转染VEGF165基因的内皮祖细胞移植治疗慢性深静脉血栓的实验研究
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摘要
目的:研究细胞移植与基因转染对慢性深静脉血栓的协同治疗作用,为慢性深静脉血栓的治疗提供一种新的治疗思路。
方法:首先用pAdEasy系统构建携有血管内皮生长因子165(VEGF165)的重组腺病毒载体。HEK293A细胞包装、重组、扩增腺病毒颗粒,荧光显微镜观察绿色荧光表达。PCR、双酶切、基因测序进行鉴定。同时用Ficoll法分离大鼠骨髓单个核细胞,EBM-2MV培养基培养扩增骨髓源性EPCs并进行鉴定。MTT法描绘生长曲线。再用Ad-VEGF165体外转染EPCs,荧光显微镜观察绿色荧光表达,激光共聚焦测转染效率,MTT法测对生长活性的影响,ELISA检测上清中VEGF的表达。转染后行Dil体外标记EPCs。然后结扎大鼠肾下段下腔静脉建立慢性深静脉血栓模型,分四组:A组(n=25只),基因转染的EPCs移植组,移植1ml含有106个带有VEGF165的EPCs细胞悬液;B组((n=25只),单纯EPCs组,移植1ml含有个106EPCs细胞悬液;C组((n=25只),转染空病毒的EPCs组,移植1ml含有106个转染空病毒的EPCs细胞悬液;D组((n=25只),空白对照组,移植1ml培养基。移植后分时间段取出血栓段下腔静脉及血栓,应用实时荧光定量PCR技术检测VEGF、ANG-1 mRNA的表达变化,同时应用western blot测下腔静脉及血栓内VEGF、ANG-1蛋白表达变化;HE染色及vWF免疫组化染色观察血栓机化再通情况,扫描电镜观察血栓中新生血管通道。高倍显微镜下计数血栓再通毛细血管密度。采用SPSS11.5软件进行分析,P<0.05,有统计学意义。
结果:成功构建了Ad-VEGF165,成功培养了大鼠骨髓源性内皮祖细胞。Ad-VEGF165能有效转染EPCs,合适比率的转染对EPCs生长具有促进作用,转染后的EPCs能高分泌VEGF蛋白。移植后通过标记观察EPCs在体内存活并分化为内皮细胞。EPCs移植后,A、B、C、D四组VEGF、ANG-1 mRNA均有表达,A组较B、C、D组明显上调(P<0.05),B、C组较D组明显上调(P<0.05),B组和C组之间无统计学意义(P>0.05)。移植后四组血栓再通毛细血管密度A组明显高于B、C组(P<0.05)及D组(P<0.01),B、C组之间无统计学意义,B、C组明显高于D组(P<0.05),有统计学意义。免疫组化染色vWF确定再通管道为新生血管,管道为内皮细胞组成。
结论: Ad-VEGF165能成功转染EPCs,合适比率的转染能改进EPCs质量,移植后促血管新生能力明显增强,显著加快血栓机化再通。
Objective: To study the co-therapeutic effect of stem cell transplantation and gene transfection on the chronic deep venous thrombosis.
Methods: The recombinant adenoviral vector carrying the VEGF165 gene was constructed by pAdEasy system.pAdEasy-VEGF165 transfected into HEK293A cells to package the adenovirus,followed by identification by means of GFP.PCR and gene sequencing identify the recombinant.EPCs were isolated from rat bone marrow by Ficoll and cultured with EBM-2MV medium cultivation.MTT assay were used to draw growth curve of cells.After infection with the recombinant Ad-VEGF165,EPCs were examined with a fluorescent microscopy and ELISA for their expression of VEGF,MTT assay was employed to assess the growth character,and Laser scanning confocal microscope for transduction efficiency.Experimental rat model of chronic deep vein thrombosis were obtained by partial ligation of inferior vena cava.The rats were randomly divided into four groups:A(n=25),Ad-VEGF165 transfection group,1ml 106 Ad-VEGF165-EPCs transplantation;B(n=25),EPCs group,1ml 106 EPCs transplantation;C(n=25),Ad-GFP transfection Group, 1ml 106 Ad-GFP-EPCs transplantation; D(n=25),control group, 1 ml of medium transplantation.After transplantation, real-time quantitative PCR was used to detect VEGF, ANG-1 mRNA expression level and western blot of inferior vena cava thrombosis, and VEGF,ANG-1 protein expression changes.HE staining and Immunohistochemical staining was performed to detect recanalization and the neovascularization was observed by scanning electron microscopy.The capillary density was determined quantitatively by counting the capillaries under high-power microscope.SPSS11.5 software used for analysis, P <0.05 for the difference was significant.
Results: Ad-VEGF165 was constructed and successfully cultivated a bone marrow-derived endothelial progenitor cell(sEPCs).Appropriate ratio of transfection had promotion of the growth of EPCs.After transfection, EPCs had secretioned VEGF protein.EPCs in vivo survival,differentiated into endothelial cells was viewed by marker of Dil stain after transplantation. VEGF, ANG-1 mRNA were expressed in A, B, C and D group after transplantation, and that were significantly increased in group A than group B, C and D (P<0.05), were significantly increased in group B and group C than group D (P<0.05), were no statistical significance in between group B and group C. The recanalization capillary density were significantly higher in group A than that in group B,C(P<0.05),than in group D(P<0.01) ,were significantly higher in group B and group C than in group D(P<0.05).And there was no statistical significance in between group B and group C.The neovascularization were identified by immunohistochemical staining of vWF antibody, that were compose of endothelial cells.
Conclusion: EPCs were transfected by Ad-VEGF165 successfully.A suitable ratio of transfection can improve the efficiency of EPCs.That can improve potentiality of promotion of angiogenesis after transplantation. And had accelerate organization and recanalization of vein thrombus.
方法:首先用pAdEasy系统构建携有血管内皮生长因子165(VEGF165)的重组腺病毒载体。HEK293A细胞包装、重组、扩增腺病毒颗粒,荧光显微镜观察绿色荧光表达。PCR、双酶切、基因测序进行鉴定。同时用Ficoll法分离大鼠骨髓单个核细胞,EBM-2MV培养基培养扩增骨髓源性EPCs并进行鉴定。MTT法描绘生长曲线。再用Ad-VEGF165体外转染EPCs,荧光显微镜观察绿色荧光表达,激光共聚焦测转染效率,MTT法测对生长活性的影响,ELISA检测上清中VEGF的表达。转染后行Dil体外标记EPCs。然后结扎大鼠肾下段下腔静脉建立慢性深静脉血栓模型,分四组:A组(n=25只),基因转染的EPCs移植组,移植1ml含有106个带有VEGF165的EPCs细胞悬液;B组((n=25只),单纯EPCs组,移植1ml含有个106EPCs细胞悬液;C组((n=25只),转染空病毒的EPCs组,移植1ml含有106个转染空病毒的EPCs细胞悬液;D组((n=25只),空白对照组,移植1ml培养基。移植后分时间段取出血栓段下腔静脉及血栓,应用实时荧光定量PCR技术检测VEGF、ANG-1 mRNA的表达变化,同时应用western blot测下腔静脉及血栓内VEGF、ANG-1蛋白表达变化;HE染色及vWF免疫组化染色观察血栓机化再通情况,扫描电镜观察血栓中新生血管通道。高倍显微镜下计数血栓再通毛细血管密度。采用SPSS11.5软件进行分析,P<0.05,有统计学意义。
结果:成功构建了Ad-VEGF165,成功培养了大鼠骨髓源性内皮祖细胞。Ad-VEGF165能有效转染EPCs,合适比率的转染对EPCs生长具有促进作用,转染后的EPCs能高分泌VEGF蛋白。移植后通过标记观察EPCs在体内存活并分化为内皮细胞。EPCs移植后,A、B、C、D四组VEGF、ANG-1 mRNA均有表达,A组较B、C、D组明显上调(P<0.05),B、C组较D组明显上调(P<0.05),B组和C组之间无统计学意义(P>0.05)。移植后四组血栓再通毛细血管密度A组明显高于B、C组(P<0.05)及D组(P<0.01),B、C组之间无统计学意义,B、C组明显高于D组(P<0.05),有统计学意义。免疫组化染色vWF确定再通管道为新生血管,管道为内皮细胞组成。
结论: Ad-VEGF165能成功转染EPCs,合适比率的转染能改进EPCs质量,移植后促血管新生能力明显增强,显著加快血栓机化再通。
Objective: To study the co-therapeutic effect of stem cell transplantation and gene transfection on the chronic deep venous thrombosis.
Methods: The recombinant adenoviral vector carrying the VEGF165 gene was constructed by pAdEasy system.pAdEasy-VEGF165 transfected into HEK293A cells to package the adenovirus,followed by identification by means of GFP.PCR and gene sequencing identify the recombinant.EPCs were isolated from rat bone marrow by Ficoll and cultured with EBM-2MV medium cultivation.MTT assay were used to draw growth curve of cells.After infection with the recombinant Ad-VEGF165,EPCs were examined with a fluorescent microscopy and ELISA for their expression of VEGF,MTT assay was employed to assess the growth character,and Laser scanning confocal microscope for transduction efficiency.Experimental rat model of chronic deep vein thrombosis were obtained by partial ligation of inferior vena cava.The rats were randomly divided into four groups:A(n=25),Ad-VEGF165 transfection group,1ml 106 Ad-VEGF165-EPCs transplantation;B(n=25),EPCs group,1ml 106 EPCs transplantation;C(n=25),Ad-GFP transfection Group, 1ml 106 Ad-GFP-EPCs transplantation; D(n=25),control group, 1 ml of medium transplantation.After transplantation, real-time quantitative PCR was used to detect VEGF, ANG-1 mRNA expression level and western blot of inferior vena cava thrombosis, and VEGF,ANG-1 protein expression changes.HE staining and Immunohistochemical staining was performed to detect recanalization and the neovascularization was observed by scanning electron microscopy.The capillary density was determined quantitatively by counting the capillaries under high-power microscope.SPSS11.5 software used for analysis, P <0.05 for the difference was significant.
Results: Ad-VEGF165 was constructed and successfully cultivated a bone marrow-derived endothelial progenitor cell(sEPCs).Appropriate ratio of transfection had promotion of the growth of EPCs.After transfection, EPCs had secretioned VEGF protein.EPCs in vivo survival,differentiated into endothelial cells was viewed by marker of Dil stain after transplantation. VEGF, ANG-1 mRNA were expressed in A, B, C and D group after transplantation, and that were significantly increased in group A than group B, C and D (P<0.05), were significantly increased in group B and group C than group D (P<0.05), were no statistical significance in between group B and group C. The recanalization capillary density were significantly higher in group A than that in group B,C(P<0.05),than in group D(P<0.01) ,were significantly higher in group B and group C than in group D(P<0.05).And there was no statistical significance in between group B and group C.The neovascularization were identified by immunohistochemical staining of vWF antibody, that were compose of endothelial cells.
Conclusion: EPCs were transfected by Ad-VEGF165 successfully.A suitable ratio of transfection can improve the efficiency of EPCs.That can improve potentiality of promotion of angiogenesis after transplantation. And had accelerate organization and recanalization of vein thrombus.
引文
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