GCA-HIF-1α-GCC提高移植骨髓干细胞存活率及治疗缺血性心肌病的实验研究
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摘要
背景:近年来,随着干细胞工程的兴起,骨髓间充质干细胞在治疗缺血性心脏病方面备受关注。研究表明,干细胞可以分化为心肌细胞、增加有功能的心肌细胞数量,增加心梗区域血管的密度,改善心功能,干细胞的存活依赖于所种植的微环境,移植到缺血心肌的干细胞存活率极低,严重制约了其疗效。实验证实低氧诱导因子-1α( hypoxia-inducible factor 1α,HIF-1α)能明显抑制细胞的凋亡,虽然缺氧心肌能够产生HIF-1α,但由于降解极快,难以在心肌缺血缺氧中发挥作用。因此我们运用双定点基因突变的方法重组HIF-1α,转染干细胞,研究在体内外的缺氧条件下,基因突变的HIF-1α能否抑制干细胞的凋亡,提高治疗效果,并在此基础上进一步探讨干细胞自体移植联合基因转染治疗缺血性心肌病的机理。
第一部分猪骨髓间充质干细胞(MSCs)的分离、培养和心梗模型的建立
目的:从猪的骨髓中分离出具有多向分化潜能的骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)以及建立猪的心梗动物动物模型。
第一节猪骨髓间充质干细胞(MSCs)的分离、培养
目的:建立猪骨髓间充质干细胞体外培养方法,为细胞心肌成形术提供新材料。
方法:猪的骨髓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达到融合,细胞周期显示73%的细胞处于G0/G1期。
结论:根据黏附特性分离的猪骨髓MSCs在体外条件下可生长扩增,可用于细胞心肌成形术。
第二节猪心肌梗死模型的建立
目的:经心导管用明胶海绵栓子栓塞猪的冠状动脉左前降支(LAD),建立心肌梗死的模型。
方法:麻醉后经股动脉置入心导管至冠状动脉左前降支远端用明胶海绵栓塞LAD。8周后行血流动力学、核磁共振、冠状动脉造影及组织学检查。
结果:与对照组相比,心梗(MI)组左室发展压(LVDP)降低(P<0.05),±dp/dtmax下降(P<0.05)。与对照组相比,心梗(MI)组左室每搏量(SV)、舒张末期室壁厚度(EDWT)下降(P<0.05)。复查冠脉造影提示LAD栓塞仍存在。组织学检查MI组前室壁、室间隔形成了透壁梗死灶。
结论:运用明胶海绵封堵冠状动脉成功建立猪心肌梗死模型,符合临床病理生理过程,稳定可靠。
第二部分pcDNA3.1+-GCA-HIF-1α-GCC慢病毒表达载体构建
目的:构建低氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)真核表达载体pcDNA3.1+-HIF-1α的突变体pcDNA3.1+-GCA-HIF-1α-GCC,并用慢病毒包装。
方法:用定点突变的方法将pcDNA3.1+-HIF1α的HIF1α第567位脯氨酸HIF1αPro密码子CCA突变为丙氨酸(Ala)密码子GCA,构建成单个突变HIF1α真核表达载体pcDNA3.1+HIF1α-567Ala在第一次突变的基础上仍然用定点突变的方法将pcDNA3.1+- HIF1α-567Ala的第811位天冬酰胺(Asn)密码子AAT突变为丙氨酸(Ala)密码子GCC,构建成双定点突变HIF1α真核表达载体pcDNA3.1+-GCA-HIF-1α-GCC,重组成HIF1α慢病毒质粒,经酶切及测序鉴定正确后,在293细胞中包装成为重组HIF1α慢病毒,并进行PCR鉴定及滴度测定,以RT-PCR和Western blot法分别对转染细胞进行表达分析。
结果:经酶切鉴定及基因测序证实人双突变型低氧诱导因子1α真核表达载体GCA-HIF-1α-GCC构建成功,PCR检测重组慢病毒包装成功,病毒滴度为2.5×108tu /ml。
结论:成功构建慢病毒包装重组人双突变型低氧诱导因子1α真核表达载体GCA-HIF-1α-GCC,能稳定表达相应的蛋白和HIF-1αmRNA。
第三部分慢病毒系统介导的GCA-HIF-1α-GCC基因转染猪骨髓干细胞及凋亡检测
目的:观察慢病毒包装系统(pPACKH1-Lentivector Packaging Kit)介导的GCA-HIF-1α-GCC基因转染猪MSCs,细胞内GCA-HIF-1α-GCC表达情况以及对MSCs生长的影响,在低氧的环境中检测转GCA-HIF-1α-GCC基因的骨髓干细胞和原代细胞、转慢病毒空载体细胞的凋亡情况。
方法:取生长良好的第2代MSCs接种,培养至生长汇合70-80%时,加入慢病毒载体GCA-HIF-1α-GCC,设定转染复数(multiplicities of infection,MOI )为5,以只含GFP基因的空质粒进行对照。荧光显微镜观察绿色荧光。提取蛋白后进行western blot蛋白鉴定。用FITC- Annexin V和PI双染法行流式细胞仪分析比较MSCs凋亡,转慢病毒空载体的MSCs和转GCA-HIF-1α-GCC的MSCs三种细胞在1% O2 37℃的培养箱中培养24 h后细胞的凋亡和死亡情况。Western blot检测Bax、HO-1蛋白表达。
结果:转染MSCs 48小时后,可检测到强荧光出现。Western blot免疫印迹技术表明HIF-1α蛋白表达产物的分子量与已知分子量相符。原代细胞在缺氧和乏营养状态下发生大量的凋亡,其凋亡率为61.3%,转空载体的细胞死亡率达67.3%,而转GCA-HIF-1α-GCC基因的MSC的凋亡率仅为7.1%。Western blot表明转基因的MSCs,HO-1蛋白表达增高而Bax表达降低。
结论:使用携带目的基因GCA-HIF-1α-GCC的慢病毒系统转染猪的骨髓干细胞可使其稳定表达HIF-1α。转GCA-HIF-1α-GCC的MSCs在体外较MSCs更能耐受缺氧和乏营养所诱发的凋亡,其原因与HIF-1α稳定表达促使干细胞分泌HO-1抑制Bax有关。
第四部分GCA-HIF-1α-GCC修饰自体骨髓干细胞移植治疗缺血性心肌病的实验研究
目的:探讨转染GCA-HIF-1α-GCC基因的MSCs自体移植治疗缺血性心肌病的疗效及机理,为治疗缺血性心肌病提供新的方法。
方法:选用15～20㎏梅山小型猪24头,随机分4组:A组:空白组、B组:DMEM组、C组:MSCs组和D组:转基因组,应用明胶海绵栓塞LAD建立心梗模型。采用密度梯度离心与贴壁筛选相结合的方法分离纯化、体外扩增MSCs,D组MSCs体外转染GCA-HIF-1α-GCC基因。干细胞(1×107/5 mL)经心导管注射到LAD栓塞处远端。分别于MSCs注射1周、2周、4周、6周和8周,行Powerlab及MRI检查,评估心功能和活体内干细胞标记情况,采用ELISA法检测血浆BNP、VEGF在梗死后和干细胞移植前后的变化;组织学观察各组移植细胞的情况,测算心梗面积,以及Western blot检测过氧化酶体增殖物激活型受体α(peroxisomeproliferator activated receptorα, PPARα)。
结果:移植前,磁共振示梗死心脏的左室舒张末径(EDWT)增加,每搏量(SV)明显下降,血流动力学LVDP、±dp/dtmax下降明显。移植后,C、D组的心功能较移植前有改善(P<0.05),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),移植组的心梗面积缩小(P<0.05);这种心功能的改善在D组更为明显(与C组比较,P<0.05)。Western blot检测PPAR,与A、B和C组相比, D组PPARα蛋白表达明显增加(P<0.05)。D组的增加比C组显著。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),D组的增加比C组显著,同样比较,BNP降低明显(P<0.05),D组的降低比C组显著。C、D组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:MSCs能在宿主心肌组织中存活,通过防止梗死区变薄、抑制收缩功能异常而改善左室功能。GCA-HIF-1α-GCC转染MSCs可以使移植的干细胞更能耐受移植早期的缺氧和乏营养的微环境,减少细胞的凋亡,增加参与心肌修复的干细胞的绝对数量,从而可更加显著改善心脏功能,PPAR在其中起非常重要的作用。
应用GCA-HIF-1α-GCC基因和MSCs自体移植是治疗缺血性心肌病的一种有效的方法。
Background: In recent years, with the emergence of stem cell engineering, more and more attention was paid to mesenchymal stem cells in areas of the treatment of ischemic heart disease. Research shows that stem cells can differentiate into cardiac cells, a function of increasing the number of cardiac cells, increasing the density of blood vessels regional myocardial infarction, improving heart function. While the survival of stem cells depends on the cultivation of the micro-environment, the low stem cells survived rate of myocardial ischemia transplant is one of the important factors on the clinical efficacy. It was confirmed that HIF--1α(hypoxia-inducible factor 1α, HIF-1α) generated by hypoxia can inhibit apoptosis. However HIF-1αdegrades very fast, and can not meet the needs of myocardial ischemia and hypoxia. Therefore, we use two-way fixed-point mutation in the reorganization of HIF-1α, then transfer stem cells in vitro and in vivo studies in the hypoxic conditions. We investigated whether the gene mutation in HIF-1αstem cells can inhibit apoptosis, improve treatment efficacy. Furthermore the mechanism of autologous stem cell transplantation of gene transfer treatment in ischemic cardiomyopathy was also investigated.
PartⅠSection A The isolation, and purification of swine autogenous bone marrow-derived mesenchymal stem cells
Objective : To evaluate the culture and purification of bone marrow-derived mesenchymal stem cells in vitro.
Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer.
Results The adherent, fibroblast-like cells were confluent in single layer after plating for 10～14 days. The cell cycle analysis showed that 75% of MSCs was in G0/G1 phase.
Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. It is suggesting that MSCs may be a new cell source for the cellar cardiomyoplasty.
Section B A Model of Myocardial Infarction by Intracoronary Embolization With Gelatin Sponges In Swines
Object: Producing a swine model of myocardial infarction(MI) by transcatheter embolization of the left coronary artery(LAD) with gelatin sponges.
Method: Six swines were underwent transcatheter embolization of LAD using gelatin sponge to produce anteroapical myocardial infarction. Coronary angiography、and Pathological examination were performed 4 weeks later.
Result: In myocardial infarction (MI) group, left ventricular developed pressure (LVDP) decreased compared with the control group (P<0.05),while±dp/dtmax also declined(P <0.05). LAD coronary angiography review prompted embolization still exists. In MI group, transmural infarction was formed in anterior and septal.
Conclusion The model is stable, reliable and much closer to clinical pathology physiological processes. Use of gelatin sponge can successfully establish coronary occlusion swine myocardial infarction model. It has the advantages of minor trauma, animal survival for a long time and easily feeding after surgery.
PartⅡConstruction of pcDNA3.1+-GCA-HIF-1α-GCC
Object: Construct HIF--1α(hypoxia inducible factor-1, HIF-1α) eukaryotic expression vector pcDNA3.1 +-GCA-HIF-1α-GCC, the mutant of pcDNA3.1 +-HIF-1α, and pack it with Lentivirus.
Method: PcDNA3.1 +-HIF1αof HIF1αNo. 567 Proline HIF1αPro codon was mutated into the CCA alanine (Ala) codon GCA with site-directed mutagenesis approach, building into a single mutation HIF1αeukaryotic expression vector pcDNA3.1 + HIF1α– 567Ala in the first mutation. On the basis of that,site-directed mutagenesis was used to mutated pcDNA3.1 + - HIF1α-567 Ala No. 811 in amide (Asn) codon for AAC alanine (Ala) codon GCC,building into dual site-directed mutagenesis HIF1αeukaryotic expression vector pcDNA3.1 +-GCA-HIF-1α-GCC.RT-PCR and Western blot were used to detect the expression of HIF1αin the transferred cells. Recomposed Lentivirus vector of HIF1αwas digested and sequenced, then packed into the reorganization HIF1αLentivirus virus in 293 cells. PCR was used to identify HIF1αexpression. Titer of virus vector was determined.
Results: Human double-mutant HIF-1αeukaryotic expression vector GCA-HIF-1α-GCC was successfully constructed by identification of digestion and gene sequencing. Reorganization of Lentivirus was packaged successfully, which was tested by PCR. The virus titer is 2 .5×108tu/ml.
Conclusion: Human double-mutant HIF-1αeukaryotic expression vector GCA-HIF-1α-GCC was successfully constructed to express the corresponding protein and mRNA, which lay the foundation for gene therapy of HIF-1αfor ischemic heart disease.
PartⅢLentivirus mediated GCA-HIF-1α-GCC gene transfer of bone marrow stem cells and the detection of apoptosis
Object: Observe the GCA-HIF-1α-GCC expression and it affection to the growth of cells in the Lentivirus mediated GCA-HIF-1α-GCC gene transfer of swine MSCs. Detection of apoptosis rates respectly in the GCA-HIF-1α-GCC gene transferred bone marrow stem cells, blank transferred bone marrow stem cells and primary cells in the hypoxic environment.
Method: After GCA-HIF-1α-GCC-cDNA shuttle plasmid transfected pCDH1 packaging cell 293T cells were amplified, gene was recombined by the four plasmid congested slow virus gene transfer system and transferred into swine bone marrow stem cells. It’s expression was detected by fluorescence ion. Western blot was used to detect protein expression. After FITC-Annexin V and PI double-stained, cell apoptosis and death of MSCs, blank transferred MSCs and GCA-HIF-1α-GCC transferred MSCs in 1% O2, 37℃in the incubator for 24 h were detected by flow cytometry analysis.Western blot was used to identificae protein expression of Bax and HO-1.
Results: 24 or 48 hours after transferring gene into 293 T cells and MSCs, strong fluorescence can be detected. Western blot showed that the molecular weight of aimed protein is consistent with the known molecular weight of HIF-1α. RT-PCR real-time quantitative results showed that two weeks after the transfer, MSCs can stable transcript HIF-1α. Primary cells in hypoxia and lack of nutritional status undergoing a lot of apoptosis, while the apoptosis rate is 61.3 percent compared to the GCA-HIF-1α-GCC transferred MSC of 7.1 percent. A large number of deaths occurred in the blank transferred cells, while the mortality rate is 67.3 percent. Western blot was used to identificae protein expression of Bax and HO-1.
Conclusion: Transfer of portable GCA-HIF-1α-GCC gene with the Lentivirus transfer of viruses into bone marrow stem cells of swine can stable express HIF-1α. The GCA-HIF-1α-GCC transferred MSCs in vitro showed more tolerance to apoptosis induced by lack of nutrition hypoxia than simple MSCs. It is because maybe that the stable expression of HIF-1αenhance the secretion of HO-1 of stem cells, which suppress Bax expression.
PartⅣThe experimental study of GCA-HIF-1α-GCC-modified autologous bone marrow stem cell transplantation in the treatment of ischemic cardiomyopathy
Object: To investigate whether GCA-HIF-1α-GCC-modified autologous bone marrow stem cell transplantation could significantly improve cardiac function in myocardial ischemic heart disease with swine MI model. Further, to discuss the MSCs and gene combined therapy in myocardial ischemia and the possible mechanism involved.
Method: 24 small Meishan swines from 15 to 20 kg , divided into four groups: A group: blank group, B group: DMEM group, C group: MSCs Group and Group D: TG group. Myocardial infarction LAD Model is established by gelatin sponge embolization. Four weeks later, hemodynamics and MRI were used to assess heart function. 30 mL bone marrow was taken from the iliac of each swine, separated and purificated by methods of density centrifugal and adherent screening. MSCs were amplified in vitro, while MSCs of D Group transfected with GCA-HIF-1α-GCC gene. Nano-iron marked autologous bone marrow stem cells (1×107 / 5 mL) injected transcatheter into the remote embolization of LAD. After one week, two weeks, four weeks, six weeks and eight weeks of injections of MSCs, Powerlab and MRI were used to assess cardiac function and stem cell marker. Plasma BNP was detected by ELISA in before infarction and stem cell transplantation or after that. We estimated risk zone of myocardial infarction and use Western blot to test PPAR.
Results: Before transplantation, MRI showed the left ventricular end-diastolic diameter (EDLVd) increased, stroke volume (SV) decreased significantly, hemodynamics LVDP and±dp / dtmax dropped significantly in cardiac infarction.Cardiac function of C, D group has improved after transplantation (P<0.05). The transplanted MSCs prevented thinning and expansion of infarct zone. Compared with Group A and B , the contract function and perfusion had improved (P <0.05). This improvement in cardiac function in Group D is more obvious (vs the C group, P<0.05). Group D increased more significantly than group C. BNP of C, D group has decrease after after transplantation (P<0.05), Group D increased more significantly than group C(P<0.05). The level of VEGF reached a high level 1 week after implanting the MSCs,then decreased gradually.
Conclusion: MSCs can survive in the host myocardium and improve left ventricular function by preventing thinning of infarction zone and inhibit contraction dysfunction. Overexpression of GCA-HIF-1α-GCC can make transplanted stem cells more tolerant of microenvironment hypoxia and lack of nutrient in the early transplantion, reduce apoptosis and increase absolute number of stem cells participation in cardiac rehabilitation, which can improve heart function more significantly. Application of GCA-HIF-1α-GCC genes combined with MSCs autologous transplantation is an effective way to the treatment of ischemic cardiomyopathy.
第一部分猪骨髓间充质干细胞(MSCs)的分离、培养和心梗模型的建立
目的:从猪的骨髓中分离出具有多向分化潜能的骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)以及建立猪的心梗动物动物模型。
第一节猪骨髓间充质干细胞(MSCs)的分离、培养
目的:建立猪骨髓间充质干细胞体外培养方法,为细胞心肌成形术提供新材料。
方法:猪的骨髓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达到融合,细胞周期显示73%的细胞处于G0/G1期。
结论:根据黏附特性分离的猪骨髓MSCs在体外条件下可生长扩增,可用于细胞心肌成形术。
第二节猪心肌梗死模型的建立
目的:经心导管用明胶海绵栓子栓塞猪的冠状动脉左前降支(LAD),建立心肌梗死的模型。
方法:麻醉后经股动脉置入心导管至冠状动脉左前降支远端用明胶海绵栓塞LAD。8周后行血流动力学、核磁共振、冠状动脉造影及组织学检查。
结果:与对照组相比,心梗(MI)组左室发展压(LVDP)降低(P<0.05),±dp/dtmax下降(P<0.05)。与对照组相比,心梗(MI)组左室每搏量(SV)、舒张末期室壁厚度(EDWT)下降(P<0.05)。复查冠脉造影提示LAD栓塞仍存在。组织学检查MI组前室壁、室间隔形成了透壁梗死灶。
结论:运用明胶海绵封堵冠状动脉成功建立猪心肌梗死模型,符合临床病理生理过程,稳定可靠。
第二部分pcDNA3.1+-GCA-HIF-1α-GCC慢病毒表达载体构建
目的:构建低氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)真核表达载体pcDNA3.1+-HIF-1α的突变体pcDNA3.1+-GCA-HIF-1α-GCC,并用慢病毒包装。
方法:用定点突变的方法将pcDNA3.1+-HIF1α的HIF1α第567位脯氨酸HIF1αPro密码子CCA突变为丙氨酸(Ala)密码子GCA,构建成单个突变HIF1α真核表达载体pcDNA3.1+HIF1α-567Ala在第一次突变的基础上仍然用定点突变的方法将pcDNA3.1+- HIF1α-567Ala的第811位天冬酰胺(Asn)密码子AAT突变为丙氨酸(Ala)密码子GCC,构建成双定点突变HIF1α真核表达载体pcDNA3.1+-GCA-HIF-1α-GCC,重组成HIF1α慢病毒质粒,经酶切及测序鉴定正确后,在293细胞中包装成为重组HIF1α慢病毒,并进行PCR鉴定及滴度测定,以RT-PCR和Western blot法分别对转染细胞进行表达分析。
结果:经酶切鉴定及基因测序证实人双突变型低氧诱导因子1α真核表达载体GCA-HIF-1α-GCC构建成功,PCR检测重组慢病毒包装成功,病毒滴度为2.5×108tu /ml。
结论:成功构建慢病毒包装重组人双突变型低氧诱导因子1α真核表达载体GCA-HIF-1α-GCC,能稳定表达相应的蛋白和HIF-1αmRNA。
第三部分慢病毒系统介导的GCA-HIF-1α-GCC基因转染猪骨髓干细胞及凋亡检测
目的:观察慢病毒包装系统(pPACKH1-Lentivector Packaging Kit)介导的GCA-HIF-1α-GCC基因转染猪MSCs,细胞内GCA-HIF-1α-GCC表达情况以及对MSCs生长的影响,在低氧的环境中检测转GCA-HIF-1α-GCC基因的骨髓干细胞和原代细胞、转慢病毒空载体细胞的凋亡情况。
方法:取生长良好的第2代MSCs接种,培养至生长汇合70-80%时,加入慢病毒载体GCA-HIF-1α-GCC,设定转染复数(multiplicities of infection,MOI )为5,以只含GFP基因的空质粒进行对照。荧光显微镜观察绿色荧光。提取蛋白后进行western blot蛋白鉴定。用FITC- Annexin V和PI双染法行流式细胞仪分析比较MSCs凋亡,转慢病毒空载体的MSCs和转GCA-HIF-1α-GCC的MSCs三种细胞在1% O2 37℃的培养箱中培养24 h后细胞的凋亡和死亡情况。Western blot检测Bax、HO-1蛋白表达。
结果:转染MSCs 48小时后,可检测到强荧光出现。Western blot免疫印迹技术表明HIF-1α蛋白表达产物的分子量与已知分子量相符。原代细胞在缺氧和乏营养状态下发生大量的凋亡,其凋亡率为61.3%,转空载体的细胞死亡率达67.3%,而转GCA-HIF-1α-GCC基因的MSC的凋亡率仅为7.1%。Western blot表明转基因的MSCs,HO-1蛋白表达增高而Bax表达降低。
结论:使用携带目的基因GCA-HIF-1α-GCC的慢病毒系统转染猪的骨髓干细胞可使其稳定表达HIF-1α。转GCA-HIF-1α-GCC的MSCs在体外较MSCs更能耐受缺氧和乏营养所诱发的凋亡,其原因与HIF-1α稳定表达促使干细胞分泌HO-1抑制Bax有关。
第四部分GCA-HIF-1α-GCC修饰自体骨髓干细胞移植治疗缺血性心肌病的实验研究
目的:探讨转染GCA-HIF-1α-GCC基因的MSCs自体移植治疗缺血性心肌病的疗效及机理,为治疗缺血性心肌病提供新的方法。
方法:选用15～20㎏梅山小型猪24头,随机分4组:A组:空白组、B组:DMEM组、C组:MSCs组和D组:转基因组,应用明胶海绵栓塞LAD建立心梗模型。采用密度梯度离心与贴壁筛选相结合的方法分离纯化、体外扩增MSCs,D组MSCs体外转染GCA-HIF-1α-GCC基因。干细胞(1×107/5 mL)经心导管注射到LAD栓塞处远端。分别于MSCs注射1周、2周、4周、6周和8周,行Powerlab及MRI检查,评估心功能和活体内干细胞标记情况,采用ELISA法检测血浆BNP、VEGF在梗死后和干细胞移植前后的变化;组织学观察各组移植细胞的情况,测算心梗面积,以及Western blot检测过氧化酶体增殖物激活型受体α(peroxisomeproliferator activated receptorα, PPARα)。
结果:移植前,磁共振示梗死心脏的左室舒张末径(EDWT)增加,每搏量(SV)明显下降,血流动力学LVDP、±dp/dtmax下降明显。移植后,C、D组的心功能较移植前有改善(P<0.05),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),移植组的心梗面积缩小(P<0.05);这种心功能的改善在D组更为明显(与C组比较,P<0.05)。Western blot检测PPAR,与A、B和C组相比, D组PPARα蛋白表达明显增加(P<0.05)。D组的增加比C组显著。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),D组的增加比C组显著,同样比较,BNP降低明显(P<0.05),D组的降低比C组显著。C、D组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:MSCs能在宿主心肌组织中存活,通过防止梗死区变薄、抑制收缩功能异常而改善左室功能。GCA-HIF-1α-GCC转染MSCs可以使移植的干细胞更能耐受移植早期的缺氧和乏营养的微环境,减少细胞的凋亡,增加参与心肌修复的干细胞的绝对数量,从而可更加显著改善心脏功能,PPAR在其中起非常重要的作用。
应用GCA-HIF-1α-GCC基因和MSCs自体移植是治疗缺血性心肌病的一种有效的方法。
Background: In recent years, with the emergence of stem cell engineering, more and more attention was paid to mesenchymal stem cells in areas of the treatment of ischemic heart disease. Research shows that stem cells can differentiate into cardiac cells, a function of increasing the number of cardiac cells, increasing the density of blood vessels regional myocardial infarction, improving heart function. While the survival of stem cells depends on the cultivation of the micro-environment, the low stem cells survived rate of myocardial ischemia transplant is one of the important factors on the clinical efficacy. It was confirmed that HIF--1α(hypoxia-inducible factor 1α, HIF-1α) generated by hypoxia can inhibit apoptosis. However HIF-1αdegrades very fast, and can not meet the needs of myocardial ischemia and hypoxia. Therefore, we use two-way fixed-point mutation in the reorganization of HIF-1α, then transfer stem cells in vitro and in vivo studies in the hypoxic conditions. We investigated whether the gene mutation in HIF-1αstem cells can inhibit apoptosis, improve treatment efficacy. Furthermore the mechanism of autologous stem cell transplantation of gene transfer treatment in ischemic cardiomyopathy was also investigated.
PartⅠSection A The isolation, and purification of swine autogenous bone marrow-derived mesenchymal stem cells
Objective : To evaluate the culture and purification of bone marrow-derived mesenchymal stem cells in vitro.
Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer.
Results The adherent, fibroblast-like cells were confluent in single layer after plating for 10～14 days. The cell cycle analysis showed that 75% of MSCs was in G0/G1 phase.
Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. It is suggesting that MSCs may be a new cell source for the cellar cardiomyoplasty.
Section B A Model of Myocardial Infarction by Intracoronary Embolization With Gelatin Sponges In Swines
Object: Producing a swine model of myocardial infarction(MI) by transcatheter embolization of the left coronary artery(LAD) with gelatin sponges.
Method: Six swines were underwent transcatheter embolization of LAD using gelatin sponge to produce anteroapical myocardial infarction. Coronary angiography、and Pathological examination were performed 4 weeks later.
Result: In myocardial infarction (MI) group, left ventricular developed pressure (LVDP) decreased compared with the control group (P<0.05),while±dp/dtmax also declined(P <0.05). LAD coronary angiography review prompted embolization still exists. In MI group, transmural infarction was formed in anterior and septal.
Conclusion The model is stable, reliable and much closer to clinical pathology physiological processes. Use of gelatin sponge can successfully establish coronary occlusion swine myocardial infarction model. It has the advantages of minor trauma, animal survival for a long time and easily feeding after surgery.
PartⅡConstruction of pcDNA3.1+-GCA-HIF-1α-GCC
Object: Construct HIF--1α(hypoxia inducible factor-1, HIF-1α) eukaryotic expression vector pcDNA3.1 +-GCA-HIF-1α-GCC, the mutant of pcDNA3.1 +-HIF-1α, and pack it with Lentivirus.
Method: PcDNA3.1 +-HIF1αof HIF1αNo. 567 Proline HIF1αPro codon was mutated into the CCA alanine (Ala) codon GCA with site-directed mutagenesis approach, building into a single mutation HIF1αeukaryotic expression vector pcDNA3.1 + HIF1α– 567Ala in the first mutation. On the basis of that,site-directed mutagenesis was used to mutated pcDNA3.1 + - HIF1α-567 Ala No. 811 in amide (Asn) codon for AAC alanine (Ala) codon GCC,building into dual site-directed mutagenesis HIF1αeukaryotic expression vector pcDNA3.1 +-GCA-HIF-1α-GCC.RT-PCR and Western blot were used to detect the expression of HIF1αin the transferred cells. Recomposed Lentivirus vector of HIF1αwas digested and sequenced, then packed into the reorganization HIF1αLentivirus virus in 293 cells. PCR was used to identify HIF1αexpression. Titer of virus vector was determined.
Results: Human double-mutant HIF-1αeukaryotic expression vector GCA-HIF-1α-GCC was successfully constructed by identification of digestion and gene sequencing. Reorganization of Lentivirus was packaged successfully, which was tested by PCR. The virus titer is 2 .5×108tu/ml.
Conclusion: Human double-mutant HIF-1αeukaryotic expression vector GCA-HIF-1α-GCC was successfully constructed to express the corresponding protein and mRNA, which lay the foundation for gene therapy of HIF-1αfor ischemic heart disease.
PartⅢLentivirus mediated GCA-HIF-1α-GCC gene transfer of bone marrow stem cells and the detection of apoptosis
Object: Observe the GCA-HIF-1α-GCC expression and it affection to the growth of cells in the Lentivirus mediated GCA-HIF-1α-GCC gene transfer of swine MSCs. Detection of apoptosis rates respectly in the GCA-HIF-1α-GCC gene transferred bone marrow stem cells, blank transferred bone marrow stem cells and primary cells in the hypoxic environment.
Method: After GCA-HIF-1α-GCC-cDNA shuttle plasmid transfected pCDH1 packaging cell 293T cells were amplified, gene was recombined by the four plasmid congested slow virus gene transfer system and transferred into swine bone marrow stem cells. It’s expression was detected by fluorescence ion. Western blot was used to detect protein expression. After FITC-Annexin V and PI double-stained, cell apoptosis and death of MSCs, blank transferred MSCs and GCA-HIF-1α-GCC transferred MSCs in 1% O2, 37℃in the incubator for 24 h were detected by flow cytometry analysis.Western blot was used to identificae protein expression of Bax and HO-1.
Results: 24 or 48 hours after transferring gene into 293 T cells and MSCs, strong fluorescence can be detected. Western blot showed that the molecular weight of aimed protein is consistent with the known molecular weight of HIF-1α. RT-PCR real-time quantitative results showed that two weeks after the transfer, MSCs can stable transcript HIF-1α. Primary cells in hypoxia and lack of nutritional status undergoing a lot of apoptosis, while the apoptosis rate is 61.3 percent compared to the GCA-HIF-1α-GCC transferred MSC of 7.1 percent. A large number of deaths occurred in the blank transferred cells, while the mortality rate is 67.3 percent. Western blot was used to identificae protein expression of Bax and HO-1.
Conclusion: Transfer of portable GCA-HIF-1α-GCC gene with the Lentivirus transfer of viruses into bone marrow stem cells of swine can stable express HIF-1α. The GCA-HIF-1α-GCC transferred MSCs in vitro showed more tolerance to apoptosis induced by lack of nutrition hypoxia than simple MSCs. It is because maybe that the stable expression of HIF-1αenhance the secretion of HO-1 of stem cells, which suppress Bax expression.
PartⅣThe experimental study of GCA-HIF-1α-GCC-modified autologous bone marrow stem cell transplantation in the treatment of ischemic cardiomyopathy
Object: To investigate whether GCA-HIF-1α-GCC-modified autologous bone marrow stem cell transplantation could significantly improve cardiac function in myocardial ischemic heart disease with swine MI model. Further, to discuss the MSCs and gene combined therapy in myocardial ischemia and the possible mechanism involved.
Method: 24 small Meishan swines from 15 to 20 kg , divided into four groups: A group: blank group, B group: DMEM group, C group: MSCs Group and Group D: TG group. Myocardial infarction LAD Model is established by gelatin sponge embolization. Four weeks later, hemodynamics and MRI were used to assess heart function. 30 mL bone marrow was taken from the iliac of each swine, separated and purificated by methods of density centrifugal and adherent screening. MSCs were amplified in vitro, while MSCs of D Group transfected with GCA-HIF-1α-GCC gene. Nano-iron marked autologous bone marrow stem cells (1×107 / 5 mL) injected transcatheter into the remote embolization of LAD. After one week, two weeks, four weeks, six weeks and eight weeks of injections of MSCs, Powerlab and MRI were used to assess cardiac function and stem cell marker. Plasma BNP was detected by ELISA in before infarction and stem cell transplantation or after that. We estimated risk zone of myocardial infarction and use Western blot to test PPAR.
Results: Before transplantation, MRI showed the left ventricular end-diastolic diameter (EDLVd) increased, stroke volume (SV) decreased significantly, hemodynamics LVDP and±dp / dtmax dropped significantly in cardiac infarction.Cardiac function of C, D group has improved after transplantation (P<0.05). The transplanted MSCs prevented thinning and expansion of infarct zone. Compared with Group A and B , the contract function and perfusion had improved (P <0.05). This improvement in cardiac function in Group D is more obvious (vs the C group, P<0.05). Group D increased more significantly than group C. BNP of C, D group has decrease after after transplantation (P<0.05), Group D increased more significantly than group C(P<0.05). The level of VEGF reached a high level 1 week after implanting the MSCs,then decreased gradually.
Conclusion: MSCs can survive in the host myocardium and improve left ventricular function by preventing thinning of infarction zone and inhibit contraction dysfunction. Overexpression of GCA-HIF-1α-GCC can make transplanted stem cells more tolerant of microenvironment hypoxia and lack of nutrient in the early transplantion, reduce apoptosis and increase absolute number of stem cells participation in cardiac rehabilitation, which can improve heart function more significantly. Application of GCA-HIF-1α-GCC genes combined with MSCs autologous transplantation is an effective way to the treatment of ischemic cardiomyopathy.
引文
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13 Mice.Valerie Blanc, Jeffrey O. Henderson, Rodney D. Newberry, et al. Deletion of the AU-Rich RNA Binding Protein Apobec-1 Reduces Intestinal Tumor Burden in Apcmin Cancer Res., 2007; 67: 8565 - 8573.
14 Juha R, Tuomas T. Rissanen, et al. Adenoviral Catheter-Mediated Intramyocardial Gene Transfer Using the Mature Form of Vascular Endothelial Growth Factor-D Induces Transmural Angiogenesis in Porcine Heart. Circulation,2004;109:1029
15 Isao K, Koji O,Akira O,et al. Treatment of acute myocardial infarction by hepatocyte growth factor gene transfer:The first demonstration of myocardial transfer of a "functional" gene using ultrasonic microbubble destruction.J. Am. Coll. Cardiol., 2004;44:644 - 653.
16 Dawn E. Telford; Sara M. A Novel Inhibitor of Oxidosqualene:Lanosterol Cyclase Inhibits Very Low–Density Lipoprotein Apolipoprotein B100 (ApoB100) Production and Enhances Low-Density Lipoprotein ApoB100 Catabolism Through Marked Reduction in Hepatic Cholesterol Content . Arterioscler. Thromb. Vasc. Biol.,2005; 25:2608 - 2614.
17 H Hamada, M Kobune, K Nakamura, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy . Cancer Sci, Mar 2005; 96(3): 149-56.
18 Massimo Dominici, Rita Sternieri, Domenico De Fazio, et al. 0.1 Grams of Sub-Cutaneous Adipose Tissue (SCAT) as Source of Adult Multipotent Mesenchymal Stromal Cells (MSC) for Cell-Based Therapies.Blood, 2007; 110: 4915.
19 Shideng Bao, Qiulian Wu, Sith Sathornsumetee, et al.Stem Cell–like Glioma Cells Promote Tumor Angiogenesis through Vascular Endothelial Growth Factor.Cancer Res., Aug 2006; 66: 7843 - 7848.
20 Betty Schwartz, Oded Shoseyov, Vladislava O. Melnikova, et al. Angiogenin and Inhibits Human Melanoma Growth, Angiogenesis, and Metastasis. Cancer Res., 2007; 67:5258-5266.
21 Vitaly Vasilevko, Feng Xu, Mary Lou Previti, et al Experimental Investigation of Antibody-Mediated Clearance Mechanisms of Amyloid-βin CNS of Tg-SwDI Transgenic Mice. J. Neurosci., 2007; 27: 13376 - 13383.
22 Ian B. Copland, E. Marc Jolicoeur, Marc-Antoine Gillis, et al. Coupling erythropoietin secretion to mesenchymal stromal cells enhances their regenerative properties. Cardiovasc Res, 2008; 10.1093-1097.
23 Ian B. Copland, Marc E. Jolicoeur, Marc-Antoine Gillis, et al.Erythropoietin (Epo) Secreting Mesenchymal Stromal Cells and Autocrine Signalling Via the Epo Receptor. A Novel, Cell-Based Paracrine Epo Delivery System for Cardiovascular Therapy. Blood (ASH Annual Meeting Abstracts), 2007; 110: 1198.
24 Wenke Feng, Frederick W. Benz, Jian Cai, et al. Metallothionein Disulfides Are Presentin Metallothionein-overexpressing Transgenic Mouse Heart and Increase under Conditions of Oxidative Stress.J. Biol. Chem., 2006; 281: 681 - 687.
25 EE Hood, R Love, J Lane, et al. Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J, 2007; 5(6): 709-19.
26 Etty Grad, Mordechai Golomb, Irit Mor-Yosef, et al. Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury. Am J Physiol Heart Circ Physiol, 2007; 293: H489 - H495.
27 VL Battula, PM Bareiss, S Treml, et al. Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation.Differentiation, 2007; 75(4): 279-91.
28 S Chebolu and H Daniell. Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis .Plant Biotechnol J,2007; 5(2): 230-9.
29 EM Dragunsky, AP Ivanov, S Abe, et al.Further development of a new transgenic mouse test for the evaluation of the immunogenicity and protective properties of inactivated poliovirus vaccine.J Infect Dis, 2006; 194(6): 804-7.
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3 Tim K,Eugenio S,Mary SB,et al. Bone Marrow–Derived Cells for Enhancing Collateral Development: Mechanisms,Animal Data,and Initial Clinical Experiences Circ. Res.,2004;95:354-363.
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5 Daniela Galli;Anna Innocenzi; Lidia Staszewsky;et al .Mesoangioblasts, Vessel-Associated Multipotent Stem Cells, Repair the Infarcted Heart by Multiple Cellular Mechanisms: A Comparison With Bone Marrow Progenitors, Fibroblasts, and Endothelial Cells. Arterioscler. Thromb. Vasc. Biol.,2005;25:692-697.
6 Meyer GP,Wollert KC,Lotz J et al. Intracoronary bone marrow cell transfer after myocardial infarction:eighteen months’follow-up data from the randomized, controlled BOOST (Bone marrow transfer to enhance ST-elevation infarct regeneration) trial. Circulation,2006; 113:1287–1294.
7 Emerson C, Hans F.R,Radovan B,Transendocardial, Autologous Bone Marrow Cell Transplantation for Severe, Chronic Ischemic Heart Failure,Cir,2003;107: 2294.
8 Cleland JG, Freemantle N, Coletta AP et al. Repair-AMI,ASTAMI,JELIS,MEGA,REVIVE-II,SURVIVE,and PROACTIVE. Eur J Heart Fail 2006; 8,105–110.
9 Tim K, Eugenio S,Mary SB, et al. Bone Marrow–Derived Cells for Enhancing Collateral Development:Mechanisms,Animal Data,and Initial Clinical Experiences Circ. Res.,2004;95:354 - 363.
10 H Piao,TJ Youn,JS Kwon, et al. Effects of bone marrow derived mesenchymal stem cells transplantation in acutely infarcting myocardium. Eur J Heart Fail,2005;7: 730-8.
11 Lei Ye,Husnain Kh Haider,and Eugene K et al. Adult Stem Cells for Cardiac Repair: A Choice Between Skeletal Myoblasts and Bone Marrow Stem Cells. Experimental Biology and Medicine,2006;231:8–19
12 David M. Lehmann, Chad A. Galloway, Mark P. Sowden, et al. Metabolic regulation of ApoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor. Nucleic Acids Res., 2006; 34: 3299 - 3308.
13 Mice.Valerie Blanc, Jeffrey O. Henderson, Rodney D. Newberry, et al. Deletion of the AU-Rich RNA Binding Protein Apobec-1 Reduces Intestinal Tumor Burden in Apcmin Cancer Res., 2007; 67: 8565 - 8573.
14 Juha R, Tuomas T. Rissanen, et al. Adenoviral Catheter-Mediated Intramyocardial Gene Transfer Using the Mature Form of Vascular Endothelial Growth Factor-D Induces Transmural Angiogenesis in Porcine Heart. Circulation,2004;109:1029
15 Isao K, Koji O,Akira O,et al. Treatment of acute myocardial infarction by hepatocyte growth factor gene transfer:The first demonstration of myocardial transfer of a "functional" gene using ultrasonic microbubble destruction.J. Am. Coll. Cardiol., 2004;44:644 - 653.
16 Dawn E. Telford; Sara M. A Novel Inhibitor of Oxidosqualene:Lanosterol Cyclase Inhibits Very Low–Density Lipoprotein Apolipoprotein B100 (ApoB100) Production and Enhances Low-Density Lipoprotein ApoB100 Catabolism Through Marked Reduction in Hepatic Cholesterol Content . Arterioscler. Thromb. Vasc. Biol.,2005; 25:2608 - 2614.
17 H Hamada, M Kobune, K Nakamura, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy . Cancer Sci, Mar 2005; 96(3): 149-56.
18 Massimo Dominici, Rita Sternieri, Domenico De Fazio, et al. 0.1 Grams of Sub-Cutaneous Adipose Tissue (SCAT) as Source of Adult Multipotent Mesenchymal Stromal Cells (MSC) for Cell-Based Therapies.Blood, 2007; 110: 4915.
19 Shideng Bao, Qiulian Wu, Sith Sathornsumetee, et al.Stem Cell–like Glioma Cells Promote Tumor Angiogenesis through Vascular Endothelial Growth Factor.Cancer Res., Aug 2006; 66: 7843 - 7848.
20 Betty Schwartz, Oded Shoseyov, Vladislava O. Melnikova, et al. Angiogenin and Inhibits Human Melanoma Growth, Angiogenesis, and Metastasis. Cancer Res., 2007; 67:5258-5266.
21 Vitaly Vasilevko, Feng Xu, Mary Lou Previti, et al Experimental Investigation of Antibody-Mediated Clearance Mechanisms of Amyloid-βin CNS of Tg-SwDI Transgenic Mice. J. Neurosci., 2007; 27: 13376 - 13383.
22 Ian B. Copland, E. Marc Jolicoeur, Marc-Antoine Gillis, et al. Coupling erythropoietin secretion to mesenchymal stromal cells enhances their regenerative properties. Cardiovasc Res, 2008; 10.1093-1097.
23 Ian B. Copland, Marc E. Jolicoeur, Marc-Antoine Gillis, et al.Erythropoietin (Epo) Secreting Mesenchymal Stromal Cells and Autocrine Signalling Via the Epo Receptor. A Novel, Cell-Based Paracrine Epo Delivery System for Cardiovascular Therapy. Blood (ASH Annual Meeting Abstracts), 2007; 110: 1198.
24 Wenke Feng, Frederick W. Benz, Jian Cai, et al. Metallothionein Disulfides Are Presentin Metallothionein-overexpressing Transgenic Mouse Heart and Increase under Conditions of Oxidative Stress.J. Biol. Chem., 2006; 281: 681 - 687.
25 EE Hood, R Love, J Lane, et al. Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J, 2007; 5(6): 709-19.
26 Etty Grad, Mordechai Golomb, Irit Mor-Yosef, et al. Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury. Am J Physiol Heart Circ Physiol, 2007; 293: H489 - H495.
27 VL Battula, PM Bareiss, S Treml, et al. Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation.Differentiation, 2007; 75(4): 279-91.
28 S Chebolu and H Daniell. Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis .Plant Biotechnol J,2007; 5(2): 230-9.
29 EM Dragunsky, AP Ivanov, S Abe, et al.Further development of a new transgenic mouse test for the evaluation of the immunogenicity and protective properties of inactivated poliovirus vaccine.J Infect Dis, 2006; 194(6): 804-7.
30 C Pereira, M Maamar-Tayeb, A Burke, et al.Immunohistochemical staining of transgenic beta-galactosidase in burned skin is a better indicator of transfection efficiency than histochemical techniques.J Immunol Methods, 2006; 315(1-2): 75-9.
31 Buom-Yong Ryu, Kyle E. Orwig, Jon M. Oatley, et al.Efficient Generation of Transgenic Rats Through the Male Germline Using Lentiviral Transduction and Transplantation of Spermatogonial Stem Cells.J Androl, 2007; 28: 353 - 360.