脉冲电流刺激对MSCs与心肌细胞共培养层分化状态的作用研究

发布时间：2018-05-01 04:16

本文选题：脉冲微电流 + S100A4　；参考：《第三军医大学》2012年博士论文

【摘要】：一、背景与目的冠状动脉粥样硬化性心脏病(简称冠心病)是临床常见病、多发病，严重的危害人类健康。冠心病的最严重类型---急性心肌梗死(AMI)，因冠状动脉急性闭塞和心肌组织急性缺血，可在15～20分钟内导致心肌细胞不可逆性损伤。心肌梗死后心室发生重构，随之而来的是心肌细胞被替换为纤维组织，导致心肌细胞收缩功能减退或消失，继之出现左心室扩张等病理改变。间充质干细胞是已经是比较获得认可的可用于心脏移植的细胞类型,在心肌梗死后，间充质干细胞移植可以减少坏死，分泌许多促进血管生成的细胞因子和改善心脏功能,还有研究认为间充质干细胞可作为支持心肌干细胞生长重要的支持细胞。如间充质干细胞可以招募许多的内源性干细胞，这些干细胞为损伤部位带了了修复，而非仅仅间充质干细胞，这其中就包括了非常有潜力的心肌干细胞。在对病窦综合征的研究中，HCN基因家族修饰的间充质干细胞用于重建起搏点。如何更好的促进间充质干细胞移植后的分化和移植后的效果也是研究人员不断探索的方向，其中分子手段是比较有效的方式，但由于慢病毒等手段的临床应用受到限制，而在心血管内科治疗中各种物理手段在临床应用较多，那么可否使用物理手段干预间充质干细胞分化过程？或者改变间充质与心肌共培养层分化状态？在正常生理状态过程中，内源性的电流、电场扮演了影响分化的重要角色，内源性的电场对于胚胎的分化作用十分重要。本课题的目的是探求外源性电干预可否作为促进间充质向心肌分化或者改变间充质与心肌共培养层分化状态的一种手段的可能性与可行性，寻找合适的电干预参数，设计制作方便实用的电干预系统，了解间充质干细胞向心脏分化潜能，寻找其作用的靶点和初步确定了解合适的干预时间。二、研究方法 1.反复结合EPCS使用效果反馈制作了EPCS培养装置。 2.膜片钳检测电流干预对共培养条件下间充质干细胞膜电位的影响， 3.将间充质干细胞分为普通培养组和EPCS处理组，检测EPCS对其增殖(cck-8andPCNA)，表面标记物，形成缝隙连接潜力影响。检测EPCS对其搏动维持，搏动频率，及肌钙蛋白，肌球蛋白表达在转录水平的影响。 4.将5-aza处理后的3w后的小鼠间充质干细胞分为普通培养组和EPCS组，检测EPCS对其诱导效果影响。 5.间充质干细胞分化情况检测：间充质干细胞与心肌共培养4-5天后分别检测CX43，Nav1.5，CaMKⅡ表达情况；将间充质干细胞和心肌细胞按4:1比例种植于培养皿中，取48h，72h和96h进行western-blotting实验，检测CaMKⅡ含量的动态变化。 6.共培养5天后，检测共培养层GATA4,MEF2c,TBX5相关转录因子表达的情况；检测蛋白质核转运受体在共培养层表达。 7.将细胞分为两组，普通共培养培养组和钙离子通道阻滞共培养组。共培养5天后，检测细胞上VSCC(电压敏感钙通道)的表达和钙瞬变情况。检测MEF2C，GATA4和TBX5表达情况。检测细胞钙调神经磷酸酶活力；western-blotting检测细胞TroponinT， NAV1.5， CACNA1G/H和CX43蛋白含量变化。 8.间充质和心肌细胞在初期接种时的比例为３：１。每组给予1h/d，3h/d和6h/d的EPCS干预，EPCS从接种后24h开始，持续4天，同期接种的未进行EPCS干预的共培养细胞作为对照组。用蛋白印迹法检测troponinT和CX43在每个不同时间处理组表达含量。 9.用免疫荧光检测EPCS连续处理5d组，及对照组在S100A4，MEF2C,troponinT,CX43和GATA4表达情况。三、研究结果 1.结合试验反馈反复摸索分析中制备了一套稳定可靠的电刺激给予和接受装置(EPCS培养系统)，我们可实时对其干预信号监测，这套系统便于使用和准确给予EPCS刺激。 2.膜片钳检测显示在共培养条件下的间充质干细胞在接受到膜片钳仪器提供直流电流刺激后产生膜电位微小改变。 3.在心肌细胞长期体外培养中EPCS可以维持少数区域较良好的搏动状态和形态。 4.安全性检测方面：EPCS对心肌细胞搏动频率未见明显影响；.EPCS不影响间充质干细胞增殖(cck-8实验，PCNA蛋白含量检测)，形成缝隙连接的潜能；EPCS对间充质干细胞表面标记物中的CD44分子具有轻度下调作用，不随每日干预时间延长增加下调幅度；EPCS处理9天，在体外培养共计12天的心肌细胞其肌钙蛋白，肌球蛋白基因转录水平未见影响。EPCS对5-aza诱导的间充质干细胞过程未见影响。 5.狗间充质干细胞心肌分化潜能鉴定：在于心肌细胞共培养后，间充质干细胞可以出现缝隙连接蛋白，钠离子通道Nav1.5，CaMKⅡ，和转录因子GATA4，MEF2C和TBX5表达。GATA4在心肌细胞核的浓度远高于间充质干细胞，是其在间充质细胞核的4.49倍，P0.01。间充质与心肌细胞培养层(4：1)中CaMKⅡ含量随共培养时间延长逐渐升高，CaMKⅡ表现出强上升趋势，尤其是共培养后72-96小时。在核转运受体中， Karyopherinα3的分布间充质干细胞和心肌细胞的分布有比较明显的差异，Karyopherinα4和Karyopherinα5在间充质干细胞和部分心肌细胞上存在差异，Karyopherin β1，Karyopherin α1/6，和Karyopherin α2在间充质干细胞和心肌细胞的分布无明显差异，其中Karyopherin α3表达更强的心肌细胞的阳性比例与核富集GATA4心肌细胞的比例相似(90%)。钙通道阻滞诱发原代培养心肌细胞内成纤维细胞强烈增生和S100A4表达升高。间充质干细胞表面钙离子离子通道的形成较强依赖于正常钙电流活动。阻断钙离子流培养对间充质干细胞钙瞬变产生幅度减小和时间缩短影响。钙离子通道阻滞导致间充质心肌细胞共培养层部分心肌转录因子升高，但未见引发下游结构蛋白含量增高。 6.CX43和TroponinT随每天EPCS干预时间的延长显示出逐渐上升趋势。CX43在3h/d组和6h/d组表现出1.5倍和1.7倍的升高(与对照组比较,p0.01)；肌钙蛋白T在3h/d和6h/d组表现出3.6倍(P0.01)和4.4倍(P0.05)的升高。 7.在5天连续的EPCS处理后，发现S100A4升高显著，其在心肌细胞是对照组的2.33倍(P0.01)在间充质干细胞是对照组的1.99倍(P0.01)。在心肌细胞MEF2C和GATA4的表达量升高了1.63倍(P0.01)和1.57倍(P0.01)，在心肌细胞EPCS可促进MEF2C在细胞核集中表达，同时我们也发现了更多数量的双核心肌细胞，MSC则对于EPCS触发出现的MEF2C升高没有反应， MEF2C在心肌细胞胞浆和细胞核的升高可以将共培养体系中的心肌细胞和间充质干细胞较清晰区分开来。也有一些指标在EPCS处理后在心肌细胞和间充质干细胞均出现升高，这包括强烈升高的troponin T和升高幅度相对弱些的CX43，其中肌钙蛋白T在心肌细胞为对照组的2倍(P0.01)，CX43的表达在心肌细胞为对照组的1.4倍(P0.05)；在间充质干细胞肌钙蛋白T为对照组1.88倍(P0.01)，CX43为对照组的1.94倍(P0.01),在共培养层中，EPCS组心肌细胞S100A4和troponin t出现了明显的核聚集现象，极少MSC上有此现象。四、结论 1.合适的EPCS干预是一种非常有效地多靶位干预间充质干细胞和心肌细胞共培养层分化程度的物理方式。 2.间充质干细胞细胞核中GATA4得含量远低于其在心肌细胞核含量。在蛋白核转运受体中，Karyopherin3的分布间充质干细胞和心肌细胞的分布有比较明显的差异，Karyopherin4和Karyopherin5在间充质干细胞和部分心肌细胞上存在差异。 3.间充质干细胞钙离子通道的形成依赖于共培养后共培养层正常钙电流活动。 4.间充质干细胞在接受EPCS方面的敏感性和广泛性上不及心肌细胞。EPCS处理后，S100A4，，TroponinT虽然有整体水平升高，但心肌出现的出现的核内较胞质更加富集现象在间充质干细胞鲜见，提示间充质干细胞部分分化障碍可能和重要分化相关蛋白质主动转运入核相关。在心肌细胞和间充质培养层中，心肌分化重要转录因子MEF2C在间充质干细胞的表达不受EPCS干预升高，而在心肌细胞升高，提示间充质干细胞本身对EPCS感受性有限。 5.EPCS引起间充质干细胞S100A4升高，而既往文献认为S100A4是顶叶内胚层分泌促进心肌分化的因子之一，说明EPCS可能增强间充质干细胞在间充质干细胞与心肌细胞共培养层的支持作用。
[Abstract]:First, background and purpose
Coronary atherosclerotic heart disease (CAD) is a common clinical disease, which is frequently occurring and seriously endangers human health. The most serious type of coronary heart disease, acute myocardial infarction (AMI), can cause myocardial irreversible damage in 15~20 minutes due to acute coronary occlusion and acute myocardial ischemia. The result of reconstruction is that the cardiomyocytes are replaced with fibrous tissue, which leads to the decrease or disappearance of the systolic function of cardiac myocytes, followed by pathological changes such as left ventricular dilatation.
Mesenchymal stem cells are already an approved cell type that can be recognized in heart transplantation. After myocardial infarction, mesenchymal stem cell transplantation can reduce necrosis, secrete many cytokines that promote angiogenesis and improve cardiac function. There is also a study that mesenchymal stem cells can be important in supporting the growth of cardiac stem cells. Support cells, such as mesenchymal stem cells, can recruit many endogenous stem cells, which have been repaired for damaged sites, not only mesenchymal stem cells, which include very potential cardiac stem cells. In the study of sinus syndrome, HCN based family modified mesenchymal stem cells are used for reconstruction. How to better promote the differentiation and post transplant effect of mesenchymal stem cells after transplantation is also the direction for researchers to explore. Molecular means are more effective, but the clinical application of lentivirus is limited, and various physical means in the treatment of cardiovascular medicine are widely used in clinical practice. Can the differentiation process of mesenchymal stem cells be interfered with physical means or change the state of differentiation between mesenchymal and myocardial co culture layer? In normal physiological state, endogenous electric current and electric field play an important role in the differentiation, and the endogenous electric field is very important for the differentiation of embryos. The possibility and feasibility of source electrical interference can be used as a means to promote differentiation of mesenchyme to myocardium or to change the differentiation state of mesenchymal and myocardial co culture layer. To find appropriate electrical interference parameters and to design a convenient and practical electrical interference system to understand the potential of mesenchymal stem cells to heart differentiation, and to find the targets and targets for its role. A preliminary determination of the appropriate intervention time.
Two, research methods
1. a EPCS culture device was produced by repeated use of EPCS feedback.
2. patch clamp technique was used to detect the effect of current intervention on the membrane potential of mesenchymal stem cells under co culture.
3. the mesenchymal stem cells were divided into the common culture group and the EPCS treatment group. The effects of EPCS on its proliferation (cck-8andPCNA), surface markers, and the potential of gap junction potential were detected. The effects of EPCS on its pulsation maintenance, pulsation frequency, and troponin and myosin expression at the transcriptional level were detected.
4. mouse mesenchymal stem cells after 3W treated by 5-aza were divided into normal culture group and EPCS group, and the effect of EPCS on its induction was detected.
The differentiation of 5. mesenchymal stem cells: the expression of CX43, Nav1.5 and CaMK II was detected for 4-5 days after the co culture of mesenchymal stem cells and myocardium, and the mesenchymal stem cells and cardiomyocytes were planted in the culture dish in proportion to 4:1, and 48h, 72h and 96h were taken for Western-blotting experiment to detect the dynamic changes of CaMK II content.
6. after 5 days of co culture, the expression of GATA4, MEF2c and TBX5 related transcription factors in the co culture layer was detected, and the protein nuclear transport receptor was detected in the coculture layer.
7. the cells were divided into two groups, the common culture culture group and the calcium ion channel block co culture group. The expression of VSCC (voltage sensitive calcium channel) and the transient state of calcium were detected for 5 days. The expression of MEF2C, GATA4 and TBX5 were detected. The activity of cell calcineurin was detected, and TroponinT, NAV1.5, C of cells were detected by Western-blotting. Changes in the content of ACNA1G/H and CX43 protein.
The proportion of 8. mesenchymal and cardiac myocytes at initial inoculation was 3:1. each group was given 1h/d, 3h/d and 6h/d EPCS intervention. EPCS started from 24h after inoculation and lasted for 4 days. Co cultured cells without EPCS intervention were used as control group during the same period. The expression of troponinT and CX43 in each time treatment group was detected by Western blot.
9. immunofluorescence was used to detect the expression of EPCS, 5D, S100A4, MEF2C, troponinT, CX43 and GATA4 in continuous treatment group.
Three, the results of the study
1. a set of stable and reliable electrical stimulation giving and receiving devices (EPCS culture system) was prepared in combination with repeated test feedback analysis. We can monitor the interference signal in real time. This system is easy to use and accurately give EPCS stimulation.
2. patch clamp test showed that the membrane potential of MSCs in co culture condition changed slightly after receiving DC current stimulation.
3. in the long-term culture of cardiac myocytes, EPCS can maintain a relatively good beating state and morphology in a few regions.
4. security detection: EPCS has no significant influence on the pulsation frequency of cardiac myocytes;.EPCS does not affect the proliferation of mesenchymal stem cells (CCK-8 test, PCNA protein content detection), and forms the potential of gap junction; EPCS has a slight downregulation effect on the CD44 molecules in the surface markers of mesenchymal stem cells, and does not increase with the daily intervention time. After 9 days of EPCS treatment, the cardiac troponin was cultured for a total of 12 days in vitro, and the transcriptional level of myosin gene did not affect the effect of.EPCS on the process of 5-aza induced mesenchymal stem cells.
5. identification of myocardial differentiation potential of mesenchymal stem cells: after co culture of cardiac myocytes, mesenchymal stem cells can appear gap connexin, sodium channel Nav1.5, CaMK II, and transcription factor GATA4, MEF2C and TBX5 expression.GATA4 in the nucleus of cardiac myocytes far higher than mesenchymal stem cells, which are 4.49 times of the mesenchyme nuclei, P The content of CaMK II in 0.01. mesenchyme and cardiomyocyte culture layer (4:1) increased gradually with the duration of co culture, and CaMK II showed a strong upward trend, especially 72-96 hours after co culture. The distribution of Karyopherin alpha 3 in the distribution of mesenchymal stem cells and cardiomyocytes in Karyopherin alpha 3 was significantly different, Karyopherin alpha 4 and Karyop There was a difference in herin alpha 5 on mesenchymal stem cells and some cardiac myocytes. There was no significant difference in the distribution of Karyopherin beta 1, Karyopherin alpha 1/6, and Karyopherin alpha 2 in mesenchymal stem cells and cardiomyocytes. The ratio of positive myocardial cells with a stronger expression of Karyopherin alpha 3 was similar to that of nuclear rich GATA4 cardiomyocytes (90%). Blocking induced strong proliferation and S100A4 expression in primary cultured cardiomyocytes. The formation of calcium ion channels on the surface of mesenchymal stem cells is strongly dependent on normal calcium current activity. The effect of calcium ion blocking on calcium transients in mesenchymal stem cells is reduced and time shortens. Calcium channel block is blocked. Some myocardial transcription factors increased in the co culture layer of MSCs, but no increase in downstream structural proteins was observed.
6.CX43 and TroponinT showed a gradual increase in the daily EPCS intervention time..CX43 showed a 1.5 and 1.7 fold increase in the 3h/d and 6h/d groups (compared with the control group, P0.01); the troponin T showed a 3.6 times (P0.01) and 4.4 times (P0.05) higher in the 3h/d and 6h/d groups.
7. after 5 days of continuous EPCS treatment, it was found that S100A4 increased significantly, and its myocardial cells were 2.33 times as much as the control group (P0.01) at 1.99 times (P0.01) in the control group. The expression of MEF2C and GATA4 in cardiac myocytes increased by 1.63 times (P0.01) and 1.57 times (P0.01), and EPCS in cardiac myocytes could promote the concentration of MEF2C in the nucleus. We also found a greater number of binuclear cardiomyocytes, and MSC did not respond to the increase of MEF2C triggered by EPCS. The elevation of MEF2C in the cytoplasm and nucleus of the cardiac myocytes could clearly distinguish the cardiomyocytes from the mesenchymal stem cells in the co culture system. There were some indicators in the cardiomyocytes and between the myocardium after EPCS treatment. Mesenchymal stem cells were elevated, including a strong increase of troponin T and a relatively weak CX43, of which cardiac troponin T was 2 times as high as that of the control group (P0.01), CX43 expression was 1.4 times (P0.05) in the myocardial cells as the control group, and 1.88 times (P0.01) in the MSCs as the control group, and CX43 was the control group. 1.94 times (P0.01), in the co culture layer, there was an obvious nuclear aggregation phenomenon of S100A4 and troponin T in myocardial cells of group EPCS, and this phenomenon was very rare on MSC.
Four. Conclusion
1. proper EPCS intervention is a very effective way to multitarget intervention in the differentiation of mesenchymal stem cells and cardiac myocytes.
The content of GATA4 in the nucleus of 2. mesenchymal stem cells was much lower than that in the nucleus of the cardiomyocytes. In the nuclear transporter receptor, the distribution of mesenchymal stem cells and cardiomyocytes in the distribution of Karyopherin3 was significantly different, and the difference between Karyopherin4 and Karyopherin5 in mesenchymal stem cells and some cardiac myocytes was found.
The formation of calcium channels in 3. mesenchymal stem cells depends on the normal calcium current activity in co culture layer after co culture.
The sensitivity and universality of 4. mesenchymal stem cells in the acceptance of EPCS were less than that of.EPCS treatment in cardiac myocytes. Although S100A4, TroponinT had an overall level of increase, the accumulation of more cytoplasm in the nucleus appeared in the myocardium, which was rare in mesenchymal stem cells, suggesting that the partial differentiation of mesenchymal stem cells may be associated with important differentiation. In cardiomyocytes and mesenchymal cultures, the expression of important transcription factor MEF2C in mesenchymal stem cells in mesenchymal stem cells is not increased by EPCS intervention, but in cardiomyocytes, suggesting that MSCs themselves have limited sensitivity to EPCS.
5.EPCS causes the increase of S100A4 in mesenchymal stem cells, while previous literature suggests that S100A4 is one of the factors that promote myocardial differentiation in the parietal endoderm, suggesting that EPCS may enhance the support of mesenchymal stem cells in the coculture layer of mesenchymal stem cells and cardiac myocytes.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【参考文献】