心肌微环境诱导间充质干细胞通道蛋白表达及丹参酮的干预作用研究
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摘要
研究背景
随着干细胞研究的逐渐深入,成体干细胞的心肌分化潜能给心肌损伤的治疗带来了新的希望。其中骨髓来源的间充质干细胞(Bone-derived Mesenchymal StemCells,BMSCs)具有取材方便、易于分离和体外培养扩增;免疫原性低、易于外源基因的导入和表达、可进行基因修饰;能分化为心肌细胞,内皮细胞和血管平滑肌细胞等诸多优势,成为治疗心脏疾患极有前景的种子细胞。诱导BMSCs向心肌分化的因素很多,其中胞嘧啶类化学物质5-氮胞苷(5-azachtine,5-aza)是最常用的化学诱导剂,除此之外,心肌的微环境是影响BMSCs在心脏局部存活和分化的重要因素。BMSCs和搏动心肌的接触,心肌细胞分泌的各种细胞因子都会影响BMSCs的分化。既往研究结果已经证实了,BMSCs移植后可以改善心功能。但是,人们更希望用有收缩力的细胞来替代坏死的心肌细胞,并且能与心肌同步收缩。同步收缩的前提是要产生同步兴奋。BMSCs是否具有产生生物电的基础,这是首先需要明确的问题,但这方面的研究较少。正常心脏的心电活动与节律性收缩,还有赖于心肌细胞之间信息通道传递的完整性,其结构基础是细胞间的间隙连接。连接蛋白43(connexin,Cx43)作为心肌细胞间隙连接组成中主要的连接蛋白,在维持心肌细胞的连接通讯功能,电信号传导和正常的节律性收缩中起重要作用。
在近几年对BMSCs的研究中,研究者发现,移植单纯的BMSCs之后,移植细胞在宿主细胞局部的存活率低,影响长期治疗效果。如果BMSCs与SCF、G-CSF以及AKT等细胞因子联合,或将BMSCs修饰后再进行移植,其治疗效果会明显提高。丹参是临床上治疗心血管病的最常用中药之一,丹参酮ⅡA是其脂溶性成分。现代药理研究显示,丹参酮ⅡA具有扩张冠脉,抗心肌急性缺氧、抗心律失常、抗心肌肥厚和抗血小板聚集等作用。鉴于以上原因,我们拟在体外条件下,模拟心肌微环境,研究正常和缺氧心肌上清液对BMSCs细胞膜上离子通道和连接蛋白43的影响;观察丹参酮ⅡA和心肌的上清液联合治疗是否有助于提高BMSCs之间Cx43的表达。
研究目的
1、研究SD大鼠BMSCs的体外分离、纯化和扩增,建立成熟而稳定的BMSCs培养体系,为后续的实验研究提供充足的细胞来源。
2、研究在未分化的BMSCs细胞膜上,5-aza诱导之后,正常心肌上清液和缺氧心肌上清液诱导之后,BMSCs细胞膜上的钠通道基因SCN2a、钾通道基因Kv1.4、Kv2.1、Kir1.1、EAG1和钙通道α亚单位基因CCHL2α的mRNA表达,推测心肌微环境对移植的BMSCs电生理特性的影响,为移植细胞在心肌局部微环境的作用下可能发生的离子通道的变化提供实验支持。
3、研究5-aza诱导后,正常心肌上清液和缺氧心肌上清液诱导后Cx43的表达量的变化;探讨心肌微环境在促进BMSCs之间电通讯连接的作用;进一步观察丹参酮ⅡA是否可促进BMSCs之间Cx43的建立以及与心肌上清液联合应用后的效果。探讨在BMSCs移植治疗心肌梗死的时候,丹参酮ⅡA是否具有协同作用,为中药联合细胞移植提高治疗效果提供实验基础。
研究方法
1、BMSCs的体外分离,纯化、扩增和鉴定。采用全骨髓贴壁筛选法分离培养SD大鼠的BMSCs,用免疫荧光染色法和流式细胞技术来检测BMSCs表面蛋白标志CD34、CD29、CD44和CD45抗原的表达情况,以鉴定BMSCs和判断BMSCs的纯化效果。
2、心肌微环境对大鼠BMSCs离子通道mRNA表达的影响。体外培养乳鼠心肌细胞,制备缺氧心肌细胞模型。取培养的正常心肌细胞和缺氧心肌细胞的上清液诱导BMSCs3周,同时用10μmol/L5-aza诱导。用逆转录-集合酶链式反应(reversetranscription-polymerase chain reaction,RT-PCR)法分别检测BMSCs细胞膜上与心肌细胞动作电位相关的离子通道的mRNA表达情况,包括钠通道基因SCN2a、钾通道基因Kv1.4、Kv2.1、Kir1.1、EAG1和钙通道α亚单位基因CCHL2α。
3、心肌微环境对BMSCsCx43的影响以及丹参酮ⅡA的干预作用。采用RT-PCR技术观察5-aza诱导后,正常心肌细胞和缺氧心肌上清液对BMSCsCx43mRNA表达的影响。采用Westernblotting和免疫荧光技术检测其Cx43蛋白的表达,分别观察高、低剂量丹参酮ⅡA是否可促进BMSCs之间Cx43的建立。将不同剂量的丹参酮ⅡA与缺氧心肌上清液联合应用,观察其促进Cx43表达的作用是否更佳。
研究结果
1、采用全骨髓贴壁筛选法分离BMSCs。原代取材后48h首次换液,即可去除未贴壁的血细胞和部分造血干细胞。3天后细胞进入对数生长期,增殖迅速,形态多为长梭形。原代第9-10天细胞可铺满瓶底,90%以上的细胞发生融合,呈放射状或漩涡形。传代后的细胞较快贴壁,无潜伏期,迅速进入生长期。5天左右即可进行下一次传代。在传代后的生长过程中,细胞排列较规律,多呈旋涡状。随着代数的增加,其他非BMSCs细胞逐渐减少,传代至第3代细胞时,细胞已比较纯化。传代到第6代之后,细胞的增殖能力开始下降,形态变扁平,折光性变差。部分细胞在形态上出现向脂肪细胞分化的趋势。取第3代细胞进行鉴定,用免疫荧光染色法检测表面蛋白标志CD44(BMSCs表面标志)和CD45抗原(造血干细胞表面标志),结果为CD44阳性表达、CD45阴性表达。用流式细胞技术检测表面蛋白标志CD29(BMSCs细胞表面标志)和CD34抗原(造血干细胞及内皮细胞表面标志),结果为送检细胞CD29阳性率占99.27%,CD34阴性率为1.28%。
2、取第3代BMSCs进行RT-PCR实验。结果显示,未分化的BMSCs细胞膜上表达与心肌动作电位相关的离子通道基因mRNA,包括钠通道基因SCN2a,钾通道基因Kv2.1、Kv1.4、Kir1.1,EAG1和钙通道α亚单位基因CCHL2α,但对于同一目的基因而言,在BMSCs上的表达量要低于心肌细胞(p＜0.01)。经10μmol/L5-aza诱导之后,BMSCs首先发生形态上的改变。细胞体积较前增大,由长梭形逐渐变为短棒状或不规则。2周后细胞开始融合,3周后形成肌管样结构,部分细胞形成球状。采用RT-PCR技术检测离子通道mRNA的改变,结果显示,SCN2a、Kv2.1、Kir1.1、EAG1和CCHL2α表达增加(p＜0.01),Kv1.4表达量变化不明显。经心肌细胞培养液和缺氧心肌细胞培养液诱导后,形态上的改变不明显。RT-PCR检测结果为,经正常心肌上清液诱导之后,SCN2a和Kv2.1mRNA的表达升高(p＜0.05),Kv1.4变化不明显,Kit1.1,EAG1和CCHL2αmRNA的表达明显升高(p＜0.01)。缺氧心肌上清液诱导后,各离子通道基因的mRNA表达与对照组相比,表达量均升高。在整个实验过程中,未见到BMSCs自发收缩现象。
3、取第3代BMSCs进行RT-PCR实验。BMSCs诱导3周后,可见未分化的BMSCs上Cx43mRNA有弱的表达;经5-aza诱导之后,正常心肌上清液以及缺氧心肌上清液诱导之后Cx43mRNA的表达量升高,P＜0.01。经丹参酮高剂量(1.5×10~(-7)mol/L)处理之后的BMSCs,其Cx43mRNA的表达量增加(P＜0.05),经丹参酮低剂量组(1.5×10~(-8)mol/L)处理之后,Cx43的表达明显升高(P＜0.01);经缺氧心肌上清液和丹参酮共同干预后,Cx43较对照组均有明显升高。但仍是丹参酮低剂量组作用更加显著(P＜0.01);缺氧心肌上清液和丹参酮低剂量组联合干预之后Cx43的表达接近心肌细胞组(p＞0.05),阳性对照组心肌细胞有强的Cx43的表达。与不同浓度丹参酮诱导BMSCs比较,缺氧心肌上清液和丹参酮的联合作用更强(p＜0.05)。Cx43mRNA在心肌细胞上有强表达。经western-blotting结果显示,各组均有不同强度的特异性条带出现,分子量约为43KD。进一步发现,在未分化的BMSCs上有Cx43蛋白的出现,经5-aza诱导之后,正常心肌上清液以及缺氧心肌上清液诱导之后Cx43的表达量升高;丹参酮ⅡA作用之后的变化与RT-PCR的结果一致,说明在蛋白质水平,丹参酮ⅡA仍可以上调Cx43的表达。采用Cx43抗体免疫荧光标记后的检测结果表明,各组细胞在荧光显微镜下均可见大致的细胞轮廓,细胞核呈蓝色(DAPI染色),胞浆和胞膜中有不同强度的点状绿色荧光。不同组间荧光强度的变化与RT-PCR和westernblotting的结果一致。
结论
1、本研究建立了成熟的SD大鼠BMSCs的体外培养方法,细胞增殖迅速,状态良好,生物学性状稳定,无过多的非BMSCs的干扰,经细胞形态和表面蛋白标志鉴定为BMSCs。
2、本研究证明了5-aza可诱导BMSCs细胞膜钠通道基因SCN2a,钾通道基因Kv2.1,Kv1.4,EAG1和Kir1.1和钙通道α亚单位CCHL2αmRNA表达量增加,从离子通道方面说明了5-aza可诱导BMSCs向心肌细胞方向分化;正常心肌上清液和缺氧心肌上清液在不依赖化学诱导剂的作用下,可促进BMSCs细胞膜上以上离子通道基因mRNA的表达,说明心肌细胞分泌的可溶性细胞因子可促进BMSCs表达离子通道蛋白,提示BMSCs移植后,心肌微环境有助于BMSCs在心脏局部分化,产生与心肌动作电位相关的离子通道蛋白。
3、本研究证实了5-aza可诱导BMSCs表达Cx43,证明了正常心肌上清液和缺氧心肌上清液均可促进Cx43在BMSCs上的表达,心肌微环境也有助于BMSCs之间连接蛋白的建立。不同浓度丹参酮ⅡA可上调BMSCs表达Cx43的作用,但丹参酮ⅡA和缺氧心肌上清液联合诱导作用更强。提示在BMSCs移植时联合应用丹参酮ⅡA可以提高细胞间的电信号通讯,在电生理改善方面提高移植效果。
成果与创新点
我们在体外条件下,从离子通道和连接蛋白角度探讨了化学诱导剂,心肌分泌的可溶性因子和中药丹参酮对BMSCs的作用。实验结果①本实验从与心肌动作电位产生相关的离子通道角度,证实了5-aza可诱导BMSCs向心肌细胞方法分化,丰富了5-aza作为心肌定向分化诱导剂的理论研究内容。②我们通过采用正常心肌/缺氧心肌的培养液对BMSCs的影响作用观察,来更加方便在体外观察心肌细胞的分泌环境对BMSCs的作用,了解移植细胞在宿主微环境下的变化。本研究证实了心肌可通过其分泌的细胞因子调节移植细胞离子通道和连接蛋白的表达,且此作用不依赖化学诱导剂的作用。这对于移植细胞在心肌局部因素作用下细胞膜离子通道和连接蛋白的改变提供了实验依据,为移植细胞在宿主局部是否会产生生物电以及具有致心律失常作用提供一定的理论参考。③国内研究中药单体或复方在BMSCs的影响方面,多集中在是否具有和5-aza同样的诱导分化作用,但诱导率比较低。本课题的创新之处在于,证明了丹参酮联合缺氧心肌上清液可明显提高BMSCsCx43的表达,作用要强于单独一种因素的作用。说明在细胞移植治疗心肌梗死过程中,联合应用活血化瘀中药,不仅有利于移植细胞在宿主局部的存活,而且可以提高细胞之间的电生理通讯,有助于移植细胞和宿主细胞发生同步兴奋,进而提高移植效果。为扩展中药的临床用途,为中医药进入细胞移植领域提供了新的切入途径。
Research background
With the deep study of stem cells,Adult stem cell for the potency which differentiate into cardiomyocyte lineage has a perspective for the therapy of myocardial damage.Bone-derived mesenchymal stem cells(BMSCs)has considered as a promising candidate for their some characteristics such as obtain easily,the less immunogenicity,easiness to separation and amplification in vitro,because it accepted exogenous gene importing and expression,gene would be modified,differentiation into the myocardial cell, endotheliocyte and vascular smooth muscle cell.Therefore,BMSCs are thought to be a suitable source of cell transplantation for the therapy of infarcted myocardium.There are many induced methods that made BMSCs differentiate into myocardial cells.5-azacytidine is frequently used the chemical inductor.Besides,the myocadial environment is the most important factor that affect the survival and differentiation of BMSCs in the local heart,including the contact between BMSCs and jumping myocardium,kinds of cytokine secreted by myocardial cell.It' s confirmed than BMSCs Transplantation improved the heart function.But,researchers hope that using the contractile cell substitute the damage myocardial cell,and then generate the synchronous contraction with myocardium,synchronous excitation would be the key foundation.This must be verified firstly whether BMSCs has this foundation for produce bioelectricity,but there were less exploration.The normal heart electrophysiological activity and rhythmic contraction depend on the integrity of message transmit between myocardial cell.The gap junction is the important message channel.Connexin 43(Cx43) is the main connexin in the gap junction,act as the structural and functional coupling of cardiomyocytes.
In recent years,researchers had discovered that transplantation cells would have lower survival rate in the local host after transplantating single BMSCs,that method might affect the long term therapeutic efficacy.The therapeutic efficacy would be improved if BMSCs transplantation combine the cytokines including SCF、G-CSF and AKT and so on,or modified BMSCs before transplantation.After BMSCs transplanting into the infarcted area of myocardium,the microenvironment might effect on the BMSCs for its survival and differentiation,as for electrophysiological property,the cell factor of myocardium secreted would be a important factor,salvia miltiorrhizae is the most commonly used Chinese materia medical to treat cardiovascular disease clinically.TanshenoneⅡA is liposoluble constituent of salvia miltiorrhizae. TanshenoneⅡA have some pharmacological action of cardiovascular system, such as relaxing coronary artery,anti-myocardium ischemia acutely, anti-arrhythmia,anti-myocardial hypertrophy and anti-platelet aggregation. So,we would aim to study the effects of myocardial microenvironment in vitro on the ion channels and Cx43 of BMSCs membrane,meanwhile,to observe the effects of TanshenoneⅡA on Cx43.
Research objective
1、To study the separation,purification and amplification of the BMSCs of Sprague Dawley(SD) rat in vitro,to establish the mature and stable culture system of BMSCs,to provide sufficient cell support for posterior experimental research.
2、To study the expression of some gene mRNA on the undifferentiated of BMSCs membrane induced by 5-aza,the normal and hypoxia myocyte supernatant during myocardium cultured,respectively,including SCN2a,sodium channel;Kv1.4, transient outward current channel;Kv2.1 and EAG-1,voltage-gated potassium channel;Kir1.1,inward rectifier potassium channel;CCHL2α,L-type calcium channelα-2 subunit.To infer the effects of myocardial microenvironment on the electrophysiological property of BMSCs,to provide the experimental evidence for the change of ionic channel on the transplanting cell affected by myocardial local infarcted environment.
3、To study the Cx43 expression of BMSCs induced by 5-aza,the normal and hypoxia myocyte supernatant respectively,to observe the effects of TanshenoneⅡA on the Cx43 expression of BMSCs and the synergistic effect between TanshenoneⅡA and BMSCs for the treatment of infarcted myocardium.
Research methods
1、The methods of the separation,purification,amplification and identification of the BMSCs of SD rat in vitro.To culture the rat BMSCs using the adherence screening method,to detect the antigenic expression of CD34,CD29,CD44 and CD45 using respectively the immunofluorescence staining method and flow cytometry.
2、The effects of myocardial supernatant on the ionic mRNA expression of BMSCs.The cadiocyte of neonate rat were isolated by enzyme and cultured,and then make the hypoxia myocardial cell model.BMSCs were induced by the normal and hypoxia myocyte supernatant during myocardium cultured.,10μmol/L 5-aza, After 3 weeks,to detect the expression of ionic channel gene mRNA that concerned with the action potential of myocardium on BMSCs,which include SCN2a,Kv1.4、Kv2.1、Kir1.1、EAG1 and CCHL2αusing RT-PCR.
3、The effects of myocardial supernatant and TanshenoneⅡA on Cx43 expression of BMSCs.RT-PCR technique was used to detect Cx43 mRNA expression of BMSCs that induced by 5-aza,the normal and hypoxia cadiocyte supernatant during myocardium cultured,to study the protein expression of Cx43 on BMSCs using westernblotting and immunofluorescence staining methods after applying the different concentration TanshinoneⅡA,and to proof the promoting effects of TanshenoneⅡA on the Cx43 establishment inside BMSCs.
Results
1、BMSCs were isolated by the whole bone marrow adherent cultivation.We operate the first medium change after 48 hours,to removal unadherent blood cells and a part of hemopoietic stem cell.The cells begin to enter the exponential phase of growth phase after 3days,cell begin rapid proliferation, as well as the majority of cells displayed spindle-like shape.The cell would spread out fully at the ninth or tenth day for original generation,above 90% cells get fusion,they have radiation or swirl shape.After the cell carried out passage,they adhere the culture dish quickly,no eclipse period,entering the growth period,at the fifth day cell might enter the next passage.The BMSCs array of shows a regular pattern,most are whirlpool shape.With continuous passage,other non-BMSCs were decreased gradually.The cell have been depurated at passage 3.The reproductive activity of BMSCs began to descend at passage 6,cell' s shape grow into thin and flat,poor refractive activity. Part of BMSCs have a trend of differentiating into adipose cell.cells of passage 3 were used identification.The results of CD44 and CD45 using the immunofluorescence staining are positive expression of CD44,negative expression of CD45(this is a cell-surface marker of hematopoietic cell) on BMSCs.The cell-surface marker of CD29(expressed BMSCs) and CD34 antigen (positive expression on the hemopoietic stem cell and endothelia cell)using flow cytometry,the results are 99.27%positive cell of CD29 and 1.28%negative cell of CD34.
2、Passage 3 were used to experiment through RT-PCR amplification.The results show that there were basic expression of SCN2a(sodium channel gene),Kv2.1,Kir1.1,EAG1 and Kv1.4 mRNA(potassium channel gene),CCHL2αmRNA of L-type calcium channelα-2 subunit on the undifferentiating BMSCs.There were lower expression level on BMSCs than myocardial cell as for the same gene(p<0.01).After 10μmol/L 5-aza induced,The morphous of BMSCs changed gradually,BMSCs were larger in size,from fusiform shape change into rod-shape or irregular form.After 2 weeks,the BMSCs began to fusion into each other,3 weeks later,myotulule-like structure formed,a part of BMSCs form some global-like shape.The results of ionic channel mRNA expression detected by RT-PCR are that SCN2a、Kv2.1、Kir1.1、EAG-1 and CCHL2αmRNA increase significantly at the expressing level(p<0.01),no obvious variance on Kv1.4 mRNA expression.The shape of BMSCs have no more change induced by normal myocyte and hypoxia myocyte culture fluid.In contrast of control group,the expression of SCN2a,Kv2.1 and Kv1.4 mRNA respectively increase induced by normal myocardial supernatant after 3 weeks,that had statistically significant(p<0.05),and the expression of Kir1.1,EAG-1 and CCHL2αmRNA also increase obviously(p<0.01).The expression of ionic channel mRNA have the same variation as the normal myocardial supernatant after induced by hypoxia myocardial supernatant,at expressing level just lower,but no statistically significant between two groups.There are no spontaneous contraction phenomenon on the BMSCs during the whole experimental process.
3、The result of RT-PCR show that the expression of Cx43 mRNA on undifferentiating of BMSCs is lower.However,the level of Cx43mRNA had been increased after induced by 5-aza,normal and hypoxia myocardial supernatant respectively(P<0.01).The effects of different TanshenoneⅡA on the Cx43mRNA of BMSCs are great,that are the Cx43 expression had more raise treated by 1.5×10~(-7)mol/L TanshenoneⅡA(P<0.05),significant increase treated by 1.5×10~(-8)mol/L TanshenoneⅡA(P<0.01).To compare with another groups,the increasing degree of Cx43 mRNA change very significantly after allying treated by 1.5×10~(-8)mol/L TanshinoneⅡA and hypoxia myocardial supernatant.The expression level of Cx43 after combining hypoxia myocadial supernatant and low-dose Tanshinone got close to the myocardium group(p>0.05).Certainly, there are obvious expression of Cx43 on myocardium.Meanwhile,The results detected by Westernblotting are there are special straps of different intensity on each group,molecular weight is 43KD.further,the Cx43 protein straps of BMSCs had increased after affected 5-aza,normal and hypoxia myocardial supernatant respectively contrast to the undifferentiating BMSCs. The effects of TanshenoneⅡA on Cx43 of BMSCs using Western blotting had same changes as the findings of RT-PCR.That to say,TanshenoneⅡA might also up regulate the expression of Cx43 on protein level.Furthermore,we observed the Cx43 variation between each group through antibody immunofluorescence labeling.That' s to found vague cell contour under fluorescence microscope in each group,cell nucleus is blue(DAPI dyeing),there are spot or strip green fluorescence on the endochylema and cell membrane,no same fluorescence were found on the blank control group(no Cx43 antibody),the results confirmed the specificity fluorescence massage from Cx43.The distribution of fluorescence on the cytoplasm and membrane of undifferentiating BMSCs were thin and weak intensity.After induced respectively by 5-aza,normal and hypoxia myocardial supernatant and therapied by Tanshinone,the fluorescence of Cx43 grew dense, increased fluorescence intensity.The change of fluorescent intensity among various groups had the same as RT-PCR and westernblotting.
Conclusion
1、We have established a successful culture system for BMSCs of SD rat in vitro.The cell were rapid proliferation,good condition and stable biological character,more purification,the cell identified by cell appearance and cell-surface marker was BMSCs.
2、5-aza would induce the gene mRNA expression increase on BMSCs membrane, which include SCN2a genefor sodium channel,Kv2.1,Kv1.4,Kir1.1 and EAG1 gene for potassium channel,CCHL2αof L-type calcium channelα-2 subunit.That indicate 5-aza would induce BMSCs differentiate into cardiomyocyte lineage on ionic channel respect.The normal and hypoxia myocardial supernatant respectively also promote the above-mentioned gene expression,and no depend on chemical induction.Our finding would infer that dissoluble cell factor secreted by local myocardium promote the ionic channel expression on BMSCs membrane,when BMSCs were transplanted into the infarcted myocardium.The occurrence of ionic channel on BMSCs membrane might contribute to generate resting potential and action potential.
3、5-aza would induce the Cx43 expression on BMSCs,meanwhile normal myocardial supernatant promote the Cx43 expression,hypoxia myocardial supernatant did alsoo Solo TanshenoneⅡA could up regulate the Cx43 expression of BMSCs,If TanshenoneⅡA and hypoxia myocardial supernatant were associated,the stimulating action would more powerful.This experimental results indicate that might improve the therapeutic efficacy of cell therapy if BMSCs transplantation would combine with TanshenoneⅡA.That would provide the new pathway for Chinese materia medical applying the cell transplantation. Achievement and innovative points
We have detected the effects of chemical inductor,soluble factor secreted myocardium and Chinese materia medica-Tanshenone on BMSCs from ionic channel and connexin aspect in vitro.The results are as follows:①many studies focus on whether or not BMSCs differentiating into the myocardial cells and expressing the specific myocardial protein on BMSCs by 5-aza.Our results confirmed that 5-aza would induce BMSCs differentiate into myocardium on ionic channels relating to action potential of myocardium.This findings raise to the level of theoretical research on 5-aza inductor.②We have observed the effects on BMSCs using the normal and hypoxia myocardial supernatant,and to detect conveniently the effects of myocardial microenvironment on BMSCs in vitro,furthermore to understand the change of grafting calls in local host microenvironment.This experiment confirmed that the cytokine secreted myocardium would regulate the expression of ionic channel and connexion43 proteins,and this action didn't depend on the chemical inductor.So,the findings would provide the experimental evidence for the change of electrophysiological proteins of BMSCs in local myocardial factor,and provide the theoreticai reference for grafting cell creating the bioelectricity and inducing arrhythmia.③The study of Chinese materia medica monomers or compound recipe on BMSCs in the domestic researcher focus on the induced effects of materia medica as so as 5-aza,but the results indicate the lower induced rate for them.The innovative points of our findings is that proved the high expression level of BMSCs Cx43 after treated Tanshenone and hypoxia myocardial supernatant,and this action has be higher than other factors.The results would indicate that the therapy for method cell transplantation combined the drug activating blood circulation to dissipate blood stasis,not only increasing the grafting cell survive,but also improving the electrical signal communication between cells,that contribute to the synchronous excitation,furthermore,to increase the transplanting effect. Also,our findings might provide a new therapy pathway for improving the effect of BMSCs.
随着干细胞研究的逐渐深入,成体干细胞的心肌分化潜能给心肌损伤的治疗带来了新的希望。其中骨髓来源的间充质干细胞(Bone-derived Mesenchymal StemCells,BMSCs)具有取材方便、易于分离和体外培养扩增;免疫原性低、易于外源基因的导入和表达、可进行基因修饰;能分化为心肌细胞,内皮细胞和血管平滑肌细胞等诸多优势,成为治疗心脏疾患极有前景的种子细胞。诱导BMSCs向心肌分化的因素很多,其中胞嘧啶类化学物质5-氮胞苷(5-azachtine,5-aza)是最常用的化学诱导剂,除此之外,心肌的微环境是影响BMSCs在心脏局部存活和分化的重要因素。BMSCs和搏动心肌的接触,心肌细胞分泌的各种细胞因子都会影响BMSCs的分化。既往研究结果已经证实了,BMSCs移植后可以改善心功能。但是,人们更希望用有收缩力的细胞来替代坏死的心肌细胞,并且能与心肌同步收缩。同步收缩的前提是要产生同步兴奋。BMSCs是否具有产生生物电的基础,这是首先需要明确的问题,但这方面的研究较少。正常心脏的心电活动与节律性收缩,还有赖于心肌细胞之间信息通道传递的完整性,其结构基础是细胞间的间隙连接。连接蛋白43(connexin,Cx43)作为心肌细胞间隙连接组成中主要的连接蛋白,在维持心肌细胞的连接通讯功能,电信号传导和正常的节律性收缩中起重要作用。
在近几年对BMSCs的研究中,研究者发现,移植单纯的BMSCs之后,移植细胞在宿主细胞局部的存活率低,影响长期治疗效果。如果BMSCs与SCF、G-CSF以及AKT等细胞因子联合,或将BMSCs修饰后再进行移植,其治疗效果会明显提高。丹参是临床上治疗心血管病的最常用中药之一,丹参酮ⅡA是其脂溶性成分。现代药理研究显示,丹参酮ⅡA具有扩张冠脉,抗心肌急性缺氧、抗心律失常、抗心肌肥厚和抗血小板聚集等作用。鉴于以上原因,我们拟在体外条件下,模拟心肌微环境,研究正常和缺氧心肌上清液对BMSCs细胞膜上离子通道和连接蛋白43的影响;观察丹参酮ⅡA和心肌的上清液联合治疗是否有助于提高BMSCs之间Cx43的表达。
研究目的
1、研究SD大鼠BMSCs的体外分离、纯化和扩增,建立成熟而稳定的BMSCs培养体系,为后续的实验研究提供充足的细胞来源。
2、研究在未分化的BMSCs细胞膜上,5-aza诱导之后,正常心肌上清液和缺氧心肌上清液诱导之后,BMSCs细胞膜上的钠通道基因SCN2a、钾通道基因Kv1.4、Kv2.1、Kir1.1、EAG1和钙通道α亚单位基因CCHL2α的mRNA表达,推测心肌微环境对移植的BMSCs电生理特性的影响,为移植细胞在心肌局部微环境的作用下可能发生的离子通道的变化提供实验支持。
3、研究5-aza诱导后,正常心肌上清液和缺氧心肌上清液诱导后Cx43的表达量的变化;探讨心肌微环境在促进BMSCs之间电通讯连接的作用;进一步观察丹参酮ⅡA是否可促进BMSCs之间Cx43的建立以及与心肌上清液联合应用后的效果。探讨在BMSCs移植治疗心肌梗死的时候,丹参酮ⅡA是否具有协同作用,为中药联合细胞移植提高治疗效果提供实验基础。
研究方法
1、BMSCs的体外分离,纯化、扩增和鉴定。采用全骨髓贴壁筛选法分离培养SD大鼠的BMSCs,用免疫荧光染色法和流式细胞技术来检测BMSCs表面蛋白标志CD34、CD29、CD44和CD45抗原的表达情况,以鉴定BMSCs和判断BMSCs的纯化效果。
2、心肌微环境对大鼠BMSCs离子通道mRNA表达的影响。体外培养乳鼠心肌细胞,制备缺氧心肌细胞模型。取培养的正常心肌细胞和缺氧心肌细胞的上清液诱导BMSCs3周,同时用10μmol/L5-aza诱导。用逆转录-集合酶链式反应(reversetranscription-polymerase chain reaction,RT-PCR)法分别检测BMSCs细胞膜上与心肌细胞动作电位相关的离子通道的mRNA表达情况,包括钠通道基因SCN2a、钾通道基因Kv1.4、Kv2.1、Kir1.1、EAG1和钙通道α亚单位基因CCHL2α。
3、心肌微环境对BMSCsCx43的影响以及丹参酮ⅡA的干预作用。采用RT-PCR技术观察5-aza诱导后,正常心肌细胞和缺氧心肌上清液对BMSCsCx43mRNA表达的影响。采用Westernblotting和免疫荧光技术检测其Cx43蛋白的表达,分别观察高、低剂量丹参酮ⅡA是否可促进BMSCs之间Cx43的建立。将不同剂量的丹参酮ⅡA与缺氧心肌上清液联合应用,观察其促进Cx43表达的作用是否更佳。
研究结果
1、采用全骨髓贴壁筛选法分离BMSCs。原代取材后48h首次换液,即可去除未贴壁的血细胞和部分造血干细胞。3天后细胞进入对数生长期,增殖迅速,形态多为长梭形。原代第9-10天细胞可铺满瓶底,90%以上的细胞发生融合,呈放射状或漩涡形。传代后的细胞较快贴壁,无潜伏期,迅速进入生长期。5天左右即可进行下一次传代。在传代后的生长过程中,细胞排列较规律,多呈旋涡状。随着代数的增加,其他非BMSCs细胞逐渐减少,传代至第3代细胞时,细胞已比较纯化。传代到第6代之后,细胞的增殖能力开始下降,形态变扁平,折光性变差。部分细胞在形态上出现向脂肪细胞分化的趋势。取第3代细胞进行鉴定,用免疫荧光染色法检测表面蛋白标志CD44(BMSCs表面标志)和CD45抗原(造血干细胞表面标志),结果为CD44阳性表达、CD45阴性表达。用流式细胞技术检测表面蛋白标志CD29(BMSCs细胞表面标志)和CD34抗原(造血干细胞及内皮细胞表面标志),结果为送检细胞CD29阳性率占99.27%,CD34阴性率为1.28%。
2、取第3代BMSCs进行RT-PCR实验。结果显示,未分化的BMSCs细胞膜上表达与心肌动作电位相关的离子通道基因mRNA,包括钠通道基因SCN2a,钾通道基因Kv2.1、Kv1.4、Kir1.1,EAG1和钙通道α亚单位基因CCHL2α,但对于同一目的基因而言,在BMSCs上的表达量要低于心肌细胞(p＜0.01)。经10μmol/L5-aza诱导之后,BMSCs首先发生形态上的改变。细胞体积较前增大,由长梭形逐渐变为短棒状或不规则。2周后细胞开始融合,3周后形成肌管样结构,部分细胞形成球状。采用RT-PCR技术检测离子通道mRNA的改变,结果显示,SCN2a、Kv2.1、Kir1.1、EAG1和CCHL2α表达增加(p＜0.01),Kv1.4表达量变化不明显。经心肌细胞培养液和缺氧心肌细胞培养液诱导后,形态上的改变不明显。RT-PCR检测结果为,经正常心肌上清液诱导之后,SCN2a和Kv2.1mRNA的表达升高(p＜0.05),Kv1.4变化不明显,Kit1.1,EAG1和CCHL2αmRNA的表达明显升高(p＜0.01)。缺氧心肌上清液诱导后,各离子通道基因的mRNA表达与对照组相比,表达量均升高。在整个实验过程中,未见到BMSCs自发收缩现象。
3、取第3代BMSCs进行RT-PCR实验。BMSCs诱导3周后,可见未分化的BMSCs上Cx43mRNA有弱的表达;经5-aza诱导之后,正常心肌上清液以及缺氧心肌上清液诱导之后Cx43mRNA的表达量升高,P＜0.01。经丹参酮高剂量(1.5×10~(-7)mol/L)处理之后的BMSCs,其Cx43mRNA的表达量增加(P＜0.05),经丹参酮低剂量组(1.5×10~(-8)mol/L)处理之后,Cx43的表达明显升高(P＜0.01);经缺氧心肌上清液和丹参酮共同干预后,Cx43较对照组均有明显升高。但仍是丹参酮低剂量组作用更加显著(P＜0.01);缺氧心肌上清液和丹参酮低剂量组联合干预之后Cx43的表达接近心肌细胞组(p＞0.05),阳性对照组心肌细胞有强的Cx43的表达。与不同浓度丹参酮诱导BMSCs比较,缺氧心肌上清液和丹参酮的联合作用更强(p＜0.05)。Cx43mRNA在心肌细胞上有强表达。经western-blotting结果显示,各组均有不同强度的特异性条带出现,分子量约为43KD。进一步发现,在未分化的BMSCs上有Cx43蛋白的出现,经5-aza诱导之后,正常心肌上清液以及缺氧心肌上清液诱导之后Cx43的表达量升高;丹参酮ⅡA作用之后的变化与RT-PCR的结果一致,说明在蛋白质水平,丹参酮ⅡA仍可以上调Cx43的表达。采用Cx43抗体免疫荧光标记后的检测结果表明,各组细胞在荧光显微镜下均可见大致的细胞轮廓,细胞核呈蓝色(DAPI染色),胞浆和胞膜中有不同强度的点状绿色荧光。不同组间荧光强度的变化与RT-PCR和westernblotting的结果一致。
结论
1、本研究建立了成熟的SD大鼠BMSCs的体外培养方法,细胞增殖迅速,状态良好,生物学性状稳定,无过多的非BMSCs的干扰,经细胞形态和表面蛋白标志鉴定为BMSCs。
2、本研究证明了5-aza可诱导BMSCs细胞膜钠通道基因SCN2a,钾通道基因Kv2.1,Kv1.4,EAG1和Kir1.1和钙通道α亚单位CCHL2αmRNA表达量增加,从离子通道方面说明了5-aza可诱导BMSCs向心肌细胞方向分化;正常心肌上清液和缺氧心肌上清液在不依赖化学诱导剂的作用下,可促进BMSCs细胞膜上以上离子通道基因mRNA的表达,说明心肌细胞分泌的可溶性细胞因子可促进BMSCs表达离子通道蛋白,提示BMSCs移植后,心肌微环境有助于BMSCs在心脏局部分化,产生与心肌动作电位相关的离子通道蛋白。
3、本研究证实了5-aza可诱导BMSCs表达Cx43,证明了正常心肌上清液和缺氧心肌上清液均可促进Cx43在BMSCs上的表达,心肌微环境也有助于BMSCs之间连接蛋白的建立。不同浓度丹参酮ⅡA可上调BMSCs表达Cx43的作用,但丹参酮ⅡA和缺氧心肌上清液联合诱导作用更强。提示在BMSCs移植时联合应用丹参酮ⅡA可以提高细胞间的电信号通讯,在电生理改善方面提高移植效果。
成果与创新点
我们在体外条件下,从离子通道和连接蛋白角度探讨了化学诱导剂,心肌分泌的可溶性因子和中药丹参酮对BMSCs的作用。实验结果①本实验从与心肌动作电位产生相关的离子通道角度,证实了5-aza可诱导BMSCs向心肌细胞方法分化,丰富了5-aza作为心肌定向分化诱导剂的理论研究内容。②我们通过采用正常心肌/缺氧心肌的培养液对BMSCs的影响作用观察,来更加方便在体外观察心肌细胞的分泌环境对BMSCs的作用,了解移植细胞在宿主微环境下的变化。本研究证实了心肌可通过其分泌的细胞因子调节移植细胞离子通道和连接蛋白的表达,且此作用不依赖化学诱导剂的作用。这对于移植细胞在心肌局部因素作用下细胞膜离子通道和连接蛋白的改变提供了实验依据,为移植细胞在宿主局部是否会产生生物电以及具有致心律失常作用提供一定的理论参考。③国内研究中药单体或复方在BMSCs的影响方面,多集中在是否具有和5-aza同样的诱导分化作用,但诱导率比较低。本课题的创新之处在于,证明了丹参酮联合缺氧心肌上清液可明显提高BMSCsCx43的表达,作用要强于单独一种因素的作用。说明在细胞移植治疗心肌梗死过程中,联合应用活血化瘀中药,不仅有利于移植细胞在宿主局部的存活,而且可以提高细胞之间的电生理通讯,有助于移植细胞和宿主细胞发生同步兴奋,进而提高移植效果。为扩展中药的临床用途,为中医药进入细胞移植领域提供了新的切入途径。
Research background
With the deep study of stem cells,Adult stem cell for the potency which differentiate into cardiomyocyte lineage has a perspective for the therapy of myocardial damage.Bone-derived mesenchymal stem cells(BMSCs)has considered as a promising candidate for their some characteristics such as obtain easily,the less immunogenicity,easiness to separation and amplification in vitro,because it accepted exogenous gene importing and expression,gene would be modified,differentiation into the myocardial cell, endotheliocyte and vascular smooth muscle cell.Therefore,BMSCs are thought to be a suitable source of cell transplantation for the therapy of infarcted myocardium.There are many induced methods that made BMSCs differentiate into myocardial cells.5-azacytidine is frequently used the chemical inductor.Besides,the myocadial environment is the most important factor that affect the survival and differentiation of BMSCs in the local heart,including the contact between BMSCs and jumping myocardium,kinds of cytokine secreted by myocardial cell.It' s confirmed than BMSCs Transplantation improved the heart function.But,researchers hope that using the contractile cell substitute the damage myocardial cell,and then generate the synchronous contraction with myocardium,synchronous excitation would be the key foundation.This must be verified firstly whether BMSCs has this foundation for produce bioelectricity,but there were less exploration.The normal heart electrophysiological activity and rhythmic contraction depend on the integrity of message transmit between myocardial cell.The gap junction is the important message channel.Connexin 43(Cx43) is the main connexin in the gap junction,act as the structural and functional coupling of cardiomyocytes.
In recent years,researchers had discovered that transplantation cells would have lower survival rate in the local host after transplantating single BMSCs,that method might affect the long term therapeutic efficacy.The therapeutic efficacy would be improved if BMSCs transplantation combine the cytokines including SCF、G-CSF and AKT and so on,or modified BMSCs before transplantation.After BMSCs transplanting into the infarcted area of myocardium,the microenvironment might effect on the BMSCs for its survival and differentiation,as for electrophysiological property,the cell factor of myocardium secreted would be a important factor,salvia miltiorrhizae is the most commonly used Chinese materia medical to treat cardiovascular disease clinically.TanshenoneⅡA is liposoluble constituent of salvia miltiorrhizae. TanshenoneⅡA have some pharmacological action of cardiovascular system, such as relaxing coronary artery,anti-myocardium ischemia acutely, anti-arrhythmia,anti-myocardial hypertrophy and anti-platelet aggregation. So,we would aim to study the effects of myocardial microenvironment in vitro on the ion channels and Cx43 of BMSCs membrane,meanwhile,to observe the effects of TanshenoneⅡA on Cx43.
Research objective
1、To study the separation,purification and amplification of the BMSCs of Sprague Dawley(SD) rat in vitro,to establish the mature and stable culture system of BMSCs,to provide sufficient cell support for posterior experimental research.
2、To study the expression of some gene mRNA on the undifferentiated of BMSCs membrane induced by 5-aza,the normal and hypoxia myocyte supernatant during myocardium cultured,respectively,including SCN2a,sodium channel;Kv1.4, transient outward current channel;Kv2.1 and EAG-1,voltage-gated potassium channel;Kir1.1,inward rectifier potassium channel;CCHL2α,L-type calcium channelα-2 subunit.To infer the effects of myocardial microenvironment on the electrophysiological property of BMSCs,to provide the experimental evidence for the change of ionic channel on the transplanting cell affected by myocardial local infarcted environment.
3、To study the Cx43 expression of BMSCs induced by 5-aza,the normal and hypoxia myocyte supernatant respectively,to observe the effects of TanshenoneⅡA on the Cx43 expression of BMSCs and the synergistic effect between TanshenoneⅡA and BMSCs for the treatment of infarcted myocardium.
Research methods
1、The methods of the separation,purification,amplification and identification of the BMSCs of SD rat in vitro.To culture the rat BMSCs using the adherence screening method,to detect the antigenic expression of CD34,CD29,CD44 and CD45 using respectively the immunofluorescence staining method and flow cytometry.
2、The effects of myocardial supernatant on the ionic mRNA expression of BMSCs.The cadiocyte of neonate rat were isolated by enzyme and cultured,and then make the hypoxia myocardial cell model.BMSCs were induced by the normal and hypoxia myocyte supernatant during myocardium cultured.,10μmol/L 5-aza, After 3 weeks,to detect the expression of ionic channel gene mRNA that concerned with the action potential of myocardium on BMSCs,which include SCN2a,Kv1.4、Kv2.1、Kir1.1、EAG1 and CCHL2αusing RT-PCR.
3、The effects of myocardial supernatant and TanshenoneⅡA on Cx43 expression of BMSCs.RT-PCR technique was used to detect Cx43 mRNA expression of BMSCs that induced by 5-aza,the normal and hypoxia cadiocyte supernatant during myocardium cultured,to study the protein expression of Cx43 on BMSCs using westernblotting and immunofluorescence staining methods after applying the different concentration TanshinoneⅡA,and to proof the promoting effects of TanshenoneⅡA on the Cx43 establishment inside BMSCs.
Results
1、BMSCs were isolated by the whole bone marrow adherent cultivation.We operate the first medium change after 48 hours,to removal unadherent blood cells and a part of hemopoietic stem cell.The cells begin to enter the exponential phase of growth phase after 3days,cell begin rapid proliferation, as well as the majority of cells displayed spindle-like shape.The cell would spread out fully at the ninth or tenth day for original generation,above 90% cells get fusion,they have radiation or swirl shape.After the cell carried out passage,they adhere the culture dish quickly,no eclipse period,entering the growth period,at the fifth day cell might enter the next passage.The BMSCs array of shows a regular pattern,most are whirlpool shape.With continuous passage,other non-BMSCs were decreased gradually.The cell have been depurated at passage 3.The reproductive activity of BMSCs began to descend at passage 6,cell' s shape grow into thin and flat,poor refractive activity. Part of BMSCs have a trend of differentiating into adipose cell.cells of passage 3 were used identification.The results of CD44 and CD45 using the immunofluorescence staining are positive expression of CD44,negative expression of CD45(this is a cell-surface marker of hematopoietic cell) on BMSCs.The cell-surface marker of CD29(expressed BMSCs) and CD34 antigen (positive expression on the hemopoietic stem cell and endothelia cell)using flow cytometry,the results are 99.27%positive cell of CD29 and 1.28%negative cell of CD34.
2、Passage 3 were used to experiment through RT-PCR amplification.The results show that there were basic expression of SCN2a(sodium channel gene),Kv2.1,Kir1.1,EAG1 and Kv1.4 mRNA(potassium channel gene),CCHL2αmRNA of L-type calcium channelα-2 subunit on the undifferentiating BMSCs.There were lower expression level on BMSCs than myocardial cell as for the same gene(p<0.01).After 10μmol/L 5-aza induced,The morphous of BMSCs changed gradually,BMSCs were larger in size,from fusiform shape change into rod-shape or irregular form.After 2 weeks,the BMSCs began to fusion into each other,3 weeks later,myotulule-like structure formed,a part of BMSCs form some global-like shape.The results of ionic channel mRNA expression detected by RT-PCR are that SCN2a、Kv2.1、Kir1.1、EAG-1 and CCHL2αmRNA increase significantly at the expressing level(p<0.01),no obvious variance on Kv1.4 mRNA expression.The shape of BMSCs have no more change induced by normal myocyte and hypoxia myocyte culture fluid.In contrast of control group,the expression of SCN2a,Kv2.1 and Kv1.4 mRNA respectively increase induced by normal myocardial supernatant after 3 weeks,that had statistically significant(p<0.05),and the expression of Kir1.1,EAG-1 and CCHL2αmRNA also increase obviously(p<0.01).The expression of ionic channel mRNA have the same variation as the normal myocardial supernatant after induced by hypoxia myocardial supernatant,at expressing level just lower,but no statistically significant between two groups.There are no spontaneous contraction phenomenon on the BMSCs during the whole experimental process.
3、The result of RT-PCR show that the expression of Cx43 mRNA on undifferentiating of BMSCs is lower.However,the level of Cx43mRNA had been increased after induced by 5-aza,normal and hypoxia myocardial supernatant respectively(P<0.01).The effects of different TanshenoneⅡA on the Cx43mRNA of BMSCs are great,that are the Cx43 expression had more raise treated by 1.5×10~(-7)mol/L TanshenoneⅡA(P<0.05),significant increase treated by 1.5×10~(-8)mol/L TanshenoneⅡA(P<0.01).To compare with another groups,the increasing degree of Cx43 mRNA change very significantly after allying treated by 1.5×10~(-8)mol/L TanshinoneⅡA and hypoxia myocardial supernatant.The expression level of Cx43 after combining hypoxia myocadial supernatant and low-dose Tanshinone got close to the myocardium group(p>0.05).Certainly, there are obvious expression of Cx43 on myocardium.Meanwhile,The results detected by Westernblotting are there are special straps of different intensity on each group,molecular weight is 43KD.further,the Cx43 protein straps of BMSCs had increased after affected 5-aza,normal and hypoxia myocardial supernatant respectively contrast to the undifferentiating BMSCs. The effects of TanshenoneⅡA on Cx43 of BMSCs using Western blotting had same changes as the findings of RT-PCR.That to say,TanshenoneⅡA might also up regulate the expression of Cx43 on protein level.Furthermore,we observed the Cx43 variation between each group through antibody immunofluorescence labeling.That' s to found vague cell contour under fluorescence microscope in each group,cell nucleus is blue(DAPI dyeing),there are spot or strip green fluorescence on the endochylema and cell membrane,no same fluorescence were found on the blank control group(no Cx43 antibody),the results confirmed the specificity fluorescence massage from Cx43.The distribution of fluorescence on the cytoplasm and membrane of undifferentiating BMSCs were thin and weak intensity.After induced respectively by 5-aza,normal and hypoxia myocardial supernatant and therapied by Tanshinone,the fluorescence of Cx43 grew dense, increased fluorescence intensity.The change of fluorescent intensity among various groups had the same as RT-PCR and westernblotting.
Conclusion
1、We have established a successful culture system for BMSCs of SD rat in vitro.The cell were rapid proliferation,good condition and stable biological character,more purification,the cell identified by cell appearance and cell-surface marker was BMSCs.
2、5-aza would induce the gene mRNA expression increase on BMSCs membrane, which include SCN2a genefor sodium channel,Kv2.1,Kv1.4,Kir1.1 and EAG1 gene for potassium channel,CCHL2αof L-type calcium channelα-2 subunit.That indicate 5-aza would induce BMSCs differentiate into cardiomyocyte lineage on ionic channel respect.The normal and hypoxia myocardial supernatant respectively also promote the above-mentioned gene expression,and no depend on chemical induction.Our finding would infer that dissoluble cell factor secreted by local myocardium promote the ionic channel expression on BMSCs membrane,when BMSCs were transplanted into the infarcted myocardium.The occurrence of ionic channel on BMSCs membrane might contribute to generate resting potential and action potential.
3、5-aza would induce the Cx43 expression on BMSCs,meanwhile normal myocardial supernatant promote the Cx43 expression,hypoxia myocardial supernatant did alsoo Solo TanshenoneⅡA could up regulate the Cx43 expression of BMSCs,If TanshenoneⅡA and hypoxia myocardial supernatant were associated,the stimulating action would more powerful.This experimental results indicate that might improve the therapeutic efficacy of cell therapy if BMSCs transplantation would combine with TanshenoneⅡA.That would provide the new pathway for Chinese materia medical applying the cell transplantation. Achievement and innovative points
We have detected the effects of chemical inductor,soluble factor secreted myocardium and Chinese materia medica-Tanshenone on BMSCs from ionic channel and connexin aspect in vitro.The results are as follows:①many studies focus on whether or not BMSCs differentiating into the myocardial cells and expressing the specific myocardial protein on BMSCs by 5-aza.Our results confirmed that 5-aza would induce BMSCs differentiate into myocardium on ionic channels relating to action potential of myocardium.This findings raise to the level of theoretical research on 5-aza inductor.②We have observed the effects on BMSCs using the normal and hypoxia myocardial supernatant,and to detect conveniently the effects of myocardial microenvironment on BMSCs in vitro,furthermore to understand the change of grafting calls in local host microenvironment.This experiment confirmed that the cytokine secreted myocardium would regulate the expression of ionic channel and connexion43 proteins,and this action didn't depend on the chemical inductor.So,the findings would provide the experimental evidence for the change of electrophysiological proteins of BMSCs in local myocardial factor,and provide the theoreticai reference for grafting cell creating the bioelectricity and inducing arrhythmia.③The study of Chinese materia medica monomers or compound recipe on BMSCs in the domestic researcher focus on the induced effects of materia medica as so as 5-aza,but the results indicate the lower induced rate for them.The innovative points of our findings is that proved the high expression level of BMSCs Cx43 after treated Tanshenone and hypoxia myocardial supernatant,and this action has be higher than other factors.The results would indicate that the therapy for method cell transplantation combined the drug activating blood circulation to dissipate blood stasis,not only increasing the grafting cell survive,but also improving the electrical signal communication between cells,that contribute to the synchronous excitation,furthermore,to increase the transplanting effect. Also,our findings might provide a new therapy pathway for improving the effect of BMSCs.
引文
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