单质粒心肌特异性缺氧调节SDF-1警戒载体在小鼠心肌梗死治疗中的应用
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摘要
间质细胞衍生因子-1(Stromal cell derived factor-1)是一种趋化因子,它和主要受体CXCR4相互作用,介导干细胞归巢、抑制细胞凋亡等。心肌梗死后,内源性SDF-1分泌在表达量和持续时间上无法满足心肌梗死后机体修复的需求。本研究构建了单质粒心肌特异性缺氧调节SDF-1警戒载体,体内体外实验论证这个单质粒警戒载体的优越性。同时,探讨SDF-1抑制缺氧引起心肌细胞凋亡的机制。
[目的]掌握分离间充质干细胞、心脏干细胞的方法；使用H9C2细胞模拟心肌细胞,研究SDF-1基因治疗后SDF-1在缺氧心肌内的表达时间曲线；研究SDF-1能否抑制缺氧应激引起的H9C2、间充质干细胞、心脏干细胞凋亡；探讨这种抑制作用与p-Bad蛋白去磷酸化为Bad的关系。
[方法]分离、培养MSCs、CSCs。构建pCMV-hSDF-1-GFP质粒,转染到缺氧的H9C2细胞,Real-time PCR、细胞染色验证转染。Western-blot法检测SDF-1的表达时间曲线。在SDF-1表达高峰时间点(转染后3天)收集条件培养基,以缺氧后内源性SDF-1表达高峰培养基为对照培养基。对比两种培养基对MSCs、CSCs迁移的影响。分析两种培养基对H2O2模拟的缺氧应激引起的H9C2、MSCs、CSCs凋亡的影响(细胞活力试验、台盼蓝拒染法实验、细胞形态),并进一步观察SDF-1条件培养基对在这个过程中p-Bad蛋白去磷酸化为Bad蛋白的影响。
[结果]pCMV-hSDF-1-GFP转染缺氧的H9C2细胞后,hSDF-1基因表达是对照组的31倍(p<0.01),转染效率在27.83%±2.61%。SDF-1蛋白在转染后3-7天达到表达高峰,即使14天后SDF-1的表达还较缺氧后内源性SDF-1表达高峰明显。条件培养基较对照培养基促进了MSCs、CSCs的迁移：CSCs34±11 vs.11±3,p < 0.001; MSCs 56±4 vs.10±1,p< 0.001。细胞活力试验：条件培养基组共聚焦显微镜下H9C2、MSCs、CSCs中绿色细胞比例较对照培养基组绿色细胞比例高。台盼蓝拒染法实验：MSCs(条件培养基组84%±7%vs.对照培养基组60%±2%,P<0.01);CSCs(条件培养基组82%±3%vs.对照培养基组62%±3%,P<0.001)；H9C2(条件培养基组89%±3%vs.对照培养基组65%±2%,P<0.001)。形态学上观察H9C2、MSCs、CSCs细胞的区别：对照培养基组可以观察到更多的圆形、光亮的细胞。不同的培养基培养后,Western-blot结果：条件培养基组培养的H9C2、MSCs、CSCs细胞p-Bad的表达较对照组明显,Bad蛋白则相反。细胞染色：在共聚焦显微镜下,条件培养基组培养的H9C2、MSCs、CSCs细胞可以观察到p-Bad的表达。[结论]pCMV-hSDF-1-GFP质粒转染缺氧H9C2细胞后,SDF-1高表达持续超过14天,高峰在3-7天。基因治疗的SDF-1条件培养基可以通过减少p-Bad去磷酸为Bad抑制缺氧引起的H9C2、MSCs、CSCs凋亡。基因治疗的SDF-1条件培养基明显促进了MSCs、CSCs的迁移。
[目的]构建单质粒心肌特异性缺氧调节SDF-1警戒载体,体外、体内试验验证这个载体系统的有效性。探讨SDF-1抑制心肌细胞凋亡的机制。
[方法]构建单、双质粒心肌特异性缺氧调节SDF-1警戒载体,Real-time PCR对比单双质粒载体hSDF-1的表达。分离原代心肌细胞,转染单质粒载体,对比正常氧、低氧下hSDF-1的表达。探寻单质粒载体启动的时间。叠氮钠模拟缺氧应激,单质粒心肌特异性缺氧调节SDF-1警戒载体转染心肌细胞后,观察心肌细胞的生存,提取条件培养基,用AMD3100阻断SDF-1和CXCR4的相互作用,分为Control、AMD3100、AMD3100+条件培养基、条件培养基组,分析SDF-1抑制心肌细胞时是否磷酸化Akt、p-38MAPK、ERK1/2。体内实验验证单质粒警戒载体能否实现：高效、心肌特异性、缺氧警戒,分为Control组、MI组、假手术+V(警戒载体)组、MI+V组,在心肌梗死后1天分析SDF-1在肝脏、假手术+V组心肌、MI+V组心肌的表达,同时Western-blot和免疫组化法分析心肌组织p-Akt、p-ERK1/2/ p-38MAPK、p-Bad的表达,TUNEL实验评价体内心肌细胞的凋亡。7天后小动物心超分析各组心功能各项参数：EF%(左室射血分数)、LV mass(左室壁心肌重量)、FS%(短轴缩短率)等,通过Masson染色评价心肌纤维化。
[结果]成功构建单质粒心肌特异性缺氧调节SDF-1警戒载体,该载体在缺氧条件下hSDF-1基因表达是双质粒载体的2.5倍(p<0.01)。单质粒SDF-1载体在低氧后30分钟,hSDF-1基因开始表达上调(是正常氧浓度下hSDF-1基因的1.5倍),缺氧24小时后对比非缺氧条件上升8.6倍(p<0.01)。单质粒载体转染原代心肌细胞效率在55%±7%；原代心肌细胞分为Normal组,Model组(叠氮钠),SDF组,SDF+叠氮钠组：Normal、Model、SDF组原代心肌细胞在第3天开始出现部分心肌细胞死亡,第5天心肌细胞完全死亡；在SDF+叠氮钠组观察到原代心肌细胞在第9天才开始出现部分心肌细胞死亡,第11天培养的原代心肌细胞完全死亡。对比Control、AMD3100、AMD3100+条件培养基组,SDF-1条件培养基组明显增加了p-Akt、p-ERK1/2、p-38MAPK的表达。体内试验：单质粒载体治疗组心肌组织的SDF-1较非治疗组表达明显,也较治疗组肝脏组织的SDF-1表达明显；Western-blot、免疫组化染色的实验结果表明治疗组p-Akt、p-ERK1/2、p-38MAPK、p-Bad都较未治疗组高表达；单质粒载体治疗组心肌组织中TUNEL阳性细胞数25±2较非治疗组43±8明显少,P<0.01。7天后,心超测定心功能：MI+V组心功能较MI组明显改善,MI+V组和MI组：EF%,36.68±5.21 VS 21.1±2.12,p<0.01;LV Mass,102.85±9.90VS 151.90±9.08,p<0.01；FS%,17.42±2.22 VS 10.05±0.50,p<0.01。Masson染色结果表明：单质粒载体治疗组纤维化面积较未治疗组缩小。
[结论]本课题构建的单质粒心肌特异性缺氧调节SDF-1警戒载体具备高效、缺氧警戒、心肌特异性的特点,体外体内实验都证明这个单质粒警戒载体的有效性,是一种良好的基因工具。SDF-1通过促进Akt、ERK1/2、p38MAPK的磷酸化、减少p-Bad蛋白的去磷酸化抑制缺氧刺激引起的心肌细胞凋亡。
Stromal cell-derived factor 1 a (SDF-1a) is a homing factor that can cause stem cells to migrate from the bone marrow,which can react with its main receptor CXCR4.It can inhibit cell apoptosis,too.Although endogenous SDF-1a is secreted in the early phase of infarction in ischemic myocardium, the expression of myocardial SDF-1 is not effective for heart repair. SDF-1 has to be expressed in larger quantities and for a longer time.We try to build one single-plasmid vigilant vector.This vigilant vector has some features:high efficiency, hypoxia-regulated, vigilant,cardiac-specific.We evalute this vigilant system in cardiac myocytes. Furtherly, we evalute this vigilant system in mouse model of myocardial infarction.Another aim of this reserch is to evalute anti-apoptosis of SDF-1 and to find the inner mechanism.
Objective SDF-1 has to be expressed in larger quantities and for a longer time for the treatment of myocardial infarction. A gene therapy approach using an SDF-1 a vector can increase SDF-1 expression and mobilize and direct stem cells to the ischemic myocardium. SDF-1 can serve as a heart-protective factor through its ability to activate phosphorylated-AKT and ERK1/2. The role of the Bcl-2-associated death (BAD) and phosphorylated-BAD (p-BAD) in this process, however, has not been determined. In addition,the detailed time course of a SDF-1 a vector adminastration remains unclear.
Methods Contrust the p-CMV-hSDF-1 plasmids.H9C2 cells were transfected with the p-CMV-hSDF-1 plasmid under hypoxic stimuli. Real-time PCR and cell staining was used to confirm the transfection under hypoxic condition.Western blot analysis was used to determine the SDF-1 expression time course.In the SDF-1 peak expression,the conditioned medium was collected for further experiment.The SDF-1 conditioned medium in endogenous peak expression would be considered as control medium.Isolate mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs).Under conditioned medium or control medium,MSCs and CSCs would be migrated. H2O2 was used to induce H9C2,MSCs and CSCs apoptosis.The apoptosis of H9C2,MSCs and CSCs was evaluated with conditioned medium or control medium.Western-blot,cell staining was to confirm reduction of dephosphorylation of p-Bad.
Results The transfection efficiency was approximately 27.83%±2.61%. H9C2 cells transfected with the pCMV-hSDF-1 plasmid expressed 31-fold more h-SDF-1 than the control group (P=0.003563).Western blot analysis revealed that SDF-1 expression peaked from the third day to the 7th day after pCMV-hSDF-1 plasmid transfection. After treatment with conditioned medium, which was gathered on the third day, H9C2 cells, mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs) were more resistant to hypoxic insult than control groups, based on the results of the Live/Dead cell viability test, morphological observation and trypan blue uptake test. Live/Dead cell viability test:in confocol laser scan,there were more green H9C2,MSCs,and CSCs cells in conditioned medium treatment groups than in control medium groups. Morphological observation:there were more round,bright H9C2,MSCs and CSCs in control groups than in conditioned medium. Trypan blue uptake test: MSCs (conditioned medium 84%±7% vs. control medium60%±2%, P< 0.01); CSCs (conditioned medium 82%±3% vs. control medium 62%±3%, P< 0.001); H9C2 (conditioned medium 89%±3% vs. control medium 65%±2%, P< 0.001).Both western blot and staining results confirmed the dephosphorylation reduction of p-BAD in H9C2s, MSCs and CSCs after being treated with SDF-1 conditioned medium.The conditioned medium made more MSCs migrate compared with control medium(56±4 vs.10±1, p< 0.001).Likewise, the conditioned medium made more CSCs migrate compared with control medium(34±11 vs.11±3, p< 0.001).
Conclusions These findings indicate that, under hypoxic stimulation after SDF-1 gene delivery, the expression of SDF-1 peaks from the third day to the 7th and lasts longer than fourteen days. As a result, more MSCs and CSCs migrate into the hypoxic myocardium. In addition, treatment with SDF-1 conditioned medium causes H9C2s, MSCs and CSCs to become more resistant to hypoxic stimuli and to reduce dephosphorylation of the p-BAD proteins.
Objective To construct one single-plasmid vigilant vector,which has special features:high-efficiency,vigilant,hypoxia-regulated,cardiac-specific.This system can meet the requirement of therapy in myocardial infarction.These features would be assessed in vitro and in vivo experiments.Another aim is to prove that SDF-1 can be anti-apoptosis in cardiac myocytes.The inner mechanism would be assessed.
Methods After constructing the single-plasmid vigilant vector,this vigilant plasmid would be compared with double-plasmids vigilant vector under hypoxic stimuli.In addition,the on-set time of this single-plasmid vector would be evaluated. Furthermore,the hypoxia-regulated feature was appraised. Isolate and culture cardiac myocytes.The cardaic myocytes was transfected with single-vigilant plasmid. NaN3 was used to stimulate hypoxia insult. Cell apoptosis or death could be observed from cell morphology. Collect the used medium in the 5th day as conditioned medium.The cardiac myocytes would be divided into four groups:Control,AMD3100,AMD3100 with conditioned medium,conditioned medium.Phosphorylation of ERK1/2,p-38MAPK and Akt were detected by Westen blot. Then this single-plasmid was injected into myocardium in left ventricular apical.The animal experiment would be divided into four groups:MI,Sham+V,MI+V,Control.After 3 days,the left anterior descending coronary artery (LAD) was ligated as myocardial infarction model. 24 houres later,the SDF-1, p-ERK1/2, p-p38MAPK and p-Akt expression of MI or MI+V groups were detected by Western-blot and immunohistochemistry.
TUNEL test was performed to assess cardiac myocytes apoptosis in vivo.7 days later, the echocardiographic assessment(EF%,LV mass and FS%) of myocardial infarction and heart function was performed.Masson staining was performed to identify collagen fibers.
Results Under hypoxic stimuli,single-plasmid vigilant vector can express more 2.5 folds hSDF-1 gene than double-plasmids vigilant vector(p< 0.01).After 30 mins of hypoxia stimuli,SDF-1gene can be on-set.Compared with nomoxia,hypoxia stimuli can make more 8.6 foldes hSDF-1 be expressed in cardiac myocytes transfected with single vigilant vector.The transfection efficiency is about 55%±7%.The cardiac myocytes transfected with single-plasmid vigilant vector can be more resistant to hypoxic stimuli induced by NaN3.Compared with other groups starting dying in 3rd day and dying totally in 5th day.Part of these cardiac myocytes transfected with single plasmid would start to die in the 9th day.Most of them would die in eleven day.The conditioned medium can inhibit apoptosis of cardaic myocytes via increasing phosphorylation of Akt, p38MAPK and ERK1/2.After 24 houres,MI+V group has a stronger SDF-1,p-Akt,p-p38MAPK,p-ERK1/2 and p-Bad expression than MI group. MI+V group had less TUNEL positive cells than MI group(25±2 vs 43±8,P<0.01).The SDF-1 expression in MI+V group was stronger than liver organ.The results of echocardiographic assessment showed that MI+V group has better EF%,FS% and LV mass than MI group(EF%,36.68±5.21 VS 21.1±2.12, p<0.01; LV Mass,102.85±9.90 VS 151.90±9.08,p<0.01; FS%,17.42±2.22 VS 10.05±0.50,p< 0.01).Masson staining result showed that MI+V group has less collagen fibers.
Conclusions The hypoxia-regulated single-plasmid vigilant vector has special features:high-efficiency,vigilant,hypoxia-regulated,cardiac-specific.In addition,SDF-1 can inhibit apoptosis of cardiac myocytes via increasing phosphorylation of Akt,p38MAPK and ERK1/2.
[目的]掌握分离间充质干细胞、心脏干细胞的方法；使用H9C2细胞模拟心肌细胞,研究SDF-1基因治疗后SDF-1在缺氧心肌内的表达时间曲线；研究SDF-1能否抑制缺氧应激引起的H9C2、间充质干细胞、心脏干细胞凋亡；探讨这种抑制作用与p-Bad蛋白去磷酸化为Bad的关系。
[方法]分离、培养MSCs、CSCs。构建pCMV-hSDF-1-GFP质粒,转染到缺氧的H9C2细胞,Real-time PCR、细胞染色验证转染。Western-blot法检测SDF-1的表达时间曲线。在SDF-1表达高峰时间点(转染后3天)收集条件培养基,以缺氧后内源性SDF-1表达高峰培养基为对照培养基。对比两种培养基对MSCs、CSCs迁移的影响。分析两种培养基对H2O2模拟的缺氧应激引起的H9C2、MSCs、CSCs凋亡的影响(细胞活力试验、台盼蓝拒染法实验、细胞形态),并进一步观察SDF-1条件培养基对在这个过程中p-Bad蛋白去磷酸化为Bad蛋白的影响。
[结果]pCMV-hSDF-1-GFP转染缺氧的H9C2细胞后,hSDF-1基因表达是对照组的31倍(p<0.01),转染效率在27.83%±2.61%。SDF-1蛋白在转染后3-7天达到表达高峰,即使14天后SDF-1的表达还较缺氧后内源性SDF-1表达高峰明显。条件培养基较对照培养基促进了MSCs、CSCs的迁移：CSCs34±11 vs.11±3,p < 0.001; MSCs 56±4 vs.10±1,p< 0.001。细胞活力试验：条件培养基组共聚焦显微镜下H9C2、MSCs、CSCs中绿色细胞比例较对照培养基组绿色细胞比例高。台盼蓝拒染法实验：MSCs(条件培养基组84%±7%vs.对照培养基组60%±2%,P<0.01);CSCs(条件培养基组82%±3%vs.对照培养基组62%±3%,P<0.001)；H9C2(条件培养基组89%±3%vs.对照培养基组65%±2%,P<0.001)。形态学上观察H9C2、MSCs、CSCs细胞的区别：对照培养基组可以观察到更多的圆形、光亮的细胞。不同的培养基培养后,Western-blot结果：条件培养基组培养的H9C2、MSCs、CSCs细胞p-Bad的表达较对照组明显,Bad蛋白则相反。细胞染色：在共聚焦显微镜下,条件培养基组培养的H9C2、MSCs、CSCs细胞可以观察到p-Bad的表达。[结论]pCMV-hSDF-1-GFP质粒转染缺氧H9C2细胞后,SDF-1高表达持续超过14天,高峰在3-7天。基因治疗的SDF-1条件培养基可以通过减少p-Bad去磷酸为Bad抑制缺氧引起的H9C2、MSCs、CSCs凋亡。基因治疗的SDF-1条件培养基明显促进了MSCs、CSCs的迁移。
[目的]构建单质粒心肌特异性缺氧调节SDF-1警戒载体,体外、体内试验验证这个载体系统的有效性。探讨SDF-1抑制心肌细胞凋亡的机制。
[方法]构建单、双质粒心肌特异性缺氧调节SDF-1警戒载体,Real-time PCR对比单双质粒载体hSDF-1的表达。分离原代心肌细胞,转染单质粒载体,对比正常氧、低氧下hSDF-1的表达。探寻单质粒载体启动的时间。叠氮钠模拟缺氧应激,单质粒心肌特异性缺氧调节SDF-1警戒载体转染心肌细胞后,观察心肌细胞的生存,提取条件培养基,用AMD3100阻断SDF-1和CXCR4的相互作用,分为Control、AMD3100、AMD3100+条件培养基、条件培养基组,分析SDF-1抑制心肌细胞时是否磷酸化Akt、p-38MAPK、ERK1/2。体内实验验证单质粒警戒载体能否实现：高效、心肌特异性、缺氧警戒,分为Control组、MI组、假手术+V(警戒载体)组、MI+V组,在心肌梗死后1天分析SDF-1在肝脏、假手术+V组心肌、MI+V组心肌的表达,同时Western-blot和免疫组化法分析心肌组织p-Akt、p-ERK1/2/ p-38MAPK、p-Bad的表达,TUNEL实验评价体内心肌细胞的凋亡。7天后小动物心超分析各组心功能各项参数：EF%(左室射血分数)、LV mass(左室壁心肌重量)、FS%(短轴缩短率)等,通过Masson染色评价心肌纤维化。
[结果]成功构建单质粒心肌特异性缺氧调节SDF-1警戒载体,该载体在缺氧条件下hSDF-1基因表达是双质粒载体的2.5倍(p<0.01)。单质粒SDF-1载体在低氧后30分钟,hSDF-1基因开始表达上调(是正常氧浓度下hSDF-1基因的1.5倍),缺氧24小时后对比非缺氧条件上升8.6倍(p<0.01)。单质粒载体转染原代心肌细胞效率在55%±7%；原代心肌细胞分为Normal组,Model组(叠氮钠),SDF组,SDF+叠氮钠组：Normal、Model、SDF组原代心肌细胞在第3天开始出现部分心肌细胞死亡,第5天心肌细胞完全死亡；在SDF+叠氮钠组观察到原代心肌细胞在第9天才开始出现部分心肌细胞死亡,第11天培养的原代心肌细胞完全死亡。对比Control、AMD3100、AMD3100+条件培养基组,SDF-1条件培养基组明显增加了p-Akt、p-ERK1/2、p-38MAPK的表达。体内试验：单质粒载体治疗组心肌组织的SDF-1较非治疗组表达明显,也较治疗组肝脏组织的SDF-1表达明显；Western-blot、免疫组化染色的实验结果表明治疗组p-Akt、p-ERK1/2、p-38MAPK、p-Bad都较未治疗组高表达；单质粒载体治疗组心肌组织中TUNEL阳性细胞数25±2较非治疗组43±8明显少,P<0.01。7天后,心超测定心功能：MI+V组心功能较MI组明显改善,MI+V组和MI组：EF%,36.68±5.21 VS 21.1±2.12,p<0.01;LV Mass,102.85±9.90VS 151.90±9.08,p<0.01；FS%,17.42±2.22 VS 10.05±0.50,p<0.01。Masson染色结果表明：单质粒载体治疗组纤维化面积较未治疗组缩小。
[结论]本课题构建的单质粒心肌特异性缺氧调节SDF-1警戒载体具备高效、缺氧警戒、心肌特异性的特点,体外体内实验都证明这个单质粒警戒载体的有效性,是一种良好的基因工具。SDF-1通过促进Akt、ERK1/2、p38MAPK的磷酸化、减少p-Bad蛋白的去磷酸化抑制缺氧刺激引起的心肌细胞凋亡。
Stromal cell-derived factor 1 a (SDF-1a) is a homing factor that can cause stem cells to migrate from the bone marrow,which can react with its main receptor CXCR4.It can inhibit cell apoptosis,too.Although endogenous SDF-1a is secreted in the early phase of infarction in ischemic myocardium, the expression of myocardial SDF-1 is not effective for heart repair. SDF-1 has to be expressed in larger quantities and for a longer time.We try to build one single-plasmid vigilant vector.This vigilant vector has some features:high efficiency, hypoxia-regulated, vigilant,cardiac-specific.We evalute this vigilant system in cardiac myocytes. Furtherly, we evalute this vigilant system in mouse model of myocardial infarction.Another aim of this reserch is to evalute anti-apoptosis of SDF-1 and to find the inner mechanism.
Objective SDF-1 has to be expressed in larger quantities and for a longer time for the treatment of myocardial infarction. A gene therapy approach using an SDF-1 a vector can increase SDF-1 expression and mobilize and direct stem cells to the ischemic myocardium. SDF-1 can serve as a heart-protective factor through its ability to activate phosphorylated-AKT and ERK1/2. The role of the Bcl-2-associated death (BAD) and phosphorylated-BAD (p-BAD) in this process, however, has not been determined. In addition,the detailed time course of a SDF-1 a vector adminastration remains unclear.
Methods Contrust the p-CMV-hSDF-1 plasmids.H9C2 cells were transfected with the p-CMV-hSDF-1 plasmid under hypoxic stimuli. Real-time PCR and cell staining was used to confirm the transfection under hypoxic condition.Western blot analysis was used to determine the SDF-1 expression time course.In the SDF-1 peak expression,the conditioned medium was collected for further experiment.The SDF-1 conditioned medium in endogenous peak expression would be considered as control medium.Isolate mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs).Under conditioned medium or control medium,MSCs and CSCs would be migrated. H2O2 was used to induce H9C2,MSCs and CSCs apoptosis.The apoptosis of H9C2,MSCs and CSCs was evaluated with conditioned medium or control medium.Western-blot,cell staining was to confirm reduction of dephosphorylation of p-Bad.
Results The transfection efficiency was approximately 27.83%±2.61%. H9C2 cells transfected with the pCMV-hSDF-1 plasmid expressed 31-fold more h-SDF-1 than the control group (P=0.003563).Western blot analysis revealed that SDF-1 expression peaked from the third day to the 7th day after pCMV-hSDF-1 plasmid transfection. After treatment with conditioned medium, which was gathered on the third day, H9C2 cells, mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs) were more resistant to hypoxic insult than control groups, based on the results of the Live/Dead cell viability test, morphological observation and trypan blue uptake test. Live/Dead cell viability test:in confocol laser scan,there were more green H9C2,MSCs,and CSCs cells in conditioned medium treatment groups than in control medium groups. Morphological observation:there were more round,bright H9C2,MSCs and CSCs in control groups than in conditioned medium. Trypan blue uptake test: MSCs (conditioned medium 84%±7% vs. control medium60%±2%, P< 0.01); CSCs (conditioned medium 82%±3% vs. control medium 62%±3%, P< 0.001); H9C2 (conditioned medium 89%±3% vs. control medium 65%±2%, P< 0.001).Both western blot and staining results confirmed the dephosphorylation reduction of p-BAD in H9C2s, MSCs and CSCs after being treated with SDF-1 conditioned medium.The conditioned medium made more MSCs migrate compared with control medium(56±4 vs.10±1, p< 0.001).Likewise, the conditioned medium made more CSCs migrate compared with control medium(34±11 vs.11±3, p< 0.001).
Conclusions These findings indicate that, under hypoxic stimulation after SDF-1 gene delivery, the expression of SDF-1 peaks from the third day to the 7th and lasts longer than fourteen days. As a result, more MSCs and CSCs migrate into the hypoxic myocardium. In addition, treatment with SDF-1 conditioned medium causes H9C2s, MSCs and CSCs to become more resistant to hypoxic stimuli and to reduce dephosphorylation of the p-BAD proteins.
Objective To construct one single-plasmid vigilant vector,which has special features:high-efficiency,vigilant,hypoxia-regulated,cardiac-specific.This system can meet the requirement of therapy in myocardial infarction.These features would be assessed in vitro and in vivo experiments.Another aim is to prove that SDF-1 can be anti-apoptosis in cardiac myocytes.The inner mechanism would be assessed.
Methods After constructing the single-plasmid vigilant vector,this vigilant plasmid would be compared with double-plasmids vigilant vector under hypoxic stimuli.In addition,the on-set time of this single-plasmid vector would be evaluated. Furthermore,the hypoxia-regulated feature was appraised. Isolate and culture cardiac myocytes.The cardaic myocytes was transfected with single-vigilant plasmid. NaN3 was used to stimulate hypoxia insult. Cell apoptosis or death could be observed from cell morphology. Collect the used medium in the 5th day as conditioned medium.The cardiac myocytes would be divided into four groups:Control,AMD3100,AMD3100 with conditioned medium,conditioned medium.Phosphorylation of ERK1/2,p-38MAPK and Akt were detected by Westen blot. Then this single-plasmid was injected into myocardium in left ventricular apical.The animal experiment would be divided into four groups:MI,Sham+V,MI+V,Control.After 3 days,the left anterior descending coronary artery (LAD) was ligated as myocardial infarction model. 24 houres later,the SDF-1, p-ERK1/2, p-p38MAPK and p-Akt expression of MI or MI+V groups were detected by Western-blot and immunohistochemistry.
TUNEL test was performed to assess cardiac myocytes apoptosis in vivo.7 days later, the echocardiographic assessment(EF%,LV mass and FS%) of myocardial infarction and heart function was performed.Masson staining was performed to identify collagen fibers.
Results Under hypoxic stimuli,single-plasmid vigilant vector can express more 2.5 folds hSDF-1 gene than double-plasmids vigilant vector(p< 0.01).After 30 mins of hypoxia stimuli,SDF-1gene can be on-set.Compared with nomoxia,hypoxia stimuli can make more 8.6 foldes hSDF-1 be expressed in cardiac myocytes transfected with single vigilant vector.The transfection efficiency is about 55%±7%.The cardiac myocytes transfected with single-plasmid vigilant vector can be more resistant to hypoxic stimuli induced by NaN3.Compared with other groups starting dying in 3rd day and dying totally in 5th day.Part of these cardiac myocytes transfected with single plasmid would start to die in the 9th day.Most of them would die in eleven day.The conditioned medium can inhibit apoptosis of cardaic myocytes via increasing phosphorylation of Akt, p38MAPK and ERK1/2.After 24 houres,MI+V group has a stronger SDF-1,p-Akt,p-p38MAPK,p-ERK1/2 and p-Bad expression than MI group. MI+V group had less TUNEL positive cells than MI group(25±2 vs 43±8,P<0.01).The SDF-1 expression in MI+V group was stronger than liver organ.The results of echocardiographic assessment showed that MI+V group has better EF%,FS% and LV mass than MI group(EF%,36.68±5.21 VS 21.1±2.12, p<0.01; LV Mass,102.85±9.90 VS 151.90±9.08,p<0.01; FS%,17.42±2.22 VS 10.05±0.50,p< 0.01).Masson staining result showed that MI+V group has less collagen fibers.
Conclusions The hypoxia-regulated single-plasmid vigilant vector has special features:high-efficiency,vigilant,hypoxia-regulated,cardiac-specific.In addition,SDF-1 can inhibit apoptosis of cardiac myocytes via increasing phosphorylation of Akt,p38MAPK and ERK1/2.
引文
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