【作者】 孔宏亮；

【导师】 齐国先；

【作者基本信息】 中国医科大学 ， 内科学， 2008， 博士

【摘要】 间充质干细胞（mesenchymal stem cells,MSCs）自我复制性、非特异弱免疫原性以及可向心肌、神经元、内皮等不同胚层细胞分化的多潜能性等使其成为当今心脑血管等领域研究的热点。心肌细胞成形治疗（celularcardiomyoplasty,CCM）研究发现:MSCs尤其是骨髓MSCs可能通过分化成功能性心肌细胞、刺激梗死区血管新生、提高神经密度、增强心肌厚度和弹性、分泌细胞因子或表达细胞因子受体、抑制心肌细胞凋亡和防治心肌重构等机制而使CCM成为当今热点,不过,许多CCM报告图片显示:1.直接注入到心肌梗死区的标记MSCs仅散在分布于分化良好、结构完整的大血管壁内和成熟心肌之间而不在或偶在肉芽组织替代的梗死区（此区域内血管稀少,血液供应最差）和其毛细血管周围（原缺血区,血液供应较差）出现,更没有足够多的细胞数目替代瘢痕组织;2.标记细胞散在分布于正常组织、细胞之间而不似培养条件下以种子细胞为中心的扩展性生长;3.瘢痕组织中动脉和小动脉密度不增加,而毛细血管密度的增加并不足以改善局部区域血流;4.MSCs在梗死区域并不充分出现心肌细胞表型等;这些结果提示区域性缺氧环境不利于MSCs生存、增殖和分化等,其原因是否因为这些区域的缺氧环境不利于MSCs增殖、分化甚至导致其凋亡、死亡等国内外罕见报道。如果缺氧可导致MSCs凋亡,那么缺氧是否可致骨髓MSCs表达Bax、Bcl—2、Fas-L、Fas、Caspase-3等凋亡相关mRNA和蛋白以及它们在MSCs凋亡中所起的作用如何等迄今亦罕见研究。参予介导细胞生长、增殖的Akt蛋白既是细胞存活的信号介质又是细胞存活的必要因素,持续存在可以避免细胞损伤,那么Akt基因转染骨髓MSCs是否可减轻MSCs缺氧凋亡和提高缺氧时MSCs增殖能力（即耐缺氧能力）尚不清楚。鉴于此,本研究拟对体外培养的第三代（Passage 3,P₃）Wistar大鼠骨髓MSCs在缺氧箱（94%N₂、1%O₂和5%CO₂）中孵育,探讨下述三个方面:1.缺氧不同时间点对MSCs凋亡和增殖等的影响,明确心肌梗死后移植的MSCs能否在相对缺氧的移植区生存,为CCM成功实现提供新思维;2.MSCs在缺氧环境下凋亡相关mRNA和蛋白与凋亡的相关性,确立其是否在骨髓MSCs缺氧凋亡中发挥作用;3.探讨Akt基因是否提高MSCs耐缺氧能力,从而为更佳的CCM效应提供理论依据。另外,在这些研究的基础上,我们对Akt-MSCs是否可调整心脏交感神经和胆碱能神经以及促进神经纤维再生进行了初步探讨。论文一:缺氧对大鼠骨髓间充质干细胞凋亡、增殖和超微结构的影响目的探讨梯度离心贴壁法培养的大鼠骨髓单核细胞是否MSCs以及缺氧对其凋亡、增殖和超微结构的影响。方法1.无菌条件下取出Wistar大鼠股骨及胫骨骨髓,于Percoll（比重1.074g/mL和1.070g/mL）分离液上梯度离心（500g,20 mins）后取界面层细胞培养、传代。将P3 MSCs免疫细胞化学鉴定CD29、CD34、CD44和CD71受体,5-aza（浓度10umol/L）培养液常规孵育2周后肌钙蛋白T（troponin,Tn T）表达和H-DMEM成骨细胞诱导分化培养液（DEX 100nmol/L,β-GP 10mmol/L,AsA0.25mmol/L）常规孵育2周后ALP表达。2.将P3 MSCs置于94%N₂、1%O₂和5%CO₂缺氧箱中37℃孵育不同时间点（常氧、缺氧0.5 h、1h、2 h、4 h和8 h）。3.应用Annexin V/PI双染法进行流式细胞仪（flow cytometry,FCM）分析MSCs凋亡率（Apoptotic Rate,AR）和死亡率（dead rate,DR）、应用四唑盐比色试验（MTT比色试验）分析细胞增殖状态以及透射电镜观察MSCs缺氧时超微结构改变。结果1.骨髓单核细胞免疫细胞化学鉴定CD29、CD44和CD71免疫细胞化学染色均可见棕黄色颗粒沉积于胞膜,而CD34免疫细胞化学染色后未见棕黄色颗粒沉积于胞膜;5-aza诱导后Tn T免疫细胞化学染色可见棕黄色颗粒沉积胞质中,成骨诱导后改良Gomori法染色可见胞浆内深褐色甚或黑色颗粒呈片状沉积。2.MSCs不同缺氧时间点AR和DR MSCs常氧、缺氧0.5 h、1 h、2 h、4 h、和8 h时的AR均较缺氧前显著增高（P＜0.01）、DR亦显著增高（P＜0.05）,不过,随着缺氧时间延伸,AR显著增加（P＜0.05）,而DR没有统计学意义（P＞0.05）。3.MSCs不同缺氧时间点增殖状态缺氧不同时间点MTT法0D值较常氧（缺氧结束同步时间点）均显著性降低（P＜0.01）,并随缺氧时间延伸显著降低（P＜0.05）,不过,与常氧（缺氧开始时间点）相比,均显著升高（P＜0.05）。4.缺氧对MSCs超微结构的影响常氧时,MSCs呈卵圆形或不规则多边形等,表面微绒毛较多且向细胞内凹陷,内质网和游离核糖体较丰富而线粒体小,多为单核,双核偶见,核仁大而形态不规则,可见核裂。缺氧0.5 h即见微绒毛脱落、胞浆空泡化、线粒体肿胀等,缺氧4 h可见肿胀线粒体（伴嵴消失）明显增多。讨论MSCs在骨髓中含量极低,而其鉴定必须具有骨髓来源、体外培养贴附于培养皿上且形态呈成纤维样集落形成单位（colony forming unit,CFU-F）和能够自主增殖至少分化为三种以上间质细胞系等特性逆推得知是否为MSCs。我们通过梯度离心贴壁法培养的骨髓单核细胞具有MSCs的生长特性并且5-aza诱导后形态学接近心肌细胞并表达Tn T蛋白提示向心肌样细胞分化、成骨诱导后形态学发生改变并表达ALP提示向成骨细胞分化,所有这些特征均支持我们采用梯度离心贴壁法所培养的骨髓单核细胞是骨髓MSCs。MSCs不同缺氧时间点AR、DR较缺氧前均显著性增高提示缺氧可诱导MSCs凋亡、死亡,不过,缺氧0.5 h时AR即显著、迅速增高而随着缺氧时间延伸,MSCs AR和DR却呈缓慢上升趋势的结果提示MSCs在进入缺氧环境后即刻的不适应性以及其随着缺氧时间延伸而可能适应缺氧状态,“死亡”可能是MSCs凋亡的后期表现。结果3提示MSCs于缺氧时仍可增殖但其“增殖”滞缓。总之,我们的研究显示缺氧可导致MSCs凋亡、增殖滞缓及超微结构改变,而MSCs凋亡及增殖滞缓可能是移植的MSCs难以在心肌梗死后相对缺氧的移植区生存、增殖和分化等原因之一。结论梯度离心贴壁法培养的骨髓单核细胞是骨髓MSCs;缺氧可引起MSCs凋亡、增殖滞缓及超微结构改变。论文二:缺氧环境下大鼠骨髓间充质干细胞凋亡相关基因和蛋白的表达目的探讨MSCs在缺氧环境下凋亡相关基因和蛋白的表达以及它们在凋亡中的潜在作用。方法1.将接种的P₃ MSCs置于94%N₂、1%O₂和5%CO₂缺氧箱中37℃孵育。2.分别于0.5 h、1 h、2 h、4 h、6 h、8 h和12 h取出。3.应用Annexin V/PI双染法FCM分析MSCs凋亡率（AR）和死亡率（DR）。4.免疫细胞化学、Rt-PCR和western blotting检测Bax/Bcl-2,Fas/FasL和Caspase-3 mRNA和蛋白的表达。5.分析AR与凋亡相关mRNA和蛋白相关性。结果1.缺氧前,免疫细胞化学法未检测到Bcl-2、Bax、Fas和Fas L蛋白表达,缺氧0.5 h后均可较强表达,各缺氧时间点Caspase-3表达率较常氧显著增加。2.各缺氧时间点Bcl-2、Bax、Fas、Fas L、Caspase-3 mRNA和蛋白表达较缺氧前均显著增高（P均＜0.05）;随缺氧时间延伸,Bcl-2 mRNA和蛋白表达不显著增加（P＞0.05）,而Bax、Fas、Fas L、Caspase-3 mRNA和蛋白表达均显著增加（P均＜0.05）,但缺氧6 h～12 h时间点之间表达均没有统计学意义（P均＞0.05）。3.AR和Bcl-2/Bax mRNA（r₁=-0.435,P=0.040）及蛋白（r₂=-0.417,P=0.043）呈显著负相关,而和Fas（r₁=0.711,P=0.018:r₂=0.639,P=0.025）、Fas-L（r₁=0.605,P=0.037;r₂=0.581,P=0.022）、Caspase-3（r₁=0.657,P=0.026;r₂=0.704,P=0.014）mRNA及蛋白均呈显著正相关。讨论在细胞凋亡过程中,Bcl-2基因为抗凋亡基因,而Bax基因为促凋亡基因。在常氧,大鼠MSCs不表达Bcl-2、Bax mRNA和蛋白,而在本缺氧条件下,二者均表达增强,并且Bcl-2/Bax与AR显著负相关,提示Bcl-2、Bax mRNA和蛋白可能参予MSCs缺氧时的凋亡调控,并且Bax mRNA和蛋白可能促进凋亡、而Bcl-2 mRNA和蛋白抑制凋亡。Fas及其配体Fas-L共同参予调节细胞凋亡过程,在常氧,MSCs不表达Fas-L、Fas mRNA和蛋白,而在本缺氧条件下,二者均表达增强,并且Fas、Fas-L mRNA和蛋白均与AR显著正相关,提示Fas-L、Fas mRNA和蛋白参予并可能促进MSCs的缺氧凋亡调控。细胞凋亡信号途径中最关键的效应酶caspase-3在细胞凋亡过程中处于核心位置,在本缺氧条件下,caspase-3 mRNA和蛋白均与AR显著正相关,提示caspase-3 mRNA和蛋白参予MSCs缺氧凋亡的调控。总之,我们研究提示MSCs在缺氧状态下发生凋亡的过程中,线粒体途径和受体途径均参予细胞凋亡,并且可能呈caspase-3依赖性凋亡。结论在缺氧促进MSCs凋亡过程中,Bcl-2 mRNA和蛋白可能起着保护作用,而Bax、Fas-L、Fas、Caspase-3 mRNA和蛋白可能在MSCs凋亡的进程中起着促进作用。论文三:Akt基因转染对骨髓间充质干细胞缺氧时凋亡和增殖的影响目的探讨Akt基因是否改善MSCs耐缺氧能力。方法将转染Akt基因和未转染Akt基因的MSCs置于94%N₂、1%O₂和5%CO₂缺氧箱中37℃孵育不同时间点（常氧、缺氧0.5 h、1h、2 h、4 h和8 h）。Annexin V/PI双染法FCM分析MSCs AR和DR。应用MTT比色试验分析细胞增殖状态。免疫荧光细胞化学染色、Rt-PCR和western blot检测Akt和p-Akt表达。结果1.Akt基因显著降低MSCs缺氧时的AR和DR（P＜0.01）,而在各缺氧时间点没有统计学意义（P＞0.05）。2.与未转染Akt基因的MSCs比较,Akt基因显著增高MSCs常氧培养条件下（P＜0.01）和缺氧培养条件下的增殖能力（P＜0.01）;转染Akt基因的MSCs缺氧时的增殖能力显著低于其在常氧培养时的增殖能力（P＜0.01）。3.转染Akt基因显著增高常氧培养条件下MSCs Akt mRNA（P＜0.01）和蛋白（P＜0.01）表达,而并不增高p-Akt mRNA（P＞0.05）和蛋白（P＞0.05）的表达;在常氧、缺氧时Akt mRNA（P＞0.05）和蛋白（P＞0.05）表达均没有统计学意义,而提高缺氧时p-Akt mRNA（P＜0.01）和蛋白（P＜0.01）表达;在缺氧前、后,免疫荧光细胞化学法均可检测到转染和未转染Akt基因MSCs表达Akt和p-Akt蛋白。讨论MSCs转染Akt基因后在常氧时可显著增高Akt mRNA和蛋白表达,而不改变p-Akt蛋白表达,一旦暴露于缺氧环境可同时显著提高Akt mRNA和蛋白及p-Akt蛋白表达等可能是由于转染Akt基因的MSCs在尚未得到缺氧刺激时不能充分“触发”Akt蛋白磷酸化或其功能已足够满足细胞生存需求而“负反馈”抑制其磷酸化,当“缺氧”刺激时即可“触发”Akt蛋白磷酸化即产生p-Akt。Akt基因可显著降低MSCs在各缺氧时间点的AR和DR提示Akt基因可提高MSCs耐缺氧能力。不过,常氧培养条件下,转染Akt基因并不影响MSCs的AR和DR提示常氧状态下Akt的作用已能够满足MSCs生存、增殖,而过多表达并不在抗细胞凋亡中发生作用;MSCs缺氧培养较常氧培养的AR和DR仍然升高提示缺氧可能通过其它机制引起MSCs凋亡,而Akt基因转染并不足够完全抑制细胞凋亡。总之,研究提示Akt基因转染MSCs可显著提高MSCs耐缺氧能力,此可能是转染Akt基因MSCs的CCM效应较单纯MSCs的CCM效应更佳化的原因之一。结论Akt基因转染可显著提高MSCs耐缺氧能力论文四:Akt基因转染的间充质干细胞对阿霉素诱导的慢性心力衰竭的心脏神经的保护作用目的Akt-MSCs是否可调整心脏交感神经和胆碱能神经以及促进神经纤维再生。方法阿霉素诱导慢性心力衰竭模型完成后,大鼠被随机分为Akt-MSCs组（n=11）,s-MSCs（n=11）组和对照组（n=12）。各组分别经尾静脉给予Akt-MSCs、s-MSCs（2x106 cells in 100ul PBS）或等量PBS。第四周检测心脏超声、去甲肾上腺素（NE）,胆碱乙酰基转移酶（ChAT）、突触素（SYN）生长相关蛋白43（GAP-43）。结果1.在Akt-MSCs组和s-MSCs组,死亡率和射血分数均显著低于对照组。2.Akt-MSCs和s-MSCs均可显著降低组织NE水平。3.在Akt-MGCs组和s-MSCs组,ChAT水平没有统计学意义,但均显著高于对照组。4.在Akt-MSCs组和s-MSCs组,SYN或GAP-43染色阳性的神经密度均显著高于对照组,而以Akt-MSCs组神经密度最高。结论MSCs尤其是Akt基因转染的MSCs移植可促进心脏神经纤维的再生,该作用可能被GAP-43介导。更多还原

【Abstract】 IntroduceThe characteristics of mesenchymal stem cells（MSCs）,which includes self-duplication,non-specific weak immunogenicity and multipotency for different embryonic layer such as cardiocyte,neuron,endotheliocyte and so on make MSCs have been the present spotlight.A lot of investigation in celular cardiomyoplasty （CCM） found that the reasons for CCM being nowadays hot spot included mainly MSCs particular bone marrow MSCs,differentiating functionality cardiocyte, stimulating angio-neogenesis in infarction area,elevating the density of nerves, reinforcing the thickness and the elasticity of myocardium,excreting cytokine or expressing its receptor,inhibiting cardiocyte apoptosis and preventing cardiocyte from reconstitution and so on.However,lots of pictures about CCM have shown as followed:1.MSCs marked,which was directedly injected into the area of myocardial infarction,distributed just diffusedly in the wall of great vessels and/or ripening cardiocyte,and was rarely seen in the infarction area replaced by granulation tissue and the periphery of blood capillary,let alone enough MSCs take the place of scar tissue;2.there exists rather multitude discrepancy on growth pattern;3.the density of medium-sized artery and arteriole was not increased,and the augment of capillary improved not enough the region bloodstream;4.It is difficult of MSCs in the infarction region to appear the phenotype of cardiocyte.All the above results suggest that the regionality hypoxic microenvironment have disadvantages in the survival,multiplication and differention of MSCs.Whether the hypoxic microenvironment influences MSCs on multiplication and differentiation and even results them in apoptosis and death? So far,these have rarely been reported at domestic and oversea.If hypoxia may lead to the apoptosis of MSCs, then whether hypoxia may induce MSCs expressing Bcl-2,Bax,Fas,Fas L, Caspase-3 mRNA and protein and what role they play in MSCs apoptosis have not been found nowadays.Akt protein mediating cell growth and multiplication,which is a kind of signal essential mediator keeping cell survival,may protect cell from damage,however,whether Akt gene transfection may relieve the hypoxic apoptosis and enhance the multiplication capability of MSCs have not been conformed.In view of the above,our study aimed at elucidating the following aspect by Passage 3 bone marrow MSCs of Wistar rat,which were cultured in 94%N₂,1%O₂ and 5% CO₂:1.whether cell multiplication,apoptosis,and ultramicrostructure change of bone marrow mesenchymal stem cells of Rat were influenced by hypoxia environment ex vivo;2.the expression of apoptosis related mRNA and protein, such as Bcl-2,Bax,Fas,Fas-L and Caspase-3,of bone marrow MSCs of rat under hypoxia environment ex vivo.3.Whether Akt gene improved the capacity of both anti-apoptosis and multiplication of bone marrow MSCs of Wistar rat,ie.bearing hypoxia capacity of MSCs under hypoxic environment ex vivo.In addition,on the basis of those analysis,we elucidated further whether Akt-MSCs adjust sympathetic and cholinergic nerve and whether the beneficial effects of Akt-MSCs and MSCs are mediated by their nerve fiber sprouting in vivo.Article 1:Apoptosis And Multiplication of Bone Marrow MSCs Once Hypoxia Ex VivoObjectiveAimed at elucidating whether bone marrow monomuclear cells are mesenchymal stem cells（MSCs）,which were cultured by combining gradient centrifugation and adherence wall methods and elucidating whether cell multiplication,apoptosis,and ultramicrostructure change of bone marrow mesenchymal stem cells of Rat were influenced by hypoxia environment ex vivo.Materials and Methods1.The bone marrow,which were washed out and gathered from femoral bone and shinbone of Wistar rat in the sterilitas condition,were gradiently centrifugated （500g,20 mins） on Percoll separating medium（specific gravity:1.074 g/ml and 1.070 g/ml）,and then gathered boundary layer to culture and go down to passage 3. Immunocytochemistry identified the expression of CD29,CD34,CD44,CD71 receptor and cardiac-specific troponin T（Tn T） after two weeks incubated with L-DMEM containing 5-aza（10μmol/L） and ALP Gomori reformed methods identified sclerotomal-like cell incubated with H-DMEM containing DEX （100nmol/L）,β-GP（10mmol/L） and AsA（0.25mmol/L） for two weeks.2.Passage 3（P3） of bone marrow MSCs of wistar rat were cultured in culturing chamber with 94%N₂,1%O₂,5%CO₂ at 37℃.3.At different hypoxia time points ie 0,0.5,1,2,4 and 8h,apoptotic rate（AR） and dead rate（DR） were analyzed by Flow cytometry（FCM） after Annexin V/PI staining,cell multiplication by MTT methods and ultramicrostructure by transmission electron microscope（TEM）.Results1.The Identification Of Bone Marrow MSCs Bone marrow P₃ mononuclear cells showed positive staining of CD29,CD44 and CD71,negative staining of CD34,expressed Tn T protein（differentiating cardiac-like myocyte） after 5-aza inducing and ALP（differentiating sclerotomal-like cell） after sclerotomal cell liquid inducing.All those suggested bone marrow mononuclearcell were MSCs.2.Both AR and DR at different hypoxia time points The AR（0.09±2.03%, 12.9±1.72%,13.7±2.26%,13.8±3.01%,14.1±2.78%and 14.7±4.01%at 0,0.5,1, 4 and 8h,respectively,P＜0.01） and DR（0.04±1.79%,0.93±1.85%,3.11±2.14%, 4.09±2.36%,4.72±2.05%and 4.91±3.72%at 0,0.5,1,4 and 8h,respectively, P＜0.05） at different hypoxia time points significantly increased than normoxia; The AR further went up with time stretching（P＜0.05）,however,there was not statistic significance（P＞0.05） for the DR.3.Multiplication capability of MSCs at different hypoxia time points OD value of MTT methods at different hypoxia time points significantly decreased than normoxia（P＜0.01,synchronizing the end of hypoxia） and further aggravated, however,all significantly increased than normoxia（P＜0.05,synchronizing the begin of hypoxia 8h）.4.The change of ultrastructure of MSCs at different hypoxia time points Under normal state,there are abundant microvilli on the surface of MSCs,abundant endocytoplasmic reticulum and free ribosome in MSCs,however,microvilli of MSCs fell off,mitochondria appeared swelling（along with crista lost） once hypoxia,which were aggravated with hypoxia time extending.DiscussionThere are a little MSCs in bone marrow,its identify need these features such as stemming from bone marrow,adherence wall growth similar to desmocyte colony forming unit and at least differentiation for three kind cells and then were concluded inversely as MSCs.The bone marrow monomuclear cells,cultured by combining gradient centrifugation and adherence wall methods,were similar to the morphology of MSCs and were capability of differentiation for cardiac-like-myocytes after 5-aza inducing and sclerotomal cell after sclerotomal cell liquid inducing,all the above suggested that these bone marrow monomuclear cells were MSCs.The result that AR and DR at different hypoxia time points significantly increased than normoxia,no doubt,suggested that hypoxia may induce apoptosis and death of MSCs,however,the result that the AR was significantly, rapidly increased at hypoxia 0.5 h and further went slowly up with time stretching suggested that MSCs showed instantly inadaptability after hypoxia and adaptability with time stretching,the death may be a kind late phase of apoptosis.The result that the multiplication of MSCs at different hypoxia time points significantly decreased than normoxia（synchronizing the end of hypoxia） and further aggravated,all significantly increased than normoxia（synchronizing the begin of hypoxia 8h） suggested that hypoxia may make MSCs be "creep".In a word,our results strongly supported that hypoxia may results in apoptosis and the creep of multiplication ex vivo,which may be one of reasons for MSCs difficult to be survival in transplanted area after CCM.ConclusionsBone marrow monomuclear cells,which were cultured by combining gradient centrifugation and adherence wall methods,were mesenchymal stem cells;Hypoxia may results in apoptosis,the creep of cell multiplication,and the change of ultramicrostructure of MSCs ex vivo.Article 2:The Role Of Apoptosis Related mRNA And Protein of Bone Marrow MSCs When Hypoxia In VitroObjectiveTo elucidate the expression of apoptosis related mRNA and protein,such as Bcl-2,Bax,Fas,Fas L and Caspase-3,of bone marrow mesenchymal stem cells（MSCs） of Rat under hypoxia environment ex vivo.Materials and Methods1.The P3 MSCs were cultured in culturing chamber with 94%N₂,1%O₂,5% CO at 37℃.2.MSCs were cultured different hypoxia time points ie 0,0.5,1,2,4,6,8 and 12 h,respectively.3.AR were analyzed by FCM after Annexin V/PI staining.4.The expression of mRNA and protein,including Bcl-2,Bax,Fas,Fas L and Caspase-3,were observed by immunocytochemistry,Rt-PCR and western blot.4.The correlation between AR and the above mRNA/protein was analyzed by spss software.Results1.At normoxia,the protein of Bcl-2,Bax,Fas,Fas L and Caspase-3 were not found by immunocytochemistry methods,however,all of them were found after undergoing hypoxia.2.At above different hypoxia time points,the expression of the above protein and mRNA were significantly increased than normoxia（P＜0.05）;With hypoxia time stretching,there was not statistic significance（P＞0.05） in the expression of Bcl-2, the protein and mRNA of Bax,Fas,Fas L and Caspase-3 were further go up （P＜0.05）,however,there was not statistic significance（P＞0.05） at hypoxia 6 h～12 h（P＞0.05）.3.There was a significant negative correlation between the AR and the ratio of Bcl-2/Bax（mRNA:r₁=-0.435,P=0.040;protein:r₂=-0.417,P=0.043） and positive correlation between the AR and Fas（r₁=0.711,P=0.018;r₂=0.639,P=0.025）、Fas-L（r₁=0.605,P=0.037;r₂=0.581,P=0.022）、Caspase-3（r₁=0.657,P=0.026; r₂=0.704,P=0.014）.DiscussionIn the course of apoptosis,Bcl-2 gene is a kind of anti-apoptosis gene,and Bax gene is a kind of promoting apoptosis.Our result showed that the protein of Bcl-2,Bax,Fas,Fas L and Caspase-3 were not found by immunocytochemistry methods at normoxia and were found after undergoing hypoxia.The negative positive correlation between the AR and the ratio of Bcl-2/Bax mRNA and protein suggested that Bcl-2,Bax mRNA and protein may mediate hypoxic apoptpsis of MSCs,in which Bcl-2 inhibit apoptosis and Bax is contrary to Bcl-2.The positive correlation between the AR and Fas,Fas-L supported,no doubt,that both Fas and its ligand Fas-L take part in the regulation of apoptosis of MSCs,which are a kind of factors facilitating apoptosis.Caspase-3,the most key enzyme in apoptosis signal pathway,plays core role in regulating cell apoptosis and is a kind of promoting factors.On the whole,our results supported that both chondrosome pathway and receptor pathway took part in MSCs apoptotic process and that MSCs apoptosis may be dependent on caspase-3.ConclusionsDuring hypoxia promoting apotosis proceeding of MSCs,the mRNA and protein of Bcl-2 may be a kind of protect factors,however,the mRNA and protein of Bax,Fas-L,Fas and Caspase-3 may provoke apoptosis. Article 3:Both Apoptosis And Multiplication Of Bone Marrow MSCs Transfected By Akt Gene Under Hypoxic Environment In VitroObjectiveTo elucidate whether Akt gene improved the capacity of both anti-apoptosis and multiplication of bone marrow MSCs of Wistar rat,ie.bearing hypoxia capacity of MSCs under hypoxic environment ex vivo.Materials and Methods1.Both Akt gene transfection and non-Akt gene transfection MSCs of bone marrow of wistar rat were cultured in culturing chamber with 94%N₂,1%O₂, 5%CO₂ at 37℃for different hypoxia time points（normxia,hypoxia 0.5h,1h,2h, 4h and 8h）.2.Both AR and DR were analyzed by FCM after Annexin V/PI staining,cell multiplication by MTT methods,the expression of both Akt and p-Akt by immunofluorescence cytochemistry,Rt-PCR and western blot.Results1.Akt gene significantly decreased both AR and DR of MSCs under hypoxic environment（P＜0.01） and there was not statistic significance（P＞0.05） at different hypoxia time point.2.Akt gene significantly enhanced the capacity of multiplication of MSCs despite normoxic or hypoxic environment compared to non-Akt gene transfection,the capacity of multiplication of MSCs was significantly down-regulated in hypoxia than in nomoxia（P＜0.01）.3.Compared to non-Akt gene transfection,Akt gene significantly increased the expression of Akt mRNA （P＜0.01） and protein（P＜0.01） despite normoxia or hypoxia,and increased the expression of p-Akt protein（P＜0.01） in hypoxia and was not changed its expression in normoxia（P＞0.05）.Despite Akt gene transfection,both Akt and p-Akt protein were expressed in MSCs by immunocytochemistry while normoxia or hypoxia.DiscussionThe results,Akt gene significantly increasing the expression of Akt mRNA and protein and doing not change the expression of p-Akt protein at normoxia and being significantly increased once at hypoxia,suggested that Akt protein phosphorylation may not be enough "triggered" and/or its function is satisfied with the survival need of MSCs at normoxia,which inhibits its phosphorylation by "degenerative feedback" mechanism in return,and it is rapidly activated once being stimulated by hypoxia.The result,Akt gene significantly decreasing both AR and DR of MSCs under hypoxia and there being not statistic significance at different hypoxia time point,suggested that Akt gene may significantly enhanced bearing-hypoxia capacity of MSCs ex vivo,however,which mechanism has been suspended at present.The phenomenon,Akt gene did not influence both AR and DR at normoxia,implied that the intrinsic Akt concentration may provide the need of survival and multiplication for MSCs and do not play role in anti-apoptosis by overexpression at all.The results,the AR（DR） being increased and multiplication capability being decreased when MSCs transfected by Akt gene at hypoxia compared to MSCs at normoxia,hinted that there may exist other pathway influencing on the apoptosis and multiplication of MSCs.Briefly,Akt gene transfection may enhanced bearing-hypoxia capacity of MSCs in hypoxia ex vivo, which may be the reason for the effect of CCM by transplanting Akt gene transfection MSCs beyond the effect of simply MSCs transplant.ConclusionAkt gene transfection may significantly enhanced bearing-hypoxia capacity of MSCs in hypoxia ex vivo.Article 4:Cardiac Neuroprotective Effect of Mesenchymal Stem Cells Transduced with Akt in Adriamycin-Inducd Heart Failure ObjectiveTo elucidate whether Akt-MSCs adjust sympathetic and cholinergic nerve and whether the beneficial effects of Akt-MSCs and MSCs are mediated by their nerve fiber sprouting in vivo.Materials and Methodshealthy Wistar rat were adopted to draw out MSCs and build CHF model.Rats with CHF were randomized into three groups and then some index including myocardial norepinephrine （NE）,choline acetyltransferase（ChAT）,synaptophysin（SYN）,and growth-associated protein 43（GAP-43） was analyzed.Rats with CHF,induced by adriamycin,were randomized into Akt-MSCs group（n=11）, simple MSCs（s-MSCs,n=11） group and control group（n=12）.Each group was administered intravenously Akt-MSCs or s-MSCs（2×10⁶ cells in 100ul PBS,respectively） or the equal PBS,once a day for 3 times via tail vein,respectively.At the 4^th week,echocardiogrphic examine,contents of myocardial NE,ChAT,SYN and GAP-43 was analyzed.Results1.EF（P<0.01） was significantly improved in Akt-MSCs group and s-MSCs group than in control group,in which EF of the Akt-MSCs group was significant（P=0.001） in particularly. Mortality rate was not significant among three groups（P>0.05）.2.Tissue NE was lower in Akt-MSCs group and s-MSCs group than in control group （P=0.000）,in Akt-MSCs group than in s-MSCs group（P=0.000）.3.The expression of ChAT was not significant difference between Akt-MSCs group and s-MSCs group（P=0.851） but it was higher compared with control group（P<0.05）.4.The density of nerves,stained positive for SYN or GAP-43,were significantly higher in Akt-MSCs group than in s-MSCs group and control group,and in s-MSCs group than in control group,so is their semi-quantitative analysis.In the above comparisons,the P value was <0.01.ConclusionTransplantation of Akt-MSCs and MSCs,Akt-MSCs in particular,promoted cardiac nervous regeneration in fail heart,which might be mediated by growth-associated protein 43.更多还原