【作者】 向飞宇；

【导师】 葛均波；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 课题Ⅰ:SCN5A点突变导致扩心病发生的机制研究背景:心脏钠通道基因（cardiac sodium channel,SCN5A）突变可导致各类心律失常,现有观点认为SCN5A突变与扩心病有关,但其机理还不清楚并且争议很大。我们曾在一个进行性心脏传导障碍伴扩心病的遗传家系中,发现了一个新的SCN5A基因A1180V突变。本课题即试图在细胞及分子水平上观察该突变位点是否影响钙平衡从而造成心肌损伤。方法:以PacⅠ线性化重组腺病毒质粒pAdGFP-SCN5A（MT/WT）,经线性化后转染到HEK 293细胞中包装成病毒,随后提取DNA进行PCR鉴定和测序鉴定。重组腺病毒Ad-GFP-SCN5A（MT/WT）经HEK293传代、扩增、浓缩与纯化,并测定病毒滴度(TCID₅₀和PFU)。将这些病毒按如下分组接种新生大鼠心肌细胞:对野生型SCN5A和突变型SCN5A分别设置缺氧组、缺氧并加xamoterol处理组、xamoterol组和空白组。接种病毒48小时后,以缺氧和xamoterol处理2小时后,随后进行如下实验:（1）提取RNA,经realtime PCR,观察钠和钙相关基因(L-Ca²⁺、CaMKⅡ、NaCX、PLB、CaM、SERCA、RyR2)的表达情况;（2）加载钙荧光指示剂X—rhod—1,以激光共聚焦显微镜测定心肌细胞内钙信号的变化情况;（3）用Annexin-V-PE联合碘化丙锭（PI）流式细胞仪检测心肌细胞凋亡率。对数据采用SPSS10.0统计软件进行ANOVA方差分析,p＜0.05为差异显著,p＜0.01为差异极显著。结果:经PacI酶切线性化的腺病毒质粒pAd-GFP-SCN5A（MT/WT）用脂质体介导转染HEK293细胞后,产生的重组腺病毒对HEK 293细胞有致病作用。用目的基因片段SCN5A引物进行PCR扩增,得到一条约5006p大小片段,测序后经序列分析表明SCN5A未发生新的突变,表明带有目的基因的重组腺病毒Ad-GFP-SCN5A（MT/WT）构建成功。重组腺病毒Ad-GFP-SCN5A（MT/WT）经HEK293传代、扩增、浓缩与纯化,获得滴度分别为PFU 7×10^8.4的Ad-GFP-SCN5A（MT）和PFU 7×10^7.8的Ad-GFP-SCN5A（WT）。分别接种HEK293和新生鼠心肌细胞,GFP阳性率在90%以上,说明病毒能够感染大部分细胞。从与钠和钙相关基因表达的情况来看,CaM和SERCA其表达量各组之间差异不显著;PLB在SCN5A野生型空白组表达显著高于其它各组（p＜0.05）,其它基因其表达规律性不强。SCN5A MT空白组和SCN5A WT Xamoterol处理组之间钙瞬变差异显著（p＜0.05）,各组间钙容量差异不显著,钙瞬变和钙容量均在数量上表现出SCN5A MT各组高于SCN5A WT各组的趋势。SCN5A MT缺氧并Xamoterol处理组和SCN5A MT空白组钙升高时间显著高于除这两组之外的其它各组（p＜0.05）,除空白组间WT和MT间钙衰减时间差异显著外,其它对应组间差异不显著（p＜0.05）,钙升高和钙衰减均在数量上表现出SCN5A MT各组低于SCN5A WT各组的趋势。各SCN5A MT组其细胞凋亡均显著高于相应的SCN5A WT组（p＜0.05）。结论:钙信号的变化和细胞凋亡的结果一致表现出SCN5A A1180突变可增强心肌细胞内钙信号。这说明SCN5A A1180V突变导致的非失活内向钠电流增加引起了细胞内钙信号加强。这将支持SCN5A A1180V突变可导致心肌细胞内钙稳态失衡从而引起扩心病的假设。课题Ⅱ:BMP2诱导人MSCs向心肌样细胞分化的可能性及其机制背景骨形态发生蛋白2（BMP2）可诱导间充质干细胞（MSCs）成骨和脂肪细胞,尚未有经BMP2诱导MSCs向心肌样细胞方向分化的报道。RUNX2和PPARγ分别是向骨和脂肪细胞分化下游通路上两个关键转录因子。BMP2诱导胚胎干细胞ESC和P19CL6可分化成心肌样细胞,MSCs经5-aza诱导也可分化成心肌样细胞,在BMP2通路上,成骨和成脂肪细胞分化是可以切换的,因此,有理由设想也可向心肌细胞方向切换。本课题试图通过下调转录因子RUNX2和PPARγ的表达来观察BMP2诱导下的MSCs可否向心肌样细胞方向分化。方法第一章:抽取人髂骨骨髓,经Ficoll密度梯度离心分离获得单个核细胞,分别经不同的培养基传代培养。以流式细胞仪鉴定其表面抗原,同时,对这些细胞经向脂肪细胞分化的化学诱导剂诱导。通过上述方法,获得生长性能良好的间充质干细胞株。第二章:以pEGFP-N1构建PPARγ和RUNX2基因的真核表达质粒,将这些基因通过脂质体转染或电转的方式转染到MSCs和HEK293细胞,选择转染率高的方法和高转染的细胞。将经化学合成的PPARγ和RUNX2siRNA与相应的质粒用lipo2000转染到HEK293细胞中,筛选下调基因表达最低的PPARγ和RUNX2 siRNA片段及相应片段的最佳浓度。第三章:以Lipofectamine RNAi MAX将BLOCK-iT^TM Alexa Fluor^? Red Fluorescent Oligo（Invitrogen USA）转染到MSCs,以评价RNA传送到MSCs的效率。按如下分组诱导MSCs的分化:（1）5-aza、BMP2、PPARγ和RUNX2的siRNA,（2）BMP2、PPARγ和RUNX2的siRNA,（3）5-aza、BMP2,（4）BMP2,（5）5-aza,（6）Control（Blank）。培养基中5-aza的终浓度为0.01uM,BMP2终浓度为100ng/ml,PPARγ和RUNX2的siRNA所加量分别为10pmol/孔（六孔板）。MSCs生长到70%的汇合度时,以5-aza处理细胞24小时,然后换液培养24小时即转染PPARγ和RUNX2的siRNA,6天后再转染一次,BMP2在70%的汇合度时即开始添加,每2天换液一次,12天后收细胞,提取RNA和蛋白,经RT—PCR、realtime PCR和western blotting,检测PPARγ、RUNX2、NKX2.5、GATA4、α-MHC和cTnT基因的表达情况。对数据采用SPSS10.0统计软件进行ANOVA方差分析,p＜0.05为差异显著,p＜0.01为差异极显著。结果第一章:对抽取的骨髓分离纯化,传代培养,15天即铺满培养瓶底,梭形细胞比例增加,细胞得到纯化,但传至P3,细胞由梭形变为平坦、宽大,细胞立体感逐渐消失,细胞的形状趋向多样,说明细胞很快衰老。换用添加有补充物的MSCs专用培养基培养,MSCs则生长状态良好。样本经流式细胞仪鉴定表面抗原,MSCs高表达CD29（97.69%）和CD166（97.54%）,但不表达CD34和CD45。同时,以化学诱导剂（MDI）成功的诱导这些细胞分化成为脂肪细胞。经诱导和鉴定表面抗原,说明所获得的细胞是高纯度的MSCs。第二章:经PCR鉴定和测序鉴定,成功的构建了以pEGFP-N1为载体的PPARγ和RUNX2基因真核表达质粒。将这些质粒与PPARγ和RUNX2 siRNA共转到HEK293,其中PPARγsiRNA的P2片段（5’AGAAUAAUAAGGUGGAGAUGCAGGC3’）在mRNA水平和蛋白水平上均显著下调PPARγ的表达（p＜0.05）,而RUNX2 siRNA的3片段（R1:5’UUUAAUAGCGUGCUGCCAUUCG AGG3’,R2:5’AACAGAUUCAUCCAUUCUGCCACUA3’,R3:5’UCAAGCUUCUGUCUGUGCCUUCUGG3’）均显著下调RUNX2的表达（p＜0.05）,同时,筛选得P2片段的最佳下调浓度为10pmol/孔（六孔板）（p＜0.05）,R2片段两个最佳下调浓度为10pmol╱孔和25pmol/孔（p＜0.05）。第三章:将Fluor Red RNA转染到MSCs,发现60%以上的RNA传送到了MSCs,说明有较高的传送效率经BMP2诱导,发现第4组（BMP2）PPARγ的表达显著高于其它各组,RUNX2的表达也在第4组最高,但无统计学上的差异。转录因子Nkx2.5和GATA4和心肌特异性基因TnT和MHC的表达在第1组（5-aza、siRNA和BMP2）一致性的显著升高（p＜0.05）,并且显著高于第5组（5-aza）,而第5组这些基因的表达也显著高于除第1组之外的其它各组（p＜0.05）。说明在诱导过程中,PPARγ和RUNX2被相应siRNA抑制,单因素5-aza和BMP2均不占主导作用,BMP2与5-aza产生了协同效应,促进了MSCs向心肌样细胞分化。结论抑制转录因子RUNX2和PPARγ的表达使得BMP2诱导MSCs向心肌样细胞分化是可行的,其机制可能在于BMP2下游信号通路间的串联和切换。课题Ⅲ:CTSS基因启动子区-25G/A多态对中国人群冠心病的预测背景:动脉粥样硬化是一种以动脉管壁细胞外基质结构广泛重构为特征的炎症性疾病。最近许多研究显示诸如组织蛋白酶S（CTSS）等的溶酶体半胱氨酸蛋白酶参与动脉粥样硬化形成过程。在CTSS基因的启动子区域核苷酸-25处存在G/A基因多态现象。基于CTSS在冠状动脉粥样硬化中发挥重要作用,而-25G/A多态位于CTSS基因启动子区域,因此,我们推测CTSS -25G/A多态性可能与冠心病之间存在密切联系。本项研究的目的即为观察中国人群中,CTSS-25G/A多态分布及其多态性与冠心病（CAD）危险性之间的关联。方法:CTSS基因-25G/A多态性由多聚酶链式反应PCR及限制性内切酶降解方法获得。经冠脉造影检查明确的共659名冠心病患者（冠脉狭窄≥50%）及352名非冠心病患者（对照组）纳入本项研究。我们同时收集患者的吸烟史、高血压史及糖尿病史,并测量他们的血脂水平。结果:所有研究对象中的等位基因G和A的频率分别为0.630及0.370。在病例组及对照组之间未发现其CTSS -25G/A多态性频率（G:0.626 vs.0.633,p＞0.05;A:0.374 vs.0.367,p＞0.05）及基因型分布（G:83.4 vs.85.3,p＞0.05;A:58.0vs.58.5,p＞0.05）存在显著性差异。此外,即使在使用逻辑回归对其他危险因素校正后,CTSS基因型与冠脉狭窄严重程度之间亦未发现存在关联（p＞0.05）。结论:在中国人群中,基因型AA较基因型GG和GA更为少见。对照组及冠心病组之间的等位基因频率及基因型分布均无显著性差异。CTSS -25G/A多态性与冠状动脉狭窄的发生率及严重程度不相关。更多还原

【Abstract】 Ⅰ.Novel SCN5A Mutant-Induced Intracellular Calcium Shift in Dilated Cardiomyopathy PathogenesisBackground Mutations of SCN5A,a cardiac sodium channel gene, have been implicated in the pathogenesis of dilated cardiomyopathy（DCM）.Our previous study indicated a role of a novel mutant,SCN5A A1180V,in the onset of DCM in a Chinese family.However,the underlying mechanisms remain unknown. The present study was to investigate the biological significance of SCN5A A1180V in intracellular calcium shift and consequent myocardial damage.Materials and Methods Recombinant adenoviral vector pAdGFP-SCN5A（MT/WT） linearized by PacⅠenzyme was amplified and confirmed by PCR and DNA sequencing after transfection into HEK 293 by lipofectamine 2000.Amplification was verified by titering analysis.Cardiomyocytes of neonatal rats transfected with Ad-GFP-SCN5A（MT/WT） were then treated with hypoxia,xamoterol,hypoxia+xamoterol respectively.Total RNA were extracted after 2h treatment.Both sodium and calcium-associated genes(L-Ca²⁺,CaMKⅡ,NaCX,PLB,CaM, SERCA and RyR2) were detcted by realtime-PCR.Changes of intracellular calcium loaded with X-rhod-1 were measured by laser confocal microscopy（LCM）.Cell apoptosis was analyzed by flow cytometry with Annexin V/PI labeling.Multiple group comparison was done by a one-way analysis of variance（ANOVA） by use of SPSS10.0.Values of P＜0.05 were considered statistically significant. Results The recombinanta denovirus Ad-SCN5A（MT/WT） with GFP report gene was successfully constructed in 293cell.PCR test proved SCN5A（MT/WT） contained the insertion of SCN5A（MT/WT）. The titre of purified recombinant adenovirus of Ad-GFP-SCN5A-MT,and Ad-GFP-SCN5A-WT was 7×10^8.4 pfu/ml and 7×10^7.8/mi,respectvely.GFP posivitve HEK 293 and cardiomyocytes reached 90%or above.Realtime-PCR indicated significant elevation of PLB expression in SCN5A WT control group compared with other groups（p＜0.05）.However,no other significant changes were detected in genes expression in SCN5A MT/WT groups.LCM indicated notable calcium transient difference between SCN5A MT control group and SCN5A WT xamoterol group（p＜0.05）.SCN5A MT tended to be higher than its SCN5A WT counterparts in both calcium transient and content.However,no significant differences were observed. Time of intracellular calcium increase in SCN5A MT hypoxia+xamoterol and SCN5A MT control were markedly higher than any other group.However,no significant difference were observed in calcium decay time between its SCN5A WT counterparts except in the two control groups of SCN5A MT and SCN5A WT.Apoptosis were significantly higher in SCN5A MT than that in SCN5A WT counterparts（p＜0.05）.Conclusion SCN5A A1180V mutation could induce intracellular calcium overload and consequent cardiac apoptosis via Na+ current increase by Na+ channel inactivation,which may contribute to DCM pathogenesis. Ⅱ.Differentiation of Bone Marrow derived Human Mesenchymal Stem Cells into Cardiomyocyte-like Cell Induced by BMP2Background:Pluripotent MSCs can differentiate into several distinct cell types induced by Bone morphogenetic protein 2（BMP2）,including osteoblasts and adipocytes.Two key transcription factors,RUNX2 and peroxisome proliferators-activated receptorγ（PPARγ）,drive MSCs to differentiate into either osteoblasts or adipocytes, respectively.Also,MSCs can differentiate into cardiomyocytes induced by 5-azacytidine,and ESC or P19CL6 can differentiate into cardiomyocytes induced by BMP2.BMP2 signal pathway appears to play a crucial role in this decision of cellular fate by dose effect,cross talk or molecular switch. Therefore,its reasonable to postulate that it also exists possibility to lead MSCs differentiating into cardiomyocytes induced by BMP2.Methods:Chapter 1.MSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time method.Morphology and growth characteristics were examined by phase contrast microscopy. Cell surface markers CD166,CD29,CD34 and CD45 were tested by flow cytometer.To invest the potential of differentiation, MSCs were induced to adipocytes combined with dexamethasone （DM）,1-methy-3-isobutylxanthine（IBMX）,insulin（IS） and indomethacin（ID） in vitro.Chapter 2.The genes PPARγand RUNX2 were amplified by PCR and cloned into eukaryotic expression vector pEGFP-N1 with EGFP reported gene encoding enhanced green florescence protein,and then identified by PCR and sequeing.The recombinant plasmids pEGFP-N1-PPARγand pEGFP-N1-RUNX2 were transfected into MSCs and HEK293 by the methods mediated by lipofectin and electroporation to investigate the transfection efficiency.To select the better small interfering RNA siRNA segment down-regulating the expression of PPARγand RUNX2,3 pairs of chemically synthetical PPARγsiRNA（P1,P2,P3） and corresponding plasmid pEGFP-N1-PPARγ,RUNX2 siRNA（R1,R2,R3） and corresponding plasmid pEGFP-N1-RUNX2 were co-transfected into HEK293 by use of liposome,respectively.And negative group,positive group and blank were designed as control.The effect of silence was detected by RT-PCR,Real-time PCR,and Western blotting.Chapter 3.To access the delivery ratio of siRNA to MSCs,Fluor Red RNA was ransfected into MSCs with Lipofectamine RNAi MAX.When growing to 70%confluence,MSCs were induced by BMP2 as the following groups（groupl:5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA,group2:BMP2,PPARγsiRNA combined with RUNX2 siRNA,group3:5-aza combined with BMP2,group4:BMP2,group5:5-aza,group6:blank）.The dosage of 5-azacytidine（5-aza） and the siRNA was 0.01μM and 10pmol/well（6 well plates）,respectively.MSCs were treated by 5-aza for 24 hours in induced dayl,by siRNA in day 2 and day8 for 4 hours.When induecd 12 days,the mRNA of PPARγ,RUNX2, NKX2.5,GATA4,α-MHC and cTnT in hMSCs were tested by RT-PCR and Realtime PCR.Multiple group comparison was done by a one-way analysis of variance（ANOVA） by use of SPSS10.0. Values of P＜0.05 were considered statistically significant.Results:Chapter 1.After 24 hours in primary culture,the cells adhered to the plastic surface.After 2-3 days,the cells were shuttle-shape.After 15 days,the cells overspread the culture bottle bottom,and kept the same figure characteristics of primary culture.But when the cells grew up to the third passages,shuttle-shape of them changed to plainness and bounty,cytokinesis decreased,and cytoplasm was loosen and there were vacuoles in cytoplasm,and the surface area add up to 5 to 50 fold larger than the normal MSCs.The ageing cells could restore to normal growth status and morphology developed with the special Mesenchymal Stem Cell Medium with Growth Supplement.hMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 （97.69%）,CD166（97.54%）,and negative for CD34,CD45.After induced with MDI medium,MSCs differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O.Chapter 2.The expression vectors of recombinant plasmid pEGFP-N1-PPARγand pEGFP-N1-RUNX2 were successfully constructed and it can be steadily expressed in HEK293 cells.It provided a platform for the next experiments. plasmid pEGFP-N1-RUNX2 were co-transfected into HEK293 cells.When co-transfected siRNA and recombinant plasmid pEGFP-N1 into HEK293,the expression of mRNA and protein was checked.It resulted that the P2 segment of PPARγsiRNA （5’AGAAUAAUAAGGUGGAGAUGCAGGC3’） significantly down-regulated the expression of PPARγgene in mRNA and protein level（p＜0.05）. And all 3 RUNX2 siRNA segments（R1:5’UUUAAUAGCGUGCUGCCAUUCG AGG3’,R2:5’AACAGAUUCAUCCAUUCUGCCACUA3’,R3:5’UCAAGCUUCUGUC UGUGCCUUCUGG3’） significantly down-regulated the expression of PPARG gene in mRNA and protein level（p＜0.05）.The best concentration of P2 PPARG siRNA was 10pmol/well（6 well plate） （p＜0.05）,and the best concentration of R2 RUNX2 siRNA was 10pmol/well or 25pmol/well（6 well plate）（p＜0.05）.Chapter 3.Over 60%Fluor Red RNA was transfected into MSCs with Lipofectamine RNAi MAX.It gave a good indication of transfection efficiency and facilitated assessment and optimization of siRNA delivery into MSCs using cationic lipids. The expression of some selective genes was examined when MSCs induced for 12 days.It led that the expression of PPARγgene in group 4（BMP2）increased significantly than the other groups （p＜0.05）.Also,the expression of RUNX2 was higher in group 4（BMP2）.It was interesting that the expression of cardiac core transfacors Nkx2.5 and GATA4,cardiac structure protein cTnT andαMHC in group 1（5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA） was significantly higher than the other groups including group 5（5-aza） concurrently（p＜0.05）,also,the expression of these 4 genes in group 5（5-aza） was significantly higher than the other groups except group 1 （5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA）（p＜0.05）. It suggested that PPARγand RUNX2 gene were inhibited.Both 5-aza and BMP2 didn’t play a dominant role during the differentiation.However,5-aza combined BMP2 together with siRNA produced synergy action to promote MSCs to differentiate into cardiomyocyte-like cell.Conclusion:It was feasible that MSCs differentiate into cardiomyocye-like cell induced with BMP2 by down-regulating the the expression of PPARγand RUNX2 gene synchronously.BMP2 signal pathway may act as a molecular rheostat to fine-tune the balance among distinct lineages by dose effect,cross talk or molecular switch. Ⅲ.CTSS promoter -25G/A:not a risk factor for CHD in ChineseBackground Atherosclerosis（AS） is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Recent data suggested the participation of lysosomal cysteine proteases such as cathepsin S（CTSS） in atherogenesis.The G/A polymorphism at nucleotide -25 was reported to locate in the promoter of the CTSS gene.Because of the importance of CTSS in atherosclerosis and the special location of G/A polymorphism,we supposed that CTSS -25G/A polymorphism maybe have relationship with coronary heat disease.Therefore,the aim of this study is to observe the association between CTSS -25G/A polymorphism and the risk of CHD in Chinese population.Methods Polymerase chain reaction（PCR） and restriction digestion method were performed to screen the CTSS gene -25G/A polymorphism.Histories of smoking,hypertension and diabetes were investigated and blood lipids were simultaneously measured in patients with coronary heart disease（CHD,coronary artery narrow≥50%,n= 659） and without CHD（control,n = 352）.Results The frequencies of G and A allele in the total population was 0.630 and 0.370 respectively.No significant difference was found in the frequencies of -25G/A polymorphism in CTSS gene（G:0.626 vs.0.633,p＞0.05;A:0.374 vs.0.367, p＞0.05） and genotype distribution（G:83.4 vs.85.3,p＞0.05; A:58.0 vs.58.5,p＞0.05） between CHD patients and control group.Furthermore,CTSS genotype is not associated with severity of coronary stenosis（p＞0.05）,even though other risk factors were adjusted by means of logistic regression.Conclusion The genotype AA was less common than GG and GA in this Chinese population.There was no significant difference in the allele frequencies as well as genotype distribution among the CHD and the Controls.The CTSS -25G/A polymorphism was not related with the vulnerability and the severity of coronary stenosis.更多还原