Hypoxic Apoptotic Mechanism Of Rat Bone Marrow Mesenchymal Stem Cells And Akt Gene Influencing On Its Hypoxic Apoptosis In Vitro

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.