转染HRE-VEGF基因的骨髓间充质干细胞自体移植治疗缺血性心肌病的研究
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摘要
背景:研究表明干细胞移植可以取代坏死心肌细胞、增加有功能的心肌细胞数量,增加血管密度,改善心功能,从而为治疗缺血性心肌病开辟了一条崭新的途径。改善缺血心肌的首要任务就是改善血供,血管内皮生长因子(vascu lar endothelium growth factor,VEGF)在血管新生方面具有重要意义。虽然通过过表达VEGF的基因治疗可以改善心肌缺血,但是却不能控制VEGF的过度表达而产生癌变。本实验通过低氧反应元件(hypoxic response element,HRE)控制VEGF的表达,使骨髓间充质干细胞在低氧下表达VEGF,而常氧下VEGF表达关闭,研究受HRE调控的VEGF基因转染的骨髓间充质干细胞能否在缺血、缺氧状态下保护心肌细胞,促进血管再生,同时又防止VEGF基因过度表达,从而为基因修饰干细胞治疗缺血性心肌病提供理论依据。
第一部分大鼠骨髓间充质干细胞(MSCs)的分离、培养及心梗模型的建立
第一节大鼠骨髓间充质干细胞(MSCs)的分离、培养
目的:建立大鼠骨髓间充质干细胞体外培养方法,为自体骨髓移植提供材料。方法:大鼠的骨髓MSCs分离纯化后,流式细胞仪检测细胞周期。结果:体外培养的原代MSCs 10～14d达到汇合,细胞周期显示75 %的细胞处于G0/G1期。
结论:根据黏附特性分离的大鼠骨髓MSCs在体外条件下可生长扩增,可用于自体骨髓移植。
第二节心梗模型的建立
目的:用液氮冷冻法建立大鼠心梗模型。
方法:对大鼠用液氮中浸泡过的金属棒直接接触左室前壁,建立心梗模型。
结果:心超资料显示建模前心功能与建模后心功能比有统计学差异,建模前的心功能明显好于建模后。
结论:液氮冷冻法可以成功构建心梗模型。
第二部分HRE-VEGF表达载体的构建
第一节VEGF表达载体的构建
目的:克隆VEGF的cDNA,将其与表达载体结合,构建成VEGF -cDNA表达载体。方法:根据已经公布的VEGF的CDS功能区的基因序列,应用RT-PCR技术从Hep G2细胞中扩增VEGF基因(1239bp),并将其克隆至载体pCDH1-MCS1-EF1- copGFP,构建VEGF表达载体,并转化DH5α感受态细菌。选择阳性克隆菌,行双酶切鉴定和测序验证所获取的cDNA。
结果:双酶切鉴定和测序结果均表明所获取的cDNA为VEGF的CDS功能区基因。
结论:使用RT-PCR法成功构建VEGF表达载体。
第二节HRE-VEGF表达载体的构建
目的:人工设计低氧反应元件串联重复序列,克隆后插入VEGF目的基因表达载体并经测序证实。
方法:按照人促红细胞生成素基因3′末端HRE增强子序列,人工设计合成首尾末端部分互补的单链DNA通过3次酶切、连接的方法获得9HRE,然后通过退火及延伸合成两端带有酶切位点的包含9个串联排布的HRE的双链DNA,克隆到VEGF表达载体后测序。
结果:双酶切鉴定和测序结果均表明所获取的经测序证实9HRE-VEGF和设计相符。
结论:运用同尾酶法可成功构建9拷贝缺氧反应元件,构建9HRE-VEGF表达载体。
第三部分慢病毒介导HRE-VEGF基因转染MSCs及HRE对转基因MSCs在缺氧、复氧心肌细胞中的调控作用
第一节慢病毒介导HRE-VEGF基因转染骨髓间充质干细胞
目的:用慢病毒系统(pPACKH1- Lentivector Packaging Kit)将目的基因HRE-VEGF包装并转染干细胞。
方法:结合HRE-VEGF的穿梭质粒pCDH1转染包装细胞293T细胞进行扩增后,使用由三个质粒组成的慢病毒转染系统进行基因重组,并以其为载体转染鼠的骨髓间充质干细胞,检测其荧光的表达。
结果:293T细胞和MSCs24转染小时和48小时后,均可检测到强荧光出现。
结论:慢病毒载体系统携带目的基因HRE-VEGF基因转染鼠的骨髓间充质干细胞具有较高的转染效率。
第二节转基因骨髓间充质干细胞对缺氧心肌细胞的影响
目的:低氧反应元件对缺氧、复氧心肌细胞转导的VEGF基因表达的调控。
方法:原代培养心肌细胞,按不同的干细胞加入不同缺氧、复氧时间将心肌细胞分组,每组细胞固定后做免疫荧光染色测细胞内VEGF蛋白的表达情况;RT-PCR测细胞内人VEGF–mRNA含量;取细胞培养液ELISA法检测人VEGF含量。
结果:在缺氧组检测到VEGF–mRNA和VEGF蛋白的表达,而常氧组未检测到VEGF–mRNA和VEGF蛋白的表达。
结论:离体培养大鼠心肌细胞在低氧、复氧过程中加入携带HRE-VEGF基因的骨髓间充质干细胞时,其中的HRE对转导的VEGF基因表达具有调控作用。
第四部分转HRE-VEGF基因的MSCs自体移植对大鼠缺血性心肌病的实验研究
目的:以心梗的大鼠为模型,研究移植转染HRE-VEGF基因的自体MSCs能否在体内较未转基因的MSCs更显著地改善缺血性心肌病大鼠的心脏功能,进一步探讨MSCs联合基因转染用于治疗缺血性心肌病的可行性。
方法:选用雄性Fischer 344大鼠,体重200～250g,应用液氮冷冻法建立心梗模型,行血流动力学检测心功能。动物分成4组:空白组(A组,n=20)、DMEM组(B组,n=20)、MSCs组(C组,n=20)和转基因组(D组,n=20)。从大鼠股骨取骨髓,用贴壁筛选方法分离纯化、体外扩增MSCs,MSCs体外转染慢病毒包装的HRE-VEGF基因。将BrdU标记的自体MSCs直接注射到梗死区。4周后,重复心功能检查,评估心功能和梗死区的灌注;并观察心脏组织的病理学变化。采用ELISA法检测血浆VEGF在梗死后和干细胞移植前后的变化,以及检测Cx-43,Ⅷ因子等指标观察不同的处理对血管新生的影响。
结果:大鼠MSCs移植后4周,血流动力学测定显示,C、D组LVDP较A、B组升高(P<0.05),左心室压力变化率±dp/dtmax C、D组明显优于A、B组(P<0.05)。D组与C组相比:D组±dp/dtmax较高。移植的MSCs能防止梗塞区变薄和扩张;与A、B两组和移植前相比,梗死区的收缩功能和血流灌注均有所改善(P<0.05),心功能的改善在D组更为明显(与C组比较,P<0.05)。与A、B两组相比,C、D组梗死区的毛细血管密度明显增加(P<0.05),其中D组较C组增加明显(P<0.05)。VEGF的水平在梗死后有所增加,在细胞移植后一周出现高峰,后逐渐下降。
结论:转染HRE-VEGF的MSCs可以改善缺血心肌的左室功能,促进血管新生。这种改善可能是MSCs通过心肌再生防止梗死区变薄、抑制收缩功能异常有关。一些细胞因子在其中起非常重要的作用。VEGF的表达可以减少细胞的凋亡,提高干细胞再生血管的作用,从而可显著改善心脏功能。应用HRE-VEGF基因转染MSCs不仅理论上可以防止癌变而且能够改善心功能。
Background:Some research indicated that stem cells could not only replace the necrosis cardiomyocytes and increase the number of functional cardiomyocytes , but also raise the density of vascellum in the post infarction scars and improve the cardiac function. These results observed before would introduce a new therapeutic strategy to the field of cardiovascular diseases. It has been proved in several studys in vitro or in vivo that autologous bone marrow stem cells could rescue ischaemic myocardium、induce neovascularization and preserve left ventricular function. In this study we transfected HRE-VEGF into bone marrow stem cells, in order to make clear whether BMSCs transfected with HRE-VEGF could protecte cardiomyocytes、induce neovascularization、prevent VEGF overexpression and greatly improve the cardiac fuction after ischemic and hypoxia。
Part I: Separation and cultivation of rats bone marrow mesenchymal stem cells and establishment of animal model
Section one: Separation and cultivation of rats bone marrow mesenchymal stem cells
Objective:To build a method to cultivate rat MSCs in vitro, provide the cell resource for autologous bone marrow stem cells transplantation.
Methods: After rat MSCs have been separated and depurated, the cell cycle were detected by flow cytometry
Results: when MSCs were cultivated in vitro for 10～14 days, they confluensed and 75 percent of these cells were in the phase of G0/G1.
Conclusion: rat MSCs could be separated and amplified through the adhesiveness character in vitro grew and amplified, these cells could be used in cell transplantation.
Section two: Establishment Of Animal Model
Objective: to establish rat myocardial infarction model with liquid nitrogen frozen
Methods: to establish myocardial infarction animal model with the method that anterior wall of left ventricle of rat was contacted directly by metal stick soaked in liquid nitrogen.
Results: cordis hypersound data showed that there was significant difference between heart function before modeling and after modeling, the heart function before modeling was better than that after modeling.
Conclusion: this kind of myocardial infarction animal model is reliable because of the fixed location.
Part II: Construction of HRE-VEGF-Expression Vector
Section one: The Construction of VEGF-Expression Vector
Objective: Clone the cDNA of VEGF, combine cDNA with the expression vector to get the HRE-VEGF -Expression Vector
Methods: According to CDS domain of VEGF published in genebank we amplified the HRE-VEGF gene with RT-PCR from Hep G2 cells and clone the gene to pCDH1-MCS1-EF1- copGFP, then we transfected VEGF- expression vector into DH5αcompetence bacterium, The masculine clone bacterium were choosed, double enzyme-cutting and sequencing were conducted to verify the cDNA
Results: double enzyme cutting and sequencing indicated the cDNA we got were the same sequenc to CDS domain of VEGF
Conclusion: Using RT-PCR could successfully construct VEGF-expression vector
Section two: The Construction of HRE-VEGF Expression Vector
Objective: To clone the hypoxia response element tandem repetitive sequence, and to insert it into the objective gene expression vector which was proved by sequencing
Methods: complementary single strand DNA on head and end section was designed artificial by the end of erythropoietin of human being, and 9HRE was gotten by enzyme-cutting 3 times and connecting. Then double strands DNA of HRE with enzyme-cutting site and 9 cascade connections was gained by renaturation and elongation. The double strands DNA was sequenced after cloned to the expression vector.
Result: both the results of double enzyme-cutting verification and sequencing indicated that 9HRE-VEGF which was proved by sequencing matched to the designed one.
Conclusion: the method of isocaudarner can construct 9 copy hypoxia response element and 9HRE-VEGF expression vector successfully.
Part III: lentivirus mediated HRE-VEGF gene transfection of MSCs and the regulation effect of HRE on the transgenic stem cells in hypoxia and oxygen recovery myocardial cells
Section one: HRE-VEGF gene transfection bone marrow stem cells which was mediated by lentivirus
Objective: to transfect stem cells with HRE-VEGF gene, and to establish gene transfected bone marrow stem cells with lentivirus, in order to study the regulation effect of HRE to the cells with transduction VEGF gene in hypoxia and oxygen recovery myocardial cells.
Methods: after amplification of 293T cells which were transfected by shuttle plasmid PCDH1 combined with HRE-VEGF gene, gene recombination was carried out by the system of lentivirus transfection composed by 3 plasmids . Bone marrow stem cells of rat were then transfected by that recombination gene as trager and were detected its fluorescent expression.
Results: On 24 hours and 48 hours after transfection, the strong fluorescent expression can also be detected in 293 T cells and MSCs.
Conclusion: Lentivirus trager system with HRE-VEGF gene has high transfection efficiency in transfecting MSCs of rat.
Section two: Effects of Transgenic Stem Cells On Hypoxia Cardiomyocytes
Objective: to study the regulation of hypoxia response element to the gene expression of hypoxia and oxygen-recovery myocardial cells transfected VEGF gene of human being.
Motheds: to primary culture myocardial cells, and divided them into different groups with the type of stem cells and the time of hypoxia and oxygen recovery. After being fixed, the cells in each group were stained with immunofluorescence to detect the expression of VEGF proteinum; RT-PCR was used to detect the content of VEGF-mRNA of human being; content and type of VEGF was detect by ELISA from cell culture fluid.
Results: VEGF- mRNA and VEGF were detected in hypoxia group, and VEGF- mRNA and VEGF were found in the group without hypoxia.
Conclusion: the regulation effect of HRE to the transfected VEGF gene has been proved, during MSCs taking along HRE-VEGF gene were added into rat myocardial cells isolated cultured during the process of hypoxia and oxygen recovery.
Part IV: Gene-modified bone marrow stem cells autografting therapy in rat ischemic cardiomyopathy model
Objective: to investigate if MSCs transfected HRE-VEGF gene by autografting has more significant effect than MSCs without transfection in improving the heart function of ischemic cardiomyopathy, and to approach the therapeutic possibility about MSCs and gene used to treat myocardial ischemia.
Methods: male Fischer 344 rats with weight from 200g to 250g were put into research, MI scar were made by liquid nitrogen frozen, cardiac function were observed by hemodynamics monitoring. These rats were divided into four groups: control group(A groups, n=20)、DMEM group(B group, n=20)、MSCs group (C group, n=20)、transgene group (D group, n=20).
Bone marrow were taken from rats’femur. adherence screening technique were adopted to separate、purify and amplify MSCs in vitro. Then MSCs were transfected by HRE-VEGF.
Autologous bone marrow stem cells labeled by BrdU were injected into MI scar. After 4 weeks, cardiac function was monitored again in order to evaluate the heart function and scar infusion. Histology research were done to know the distribution of MSCs, otherwise, Changes in hematoplasma concentration of VEGF between post MI and posttransplant were got through the methods of ELISA. Cx-43,VEGF were also detected by ELISA to knowledge the effect of MSCs on neovascularization.
Results: Rats’MSCs were transplanted into MI scar, after 4 weeks, data from hemodynamics monitoring indicated that there is no obviously different in LVSP between experiment groups and control groups. In C、D group, LVEDP were lower, the±dp/dtmax were more better than that of A、B group. In D group and C group, +dp/dtmax in D group were higher that of C group. MSCs transplantion could protect scar from being thin and dilate, moreover, cardiac function and scar infusion were more improved compared with A、B group and pretransplante. The improvement of cardiac function were more successful in D group (to C group , P<0.05).The capillary density in C、D group increased greatly in MI scar compared with A、B group(P<0.05),and the increasing of D group were more obvious than C group. Concentration of VEGF also increased after MI and came up to peak 1 week later, after 1 week, the data showed the decrease in concentration of VEGF.
Conclusion: MSCs were observed still survived in the host myocardium, protected MI scar from thinning, inhibited the dysfunction of cardiac and improved the left ventricle function; these changes might attribute to the myocardium regenerate、neovascularization、extracellular matrix hyperplasy;Some cytokines might play very important role on these changes. Expression of VEGF could also release the cell apoptosis, reinforce neovascularization and improve the cardiac function.Transplantation of autologous bone marrow stem cells combined with HRE-VEGF was an effective treatment to ischemia myocardial disease
第一部分大鼠骨髓间充质干细胞(MSCs)的分离、培养及心梗模型的建立
第一节大鼠骨髓间充质干细胞(MSCs)的分离、培养
目的:建立大鼠骨髓间充质干细胞体外培养方法,为自体骨髓移植提供材料。方法:大鼠的骨髓MSCs分离纯化后,流式细胞仪检测细胞周期。结果:体外培养的原代MSCs 10～14d达到汇合,细胞周期显示75 %的细胞处于G0/G1期。
结论:根据黏附特性分离的大鼠骨髓MSCs在体外条件下可生长扩增,可用于自体骨髓移植。
第二节心梗模型的建立
目的:用液氮冷冻法建立大鼠心梗模型。
方法:对大鼠用液氮中浸泡过的金属棒直接接触左室前壁,建立心梗模型。
结果:心超资料显示建模前心功能与建模后心功能比有统计学差异,建模前的心功能明显好于建模后。
结论:液氮冷冻法可以成功构建心梗模型。
第二部分HRE-VEGF表达载体的构建
第一节VEGF表达载体的构建
目的:克隆VEGF的cDNA,将其与表达载体结合,构建成VEGF -cDNA表达载体。方法:根据已经公布的VEGF的CDS功能区的基因序列,应用RT-PCR技术从Hep G2细胞中扩增VEGF基因(1239bp),并将其克隆至载体pCDH1-MCS1-EF1- copGFP,构建VEGF表达载体,并转化DH5α感受态细菌。选择阳性克隆菌,行双酶切鉴定和测序验证所获取的cDNA。
结果:双酶切鉴定和测序结果均表明所获取的cDNA为VEGF的CDS功能区基因。
结论:使用RT-PCR法成功构建VEGF表达载体。
第二节HRE-VEGF表达载体的构建
目的:人工设计低氧反应元件串联重复序列,克隆后插入VEGF目的基因表达载体并经测序证实。
方法:按照人促红细胞生成素基因3′末端HRE增强子序列,人工设计合成首尾末端部分互补的单链DNA通过3次酶切、连接的方法获得9HRE,然后通过退火及延伸合成两端带有酶切位点的包含9个串联排布的HRE的双链DNA,克隆到VEGF表达载体后测序。
结果:双酶切鉴定和测序结果均表明所获取的经测序证实9HRE-VEGF和设计相符。
结论:运用同尾酶法可成功构建9拷贝缺氧反应元件,构建9HRE-VEGF表达载体。
第三部分慢病毒介导HRE-VEGF基因转染MSCs及HRE对转基因MSCs在缺氧、复氧心肌细胞中的调控作用
第一节慢病毒介导HRE-VEGF基因转染骨髓间充质干细胞
目的:用慢病毒系统(pPACKH1- Lentivector Packaging Kit)将目的基因HRE-VEGF包装并转染干细胞。
方法:结合HRE-VEGF的穿梭质粒pCDH1转染包装细胞293T细胞进行扩增后,使用由三个质粒组成的慢病毒转染系统进行基因重组,并以其为载体转染鼠的骨髓间充质干细胞,检测其荧光的表达。
结果:293T细胞和MSCs24转染小时和48小时后,均可检测到强荧光出现。
结论:慢病毒载体系统携带目的基因HRE-VEGF基因转染鼠的骨髓间充质干细胞具有较高的转染效率。
第二节转基因骨髓间充质干细胞对缺氧心肌细胞的影响
目的:低氧反应元件对缺氧、复氧心肌细胞转导的VEGF基因表达的调控。
方法:原代培养心肌细胞,按不同的干细胞加入不同缺氧、复氧时间将心肌细胞分组,每组细胞固定后做免疫荧光染色测细胞内VEGF蛋白的表达情况;RT-PCR测细胞内人VEGF–mRNA含量;取细胞培养液ELISA法检测人VEGF含量。
结果:在缺氧组检测到VEGF–mRNA和VEGF蛋白的表达,而常氧组未检测到VEGF–mRNA和VEGF蛋白的表达。
结论:离体培养大鼠心肌细胞在低氧、复氧过程中加入携带HRE-VEGF基因的骨髓间充质干细胞时,其中的HRE对转导的VEGF基因表达具有调控作用。
第四部分转HRE-VEGF基因的MSCs自体移植对大鼠缺血性心肌病的实验研究
目的:以心梗的大鼠为模型,研究移植转染HRE-VEGF基因的自体MSCs能否在体内较未转基因的MSCs更显著地改善缺血性心肌病大鼠的心脏功能,进一步探讨MSCs联合基因转染用于治疗缺血性心肌病的可行性。
方法:选用雄性Fischer 344大鼠,体重200～250g,应用液氮冷冻法建立心梗模型,行血流动力学检测心功能。动物分成4组:空白组(A组,n=20)、DMEM组(B组,n=20)、MSCs组(C组,n=20)和转基因组(D组,n=20)。从大鼠股骨取骨髓,用贴壁筛选方法分离纯化、体外扩增MSCs,MSCs体外转染慢病毒包装的HRE-VEGF基因。将BrdU标记的自体MSCs直接注射到梗死区。4周后,重复心功能检查,评估心功能和梗死区的灌注;并观察心脏组织的病理学变化。采用ELISA法检测血浆VEGF在梗死后和干细胞移植前后的变化,以及检测Cx-43,Ⅷ因子等指标观察不同的处理对血管新生的影响。
结果:大鼠MSCs移植后4周,血流动力学测定显示,C、D组LVDP较A、B组升高(P<0.05),左心室压力变化率±dp/dtmax C、D组明显优于A、B组(P<0.05)。D组与C组相比:D组±dp/dtmax较高。移植的MSCs能防止梗塞区变薄和扩张;与A、B两组和移植前相比,梗死区的收缩功能和血流灌注均有所改善(P<0.05),心功能的改善在D组更为明显(与C组比较,P<0.05)。与A、B两组相比,C、D组梗死区的毛细血管密度明显增加(P<0.05),其中D组较C组增加明显(P<0.05)。VEGF的水平在梗死后有所增加,在细胞移植后一周出现高峰,后逐渐下降。
结论:转染HRE-VEGF的MSCs可以改善缺血心肌的左室功能,促进血管新生。这种改善可能是MSCs通过心肌再生防止梗死区变薄、抑制收缩功能异常有关。一些细胞因子在其中起非常重要的作用。VEGF的表达可以减少细胞的凋亡,提高干细胞再生血管的作用,从而可显著改善心脏功能。应用HRE-VEGF基因转染MSCs不仅理论上可以防止癌变而且能够改善心功能。
Background:Some research indicated that stem cells could not only replace the necrosis cardiomyocytes and increase the number of functional cardiomyocytes , but also raise the density of vascellum in the post infarction scars and improve the cardiac function. These results observed before would introduce a new therapeutic strategy to the field of cardiovascular diseases. It has been proved in several studys in vitro or in vivo that autologous bone marrow stem cells could rescue ischaemic myocardium、induce neovascularization and preserve left ventricular function. In this study we transfected HRE-VEGF into bone marrow stem cells, in order to make clear whether BMSCs transfected with HRE-VEGF could protecte cardiomyocytes、induce neovascularization、prevent VEGF overexpression and greatly improve the cardiac fuction after ischemic and hypoxia。
Part I: Separation and cultivation of rats bone marrow mesenchymal stem cells and establishment of animal model
Section one: Separation and cultivation of rats bone marrow mesenchymal stem cells
Objective:To build a method to cultivate rat MSCs in vitro, provide the cell resource for autologous bone marrow stem cells transplantation.
Methods: After rat MSCs have been separated and depurated, the cell cycle were detected by flow cytometry
Results: when MSCs were cultivated in vitro for 10～14 days, they confluensed and 75 percent of these cells were in the phase of G0/G1.
Conclusion: rat MSCs could be separated and amplified through the adhesiveness character in vitro grew and amplified, these cells could be used in cell transplantation.
Section two: Establishment Of Animal Model
Objective: to establish rat myocardial infarction model with liquid nitrogen frozen
Methods: to establish myocardial infarction animal model with the method that anterior wall of left ventricle of rat was contacted directly by metal stick soaked in liquid nitrogen.
Results: cordis hypersound data showed that there was significant difference between heart function before modeling and after modeling, the heart function before modeling was better than that after modeling.
Conclusion: this kind of myocardial infarction animal model is reliable because of the fixed location.
Part II: Construction of HRE-VEGF-Expression Vector
Section one: The Construction of VEGF-Expression Vector
Objective: Clone the cDNA of VEGF, combine cDNA with the expression vector to get the HRE-VEGF -Expression Vector
Methods: According to CDS domain of VEGF published in genebank we amplified the HRE-VEGF gene with RT-PCR from Hep G2 cells and clone the gene to pCDH1-MCS1-EF1- copGFP, then we transfected VEGF- expression vector into DH5αcompetence bacterium, The masculine clone bacterium were choosed, double enzyme-cutting and sequencing were conducted to verify the cDNA
Results: double enzyme cutting and sequencing indicated the cDNA we got were the same sequenc to CDS domain of VEGF
Conclusion: Using RT-PCR could successfully construct VEGF-expression vector
Section two: The Construction of HRE-VEGF Expression Vector
Objective: To clone the hypoxia response element tandem repetitive sequence, and to insert it into the objective gene expression vector which was proved by sequencing
Methods: complementary single strand DNA on head and end section was designed artificial by the end of erythropoietin of human being, and 9HRE was gotten by enzyme-cutting 3 times and connecting. Then double strands DNA of HRE with enzyme-cutting site and 9 cascade connections was gained by renaturation and elongation. The double strands DNA was sequenced after cloned to the expression vector.
Result: both the results of double enzyme-cutting verification and sequencing indicated that 9HRE-VEGF which was proved by sequencing matched to the designed one.
Conclusion: the method of isocaudarner can construct 9 copy hypoxia response element and 9HRE-VEGF expression vector successfully.
Part III: lentivirus mediated HRE-VEGF gene transfection of MSCs and the regulation effect of HRE on the transgenic stem cells in hypoxia and oxygen recovery myocardial cells
Section one: HRE-VEGF gene transfection bone marrow stem cells which was mediated by lentivirus
Objective: to transfect stem cells with HRE-VEGF gene, and to establish gene transfected bone marrow stem cells with lentivirus, in order to study the regulation effect of HRE to the cells with transduction VEGF gene in hypoxia and oxygen recovery myocardial cells.
Methods: after amplification of 293T cells which were transfected by shuttle plasmid PCDH1 combined with HRE-VEGF gene, gene recombination was carried out by the system of lentivirus transfection composed by 3 plasmids . Bone marrow stem cells of rat were then transfected by that recombination gene as trager and were detected its fluorescent expression.
Results: On 24 hours and 48 hours after transfection, the strong fluorescent expression can also be detected in 293 T cells and MSCs.
Conclusion: Lentivirus trager system with HRE-VEGF gene has high transfection efficiency in transfecting MSCs of rat.
Section two: Effects of Transgenic Stem Cells On Hypoxia Cardiomyocytes
Objective: to study the regulation of hypoxia response element to the gene expression of hypoxia and oxygen-recovery myocardial cells transfected VEGF gene of human being.
Motheds: to primary culture myocardial cells, and divided them into different groups with the type of stem cells and the time of hypoxia and oxygen recovery. After being fixed, the cells in each group were stained with immunofluorescence to detect the expression of VEGF proteinum; RT-PCR was used to detect the content of VEGF-mRNA of human being; content and type of VEGF was detect by ELISA from cell culture fluid.
Results: VEGF- mRNA and VEGF were detected in hypoxia group, and VEGF- mRNA and VEGF were found in the group without hypoxia.
Conclusion: the regulation effect of HRE to the transfected VEGF gene has been proved, during MSCs taking along HRE-VEGF gene were added into rat myocardial cells isolated cultured during the process of hypoxia and oxygen recovery.
Part IV: Gene-modified bone marrow stem cells autografting therapy in rat ischemic cardiomyopathy model
Objective: to investigate if MSCs transfected HRE-VEGF gene by autografting has more significant effect than MSCs without transfection in improving the heart function of ischemic cardiomyopathy, and to approach the therapeutic possibility about MSCs and gene used to treat myocardial ischemia.
Methods: male Fischer 344 rats with weight from 200g to 250g were put into research, MI scar were made by liquid nitrogen frozen, cardiac function were observed by hemodynamics monitoring. These rats were divided into four groups: control group(A groups, n=20)、DMEM group(B group, n=20)、MSCs group (C group, n=20)、transgene group (D group, n=20).
Bone marrow were taken from rats’femur. adherence screening technique were adopted to separate、purify and amplify MSCs in vitro. Then MSCs were transfected by HRE-VEGF.
Autologous bone marrow stem cells labeled by BrdU were injected into MI scar. After 4 weeks, cardiac function was monitored again in order to evaluate the heart function and scar infusion. Histology research were done to know the distribution of MSCs, otherwise, Changes in hematoplasma concentration of VEGF between post MI and posttransplant were got through the methods of ELISA. Cx-43,VEGF were also detected by ELISA to knowledge the effect of MSCs on neovascularization.
Results: Rats’MSCs were transplanted into MI scar, after 4 weeks, data from hemodynamics monitoring indicated that there is no obviously different in LVSP between experiment groups and control groups. In C、D group, LVEDP were lower, the±dp/dtmax were more better than that of A、B group. In D group and C group, +dp/dtmax in D group were higher that of C group. MSCs transplantion could protect scar from being thin and dilate, moreover, cardiac function and scar infusion were more improved compared with A、B group and pretransplante. The improvement of cardiac function were more successful in D group (to C group , P<0.05).The capillary density in C、D group increased greatly in MI scar compared with A、B group(P<0.05),and the increasing of D group were more obvious than C group. Concentration of VEGF also increased after MI and came up to peak 1 week later, after 1 week, the data showed the decrease in concentration of VEGF.
Conclusion: MSCs were observed still survived in the host myocardium, protected MI scar from thinning, inhibited the dysfunction of cardiac and improved the left ventricle function; these changes might attribute to the myocardium regenerate、neovascularization、extracellular matrix hyperplasy;Some cytokines might play very important role on these changes. Expression of VEGF could also release the cell apoptosis, reinforce neovascularization and improve the cardiac function.Transplantation of autologous bone marrow stem cells combined with HRE-VEGF was an effective treatment to ischemia myocardial disease
引文
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