Background The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic ...Background The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 （VEGF165） gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells （EPCs） were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1 ,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine （Dil） before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups （n=25, each）： A, Ad-VEGF165/EPC-transplantation group received 1 ml （10^6） of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml （10^6） of EPCs; C, Ad/EPC-transplantation group received 1 ml （10^6） of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor （VEGF） mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor （vWF）, which is a component of endothelial cells.The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. Results The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D （P〈0.05）; the VEGF mRNA levels in groups B and C were significantly higher than those in group D （P〈0.05）, and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C （P 〈0.05） and D （P 〈0.01）; the recanalization capillary densities in groups B and C were significantly higher than that in group D （P 〈0.05）. Moreover, there was no statistical significant difference between the values for groups B and C. Conclusions The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.展开更多
背景:目前已将NIPBL基因突变作为诊断Cornelia de Lange综合征的首选指标,但由于该病的遗传异质性,显著增加了临床诊疗难度,尤其患儿骨骼发育畸形的发生率高,其发病机制尚不明确,目前无有效治疗方案,患儿平均寿命较一般人群显著缩短。目...背景:目前已将NIPBL基因突变作为诊断Cornelia de Lange综合征的首选指标,但由于该病的遗传异质性,显著增加了临床诊疗难度,尤其患儿骨骼发育畸形的发生率高,其发病机制尚不明确,目前无有效治疗方案,患儿平均寿命较一般人群显著缩短。目的:探究敲低NIPBL基因对小鼠骨髓间充质干细胞成软骨分化能力的影响及其可能的分子调控机制。方法:将慢病毒转染NIPBL sh RNA的小鼠骨髓间充质干细胞作为sh-NIPBL组,慢病毒空载体转染的骨髓间充质干细胞作为sh-NC组,无慢病毒干扰的骨髓间充质干细胞作为空白对照组,对上述3组细胞进行成软骨诱导培养,诱导21 d后测量软骨微球最大横截面的周长,行阿利辛蓝染色鉴定其诱导分化结果,免疫荧光法检测软骨细胞Ⅱ型胶原的表达水平,应用实时荧光定量PCR法检测软骨细胞Sox-9、TGF-β1、Smad2、Smad4 m RNA表达水平。结果与结论:(1)成软骨诱导第21天,sh-NIPBL组的软骨微球最大横截面的周长明显小于对照组(P <0.05),阿利辛蓝染色后在倒置显微镜下观察sh-NC组较sh-NIPBL组可见更多的蓝色软骨内酸性黏多糖;(2)诱导成软骨分化后,sh-NIPBL组骨髓间充质干细胞中Ⅱ型胶原、Sox-9的表达低于sh-NC组和空白对照组(P <0.05);(3)成软骨诱导过程中,sh-NIPBL组TGF-β1、Smad2、Smad4基因的表达水平低于sh-NC组和空白对照组(P <0.05);(4)结果表明,慢病毒敲低NIPBL基因表达降低了骨髓间充质干细胞的成软骨分化能力,且该过程可能由TGF-β1/Smad信号通路参与介导。展开更多
基金This work was supported by a grant from the Natural Science Foundation of Jiangsu Province （No. BK2007055）.
文摘Background The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 （VEGF165） gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells （EPCs） were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1 ,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine （Dil） before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups （n=25, each）： A, Ad-VEGF165/EPC-transplantation group received 1 ml （10^6） of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml （10^6） of EPCs; C, Ad/EPC-transplantation group received 1 ml （10^6） of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor （VEGF） mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor （vWF）, which is a component of endothelial cells.The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. Results The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D （P〈0.05）; the VEGF mRNA levels in groups B and C were significantly higher than those in group D （P〈0.05）, and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C （P 〈0.05） and D （P 〈0.01）; the recanalization capillary densities in groups B and C were significantly higher than that in group D （P 〈0.05）. Moreover, there was no statistical significant difference between the values for groups B and C. Conclusions The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.
文摘背景:目前已将NIPBL基因突变作为诊断Cornelia de Lange综合征的首选指标,但由于该病的遗传异质性,显著增加了临床诊疗难度,尤其患儿骨骼发育畸形的发生率高,其发病机制尚不明确,目前无有效治疗方案,患儿平均寿命较一般人群显著缩短。目的:探究敲低NIPBL基因对小鼠骨髓间充质干细胞成软骨分化能力的影响及其可能的分子调控机制。方法:将慢病毒转染NIPBL sh RNA的小鼠骨髓间充质干细胞作为sh-NIPBL组,慢病毒空载体转染的骨髓间充质干细胞作为sh-NC组,无慢病毒干扰的骨髓间充质干细胞作为空白对照组,对上述3组细胞进行成软骨诱导培养,诱导21 d后测量软骨微球最大横截面的周长,行阿利辛蓝染色鉴定其诱导分化结果,免疫荧光法检测软骨细胞Ⅱ型胶原的表达水平,应用实时荧光定量PCR法检测软骨细胞Sox-9、TGF-β1、Smad2、Smad4 m RNA表达水平。结果与结论:(1)成软骨诱导第21天,sh-NIPBL组的软骨微球最大横截面的周长明显小于对照组(P <0.05),阿利辛蓝染色后在倒置显微镜下观察sh-NC组较sh-NIPBL组可见更多的蓝色软骨内酸性黏多糖;(2)诱导成软骨分化后,sh-NIPBL组骨髓间充质干细胞中Ⅱ型胶原、Sox-9的表达低于sh-NC组和空白对照组(P <0.05);(3)成软骨诱导过程中,sh-NIPBL组TGF-β1、Smad2、Smad4基因的表达水平低于sh-NC组和空白对照组(P <0.05);(4)结果表明,慢病毒敲低NIPBL基因表达降低了骨髓间充质干细胞的成软骨分化能力,且该过程可能由TGF-β1/Smad信号通路参与介导。
