摘要
心肌梗死后存活心肌数量下降而导致最终的心力衰竭已成为心肌梗死患者死亡的主要原因。目前认为可通过细胞移植治疗缺血和心肌梗死等各种原因所致心肌细胞减少从而治疗心力衰竭。其中骨骼肌成肌细胞(myoblast)移植的研究时间较长,安全性最高,研究进展最快。以往大量的动物实验及临床研究已证明骨骼肌成肌细胞可以在正常的和受损伤的心肌中成活并改善受损心脏的功能,目前也已进入临床实验,但其改善心功能的机制尚不甚清楚。移植后“心肌细胞化”的骨骼肌成肌细胞相互之间以及骨骼肌成肌细胞与心肌细胞间的连接,与心肌细胞间的闰盘连接有何异同尚不清楚;骨骼肌成肌细胞“心肌细胞化”的影响因素也不清楚。国内外以往对骨骼肌成肌细胞与心肌细胞间连接的研究多为形态学研究,电生理方面的研究较少。本研究将新生SD大鼠的骨骼肌成肌细胞与心肌细胞在体外共同培养,从形态学和电生理学两个层面研究二者间连接的性质,并对可能的影响因素进行初步分析。整个实验共分五个部分。
第一部分 细胞的获取、培养及共同培养
目的 建立合适的骨骼肌成肌细胞与心肌细胞体外共同培养的模型。方法 新生第2-4天SD大鼠乳鼠8-12只/次,采用分次消化法获得心肌细胞;一次消化法获得骨骼肌成肌细胞;采用贴壁分离技术纯化;培养均选用含20%胎牛血清DMEM液(生长培养液)。在相差显微镜下观察、计数并绘制生长曲线。结果 在含20%胎牛血清的DMEM液中与心肌细胞共培养一周时,骨骼肌成肌细胞仍未出现融合、成熟的现象;经细胞生长曲线测算,与心肌细胞共同培养的骨骼肌成肌细胞数量增长速度要快于其单独培养时。结论 在含20%胎牛血清DMEM液中,骨骼肌成肌细胞与心肌细胞可共同培养,适合本研究需要;心肌细胞主要对骨骼肌成肌细胞增殖有促进作用,而对骨骼肌成肌细胞的融合、成熟无促进作用。
第二部分 共同培养的骨骼肌成肌细胞与心肌细胞间连接的形态学研究
Abundant clinical trial and animal research have demonstrated that skeletal myoblasts could survive among normal and infarcted myocardium and improve the function of damaged heart. Although clinical trial has been done in this field, the mechanism is still unclear. One of the key problems is whether the base of electronic conduction between transplanted myoblasts and myocytes is through intercellular contact or some effective connection such as gap junction. Previous research about the connection between myoblasts and myocytes are mainly morphological, with limiting data on electrophysiology. In this study, the myoblasts and myocytes of newborn SD rat were co-cultured in vitro, immuno-histochemistry, RT-PCR, transmission electroscopy were used to investigate morphologically the possible connection between the two cells consecutively; and Multiple Electrodes Array (MEA) was applied to investigate the electro-activity among the cells. Western blot was also used to investigate the possible affecting factors.Part I Cell obtainment, culture and co-cultureObjective Establish an in vitro co-culture model of skeletal myoblasts and cardiac myocytes. Methods Cardiac myocytes were obtained by multi-step digestion from newly born dayl-2 SD rats (n=8-12). Myoblast was got by one step digestion. Cell was purified by adhesion-seperation technique and cocultured in DMEM containing 20% fetal bovine serum. Phase contrast microscope was used for observation and calculation. Growth curve was thus made. Results Cells fusion and maturation were not observed among the myoblasts which were co-cultured with myocytes for 7 days in DMEM containing 20% fetal bovine serum. The growth speed of myoblasts which co-cultured with myocyte was faster than those cultured alone. Conclusion Myoblasts and myocytes could be co-cultured in DMEM containing 20% fetal bovine serum. The model was fit for our further study. Myocytes might improve the cell proliferation of myoblasts but has little effect on its differentiation and maturation.
Part II Morphology Study of Co-cultured Skeletal Myoblasts and Cardiac MyocytesObjective Investigate the morphological evidence for the connection of co-cultured myoblasts and myocytes. Methods Cell suspension of rat myocytes and myoblasts were prepared in DMEM (containing 20% fetal bovine serum)with the ratio 4:1. Cadherin、 Connexin43、 NCAM were stained by ABC-AEC staining when the cells were co-cultured for 2,4,7,14 days respectively. The staining results were studied under microscope. Cells were observed by transmission electroscope on day 4,7,10,14. Results Connexin43 was expressed in mypblasts from day 4 and increased with cultural duration among the co-cultured myoblasts and myocytes. The expression of connexin43 was higher in the cell membrane of myoblasts which contacted with cardiac myocytes. The expression of cadherin also increased with the prolongation of co-culture time but less significant than that of connexin43. The expression of NCAM which suggests the differentiation and maturation of myoblasts specifically remained lowly two weeks after co-culture. Transmission electroscope revealed that myoblasts remained mono-nucleus cell 2 weeks after co-cultured with myocytes, and cell fusion and maturation was not observed. Connection similar to gap junction was observed to exist between myoblasts and myocytes. Conclusion Material bases and possibility for the existence of the connection similar to gap junction do exist among the in vitro co-cultured skeletal myoblast and cardiac myocytes.Part III Junction Related Protein Expression among Skeletal Myoblasts and Cardiac Myocytes: RT-PCR StudyObjective The expression of junction related protein Connexin43、 Cadherin、 NCAM among co-cultured myoblast and myocyte were quantified. Methods 3 groups were set as followed: experiment group (myocyte and myoblast were co-cultured with ratio 4:1); control group 1 (myoblast was cultured alone with same cell account); control group 2 (myocyte was cultured alone with same cell account). Cells were digested and
gathered at day 2,4,7,14 after cultured and RT-PCR was applied. Results were compared among the 3 groups. Results Connexin43 was expressed by the co-cultured myoblast and the expression increased with the prolongation of culture time. The expression of Cadherin was not increased significantly. The expression of NCAM remained low level and increased only after 2 weeks of cocultured. Conclusion The myoblasts "myocytelized" in some characters when co-cultured with myocytes: the fusion and maturation was delayed, the expression of connexin43 was increased and it is quite possible that gap junction was formed between myocytes and skeletal myoblasts. Part IV Western blot Analysis of the Affecting Factors on the " Myocytelization " of Skeletal MyoblastsObjective The affecting factors of the increase of connexin43 expression in myoblasts were investigated. Methods Experimental groups and control groups were set. Experimental group 1: myocytes and myoblasts were co-cultured with the ratio 4:1, isoproterenol was added in 25nM. Experimental group 2: myoblasts were cultured alone with same cell account, the culture medium of myocyte was added daily (cultured under the myocytes' circumstance). Experiment group 3: myoblasts was cultured alone with same cell account, the culture medium of control group 1 was added daily (cultured under the co-culture's circumstance). Control group 1: the same as experiment group 1 except the isoproterenol. Control group 2: myoblasst were cultured alone with same cell account. Control group 3: myocytes were cultured alone with same cell account. Each group was repeated 4 times and the culture medium was changed daily. Cells were digested on day 5 and counted. The cells were gathered after centrifugal deposition and western blot was applied. Results The expression of connexin43 was similar between experiment group and control group. The expression of NCAM was similar among experiment group 1,2,3 and control group 1 which were all at low level. The expression of NCAM was the highest in control group 2 (myoblasts cultured alone). The average cell count in experiment group 2,3 and control group 2 was (5.15 ± 0.3) ×10~6, (4.90 ± 0.33) ×10~6, (2.32 ± 0.18) ×10~6 respectively. The cell count increased 5 times in experiment group 2 and 3, increased 2 times in control group 2. P value >0.05 when comparing the increasement of cell
count between experiment group 2 and 3 by T test while P<0.01 when comparing with control group. Conclusion The myoblasts displayed the increasment of cell count and delay of cell differentiation and maturation under the circumstance of myocyte or co-culture, the expression of connexin 43 was not increased which suggested that the contaction with myocyte is one of the most important factors affecting the differentiation of myoblast. The expression of connexin43 was not increased through increasing electronic or mechanical stimulation lonely. The affection of myocytes to myoblasts under co-culture circumstance might be the combination of multi-factors. Part V Multi Electrodes Array (MEA) electrophysiological studyObjective The characters of the transmission of electronic message between co-cultured myoblasts and myocytes were investigated to help judging the type of the cell connection. Methods 3 groups were set. Experiment group 1: The myocytes and myoblasts were co-cultured in MEA. Experiment group 2: The myocytes and myoblasts were co-cultured for 2 weeks ( transferred 4-5 times during the 2 weeks and new myocytes was added to keep the ratio 4:1). Then the cells were co-cultured with myocytes in MEA. Control group: Myocytes was cultured in MEA with same amount. Myoblasts was marked by live cell fluorescence staining PKH26 Red. MEA system was connected and results were recorded. Sub-thresh hold electronic stimulation was given and the electronic activity was recorded and analyzed. Results The range of electronic message in experiment group 1 was lower than experiment group 2, which was 1/5 of the latter. That of experiment 2 was similar to control group. The cells in experiment group 2 and control group connected in network after 24 hours of culture and beated spontaneously and synchronizingly. Synchronizingly beat net work was formed 5 days after cultured in experiment group 1. The average transmision speed of electronic activity in experiment group 1 was (2.1±0.6cm/s, n=5) which was slower than that of experiment group 2 (6.8±3.1cm/s, n=8, p=0.053) and control group (9.3±3.8cm/s, n=11, P=0.003) . The local transmision speed of electronic activity was not uniform in experiment group 1 and was related to the flurescent distribution of myoblasts. There was no significant difference between experient group 2 and control group (P=0. 26), the transmision speed was fast and uniform.The time interval of electronic message was even more uniform and the
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6. Hagege AA, Carrion C, Menasche P, et al. Viability and differentiation of autologous skeletal myoblast grafts in ischaemic cardiomyopathy. Lancet 2003;361:491-2.
7. Kamelger FS, Marksteiner R, Margreiter E, et al. A comparative study of three different biomaterials in the engineering of skeletal muscle using a rat animal model. Biomaterials. 2004; 25: 1649-1655.
8. Rigatelli G, Rossini K, Vindigni V, et al. New perspectives in the treatment of damaged myocardium using autologous skeletal myoblasts Cardiovascular Radiation Medicine. 2004; 5: 84-87.
9. Formigli L, Francini F, Tani A, et al. Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment. Am J Physiol Cell Physiol. 2004 November 10.
10. Pedrotty D, Koh J, Davis B, et al. Engineering skeletal myoblasts: Roles of 3-D culture and electrical stimulation. Am J Physiol Heart Circ Physiol. 2004 November 18.
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2. Robinson SW, Cho PC, Levitsky HI, et al. Arterial delivery of genetically labeled skeletal myoblasts to the murine heart: long-term survival and phenotypic modification of implanted myoblasts. Cell Transplant. 1996; 5: 77-91
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