肝细胞生长因子基因修饰的骨髓间充质干细胞移植的促血管新生作用
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摘要
第一部分人肝细胞生长因子重组慢病毒载体的构建及病毒的制备
目的：构建能表达重组人肝细胞生长因子(rhHGF)基因的第三代慢病毒载体,为实现rhHGF基因在大鼠骨髓间充质干细胞中长期、高效的表达奠定基础。
方法：(1)通过NheI/XbaI双酶切将pRNAT-U6.2慢病毒载体质粒中GFP基因敲除,然后将目的基因hHGF连接到该载体质粒上,构建重组载体质粒pRNAT-U6.2-hHGF。通过限制性内切酶酶切、菌落PCR和DNA测序进行鉴定。(2)利用TELEVECTORTM载体试剂盒将pRNAT-U6.2-hHGF联合辅助质粒pMDLg/pRRE、pMD2G-VSVG、pRSV-Rev共转染293T细胞,收集并浓缩慢病毒上清,逐孔稀释法测病毒滴度。
结果：(1)经限制性内切酶酶切及DNA测序分析表明,目的基因hHGF-cDNA已经插入载体质粒,且基因序列与基因库序列完全一致；(2)成功制备表达hHGF的慢病毒颗粒浓缩液,滴度为4×108Tu/ml。
结论：表达人肝细胞生长因子的第三代重组慢病毒载体pRNAT-U6.2-hHGF构建成功。
第二部分人肝细胞生长因子在大鼠骨髓间充质干细胞中的表达及生物学活性观察
目的：观察慢病毒载体介导的人肝细胞生长因子(hHGF)基因在骨髓间充质干细胞(BM-MSCs)中的表达及其生物学活性。
方法：(1)利用差速贴壁法联合Percoll密度梯度离心法分离、纯化大鼠原代BM-MSCs,流式鉴定MSC的表型,茜素红染色和油红O染色鉴定BM-MSCs的成骨、成脂分化潜能。(2)用高滴度表达hHGF (Lenti-HGF)或GFP (Lenti-GFP)的慢病毒液感染第三代的BM-MSCs,通过倒置相差荧光显微镜和流式鉴定来评估转染效率。ELISA检测细胞培养基上清中的hHGF浓度。(3)分别通过MTT法、Transwell小室法和Hoechst染色法检测脐静脉内皮细胞(HUVECs)和血管平滑肌细胞(VSMCs)的增殖、迁移及凋亡,评估感染Lenti-HGF的BM-MSCs分泌的hHGF的生物学活性。
结果：(1)通过联合差速贴壁法和密度梯度离心法可获得大量高纯度、具有多向分化潜能的BM-MSCs。(2)在转染Lenti-GFP后72h,即可在荧光显微镜下见明亮的绿色荧光,最佳感染复数(MOI)为50,流式鉴定转染效率约为75%。ELISA结果显示,Lenti-HGF感染BM-MSCs的培养上清液中的hHGF可持续较高水平地表达14d以上,且明显高于感染Lenti-GFP者(P<0.01)。(3)感染Lenti-HGF的MSCs分泌的hHGF在体外显示出促HUVECs增殖、迁移并抑制其凋亡,且能促进VSMCs迁移的生物学活性。
结论：Lenti-HGF慢病毒能够高效地介导转移外源基因hHGF至BM-MSCs表达,并具有生物学活性。
第三部分肝细胞生长因子基因修饰的骨髓间充质干细胞移植的促血管新生作用
目的：比较hHGF基因修饰的BM-MSCs移植与单纯BM-MSCs移植在大鼠后肢缺血模型中的促血管新生作用及其机制。
方法：在结扎左股动脉及其分支1天后,SD大鼠随机分为3组：HGF-MSC组(即HGF基因修饰的MSCs移植,n=8),MSC组(单纯的MSCs移植,n=8)以及PBS对照组(注射PBS,n=8)。在术后7天,进行细胞移植。实验组每只大鼠接受总计5×106个同一来源的HGF-MSCs或MSCs,分成5个点注入大鼠缺血后肢的股部肌肉；对照组注入同等体积的无菌PBS。分别在处理后1周或3周时处死大鼠,比较各组缺血股部肌肉中的毛细血管密度,移植细胞的分化转归,T淋巴细胞浸润以及生长因子HGF、VEGF蛋白表达等。ELISA检测HGF-MSC移植前和移植3周后大鼠血清HGF的水平。
结果：细胞移植3周后,MSC组和HGF-MSC组缺血肌肉组织的血管新生均明显增强,以HGF-MSC组的毛细血管密度最高(P<0.05)。从移植后1周开始,HGF-MSC组缺血组织中来源于移植细胞的内皮细胞数目明显高于MSC组(P<0.05)。在移植1周后,HGF-MSC和MSC的同种异体移植几乎不引起T淋巴细胞浸润,与PBS组相近(P>0.05)。Western-blot结果显示,MSC组和HGF-MSC组缺血肌肉组织中HGF、VEGF蛋白表达均明显高于PBS对照组,以HGF-MSC组的表达量最高(P<0.05)。HGF-MSC移植前和移植后3周,大鼠血清HGF浓度无明显变化(P>0.05)。
结论：与单纯的MSC细胞移植疗法相比,HGF基因修饰的MSC移植疗法具有更强的促进血管新生和侧枝血管形成的能力。以干细胞为基础的血管新生基因治疗策略将可能为治疗严重缺血性心血管病提供新思路。
Part I Construction of recombinant lentiviral vector carrying human hepatocyte growth factor gene
Objective:To construct a third-generation lentiviral vector carrying human hepatocyte growth factor (hHGF) for transfection.
Methods:Lentiviral vector plasmid carrying human HGF (pRNAT-U6.2-hHGF) was generated by removal of green fluorescence protein (GFP) gene from pRNAT-U6.2 plasmid by NheI/XbaI digestion and ligation of human HGF cDNA, which was identified by restriction endonuclease analysis, PCR and DNA sequence analysis. Recombinant plasmid pRNAT-U6.2-hHGF was co-transfected into 293T cells with the helper plasmids pMDLg/pRRE, pMD2G-VSVG and pRSV-Rev using the TELEVECTOR Lentiviral Vector Easy-high Production kit. The recombinant lentiviral stock (Lenti-HGF) was generated, concentrated, and then the titer was determined.
Results:The hHGF cDNA was identical to that of GeneBank. Recombinant lentiviral plasmid pRNAT-U6.2-hHGF was successfully constructed, and the titer of Lenti-HGF was 4×108Tu/ml.
Conclusion:Recombinant third-generation lentiviral vector plasmid carrying hHGF (pRNAT-U6.2-hHGF) was constructed successfully.
PartⅡExpression of human hepatocyte growth factor in primary rat bone marrow-derived mesenchymal stem cells and its biological activity
Objective:To explore the usability of lentiviral vector mediated human hepatocyte growth factor (hHGF) gene delivery into bone marrow-derived mesenchymal stem cells (BM-MSCs), and to observe its expression and biological activity.
Methods:(1) Primary rat BM-MSCs was isolated, expanded and purified by taking advantage of their differential adherence to dishes combined with Percoll density gradient centrifugation technique. MSC phenotype was analyzed by flow cytometry. Meanwhile, potency of MSCs differentiation into osteocytes and adipocytes were determined by alizarin red S staining and oil red O staining. (2) The third passage MSCs were transfected by high titer lentiviral stocks expressing human HGF (Lenti-HGF) or GFP (Lenti-GFP), and the transfection efficiency was assessed by fluorescent microscope observation and flow cytometry analysis. The concentration of hHGF in cell culture medium supernatants was determined by enzyme linked immunosorbent assay (ELISA). (3) Biological activity of hHGF secreted from Lenti-HGF infected BM-MSCs was determined by MTT cell proliferation assay, Transwell chamber migration assay and Hoechst staining.
Results:(1) Plenty of highly purified BM-MSCs were achieved by the combined methods. (2) Bright green fluorescence in the MSC was observed under the fluorescent microscope after 72h transfected with Lenti-GFP. The best multiplicity of infection (MOI) was 50, and the transfection efficiency reached about 75%. Furthermore, ELISA showed that hHGF secretion could be stably maintained more than 14 days since the level of hHGF in the culture supernatant from the Lenti-HGF transduced BM-MSCs was significantly higher than that of Lenti-GFP transduced BM-MSCs at day 5 (P<0.01). (3)The hHGF secreted from transduced MSCs showed the proproliferative, promigratory, antiapoptotic effects for human umbilical vein endothelial cells (HUVECs) and promigratory effect on vascular smooth muscle cells (VSMCs) in vitro.
Conclusion:The recombinant lentivirus (Lenti-HGF) can mediated transduction into BM-MSCs with a highly efficient and long lasting expression of the transgene. Key words:Hepatocyte growth factor; Mesenchymal stem cell; Lentivirus; Apoptosis
PartⅢHepatocyte growth factor genetically modified bone marrow-derived mesenchymal stem cells transplantation promote angiogenesis
Objective:To investigate whether a new strategy that combines MSCs transplantation and ex vivo human hepatocyte growth factor (hHGF) gene transferring with lentiviral vectors was more therapeutically efficient than MSCs cell therapy alone in a rat hindlimb ischemia model.
Methods:One day after resection of left femoral artery, Sprague-Dawley (SD) rats were randomly divided into three groups:(a) HGF genetically modified MSCs (HGF-MSCs) transplantation (HGF-MSC group, n=8), (b) BM-MSCs transplantation (MSC group, n=8), (c) PBS injection (PBS group, n=8). After creating hindlimb ischemia, an interval of 7 days was permitted for postoperative recovery. Then a total of 5×106 HGF-MSCs, MSCs obtained from same SD rats or PBS of same volume were injected into the ischemic thigh muscles of rats at five different points in each group. The ischemic thigh muscle were analyzed for capillary density, transplanted cell differentiation, T lymphocyte infiltration and the expression of HGF and VEGF protein at one or three weeks after treatment. Serum levels of HGF in HGF-MSC group before and 3 weeks after transplantation were determined by ELISA.
Results:Three weeks after transplantation, angiogenesis was significantly enhanced by both MSC group and HGF-MSC group, while capillary density was highest in the HGF-MSC group (P<0.05). The number of transplanted cell-derived endothelial cells was higher in the HGF-MSC group than in the MSC group since one week after treatment (P<0.05). In treated ischemic hind limb muscles, allogeneic HGF-MSC and MSC injection were associated with comparatively few T lymphocyte infiltration in PBS group (P>0.05). The expression of angiogenic growth factors like HGF and VEGF in local ischemic muscles undergoing intramuscular cell transplantation was more abundant in HGF-MSC group than the other two groups (P<0.05). Serum levels of HGF in HGF-MSC group before and 3 weeks after transplantation have no significant difference (P>0.05).
Conclusions:The present study shows that HGF gene-modified MSC transplantation therapy induced more potent angiogenesis and collateral vessel formation than MSC cell therapy alone. Stem cell-based angiogenic gene therapy may be a new therapeutic strategy for the treatment of severe ischemic cardiovascular disease.
目的：构建能表达重组人肝细胞生长因子(rhHGF)基因的第三代慢病毒载体,为实现rhHGF基因在大鼠骨髓间充质干细胞中长期、高效的表达奠定基础。
方法：(1)通过NheI/XbaI双酶切将pRNAT-U6.2慢病毒载体质粒中GFP基因敲除,然后将目的基因hHGF连接到该载体质粒上,构建重组载体质粒pRNAT-U6.2-hHGF。通过限制性内切酶酶切、菌落PCR和DNA测序进行鉴定。(2)利用TELEVECTORTM载体试剂盒将pRNAT-U6.2-hHGF联合辅助质粒pMDLg/pRRE、pMD2G-VSVG、pRSV-Rev共转染293T细胞,收集并浓缩慢病毒上清,逐孔稀释法测病毒滴度。
结果：(1)经限制性内切酶酶切及DNA测序分析表明,目的基因hHGF-cDNA已经插入载体质粒,且基因序列与基因库序列完全一致；(2)成功制备表达hHGF的慢病毒颗粒浓缩液,滴度为4×108Tu/ml。
结论：表达人肝细胞生长因子的第三代重组慢病毒载体pRNAT-U6.2-hHGF构建成功。
第二部分人肝细胞生长因子在大鼠骨髓间充质干细胞中的表达及生物学活性观察
目的：观察慢病毒载体介导的人肝细胞生长因子(hHGF)基因在骨髓间充质干细胞(BM-MSCs)中的表达及其生物学活性。
方法：(1)利用差速贴壁法联合Percoll密度梯度离心法分离、纯化大鼠原代BM-MSCs,流式鉴定MSC的表型,茜素红染色和油红O染色鉴定BM-MSCs的成骨、成脂分化潜能。(2)用高滴度表达hHGF (Lenti-HGF)或GFP (Lenti-GFP)的慢病毒液感染第三代的BM-MSCs,通过倒置相差荧光显微镜和流式鉴定来评估转染效率。ELISA检测细胞培养基上清中的hHGF浓度。(3)分别通过MTT法、Transwell小室法和Hoechst染色法检测脐静脉内皮细胞(HUVECs)和血管平滑肌细胞(VSMCs)的增殖、迁移及凋亡,评估感染Lenti-HGF的BM-MSCs分泌的hHGF的生物学活性。
结果：(1)通过联合差速贴壁法和密度梯度离心法可获得大量高纯度、具有多向分化潜能的BM-MSCs。(2)在转染Lenti-GFP后72h,即可在荧光显微镜下见明亮的绿色荧光,最佳感染复数(MOI)为50,流式鉴定转染效率约为75%。ELISA结果显示,Lenti-HGF感染BM-MSCs的培养上清液中的hHGF可持续较高水平地表达14d以上,且明显高于感染Lenti-GFP者(P<0.01)。(3)感染Lenti-HGF的MSCs分泌的hHGF在体外显示出促HUVECs增殖、迁移并抑制其凋亡,且能促进VSMCs迁移的生物学活性。
结论：Lenti-HGF慢病毒能够高效地介导转移外源基因hHGF至BM-MSCs表达,并具有生物学活性。
第三部分肝细胞生长因子基因修饰的骨髓间充质干细胞移植的促血管新生作用
目的：比较hHGF基因修饰的BM-MSCs移植与单纯BM-MSCs移植在大鼠后肢缺血模型中的促血管新生作用及其机制。
方法：在结扎左股动脉及其分支1天后,SD大鼠随机分为3组：HGF-MSC组(即HGF基因修饰的MSCs移植,n=8),MSC组(单纯的MSCs移植,n=8)以及PBS对照组(注射PBS,n=8)。在术后7天,进行细胞移植。实验组每只大鼠接受总计5×106个同一来源的HGF-MSCs或MSCs,分成5个点注入大鼠缺血后肢的股部肌肉；对照组注入同等体积的无菌PBS。分别在处理后1周或3周时处死大鼠,比较各组缺血股部肌肉中的毛细血管密度,移植细胞的分化转归,T淋巴细胞浸润以及生长因子HGF、VEGF蛋白表达等。ELISA检测HGF-MSC移植前和移植3周后大鼠血清HGF的水平。
结果：细胞移植3周后,MSC组和HGF-MSC组缺血肌肉组织的血管新生均明显增强,以HGF-MSC组的毛细血管密度最高(P<0.05)。从移植后1周开始,HGF-MSC组缺血组织中来源于移植细胞的内皮细胞数目明显高于MSC组(P<0.05)。在移植1周后,HGF-MSC和MSC的同种异体移植几乎不引起T淋巴细胞浸润,与PBS组相近(P>0.05)。Western-blot结果显示,MSC组和HGF-MSC组缺血肌肉组织中HGF、VEGF蛋白表达均明显高于PBS对照组,以HGF-MSC组的表达量最高(P<0.05)。HGF-MSC移植前和移植后3周,大鼠血清HGF浓度无明显变化(P>0.05)。
结论：与单纯的MSC细胞移植疗法相比,HGF基因修饰的MSC移植疗法具有更强的促进血管新生和侧枝血管形成的能力。以干细胞为基础的血管新生基因治疗策略将可能为治疗严重缺血性心血管病提供新思路。
Part I Construction of recombinant lentiviral vector carrying human hepatocyte growth factor gene
Objective:To construct a third-generation lentiviral vector carrying human hepatocyte growth factor (hHGF) for transfection.
Methods:Lentiviral vector plasmid carrying human HGF (pRNAT-U6.2-hHGF) was generated by removal of green fluorescence protein (GFP) gene from pRNAT-U6.2 plasmid by NheI/XbaI digestion and ligation of human HGF cDNA, which was identified by restriction endonuclease analysis, PCR and DNA sequence analysis. Recombinant plasmid pRNAT-U6.2-hHGF was co-transfected into 293T cells with the helper plasmids pMDLg/pRRE, pMD2G-VSVG and pRSV-Rev using the TELEVECTOR Lentiviral Vector Easy-high Production kit. The recombinant lentiviral stock (Lenti-HGF) was generated, concentrated, and then the titer was determined.
Results:The hHGF cDNA was identical to that of GeneBank. Recombinant lentiviral plasmid pRNAT-U6.2-hHGF was successfully constructed, and the titer of Lenti-HGF was 4×108Tu/ml.
Conclusion:Recombinant third-generation lentiviral vector plasmid carrying hHGF (pRNAT-U6.2-hHGF) was constructed successfully.
PartⅡExpression of human hepatocyte growth factor in primary rat bone marrow-derived mesenchymal stem cells and its biological activity
Objective:To explore the usability of lentiviral vector mediated human hepatocyte growth factor (hHGF) gene delivery into bone marrow-derived mesenchymal stem cells (BM-MSCs), and to observe its expression and biological activity.
Methods:(1) Primary rat BM-MSCs was isolated, expanded and purified by taking advantage of their differential adherence to dishes combined with Percoll density gradient centrifugation technique. MSC phenotype was analyzed by flow cytometry. Meanwhile, potency of MSCs differentiation into osteocytes and adipocytes were determined by alizarin red S staining and oil red O staining. (2) The third passage MSCs were transfected by high titer lentiviral stocks expressing human HGF (Lenti-HGF) or GFP (Lenti-GFP), and the transfection efficiency was assessed by fluorescent microscope observation and flow cytometry analysis. The concentration of hHGF in cell culture medium supernatants was determined by enzyme linked immunosorbent assay (ELISA). (3) Biological activity of hHGF secreted from Lenti-HGF infected BM-MSCs was determined by MTT cell proliferation assay, Transwell chamber migration assay and Hoechst staining.
Results:(1) Plenty of highly purified BM-MSCs were achieved by the combined methods. (2) Bright green fluorescence in the MSC was observed under the fluorescent microscope after 72h transfected with Lenti-GFP. The best multiplicity of infection (MOI) was 50, and the transfection efficiency reached about 75%. Furthermore, ELISA showed that hHGF secretion could be stably maintained more than 14 days since the level of hHGF in the culture supernatant from the Lenti-HGF transduced BM-MSCs was significantly higher than that of Lenti-GFP transduced BM-MSCs at day 5 (P<0.01). (3)The hHGF secreted from transduced MSCs showed the proproliferative, promigratory, antiapoptotic effects for human umbilical vein endothelial cells (HUVECs) and promigratory effect on vascular smooth muscle cells (VSMCs) in vitro.
Conclusion:The recombinant lentivirus (Lenti-HGF) can mediated transduction into BM-MSCs with a highly efficient and long lasting expression of the transgene. Key words:Hepatocyte growth factor; Mesenchymal stem cell; Lentivirus; Apoptosis
PartⅢHepatocyte growth factor genetically modified bone marrow-derived mesenchymal stem cells transplantation promote angiogenesis
Objective:To investigate whether a new strategy that combines MSCs transplantation and ex vivo human hepatocyte growth factor (hHGF) gene transferring with lentiviral vectors was more therapeutically efficient than MSCs cell therapy alone in a rat hindlimb ischemia model.
Methods:One day after resection of left femoral artery, Sprague-Dawley (SD) rats were randomly divided into three groups:(a) HGF genetically modified MSCs (HGF-MSCs) transplantation (HGF-MSC group, n=8), (b) BM-MSCs transplantation (MSC group, n=8), (c) PBS injection (PBS group, n=8). After creating hindlimb ischemia, an interval of 7 days was permitted for postoperative recovery. Then a total of 5×106 HGF-MSCs, MSCs obtained from same SD rats or PBS of same volume were injected into the ischemic thigh muscles of rats at five different points in each group. The ischemic thigh muscle were analyzed for capillary density, transplanted cell differentiation, T lymphocyte infiltration and the expression of HGF and VEGF protein at one or three weeks after treatment. Serum levels of HGF in HGF-MSC group before and 3 weeks after transplantation were determined by ELISA.
Results:Three weeks after transplantation, angiogenesis was significantly enhanced by both MSC group and HGF-MSC group, while capillary density was highest in the HGF-MSC group (P<0.05). The number of transplanted cell-derived endothelial cells was higher in the HGF-MSC group than in the MSC group since one week after treatment (P<0.05). In treated ischemic hind limb muscles, allogeneic HGF-MSC and MSC injection were associated with comparatively few T lymphocyte infiltration in PBS group (P>0.05). The expression of angiogenic growth factors like HGF and VEGF in local ischemic muscles undergoing intramuscular cell transplantation was more abundant in HGF-MSC group than the other two groups (P<0.05). Serum levels of HGF in HGF-MSC group before and 3 weeks after transplantation have no significant difference (P>0.05).
Conclusions:The present study shows that HGF gene-modified MSC transplantation therapy induced more potent angiogenesis and collateral vessel formation than MSC cell therapy alone. Stem cell-based angiogenic gene therapy may be a new therapeutic strategy for the treatment of severe ischemic cardiovascular disease.
引文
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