GATA-4基因转染促进骨髓间充质干细胞心肌分化和血管形成的研究及机制探讨
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摘要
第一部分MSC的分离培养及GATA-4基因的转染
目的：基因修饰骨髓间充质干细胞(MSCs)是增加细胞抗缺氧和心肌分化的新方向。分离、培养和纯化大鼠MSC，以逆转录病毒为载体将GATA-4基因转染MSC，建立稳定的GATA-4基因修饰的MSC细胞模型。
方法：从雄性大鼠骨髓中分离MSC，培养至第3代MSC，经pMSCV逆转录病毒表达系统转染GATA-4基因(MSCGATA-4)，对照组为仅表达GFP的空质粒组（MSCNull）。通过实时定量聚合酶链反应(Real-time quantitative polymerase chainreaction, Real-time PCR)、Western blot印迹和免疫组化的方法鉴定GATA-4基因转染的效果。
结果：MSCGATA-4组GATA-4mRNA和蛋白表达明显高于对照组MSCNull，P<0.05。Real-time PCR数据显示，MSCGATA-4组GATA-4mRNA的表达是MSCNull组的258倍。通过免疫组化检测发现，虽然MSCGATA-4组和MSCNull组的GFP都显示阳性，但只有MSCGATA-4组细胞核呈GATA-4阳性表达。
结论：本研究成功建立了GATA-4基因修饰MSC的培养系统，为进一步探讨GATA-4在MSC的心肌分化及血管新生中的作用奠定了基础。
第二部分GATA-4基因转染促进MSC向心肌分化的研究及机制探讨
目的：GATA-4是心肌转录因子，在心肌分化中起着重要的作用。我们从体外研究了GATA-4转染MSC是否促进其分化为心肌样细胞。
方法：
（1）分离和培养新生大鼠心室肌细胞（CM）。并通过光镜、透射电镜和免疫组化鉴定心肌细胞；
（2）MSCGATA-4与CM共培养1周，通过Real-time PCR和Western blot检测其心肌基因BNP、α-actinin和Islet-1的表达情况。
（3）通过免疫荧光α-actinin染色法检测MSCGATA-4组和MSCNull组的心肌分化情况。
（4）通过膜片钳技术检测MSCGATA-4组和MSCNull组细胞的电生理特点：分别记录内向整流性钾流（Ik1）、瞬时外向性钾流（Ito）、延迟整流性钾流（IKDR）、L-型钙离子流（ICa-L）、TTX敏感的钠离子流（INa., TTX）和动作电位（AP）的表达，
（5）通过流式细胞仪器检测MSCGATA-4组和MSCNull组心肌分化率的情况。
（6）用Microarray、Real-time PCR和Western blot检测MSCGATA-4组和MSCNull组胰岛素样生长因子结合蛋白（Insulin-Like Growth Factor Binding Protein, IGFBP-4）的表达。
（7）为了研究IGFBP-4对GATA-4调节MSC的心肌分化作用，我们进行了MSCGATA-4组的IGFBP-4基因敲除，以scrambled siRNA作为阴性对照组。并用Real-time PCR和Western blot检测IGFBP-4基因敲除效果。
（8）观察敲除IGFBP-4基因后，GATA-4对MSC心肌分化的作用。
结果：
（1）MSCGATA-4组的心肌基因BNP、α-actinin和Islet-1的表达明显高于对照组（转染空质粒组MSCNull）(P<0.05)。
（2）MSCGATA-4组记录到表达高水平的Ik1、Ito、IKDR、ICa-L和INa., TTX。与MSCNull组相比较，它们的电流密度明显增加，P<0.05。
（3）MSCGATA-4与CM和共培养时，部分细胞记录到低幅度的动作电位（AP）；而MSCNull组没有记录到AP。
（4）共培养7天后，部分GFP+的MSC细胞表达α-actinin。MSCGATA-4组（34％±4％）的阳性率与MSCNull组（15％±2％）相比较，明显增加，P<0.05。
（5）通过Microarray、Real-time PCR和Western blot检测，IGFBP-4表达在MSCGATA-4组较MSCNull组明显增加，P<0.05。
（6）经siRNA敲除IGFBP-4基因后，定量PCR显示IGFBP-4-siRNA-MSCGATA-4中IGFBP-4基因的表达仅是MSCGATA-4组的38%(P<0.05)，Western blot显示IGFBP-4-siRNA-MSCGATA-4细胞中IGFBP-4蛋白表达明显减少，只有MSCGATA-4组的28%(P<0.05)。
（7）IGFBP-4基因敲除后，IGFBP-4-siRNA-MSCGATA-4细胞中心肌基因(BNP、α-actinin和Islet-1)的表达，与MSCGATA-4组相比较，显著减低，P<0.05。
（8）通过流式细胞仪检测各组MSC的心肌分化率。结果显示，MSCGATA-4组的心肌分化率(34.09±4.65%)明显比MSCNull组心肌分化率(14.54±1.62%)增高，P<0.05。而IGFBP-4基因沉默以后，沉默组（IGFBP-4-siRNA-MSCGATA-4）的心肌分化率(16.87±1.98%)明显比MSCGATA-4组低（P<0.05）。
（9）功能研究表明当敲除IGFBP-4基因时，MSCGATA-4组分化潜能被降低。
结论：GATA-4过表达可以促进MSC分化为心肌样细胞，其机制可能与IGFBP-4的表达上调有关。
第三部分GATA-4基因转染促进MSC新生血管形成的体外和体内研究
目的：干细胞移植入受体心脏后，可以释放可溶性因子，增加心肌修复和血管再生。我们假设GATA-4过表达于MSC中可以通过增加其旁分泌作用，促进血管新生和细胞存活，从而改善心肌梗死后的心肌再生。
方法：
（1）用ELISA方法检测不同组细胞（MSCNull和MSCGATA-4组）上清中生长因子VEGF、IGF-1和b-FGF的表达。
（2）用定量PCR方法检测不同组细胞中VEGF、IGF-1和b-FGF基因的表达情况。
（3）为了检测细胞在缺氧条件下抵抗作用，细胞分别被缺氧不同时间24、48和72小时，用MTT观察细胞的增殖情况。
（4）用绿色荧光的PKH67标记人脐静脉内皮细胞（HUVEC）；用不同组MSC细胞培养上清刺激HUVEC，体外观察不同组的毛细血管样结构的形成。同时加入VEGF或和IGF-1的中和抗体，孵育12小时，观察对毛细血管样结构形成的影响。
（5）把HUVEC细胞球体接种到3D立体胶培养系统，加入细胞培养上清，同时加VEGF或和IGF-1的中和抗体作用24小时，观察对球体出芽的数量和总长度的影响。
（6）用结扎冠状动脉左前降支的方法建立雌性大鼠的急性心肌梗死模型，各组MSC被注射到梗死周边区域的心肌内。
（7）用定量PCR检测雌性大鼠中雄性Sry基因的表达，检测缺血心肌中MSC的存活。
（8）用超声心动图评估心肌功能，同时检测新生血管的形成及梗死面积等。
结果：
（1）定量PCR的结果显示，MSCGATA-4组中VEGF和IGF-1基因的表达明显比MSCNull组表达高，P<0.05。
（2）ELISA结果显示，在MSCGATA-4上清中VEGF和IGF-1的分泌明显比MSCNull上清高，P<0.05。b-FGF的分泌没有显著性差异，P＞0.05。
（3）细胞缺氧48和72小时时，MTT细胞增殖明显减小。而GATA-4过度表达可以部分预防MTT的减小。
（4）用MSCGATA-4组上清处理的HUVEC，与对照组MSCNull相比较，显著增加了血管样结构的形成和HUVEC的迁移（通过每个球体出芽的数量和出芽的总长度来评估），P<0.05。将中和抗体VEGF、IGF-1分别或同时加入到MSCGATA-4组中，可以明显减少MSCGATA-4组新生毛细血管的形成，与不加抗体的MSCGATA-4组相比较，P<0.05。
（5）通过Sry基因的检测，在MSCGATA-4治疗组的梗死及梗死周边区，Sry基因的表达明显增高；与MSCNull组或MSCbas组相比较，有显著性的差异，P<0.05。可见过度表达GATA-4可以明显增加MSC在缺血心肌的存活率。
（6）通过心脏超声检查，用MSCGATA-4移植治疗的动物组心功能明显改善；与MSCNull组或MSCbas组相比较，有显著性的差异，P<0.05。
（7）通过组织化学染色可见，MSCGATA-4组血管密度增加，心梗面积降低；与MSCNull组或MSCbas组相比较，有显著性的差异，P<0.05。
结论：GATA-4过表达于MSC，可以增加缺血心肌的MSC存活和新生血管的形成，是心肌梗死的一个有效的治疗方法。
Part ⅠIsolation and Culture of Mesenchymal Stem Cells from Rat BoneMarrow and Transduction of GATA-4Gene
Objective: Gene modified mesenchymal stem cells (MSCs) is a new direction toincrease cell resistance to hypoxia and increase myocardial transdifferentiation. Isolation,culture and purification of mesenchymal stem cells from rat bone marrow. Establishmentof stable cell model of GATA-4gene modified MSCs through retroviral transduction ofGATA-4gene into MSC.
Methods: MSCs were isolated and cultured from male rat bone marrow. GATA-4(MSCGATA-4) was overexpressed in MSC by using a murine stem cell virus (pMSCV)retroviral expression system at the third passage. Control cells were infected with emptyvector (MSCNull). Gene and protein expression of GATA-4in MSCGATA-4were analyzedusing real-time quantitative polymerase chain reaction (real-time PCR), Western blottingand immunofluorescence.
Results: Compared with control cells (MSCNull), mRNA and protein level ofGATA-4were enhanced in MSCGATA-4, as shown by real-time PCR and Western blot,P<0.05. Real-time PCR data indicated that the mRNA expression level of GATA-4was258-fold higher in MSCGATA-4vs. MSCNull. Whereas both MSCGATA-4and MSCNullwereGFP immunopositive, only MSCGATA-4stained intensely for GATA-4immunofluorescence.
Conclusions: Retroviral-mediated transduction and expression of bicistronicGATA-4/GFP construct were evaluated by the methods of immunostaining, real-time PCR,and Western blotting, as a basis for further exploration of GATA-4in regulating MSCsmyocardial transdifferentiation and angiogenesis.
Part IITransduction of GATA-4Promotes Myocardial Transdifferentiationof Mesenchymal Stem Cells and Its Related Mechanism
Objective: GATA-4is a cardiac transcription factor and plays an important role incell lineage differentiation during development. We investigated whether retroviraltransduction of GATA-4increased adult mesenchymal stromal cell (MSC)transdifferentiation into a cardiac phenotype in vitro.
Methods：
（1）Native cardiomyocytes (CM) were isolated from ventricles of neonatal rats.Cardiomyocytes were indentified through the methods of light-electricity microscopy,transmission electron microscopy and immunocytochemistry.
（2）The expression of cardiac genes, including brain natriuretic peptide (BNP),Islet-1and α-sarcomeric actinin (α-SA), was detected by real-time PCR and Western blotafter MSC were co-cultured with native CM for7days.
（3）Myocardial transdifferentiation of MSCGATA-4and MSCNullwere determined byα-sarcomeric actinin immunostaining.
（4）Electrophysiological properties of ion channels were assessed in MSC usingpatch-clamp technology. TTX-sensitive Na+current (INa.TTX), L-type calcium current (ICa.L),transient outward K+current (Ito), delayed rectifier K+current (IKDR), the inwardlyrectifying K+current (IK1) and action potential (AP) were recorded in MSCGATA-4andMSCNullafter MSC were co-cultured with native CM.
（5）The transdifferentiation rate was calculated directly from flow cytometry in MSCGATA-4and MSCNull.
（6）The gene expression level of insulin-like growth factor binding protein-4(IGFBP-4）in MSCGATA-4and MSCNullwere analyzed using real-time PCR and Westernblotting and Microarray.
（7）To study the role of IGFBP-4in GATA-4-mediated myocardialtransdifferentiation, IGFBP-4activity was knocked down in MSCGATA-4by siRNA genesilencing. Non-silencing, scrambled siRNA was used as a negative control. IGFBP-4geneexpression was analyzed using real-time PCR and Western blotting.
（8）Myocardial transdifferentiation rate of MSCGATA-4after knockdown of IGFBP-4was observed.
Results:
(1) The expression of cardiac genes, including brain natriuretic peptide (BNP),Islet-1and α-sarcomeric actinin (α-SA), was up-regulated in MSCGATA-4compared withcontrol cells that were transfected with Green Fluorescent Protein (GFP) only (MSCNull).
(2) MSCGATA-4exhibited higher levels of INa.TTX, ICa.L, Ito, IKDRand IK1channelactivities reflective of electrophysiological characteristics of CM.
(3) Low amplitude of action potential was recorded in MSCGATA-4after MSC wereco-cultured. No action potential was recorded in MSCNull.
(4) Some GFP+cells were positive for α-actinin staining after MSC were co-culturedwith native CM for7days. The transdifferentiation rate was significantly higher inMSCGATA-4(34%±4%), compared with MSCNull(15%±2%), P<0.05.
(5) At the same time, IGFBP-4was significantly up-regulated in MSCGATA-4, asshown by real-time PCR and Western blot and microarray, compared with MSCNull,P<0.05.
（6）After knocking down IGFBP-4in MSCGATA-4, real-time PCR showed that theexpression level of IGFBP-4in IGFBP-4-siRNA-MSCGATA-4(IG4-siRNA) was only38%of that in MSCGATA-4(P<0.05). IGFBP-4protein was reduced in IG4-siRNA cells by28%compared with MSCGATA-4(P<0.05).
(7) The effect of GATA-4on expressing cardiac markers (BNP、α-actinin和Islet-1) inMSC was suppressed when IGFBP-4-siRNA was transfected in MSCGATA-4, compared withMSCGATA-4, P<0.05.
(8) The transdifferentiation of various MSC was quantified using fluorescencea-activated cell sorting (FACS). The transdifferentiation rate was higher in MSCGATA-4(34.09±4.65%) than in MSCNull(14.54±1.62%; P<0.05). However, after IGFBP-4was knockeddown in the MSCGATA-4, the transdifferentiation rate was lower in IGFBP-4-siRNA (16.87±1.98%) than in MSCGATA-4(P<0.05).
（9）Functional studies indicated that the differentiation potential of MSCGATA-4wasdecreased by knockdown of IGFBP-4.
Conclusions: Retroviral transduction of GATA-4significantly increases MSCdifferentiation into a myocardial phenotype, which might be associated with theup-regulation of IGFBP-4.
Part ⅢTransduction of GATA-4Improved in vivo and in vitroAngiogenesis of Mesenchymal Stem Cells and Its RelatedMechanism
Objective: Transplanted mesenchymal stem cells (MSC) release soluble factors thatcontribute to cardiac repair and vascular regeneration. We hypothesized that retroviraltransduction of GATA-4enhances the MSC secretome, thereby increasing angiogenesisand cell survival and promoting postinfarction cardiac angiogenesis.
Methods：
(1) The concentration of VEGF, IGF-1, and basic FGF in conditioned medium (CdM)of MSC were measured using ELISA.
(2) The gene expression levels of VEGF, IGF-1, and basic FGF in transduced MSC were detected using quantitative real-time PCR.
(3) To study the resistance of MSC to oxidative stress, MSCs were exposed tohypoxia for24h,48h,72h and (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) intake was assessed.
(4) Human umbilical vein endothelial cells (HUVECs）were labeled with PKH67celltracker dye using PKH67Green Fluorescent Cell Linker kit. Conditioned medium wereadded into HUVECs and capillary-like structure formation was assessed. IGF-1-and/orVEGF neutralizing antibodies were added to CdMGATA-4. After12h of incubation,capillary-like structure formation was assessed.
(5) Spheroids of HUVEC were then embedded in3D Collagen Cell Culture Systemfor24h in the presence or absence of CdM. IGF-1-and/or VEGF neutralizing antibodieswere added to CdMGATA-4. The number of sprouts and cumulative sprout length in eachspheroid were detected and calculated.
(6) MSCs were injected into the peri-infarct region in an acute myocardial infarctionmodel in Sprague-Dawley rats developed by ligation of the left anterior descendingcoronary artery.
(7) Survival of male donor MSCs that expressed Sry gene in the female recipienthearts was assessed using real-time PCR. MSC survival in ischemic myocardium wasassayed.
(8) Cardiac function was assessed by echocardiography. Infarction size andangiogenesis were detected.
Results:
(1) Expression of IGF-1and VEGF-A in MSC was significantly upregulated inMSCGATA-4compared with MSCNull, P<0.05.
(2) levels of IGF-1and VEGF-A in CdM were assessed by ELISA and shown to besignificantly increased in CdMGATA-4, P<0.05, but b-FGF was unchanged, P＞0.05.
(3) MTT intake was significantly reduced when MSCs were exposed to hypoxia for48or72h. Overexpression of GATA-4partially prevented this reduction.
(4) HUVEC treated with MSCGATA-4conditioned medium (CdMGATA-4) exhibitedincreased formation of capillary-like structures and promoted migration (HUVECmigration was investigated by measuring both the number of sprouts per spheroid andcumulative sprout length), compared with HUVECs treated with MSCNullconditionedmedium, P<0.05. This CdMGATA-4-stimulated increase in formation of capillary-likestructures and migration were diminished by treatment with neutralizing antibodies againstIGF-1, VEGF, or both, compared with HUVECs treated with only MSCGATA-4conditionedmedium, P<0.05.
(5) Sry gene in peri-infarcted and infarcted myocardium was significantly higher inMSCGATA-4-transplanted hearts, compared with MSCNullor MSCbas, P<0.05.Overexpression of GATA-4significantly increased MSC survival in ischemic myocardium.
(6) MSCGATA-4-treated animals showed significantly improved cardiac function asassessed by echocardiography, compared with MSCNullor MSCbas-treated animals, P<0.05.
(7) Histological studies revealed increased blood vessel density and reducedinfarction size in MSCGATA-4-treated animals, compared with MSCNullor MSCbas-treatedanimals, P<0.05.
Conclusions: Transduction of GATA-4in MSCs increased both MSC survival andangiogenic potential in ischemic myocardium and may therefore represent a novel andefficient therapeutic approach for postinfarct remodeling.
目的：基因修饰骨髓间充质干细胞(MSCs)是增加细胞抗缺氧和心肌分化的新方向。分离、培养和纯化大鼠MSC，以逆转录病毒为载体将GATA-4基因转染MSC，建立稳定的GATA-4基因修饰的MSC细胞模型。
方法：从雄性大鼠骨髓中分离MSC，培养至第3代MSC，经pMSCV逆转录病毒表达系统转染GATA-4基因(MSCGATA-4)，对照组为仅表达GFP的空质粒组（MSCNull）。通过实时定量聚合酶链反应(Real-time quantitative polymerase chainreaction, Real-time PCR)、Western blot印迹和免疫组化的方法鉴定GATA-4基因转染的效果。
结果：MSCGATA-4组GATA-4mRNA和蛋白表达明显高于对照组MSCNull，P<0.05。Real-time PCR数据显示，MSCGATA-4组GATA-4mRNA的表达是MSCNull组的258倍。通过免疫组化检测发现，虽然MSCGATA-4组和MSCNull组的GFP都显示阳性，但只有MSCGATA-4组细胞核呈GATA-4阳性表达。
结论：本研究成功建立了GATA-4基因修饰MSC的培养系统，为进一步探讨GATA-4在MSC的心肌分化及血管新生中的作用奠定了基础。
第二部分GATA-4基因转染促进MSC向心肌分化的研究及机制探讨
目的：GATA-4是心肌转录因子，在心肌分化中起着重要的作用。我们从体外研究了GATA-4转染MSC是否促进其分化为心肌样细胞。
方法：
（1）分离和培养新生大鼠心室肌细胞（CM）。并通过光镜、透射电镜和免疫组化鉴定心肌细胞；
（2）MSCGATA-4与CM共培养1周，通过Real-time PCR和Western blot检测其心肌基因BNP、α-actinin和Islet-1的表达情况。
（3）通过免疫荧光α-actinin染色法检测MSCGATA-4组和MSCNull组的心肌分化情况。
（4）通过膜片钳技术检测MSCGATA-4组和MSCNull组细胞的电生理特点：分别记录内向整流性钾流（Ik1）、瞬时外向性钾流（Ito）、延迟整流性钾流（IKDR）、L-型钙离子流（ICa-L）、TTX敏感的钠离子流（INa., TTX）和动作电位（AP）的表达，
（5）通过流式细胞仪器检测MSCGATA-4组和MSCNull组心肌分化率的情况。
（6）用Microarray、Real-time PCR和Western blot检测MSCGATA-4组和MSCNull组胰岛素样生长因子结合蛋白（Insulin-Like Growth Factor Binding Protein, IGFBP-4）的表达。
（7）为了研究IGFBP-4对GATA-4调节MSC的心肌分化作用，我们进行了MSCGATA-4组的IGFBP-4基因敲除，以scrambled siRNA作为阴性对照组。并用Real-time PCR和Western blot检测IGFBP-4基因敲除效果。
（8）观察敲除IGFBP-4基因后，GATA-4对MSC心肌分化的作用。
结果：
（1）MSCGATA-4组的心肌基因BNP、α-actinin和Islet-1的表达明显高于对照组（转染空质粒组MSCNull）(P<0.05)。
（2）MSCGATA-4组记录到表达高水平的Ik1、Ito、IKDR、ICa-L和INa., TTX。与MSCNull组相比较，它们的电流密度明显增加，P<0.05。
（3）MSCGATA-4与CM和共培养时，部分细胞记录到低幅度的动作电位（AP）；而MSCNull组没有记录到AP。
（4）共培养7天后，部分GFP+的MSC细胞表达α-actinin。MSCGATA-4组（34％±4％）的阳性率与MSCNull组（15％±2％）相比较，明显增加，P<0.05。
（5）通过Microarray、Real-time PCR和Western blot检测，IGFBP-4表达在MSCGATA-4组较MSCNull组明显增加，P<0.05。
（6）经siRNA敲除IGFBP-4基因后，定量PCR显示IGFBP-4-siRNA-MSCGATA-4中IGFBP-4基因的表达仅是MSCGATA-4组的38%(P<0.05)，Western blot显示IGFBP-4-siRNA-MSCGATA-4细胞中IGFBP-4蛋白表达明显减少，只有MSCGATA-4组的28%(P<0.05)。
（7）IGFBP-4基因敲除后，IGFBP-4-siRNA-MSCGATA-4细胞中心肌基因(BNP、α-actinin和Islet-1)的表达，与MSCGATA-4组相比较，显著减低，P<0.05。
（8）通过流式细胞仪检测各组MSC的心肌分化率。结果显示，MSCGATA-4组的心肌分化率(34.09±4.65%)明显比MSCNull组心肌分化率(14.54±1.62%)增高，P<0.05。而IGFBP-4基因沉默以后，沉默组（IGFBP-4-siRNA-MSCGATA-4）的心肌分化率(16.87±1.98%)明显比MSCGATA-4组低（P<0.05）。
（9）功能研究表明当敲除IGFBP-4基因时，MSCGATA-4组分化潜能被降低。
结论：GATA-4过表达可以促进MSC分化为心肌样细胞，其机制可能与IGFBP-4的表达上调有关。
第三部分GATA-4基因转染促进MSC新生血管形成的体外和体内研究
目的：干细胞移植入受体心脏后，可以释放可溶性因子，增加心肌修复和血管再生。我们假设GATA-4过表达于MSC中可以通过增加其旁分泌作用，促进血管新生和细胞存活，从而改善心肌梗死后的心肌再生。
方法：
（1）用ELISA方法检测不同组细胞（MSCNull和MSCGATA-4组）上清中生长因子VEGF、IGF-1和b-FGF的表达。
（2）用定量PCR方法检测不同组细胞中VEGF、IGF-1和b-FGF基因的表达情况。
（3）为了检测细胞在缺氧条件下抵抗作用，细胞分别被缺氧不同时间24、48和72小时，用MTT观察细胞的增殖情况。
（4）用绿色荧光的PKH67标记人脐静脉内皮细胞（HUVEC）；用不同组MSC细胞培养上清刺激HUVEC，体外观察不同组的毛细血管样结构的形成。同时加入VEGF或和IGF-1的中和抗体，孵育12小时，观察对毛细血管样结构形成的影响。
（5）把HUVEC细胞球体接种到3D立体胶培养系统，加入细胞培养上清，同时加VEGF或和IGF-1的中和抗体作用24小时，观察对球体出芽的数量和总长度的影响。
（6）用结扎冠状动脉左前降支的方法建立雌性大鼠的急性心肌梗死模型，各组MSC被注射到梗死周边区域的心肌内。
（7）用定量PCR检测雌性大鼠中雄性Sry基因的表达，检测缺血心肌中MSC的存活。
（8）用超声心动图评估心肌功能，同时检测新生血管的形成及梗死面积等。
结果：
（1）定量PCR的结果显示，MSCGATA-4组中VEGF和IGF-1基因的表达明显比MSCNull组表达高，P<0.05。
（2）ELISA结果显示，在MSCGATA-4上清中VEGF和IGF-1的分泌明显比MSCNull上清高，P<0.05。b-FGF的分泌没有显著性差异，P＞0.05。
（3）细胞缺氧48和72小时时，MTT细胞增殖明显减小。而GATA-4过度表达可以部分预防MTT的减小。
（4）用MSCGATA-4组上清处理的HUVEC，与对照组MSCNull相比较，显著增加了血管样结构的形成和HUVEC的迁移（通过每个球体出芽的数量和出芽的总长度来评估），P<0.05。将中和抗体VEGF、IGF-1分别或同时加入到MSCGATA-4组中，可以明显减少MSCGATA-4组新生毛细血管的形成，与不加抗体的MSCGATA-4组相比较，P<0.05。
（5）通过Sry基因的检测，在MSCGATA-4治疗组的梗死及梗死周边区，Sry基因的表达明显增高；与MSCNull组或MSCbas组相比较，有显著性的差异，P<0.05。可见过度表达GATA-4可以明显增加MSC在缺血心肌的存活率。
（6）通过心脏超声检查，用MSCGATA-4移植治疗的动物组心功能明显改善；与MSCNull组或MSCbas组相比较，有显著性的差异，P<0.05。
（7）通过组织化学染色可见，MSCGATA-4组血管密度增加，心梗面积降低；与MSCNull组或MSCbas组相比较，有显著性的差异，P<0.05。
结论：GATA-4过表达于MSC，可以增加缺血心肌的MSC存活和新生血管的形成，是心肌梗死的一个有效的治疗方法。
Part ⅠIsolation and Culture of Mesenchymal Stem Cells from Rat BoneMarrow and Transduction of GATA-4Gene
Objective: Gene modified mesenchymal stem cells (MSCs) is a new direction toincrease cell resistance to hypoxia and increase myocardial transdifferentiation. Isolation,culture and purification of mesenchymal stem cells from rat bone marrow. Establishmentof stable cell model of GATA-4gene modified MSCs through retroviral transduction ofGATA-4gene into MSC.
Methods: MSCs were isolated and cultured from male rat bone marrow. GATA-4(MSCGATA-4) was overexpressed in MSC by using a murine stem cell virus (pMSCV)retroviral expression system at the third passage. Control cells were infected with emptyvector (MSCNull). Gene and protein expression of GATA-4in MSCGATA-4were analyzedusing real-time quantitative polymerase chain reaction (real-time PCR), Western blottingand immunofluorescence.
Results: Compared with control cells (MSCNull), mRNA and protein level ofGATA-4were enhanced in MSCGATA-4, as shown by real-time PCR and Western blot,P<0.05. Real-time PCR data indicated that the mRNA expression level of GATA-4was258-fold higher in MSCGATA-4vs. MSCNull. Whereas both MSCGATA-4and MSCNullwereGFP immunopositive, only MSCGATA-4stained intensely for GATA-4immunofluorescence.
Conclusions: Retroviral-mediated transduction and expression of bicistronicGATA-4/GFP construct were evaluated by the methods of immunostaining, real-time PCR,and Western blotting, as a basis for further exploration of GATA-4in regulating MSCsmyocardial transdifferentiation and angiogenesis.
Part IITransduction of GATA-4Promotes Myocardial Transdifferentiationof Mesenchymal Stem Cells and Its Related Mechanism
Objective: GATA-4is a cardiac transcription factor and plays an important role incell lineage differentiation during development. We investigated whether retroviraltransduction of GATA-4increased adult mesenchymal stromal cell (MSC)transdifferentiation into a cardiac phenotype in vitro.
Methods：
（1）Native cardiomyocytes (CM) were isolated from ventricles of neonatal rats.Cardiomyocytes were indentified through the methods of light-electricity microscopy,transmission electron microscopy and immunocytochemistry.
（2）The expression of cardiac genes, including brain natriuretic peptide (BNP),Islet-1and α-sarcomeric actinin (α-SA), was detected by real-time PCR and Western blotafter MSC were co-cultured with native CM for7days.
（3）Myocardial transdifferentiation of MSCGATA-4and MSCNullwere determined byα-sarcomeric actinin immunostaining.
（4）Electrophysiological properties of ion channels were assessed in MSC usingpatch-clamp technology. TTX-sensitive Na+current (INa.TTX), L-type calcium current (ICa.L),transient outward K+current (Ito), delayed rectifier K+current (IKDR), the inwardlyrectifying K+current (IK1) and action potential (AP) were recorded in MSCGATA-4andMSCNullafter MSC were co-cultured with native CM.
（5）The transdifferentiation rate was calculated directly from flow cytometry in MSCGATA-4and MSCNull.
（6）The gene expression level of insulin-like growth factor binding protein-4(IGFBP-4）in MSCGATA-4and MSCNullwere analyzed using real-time PCR and Westernblotting and Microarray.
（7）To study the role of IGFBP-4in GATA-4-mediated myocardialtransdifferentiation, IGFBP-4activity was knocked down in MSCGATA-4by siRNA genesilencing. Non-silencing, scrambled siRNA was used as a negative control. IGFBP-4geneexpression was analyzed using real-time PCR and Western blotting.
（8）Myocardial transdifferentiation rate of MSCGATA-4after knockdown of IGFBP-4was observed.
Results:
(1) The expression of cardiac genes, including brain natriuretic peptide (BNP),Islet-1and α-sarcomeric actinin (α-SA), was up-regulated in MSCGATA-4compared withcontrol cells that were transfected with Green Fluorescent Protein (GFP) only (MSCNull).
(2) MSCGATA-4exhibited higher levels of INa.TTX, ICa.L, Ito, IKDRand IK1channelactivities reflective of electrophysiological characteristics of CM.
(3) Low amplitude of action potential was recorded in MSCGATA-4after MSC wereco-cultured. No action potential was recorded in MSCNull.
(4) Some GFP+cells were positive for α-actinin staining after MSC were co-culturedwith native CM for7days. The transdifferentiation rate was significantly higher inMSCGATA-4(34%±4%), compared with MSCNull(15%±2%), P<0.05.
(5) At the same time, IGFBP-4was significantly up-regulated in MSCGATA-4, asshown by real-time PCR and Western blot and microarray, compared with MSCNull,P<0.05.
（6）After knocking down IGFBP-4in MSCGATA-4, real-time PCR showed that theexpression level of IGFBP-4in IGFBP-4-siRNA-MSCGATA-4(IG4-siRNA) was only38%of that in MSCGATA-4(P<0.05). IGFBP-4protein was reduced in IG4-siRNA cells by28%compared with MSCGATA-4(P<0.05).
(7) The effect of GATA-4on expressing cardiac markers (BNP、α-actinin和Islet-1) inMSC was suppressed when IGFBP-4-siRNA was transfected in MSCGATA-4, compared withMSCGATA-4, P<0.05.
(8) The transdifferentiation of various MSC was quantified using fluorescencea-activated cell sorting (FACS). The transdifferentiation rate was higher in MSCGATA-4(34.09±4.65%) than in MSCNull(14.54±1.62%; P<0.05). However, after IGFBP-4was knockeddown in the MSCGATA-4, the transdifferentiation rate was lower in IGFBP-4-siRNA (16.87±1.98%) than in MSCGATA-4(P<0.05).
（9）Functional studies indicated that the differentiation potential of MSCGATA-4wasdecreased by knockdown of IGFBP-4.
Conclusions: Retroviral transduction of GATA-4significantly increases MSCdifferentiation into a myocardial phenotype, which might be associated with theup-regulation of IGFBP-4.
Part ⅢTransduction of GATA-4Improved in vivo and in vitroAngiogenesis of Mesenchymal Stem Cells and Its RelatedMechanism
Objective: Transplanted mesenchymal stem cells (MSC) release soluble factors thatcontribute to cardiac repair and vascular regeneration. We hypothesized that retroviraltransduction of GATA-4enhances the MSC secretome, thereby increasing angiogenesisand cell survival and promoting postinfarction cardiac angiogenesis.
Methods：
(1) The concentration of VEGF, IGF-1, and basic FGF in conditioned medium (CdM)of MSC were measured using ELISA.
(2) The gene expression levels of VEGF, IGF-1, and basic FGF in transduced MSC were detected using quantitative real-time PCR.
(3) To study the resistance of MSC to oxidative stress, MSCs were exposed tohypoxia for24h,48h,72h and (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) intake was assessed.
(4) Human umbilical vein endothelial cells (HUVECs）were labeled with PKH67celltracker dye using PKH67Green Fluorescent Cell Linker kit. Conditioned medium wereadded into HUVECs and capillary-like structure formation was assessed. IGF-1-and/orVEGF neutralizing antibodies were added to CdMGATA-4. After12h of incubation,capillary-like structure formation was assessed.
(5) Spheroids of HUVEC were then embedded in3D Collagen Cell Culture Systemfor24h in the presence or absence of CdM. IGF-1-and/or VEGF neutralizing antibodieswere added to CdMGATA-4. The number of sprouts and cumulative sprout length in eachspheroid were detected and calculated.
(6) MSCs were injected into the peri-infarct region in an acute myocardial infarctionmodel in Sprague-Dawley rats developed by ligation of the left anterior descendingcoronary artery.
(7) Survival of male donor MSCs that expressed Sry gene in the female recipienthearts was assessed using real-time PCR. MSC survival in ischemic myocardium wasassayed.
(8) Cardiac function was assessed by echocardiography. Infarction size andangiogenesis were detected.
Results:
(1) Expression of IGF-1and VEGF-A in MSC was significantly upregulated inMSCGATA-4compared with MSCNull, P<0.05.
(2) levels of IGF-1and VEGF-A in CdM were assessed by ELISA and shown to besignificantly increased in CdMGATA-4, P<0.05, but b-FGF was unchanged, P＞0.05.
(3) MTT intake was significantly reduced when MSCs were exposed to hypoxia for48or72h. Overexpression of GATA-4partially prevented this reduction.
(4) HUVEC treated with MSCGATA-4conditioned medium (CdMGATA-4) exhibitedincreased formation of capillary-like structures and promoted migration (HUVECmigration was investigated by measuring both the number of sprouts per spheroid andcumulative sprout length), compared with HUVECs treated with MSCNullconditionedmedium, P<0.05. This CdMGATA-4-stimulated increase in formation of capillary-likestructures and migration were diminished by treatment with neutralizing antibodies againstIGF-1, VEGF, or both, compared with HUVECs treated with only MSCGATA-4conditionedmedium, P<0.05.
(5) Sry gene in peri-infarcted and infarcted myocardium was significantly higher inMSCGATA-4-transplanted hearts, compared with MSCNullor MSCbas, P<0.05.Overexpression of GATA-4significantly increased MSC survival in ischemic myocardium.
(6) MSCGATA-4-treated animals showed significantly improved cardiac function asassessed by echocardiography, compared with MSCNullor MSCbas-treated animals, P<0.05.
(7) Histological studies revealed increased blood vessel density and reducedinfarction size in MSCGATA-4-treated animals, compared with MSCNullor MSCbas-treatedanimals, P<0.05.
Conclusions: Transduction of GATA-4in MSCs increased both MSC survival andangiogenic potential in ischemic myocardium and may therefore represent a novel andefficient therapeutic approach for postinfarct remodeling.
引文
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