力学刺激在工程化心肌组织结构和功能改建中的作用研究

Effects Of Mechanical Stimulation On The Structural And Functional Remodeling Of Engineered Cardiac Muscle

目的探索力学刺激在工程化心肌组织结构和功能改建中的作用。方法将分离并培养的新生大鼠心肌细胞与添加了Matrigel的液态Ⅰ型胶原混合,在环形槽中铸成环状心肌细胞/胶原条带。构建的条带在环形槽中培养7天后取出,在对其施加10%的静态拉伸力的情况下,再培养7天。通过光学显微镜、常规HE染色、免疫组织化学染色和透射电镜观察等对构建的组织工程化心肌组织进行评价。结果工程化心肌组织在力学刺激组的第2天可观察到局部跳动的区域,随时间的延长跳动的区域逐渐增多,强度逐渐增大,并最终形成一致跳动。而对照组未经力学拉伸刺激的工程化心肌组织则未能形成一致跳动。组织学和免疫组织化学染色研究结果显示,构建的工程化心肌组织经力学刺激,心肌细胞比较均匀地分布在整个条带中,且多数沿拉伸方向伸展,胞核呈长梭形,其形态类似于天然成熟心肌组织。透射电镜观察显示,条带中心肌细胞含有排列整齐的肌原纤维,并沿着细胞的长轴方向伸展,肌小节结构和Z带清晰可见。结论力学刺激可以更好地促进工程化心肌组织的结构和功能改建,使其更接近正常心肌组织。

Objective To study the effects of mechanical stimulation on the structural and functional development of tissue-engineered cardiac tissue (ECT). Methods Neonatal rat cardiac myocytes were isolated, cultured, and mixed liquid collagen type I with Matrigel and then cast in circular molds to construct circular cardiac myocytes/collagen strand. After a seven-day culture in circular molds, the strands were removed from the molds and subjected to 10 % static stretch or remained in static suspension culture as control for another seven days. Microscopy and transmission electron microscopy (TEM), routine HE staining and immunohistochemical staining were used to analyze the engineered cardiac tissue. Results In stimulated group beating areas could be seen on the surface of the ECTs at the second day after stretching; More beating areas could be seen thereafter. These areas beat stronger and stronger, and finally came to synchronization. However, synchroneously beating was not observed in the control group. In stimulated group, histological and immunohistochemical analyses showed that the cardiac myocytes in the ECTs distributed evenly in the whole strand and the majority of the cells, with elongated nuclei, stretched along the stretching direction. The morphology of ECTs resembled that of the native adult cardiac tissue. TEM revealed that the cardiac myocytes in ECTs contained arranged myofibrils oriented parallel to the longitudinal cell axis. Clearly defined sarcomeres and Z lines were observed. Conclusion Mechanical stimulation can improve the structural and functional development of tissue-engineered cardiac tissue.