【作者】 何志松；

【导师】 蒋文平；

【作者基本信息】 苏州大学 ， 内科学， 2012， 博士

【摘要】 目的：观察锌指转录因子GATA-4基因过表达的骨髓间充质干细胞（MSCs）对心肌梗死后缺血心肌细胞的影响。阐明GATA-4与微小RNA（miR-195）、B-细胞淋巴瘤基因w（Bcl-w）因子之间的关系，探讨GATA-4加强MSCs细胞对梗死后缺血心肌细胞保护作用的可能机制，探寻加强缺血心脏保护的方法，为MSCs细胞移植治疗心肌梗死探索新的途径。方法：1.分离、培养健康新生Spraque-Dawley（SD）大鼠的MSCs细胞。2.构建GATA-4基因重组慢病毒载体，包装产生病毒，转染MSCs细胞并筛选稳定过表达GATA-4的MSCs细胞株。3.免疫荧光检测，蛋白免疫印迹、实时定量PCR等方法验证GATA-4基因在MSCs细胞内过量表达。4.分离、培养健康新生SD大鼠（2-5日龄）的心肌细胞，建立乳鼠心肌细胞和MSCs细胞嵌套共培养体系。5.乳鼠心肌细胞分成五组：正常组、缺氧组、与MSCs细胞共培养组、与MSC^Null细胞共培养组和与MSC^GATA-4细胞共培养组。除正常组外样本进行细胞缺氧（4%CO₂+95%N₂）24小时，Annexin V-PE免疫染色后用荧光显微镜及流式细胞术检测心肌细胞凋亡。缺氧36小时检测心肌细胞损伤指标LDH。6.结扎2-3月龄的SD大鼠冠状动脉前降支（LAD），构建心肌梗死模型。实验大鼠分为对照组、MSC^bas组、MSC^Null组和MSC^GATA-4组，每组四只，在梗死周围心肌组织上下左右四点分别注射全培养基、106细胞/50μl IMDM培养基的MSCs细胞、MSC^Null细胞和MSC^GATA-4细胞。7.细胞注射1周后收获大鼠心脏，横向等分切成0.15cm的切片，TTC染色后计算心肌梗死面积（MIA）。细胞注射4周后，收获大鼠心脏，石蜡包埋切片行Masson三色染色法染色。图像采集后测量计算梗死后纤维化面积。8.从MSC^GATA-4细胞中提取包括miRNA的总RNA，用生物芯片（microarray）筛选，发现miR-195表达受到抑制。9. miR-195前体重组慢病毒载体转导MSCs细胞，过表达miR-195；将GATA-4-siRNA转染MSCs细胞，减少MSCs细胞内源性的GATA-4表达。10.蛋白免疫印迹、实时定量PCR分别测定缺氧24小时后MSC、MSC^Null组和MSC^GATA-4组、GATA-4-siRNA组、miR-195组的GATA-4、Bcl-w蛋白及mRNA的表达。并实时定量PCR法检测MSC组、MSC^GATA-4和miR-195组的miRNA的表达水平。11.克隆Bcl-w3′UTR DNA片段，构建双荧光素报告质粒，转染HEK293TN细胞,荧光素酶活性测定及分析，明确miR-195是否通过碱基互补结合Bcl-w mRNA的3′UTR而直接调控Bcl-w的表达。结果：1.本研究成功构建了GATA-4重组慢病毒载体，获得足量、高效过量表达GATA-4基因的MSCs细胞。2.缺氧24h后，心肌细胞凋亡率达到33.7±1.92%，远远大于正常组的8.4±1.1%。嵌套共培养的MSC组和MSC^Null组，心肌细胞凋亡率较大幅度下降，分别为25.40±2.1%和26.6±1.5%（p<0.05vs缺氧组），两组之间相比无统计学差异。而共培养的MSC^GATA-4组，心肌细胞凋亡率下降为19.7±1.4%(p<0.05vs MSC^Null组)。3.缺氧36h后，心肌细胞损伤增加，释放的LDH含量明显升高。缺氧组LDH释放是正常组的420.82±20.55%，与各种MSCs嵌套共培养的MSC组和MSC^Null组，这一比值减低到320.00±22.55%和330.62±26.30%（p<0.05vs正常组）。而与转染了GATA-4基因的MSCs(MSC^GATA-4)细胞嵌套共培养组为250.87±20.39%。(p<0.05vs MSC^Null组)。4.移植后1周后，TTC染色将梗死心脏非缺血区域染为红色，缺血区域拒染而显示为苍白色，测量各个层面的缺血心肌面积，计算出MIA的比率：MSC^bas组16.38±2.51%和MSC^Null组15.45±1.81%低于对照组media组22.06±3.06%（n=4,p<0.05）。MSC^GATA-4进一步使MIA减少到9.52±1.98%(p<0.05vs MSC^Bas组和MSC^Null组, p<0.01vs MSC组)。5.心肌梗死4周后，纤维组织（主要是胶原）替代原来的死亡细胞，采用Masson三色染色法标记出蓝色的纤维组织，计算及纤维化部分的比率及左室前壁厚度与室壁厚度的比值。结果与近期试验结果类似（n=4，p<0.05）。6.经过Microarray筛选，我们发现miR-195在MSC^GATA-4中表达明显降低为MSCNul组的0.58±0.14倍（p<0.05）。RT-PCR检查， MSC^GATA-4组miR-195的表达量约为MSC^Null组的0.52±0.09倍，与Microarray结果一致。当用GATA-4-siRNA将GATA-4基因成功“沉默（silence）”而表达抑制后, miR-195的表达量与MSC组相比，升高了2.36±0.24倍（p<0.05）。7.过表达GATA-4的MSCs细胞Bcl-w表达增加；GATA-4基因静默后Bcl-w表达被逆转。MSC、MSC^Null和MSC^GATA-4三组细胞缺氧处理24小时后， MSC组和MSC^Null组Bcl-w蛋白水平相近（p>0.05）。MSC^GATA-4组Bcl-w蛋白表达水平明显升高(p<0.05vs MSC组和MSC^Null组)。GATA-4基因成功静默后，MSCs细胞GATA-4蛋白表达减少，与此同时，Bcl-w蛋白和mRNA表达水平均明显降低。8. MSC^GATA-4细胞进一步转染miR-195质粒，过表达miR-195后，细胞内高水平表达的Bcl-w蛋白随之下降(p<0.05vs MSC^GATA-4组)。9.将Bcl-W3′UTR片段作为插入子，构建双荧光素酶报告质粒。与miR-195过表达质粒共转染293T后，带有Bcl-W3′UTR报告子组的荧光活性，明显减低，而同样与miR-195过表达质粒共转染293T的阴性对照组荧光无变化。这表明：大鼠的Bcl-w3′UTR能直接被miR-195碱基配对结合。内源性的Bcl-w蛋白表达被miR-195所抑制。结论：1.本研究成功构建了GATA-4重组慢病毒载体，获得足量、高效过量表达GATA-4基因的MSCs细胞。2.体外实验结果显示： GATA-4加强MSCs细胞对心肌缺氧细胞的保护作用,使缺氧24h后的心肌细胞凋亡和缺氧36h后的心肌细胞损伤进一步减少。3.体内实验结果显示： GATA-4可加强MSCs细胞对心肌梗死后缺氧心肌细胞的保护作用,使心肌梗死面积和心肌纤维化面积进一步降低。4. miR-195通过与Bcl-w mRNA的3′UTR靶位点互补结合，抑制其翻译来降低Bcl-w蛋白水平。GATA-4抑制miR-195的表达，从而提高Bcl-w表达水平，由此加强MSCs对心肌梗死后缺血心肌的保护作用。更多还原

【Abstract】 Objective:The purpose of this study was to observe the influence of zinc finger transcriptionfactor GATA-4over-expressed mesenchymal stem cells（MSCs） on ischemic myocardiumpost myocardial infarction and to illustrate the relationship between GATA-4, miR-195andBcl-w and apoptosis as well as the possible mechanism.Methods:1. MSCs were isolated and cultured from bone marrow of the Spraque-Dawley（SD）rat.2. the GATA-4gene CDS sequence was copied and insert into the pLVX-IRES-ZsGreen1vector and the recombinant plasmid of pLVX-IRES-ZsGreen1-GATA-4wastransduced into the HEK293T cells using Lenti-X Lentiviral Expression Systems.After24h, supernatant contain the virus was filtered and incubated with MSCs for12h in the presence of10μg/mL polybrene. Stable transduced clones were obtained bypuromycin selection （3μg/mL for5days）.3. GATA-4over-expression was verified by immunostaining, RT-PCR and western blot.Negative control MSCs were transduced with pLVX-IRES-ZsGreen1(MSC^Null).4. Cardiomyocytes（CM） which was isolated and cultured from neonatal Spraque-Dawley（SD）rat（aged2-5d）,was co-cultured with MSCs on a transwell collagen-coatedmembrane inserts。5. Neonatal CMs were divided into five groups: normoxial control, hypoxia control,co-culture with MSCs group, co-culture with MSC^Nullgroup and co-culture withMSC^GATA-4group。 Apoptotic cells were labeled with annexin V-PE and examined under fluorescence microscope and flow cytometer followinghypoxia（4%CO2+95%N2） for24hrs. Cell injury estimated by LDH release from CMswas measured after hypoxia for36hrs.6. The myocardial infarction SD rats（aged2-3months） modeled by LAD permanentligation were divided into five groups:sham group, MSC^basgroup、MSC^Nullgroup andMSC^GATA-4group.50ul medium,106cells of MSCs, MSC^Nulland MSC^GATA-4in50ulIMDM medium were injected into ischemic border of the infarction area respectively.7. The hearts were harvested and sliced into0.15cm sections from the short axis whenMSCs were transplanted for1week. Myocardial infarction area（MIA） was measuredafter slices were stained by TTC. The hearts were stopped at the diastole stage andfixed in4%paraformaldhyde after MSCs transplantation for4weeks. Hearts wereembedded in paraffin and sectioned （5μm thick） for Masson’s TrichromeStaining.the MIA estimated by fibrous area was measured by Image pro plus6.0software.8. Microarray analysis of miRNA isolated from all kinds of MSCs found expression ofmiR-195were depressed in MSC^GATA-4cells.9. over-express miR-195by transduce the miR-195pre-microRNA recombinated lentivirusvector into MSCs. GATA-4expression were silenced by transducing GATA-4-siRNAinto MSCs.10. GATA-4、Bcl-w protein and miRNA levers in MSCs、MSC^Nullgroup,MSC^GATA-4group,GATA-4-siRNA group and miR-195group were measure by western blot and halfquantitative RT-PCR as well as miR-195lever in MSC group,MSC^GATA-4group andmiR-195group.11. A luciferase reporter plasmids containing Bcl-w3′UTR were constructed andco-transfected HEK293T cells with miR-195pre-microRNA recombinated lentivirusvector. The ration of fire luciferase/renilla luciferase activity was measured andanalyzed to determine whether miR-195was combined with3′UTR sites of Bcl-wmRNA and regulated the expression of Bcl-w. Results:1. The recombinated GATA-4lentivirus vector was constructed successfully, Stable MSCsclones with GATA-4over-expressed were obtained by puromycin selection.2. Treating hypoxia for24hrs，the percentage of CMs apoptosis reached33.7±1.92%，ismuch higher than that of normoxia（8.4±1.1%）。The percentage of apoptosis wasdecreased when co-culture with MSCs （25.40±2.1%）and MSC^Null（26.6±1.5%）（p<0.05vs hypoxia group）, Whereas there was no significant difference between thetwo groups。When co-culture with MSC^GATA-4cells，the apoptosis percentage wasdramatically decreased to19.7±1.4%(p<0.05vs MSC^Nullgroup)。3. After hypoxia for36hrs，Cell injury estimated by LDH release from CMs wassignificantly increased. In hypoxia group, it was420.82±20.55%folds of that innormoxia. The ratio changed to320.00±22.55%and330.62±26.30%（p<0.05vsnormoxial group） when co-culture with MSCs or MSC^Null, and250.87±20.39%whenco-cultured with MSC^GATA-4(p<0.05vs MSC^Nullgroup).4. when MSCs transplanted for1week, the none-ischemic areas of all sections of themyocardial infarction hearts were stained into red with TTC staining while theischemic area into pale. The MIA was calculated according to the measurement of allthe ischemic area. As a result, the MIA of MSC^basgroup（16.38±2.51%） and MSC^Nullgroup（15.45±1.81%） were reduced（n=4, p<0.05vs Medium group22.06±3.06%）. TheMIA of MSC^GATA-4group was reduced to9.52±1.98%进一步的(p<0.05vsMSC^Basand MSC^Null).5. when MSCs transplanted for4week, the infarction area has been replaced by fibrousscar tissue. The collagen fibers was stained blue by Masson’s Trichrome staining. Thefibrous area of MSC^basgroup and MSC^Nullgroup were reduced（n=4, p<0.05vsMedium group）. The fibrous area of MSC^GATA-4group was significantlyreduced(p<0.05vs MSC^Basand MSC^Null).6. miR-195expression in MSC^GATA-4was0.58±0.14times of that in MSC^Null（p<0.05）inmicroarray scanning, and0.52±0.09times in RT-PCR (p<0.05vs MSC^Null).whenGATA-4expression was silenced by GATA-4-siRNA, the expression of miR-195 increased to2.36±0.24times of that in MSC^Null（p<0.05）.7. Bcl-w expression in GATA-4over-expressioned MSCs(MSC^GATA-4) increased, and wasreversed when GATA-4gene was silenced by GATA-4-siRNA. When hypoxia for24hrs, Bcl-w protein lever was equal in MSC and MSC^Nullgroup（p>0.05）, butincreased dramatically in MSC^GATA-4group(p<0.05vs MSC and MSC^Nullgroup).When GATA-4gene was silenced, both expression of Bcl-w protein and mRNA weresignificantly decreased(p<0.05vs MSC^GATA-4).8. when MSC^GATA-4was transduced with miR-195pre-microRNA recombinated lentivirusvector, miR-195was over-expressed in MSC^GATA-4cells, followed by Bcl-w proteinlever decreasing(p<0.05vs MSC^GATA-4).9. A luciferase reporter plasmids containing Bcl-w3′UTR were constructed successfully.When co-transfected HEK293T cells with miR-195pre-microRNA recombinatedlentivirus vector, the luciferase activity of Bcl-w3′UTR reporter was depressed, whilethe empty vector as control failed. It was proofed that miR-195was combined with3′UTR sites of Bcl-w mRNA and regulated the expression of Bcl-w.Conclusions:1. The recombinated GATA-4lentivirus vector was constructed successfully, Stable MSCsclones with GATA-4over-expressed were obtained by puromycin selection.2. GATA-4enhanced MSCs mediated cardioprotection in hypoxia condition in vitro,decreased cardiomyocytes apoptosis after hypoxia for24hrs and ischemic injury afterhypoxia for36hrs.3. GATA-4enhanced MSCs mediated cardioprotection in Acute Myocardial Infarctionheart in vivo, decreased ischemic area and fibrous area.4. miR-195combines with3′UTR sites of Bcl-w mRNA and depresses the expression ofBcl-w. GATA-4elevates Bcl-w expression and enhances anti-apoptosis function ofMSCs in ischemic heart post MI by decreasing miR-195expression更多还原