本文選題：脈沖微電流 + S100A4　； 參考：《第三軍醫(yī)大學》2012年博士論文

【摘要】：一、背景與目的 冠狀動脈粥樣硬化性心臟病(簡稱冠心病)是臨床常見病、多發(fā)病，嚴重的危害人類健康。冠心病的最嚴重類型---急性心肌梗死(AMI)，因冠狀動脈急性閉塞和心肌組織急性缺血，可在15～20分鐘內(nèi)導致心肌細胞不可逆性損傷。心肌梗死后心室發(fā)生重構(gòu)，隨之而來的是心肌細胞被替換為纖維組織，導致心肌細胞收縮功能減退或消失，繼之出現(xiàn)左心室擴張等病理改變。 間充質(zhì)干細胞是已經(jīng)是比較獲得認可的可用于心臟移植的細胞類型,在心肌梗死后，間充質(zhì)干細胞移植可以減少壞死，分泌許多促進血管生成的細胞因子和改善心臟功能,還有研究認為間充質(zhì)干細胞可作為支持心肌干細胞生長重要的支持細胞。如間充質(zhì)干細胞可以招募許多的內(nèi)源性干細胞，這些干細胞為損傷部位帶了了修復，而非僅僅間充質(zhì)干細胞，這其中就包括了非常有潛力的心肌干細胞。在對病竇綜合征的研究中，HCN基因家族修飾的間充質(zhì)干細胞用于重建起搏點。如何更好的促進間充質(zhì)干細胞移植后的分化和移植后的效果也是研究人員不斷探索的方向，其中分子手段是比較有效的方式，但由于慢病毒等手段的臨床應用受到限制，而在心血管內(nèi)科治療中各種物理手段在臨床應用較多，那么可否使用物理手段干預間充質(zhì)干細胞分化過程？或者改變間充質(zhì)與心肌共培養(yǎng)層分化狀態(tài)？在正常生理狀態(tài)過程中，內(nèi)源性的電流、電場扮演了影響分化的重要角色，內(nèi)源性的電場對于胚胎的分化作用十分重要。本課題的目的是探求外源性電干預可否作為促進間充質(zhì)向心肌分化或者改變間充質(zhì)與心肌共培養(yǎng)層分化狀態(tài)的一種手段的可能性與可行性，尋找合適的電干預參數(shù)，設計制作方便實用的電干預系統(tǒng)，了解間充質(zhì)干細胞向心臟分化潛能，尋找其作用的靶點和初步確定了解合適的干預時間。 二、研究方法 1.反復結(jié)合EPCS使用效果反饋制作了EPCS培養(yǎng)裝置。 2.膜片鉗檢測電流干預對共培養(yǎng)條件下間充質(zhì)干細胞膜電位的影響， 3.將間充質(zhì)干細胞分為普通培養(yǎng)組和EPCS處理組，檢測EPCS對其增殖(cck-8andPCNA)，表面標記物，形成縫隙連接潛力影響。檢測EPCS對其搏動維持，搏動頻率，及肌鈣蛋白，肌球蛋白表達在轉(zhuǎn)錄水平的影響。 4.將5-aza處理后的3w后的小鼠間充質(zhì)干細胞分為普通培養(yǎng)組和EPCS組，檢測EPCS對其誘導效果影響。 5.間充質(zhì)干細胞分化情況檢測：間充質(zhì)干細胞與心肌共培養(yǎng)4-5天后分別檢測CX43，Nav1.5，CaMKⅡ表達情況；將間充質(zhì)干細胞和心肌細胞按4:1比例種植于培養(yǎng)皿中，取48h，72h和96h進行western-blotting實驗，檢測CaMKⅡ含量的動態(tài)變化。 6.共培養(yǎng)5天后，檢測共培養(yǎng)層GATA4,MEF2c,TBX5相關轉(zhuǎn)錄因子表達的情況；檢測蛋白質(zhì)核轉(zhuǎn)運受體在共培養(yǎng)層表達。 7.將細胞分為兩組，普通共培養(yǎng)培養(yǎng)組和鈣離子通道阻滯共培養(yǎng)組。共培養(yǎng)5天后，檢測細胞上VSCC(電壓敏感鈣通道)的表達和鈣瞬變情況。檢測MEF2C，GATA4和TBX5表達情況。檢測細胞鈣調(diào)神經(jīng)磷酸酶活力；western-blotting檢測細胞TroponinT， NAV1.5， CACNA1G/H和CX43蛋白含量變化。 8.間充質(zhì)和心肌細胞在初期接種時的比例為３：１。每組給予1h/d，3h/d和6h/d的EPCS干預，EPCS從接種后24h開始，持續(xù)4天，同期接種的未進行EPCS干預的共培養(yǎng)細胞作為對照組。用蛋白印跡法檢測troponinT和CX43在每個不同時間處理組表達含量。 9.用免疫熒光檢測EPCS連續(xù)處理5d組，及對照組在S100A4，MEF2C,troponinT,CX43和GATA4表達情況。 三、研究結(jié)果 1.結(jié)合試驗反饋反復摸索分析中制備了一套穩(wěn)定可靠的電刺激給予和接受裝置(EPCS培養(yǎng)系統(tǒng))，我們可實時對其干預信號監(jiān)測，這套系統(tǒng)便于使用和準確給予EPCS刺激。 2.膜片鉗檢測顯示在共培養(yǎng)條件下的間充質(zhì)干細胞在接受到膜片鉗儀器提供直流電流刺激后產(chǎn)生膜電位微小改變。 3.在心肌細胞長期體外培養(yǎng)中EPCS可以維持少數(shù)區(qū)域較良好的搏動狀態(tài)和形態(tài)。 4.安全性檢測方面：EPCS對心肌細胞搏動頻率未見明顯影響；.EPCS不影響間充質(zhì)干細胞增殖(cck-8實驗，PCNA蛋白含量檢測)，形成縫隙連接的潛能；EPCS對間充質(zhì)干細胞表面標記物中的CD44分子具有輕度下調(diào)作用，不隨每日干預時間延長增加下調(diào)幅度；EPCS處理9天，在體外培養(yǎng)共計12天的心肌細胞其肌鈣蛋白，肌球蛋白基因轉(zhuǎn)錄水平未見影響。EPCS對5-aza誘導的間充質(zhì)干細胞過程未見影響。 5.狗間充質(zhì)干細胞心肌分化潛能鑒定：在于心肌細胞共培養(yǎng)后，間充質(zhì)干細胞可以出現(xiàn)縫隙連接蛋白，鈉離子通道Nav1.5，CaMKⅡ，和轉(zhuǎn)錄因子GATA4，MEF2C和TBX5表達。GATA4在心肌細胞核的濃度遠高于間充質(zhì)干細胞，是其在間充質(zhì)細胞核的4.49倍，P0.01。間充質(zhì)與心肌細胞培養(yǎng)層(4：1)中CaMKⅡ含量隨共培養(yǎng)時間延長逐漸升高，CaMKⅡ表現(xiàn)出強上升趨勢，尤其是共培養(yǎng)后72-96小時。在核轉(zhuǎn)運受體中， Karyopherinα3的分布間充質(zhì)干細胞和心肌細胞的分布有比較明顯的差異，Karyopherinα4和Karyopherinα5在間充質(zhì)干細胞和部分心肌細胞上存在差異，Karyopherin β1，Karyopherin α1/6，和Karyopherin α2在間充質(zhì)干細胞和心肌細胞的分布無明顯差異，其中Karyopherin α3表達更強的心肌細胞的陽性比例與核富集GATA4心肌細胞的比例相似(90%)。鈣通道阻滯誘發(fā)原代培養(yǎng)心肌細胞內(nèi)成纖維細胞強烈增生和S100A4表達升高。間充質(zhì)干細胞表面鈣離子離子通道的形成較強依賴于正常鈣電流活動。阻斷鈣離子流培養(yǎng)對間充質(zhì)干細胞鈣瞬變產(chǎn)生幅度減小和時間縮短影響。鈣離子通道阻滯導致間充質(zhì)心肌細胞共培養(yǎng)層部分心肌轉(zhuǎn)錄因子升高，但未見引發(fā)下游結(jié)構(gòu)蛋白含量增高。 6.CX43和TroponinT隨每天EPCS干預時間的延長顯示出逐漸上升趨勢。CX43在3h/d組和6h/d組表現(xiàn)出1.5倍和1.7倍的升高(與對照組比較,p0.01)；肌鈣蛋白T在3h/d和6h/d組表現(xiàn)出3.6倍(P0.01)和4.4倍(P0.05)的升高。 7.在5天連續(xù)的EPCS處理后，發(fā)現(xiàn)S100A4升高顯著，其在心肌細胞是對照組的2.33倍(P0.01)在間充質(zhì)干細胞是對照組的1.99倍(P0.01)。在心肌細胞MEF2C和GATA4的表達量升高了1.63倍(P0.01)和1.57倍(P0.01)，在心肌細胞EPCS可促進MEF2C在細胞核集中表達，同時我們也發(fā)現(xiàn)了更多數(shù)量的雙核心肌細胞，MSC則對于EPCS觸發(fā)出現(xiàn)的MEF2C升高沒有反應， MEF2C在心肌細胞胞漿和細胞核的升高可以將共培養(yǎng)體系中的心肌細胞和間充質(zhì)干細胞較清晰區(qū)分開來。也有一些指標在EPCS處理后在心肌細胞和間充質(zhì)干細胞均出現(xiàn)升高，這包括強烈升高的troponin T和升高幅度相對弱些的CX43，其中肌鈣蛋白T在心肌細胞為對照組的2倍(P0.01)，CX43的表達在心肌細胞為對照組的1.4倍(P0.05)；在間充質(zhì)干細胞肌鈣蛋白T為對照組1.88倍(P0.01)，CX43為對照組的1.94倍(P0.01),在共培養(yǎng)層中，EPCS組心肌細胞S100A4和troponin t出現(xiàn)了明顯的核聚集現(xiàn)象，極少MSC上有此現(xiàn)象。 四、結(jié)論 1.合適的EPCS干預是一種非常有效地多靶位干預間充質(zhì)干細胞和心肌細胞共培養(yǎng)層分化程度的物理方式。 2.間充質(zhì)干細胞細胞核中GATA4得含量遠低于其在心肌細胞核含量。在蛋白核轉(zhuǎn)運受體中，Karyopherin3的分布間充質(zhì)干細胞和心肌細胞的分布有比較明顯的差異，Karyopherin4和Karyopherin5在間充質(zhì)干細胞和部分心肌細胞上存在差異。 3.間充質(zhì)干細胞鈣離子通道的形成依賴于共培養(yǎng)后共培養(yǎng)層正常鈣電流活動。 4.間充質(zhì)干細胞在接受EPCS方面的敏感性和廣泛性上不及心肌細胞。EPCS處理后，S100A4，，TroponinT雖然有整體水平升高，但心肌出現(xiàn)的出現(xiàn)的核內(nèi)較胞質(zhì)更加富集現(xiàn)象在間充質(zhì)干細胞鮮見，提示間充質(zhì)干細胞部分分化障礙可能和重要分化相關蛋白質(zhì)主動轉(zhuǎn)運入核相關。在心肌細胞和間充質(zhì)培養(yǎng)層中，心肌分化重要轉(zhuǎn)錄因子MEF2C在間充質(zhì)干細胞的表達不受EPCS干預升高，而在心肌細胞升高，提示間充質(zhì)干細胞本身對EPCS感受性有限。 5.EPCS引起間充質(zhì)干細胞S100A4升高，而既往文獻認為S100A4是頂葉內(nèi)胚層分泌促進心肌分化的因子之一，說明EPCS可能增強間充質(zhì)干細胞在間充質(zhì)干細胞與心肌細胞共培養(yǎng)層的支持作用。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329

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