 2, 4, or 5days after infection. The HBB protein level was determined by Western Blot.
6. Human CB CD34+ cells prestimulated with high concentrated cell factors, and infected with LV virus, then injected into peritoneal cavity of SCID neonatal mice percataneously.
7. Three weeks after transplantation, human HbF cells and adult red cells were deteced by FM and FCM in mouse blood. GFP positive cells were checked in both blood and bone marrow. GFP positive cells were also found in liver, speen, kidney and small intestine.
Results
1. Three HBB-RNAi-LV plasmids have been designed and constructed, named as PSC1, PSC2 and PSC3 respectively.
2. The relevant HBB over-expression vector has been constructed, named as HBB- pdsRED2-N1-3Flag vectors.
3. The RNAi effectiveness of PSC1 and PSC3 were confirmed by exogenous transfection experiment, and the effects were enhanced with the concentration of the RNAi plasmids increased, especially for PSC1.
4. The high titer lentiviral virus of PSC1, PSC3 and PSCNC were obtained.
5. After infection, the ratio of GFP positive cells was around 15~30% in CD34+cells. The HBB and HBG gene expressions were activated temporarily by lentivirus infection, and down-regulated relatively by PSC3 to which the degree of decrease is related to infection efficiency. In contrast, PSC1 up-regulated the HBB gene expression.
6. After transplanted human infected CB CD34+ cells, percentage of human erythrocyte cells being 0.1-1.2% in peripheral blood, percentages of GFP positive cells being 4.4-8.8% vs 3.2-10.2%( PSC3 group vs PSCNC group) in peripheral nucleated cells, 1.3-6.2% vs 1.5-5.5%( PSC3 groups vs PSCNC groups) in bone marrow cells, there weren’t statistical significance between groups. GFP positive cells were not founded in spleen, liver, kidney and small intestine tissues.
Conclusions
1. Establish a set of methods for separation, serum-free and stroma-free culture, and infection of human CB CD34+ cells.
2. Constructed three HBB-RNAi-LV vectors, confirmed the RNAi effectiveness of PSC1 and PSC3 by exogenous transfection experiment, packaged these two vectors and obtained high titer lentiviral virus.
3. Obtained one efficient RNAi constrution for down-regulation HBB gene by infection human cord blood CD34+cells.
4. After transplanted into nonmyelosuppressive SCID mouse, human CB CD34+cells with HBB shRNA may experience homing, repopulation and differentiation, in which lymphatic system were defective, and erythro-system being absence of competitive advantage.
内容和方法
1.从GeneBank查找HBB mRNA序列,设计挑选3个RNAi靶点,每个合成两条单寡核苷酸链,退火后连入酶切的pGCL-GFP载体,将连接产物转染感受态细菌,对长出的克隆进行PCR鉴定,测序比对。对阳性克隆进行质粒抽提。
2.用PCR方法从模板钓取相应的HBB序列片段,将片段与pdsRED2-N1载体进行双酶切后定向连接,转染感受态细菌,长出的克隆进行PCR鉴定,测序和比对分析后,对阳性克隆进行质粒抽提。
3.HBB-RNAi-LV质粒和HBB过表达质粒,按低浓度组(0.5:1)和高浓度组(1:1)用Lipofectamine2000共转染293T细胞,48h后FM观察GFP表达,Western Blot检测FLAG-HBB融合蛋白表达。
4.将HBB-RNAi-LV质粒和慢病毒包装载体pHelper 1.0及pHelper 2.0载体按4:3:2比例添加,转染293T细胞,培养48小时后收集培养上清,利用Plus-20离心超滤装置对病毒上清进行离心超浓缩。
5.人脐血MACS法分离的CD34+细胞,加入细胞因子进行无血清无基质预培养,慢病毒感染,48h后FM观察感染率,Real-Time PCR检测感染2、4、5天的HBB\HBG基因表达情况,Western Blot检测感染7天的HBB蛋白表达。
6.人脐血CD34+细胞,体外用高浓度细胞因子预刺激培养过夜后感染慢病毒24小时,经皮注射入SCID新生小鼠腹腔,密切观察小鼠存活情况。
7.移植后3周,用FM和FCM检测小鼠外周血中人HbF细胞和成人红细胞,外周血有核细胞中的GFP阳性细胞;骨髓细胞中的GFP阳性细胞,并观察小鼠肝、脾、肾脏、小肠等组织中的GFP阳性细胞分布。
结果
1.构建了3个HBB基因的RNAi-LV载体(PSC1、PSC2、PSC3)及一个对照质粒PSCNC。
2.构建了含有相应HBB基因片段的过表达载体HBB- pdsRED2-N1-3Flag质粒。
3.外源筛靶试验中PSC1、PSC3对HBB融合蛋白有明显敲减作用,随着浓度增加敲减作用增强,其中PSC1靶点的敲减效果最好。
4.包装并获得高滴度的PSC1、PSC3和PSCNC慢病毒。
5.慢病毒在CD34+细胞中的感染率为15~30%,病毒感染明显激活细胞中的HBB和HBG基因表达,PSC1促进HBB珠蛋白表达,PSC3则显著抑制HBB和HBG珠蛋白表达,其抑制效果与感染率有关。
6.移植CD34+细胞后,小鼠外周血中人HbF细胞及成人红细胞为0.1~1.2%;有核细胞中GFP细胞,PSCNC组为3.2~10.2%, PSC3组为4.4~8.8%;骨髓细胞中GFP细胞PSCNC组为1.5~5.5%, PSC3组为1.3~6.2%,各组间均无统计学意义。肝、脾、肾和小肠等组织均未见明显的GFP阳性细胞。
结论
1.系统建立了完整的人脐带血CD34+细胞分离、无血清无基质培养和慢病毒感染的实验方法。
2.成功构建了3个β-珠蛋白基因shRNA的慢病毒载体,通过外源筛靶方法初筛出2个有效序列,经慢病毒包装获得了高滴度的病毒颗粒。
3.成功筛选到一条对HBB基因有抑制作用的RNAi序列。
4.人脐血CD34+细胞移植入未骨髓抑制的免疫缺陷病新生小鼠腹腔,可成功归巢、增殖,主要增殖为自身缺陷的淋巴系统,红系缺乏表达优势。
Beta-hemoglobinopathies is caused by singleβ-globin genetic variation, of which the most frequent occurrence isβ-thalassemia and sickle cell disease. The patients with severe form maybe die without any treatment. For approaching the feasibility and potential value of gene RNA interference, the objective of this study is to design special RNAi target and to construct the lentiviral vector, to screen the effective target to specific bind and restrainβ-globin allele gene expression, and to observe the homing and repopulation of human infected CB CD34+ cells, and hope to conduce gene thrapy inβ-hemoglobinopathies by combination with other expression vector in follow-up experiment.
Contents and Methods
1. Based on the HBB mRNA sequence from GeneBank, we designed and selected target sequences for RNAi to HBB gene. After designed and synthesized, two single oligonucleotide DNA chains of each target were annealed and linked to enzyme lysised pGCL-GFP vector, then transferred to competence bacterial. The positive colony was amplified and extracted, and identified by PCR and sequencing.
2. The relevantβ-globin gene fragment were copied and linked with pdsRED2-N1vector, then transferred to competence bacterial. The positive colony was amplified and extracted, and identified by PCR, sequencing and Blast analysis.
3. HBB-RNAi-LV plasmid and HBB over-expression plasmid were cotransfected into 293T cells by lipofectamine 2000 with the low concentration group(0.5:1) and high concentration(1:1) experimental group. Cells were observed under fluorescence microscopy, and the Flag and HBB fusion protein were detected.
4. Packaged lentivirus particles with HBB-RNAi-LV plasmid, pHelper 1.0 vector and pHelper 2.0 vector, the viral supernatant was then concentrated by Plus-20 ultrafiltration kit.
5. CD34+ cells were isolated from human cord blood (CB) by MACS, cultured with cell factors and stroma-free SFM medium, and infected with MOI of 10 by LV particles. The ration of GFP positive cells was detected with FM and FCM. The HBB and HBG mRNA were detected by Real-Time PCR by 2, 4, or 5days after infection. The HBB protein level was determined by Western Blot.
6. Human CB CD34+ cells prestimulated with high concentrated cell factors, and infected with LV virus, then injected into peritoneal cavity of SCID neonatal mice percataneously.
7. Three weeks after transplantation, human HbF cells and adult red cells were deteced by FM and FCM in mouse blood. GFP positive cells were checked in both blood and bone marrow. GFP positive cells were also found in liver, speen, kidney and small intestine.
Results
1. Three HBB-RNAi-LV plasmids have been designed and constructed, named as PSC1, PSC2 and PSC3 respectively.
2. The relevant HBB over-expression vector has been constructed, named as HBB- pdsRED2-N1-3Flag vectors.
3. The RNAi effectiveness of PSC1 and PSC3 were confirmed by exogenous transfection experiment, and the effects were enhanced with the concentration of the RNAi plasmids increased, especially for PSC1.
4. The high titer lentiviral virus of PSC1, PSC3 and PSCNC were obtained.
5. After infection, the ratio of GFP positive cells was around 15~30% in CD34+cells. The HBB and HBG gene expressions were activated temporarily by lentivirus infection, and down-regulated relatively by PSC3 to which the degree of decrease is related to infection efficiency. In contrast, PSC1 up-regulated the HBB gene expression.
6. After transplanted human infected CB CD34+ cells, percentage of human erythrocyte cells being 0.1-1.2% in peripheral blood, percentages of GFP positive cells being 4.4-8.8% vs 3.2-10.2%( PSC3 group vs PSCNC group) in peripheral nucleated cells, 1.3-6.2% vs 1.5-5.5%( PSC3 groups vs PSCNC groups) in bone marrow cells, there weren’t statistical significance between groups. GFP positive cells were not founded in spleen, liver, kidney and small intestine tissues.
Conclusions
1. Establish a set of methods for separation, serum-free and stroma-free culture, and infection of human CB CD34+ cells.
2. Constructed three HBB-RNAi-LV vectors, confirmed the RNAi effectiveness of PSC1 and PSC3 by exogenous transfection experiment, packaged these two vectors and obtained high titer lentiviral virus.
3. Obtained one efficient RNAi constrution for down-regulation HBB gene by infection human cord blood CD34+cells.
4. After transplanted into nonmyelosuppressive SCID mouse, human CB CD34+cells with HBB shRNA may experience homing, repopulation and differentiation, in which lymphatic system were defective, and erythro-system being absence of competitive advantage.
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