心肌细胞牵张模型建立的实验研究

Establishment of a mechanical stretch model on cultured cardiac myocyte in vitro

目的:机械信号的转导与感受成为生命科学研究的理论前沿,如何对细胞施加机械刺激是进行此领域研究的前提。建立体外培养的心肌细胞牵张模型是研究的目的。方法:实验于2003-11/2004-01在解放军第三军医大学创伤烧伤复合伤国家重点实验完成。采用胰酶消化法分离培养乳鼠心肌细胞,纯化后接种于硅酮膜上,依照10%的牵张强度对心肌细胞施加机械刺激,观察心肌细胞形态、培养液中乳酸脱氢酶(lactatedehydrogenase,LDH)含量及碘化丙啶染色阳性细胞比例的变化。结果:10%牵张过程中,细胞沿受力方向排列。10%牵张后心肌细胞进一步铺展,伪足数目明显增加。牵张前后培养液中LDH含量犤培养12h(710.6±69.5)IU/L,牵张12h(769.8±66.7)IU/L犦及碘化丙啶染色阳性细胞比(牵张前0.53%,牵张12h后0.54%)无明显变化。结论:建立了较好模拟体内刺激强度的模型,并证实10%牵张对心肌细胞不是一种损伤因素,在某些方向还可能上调细胞功能。

AIM: The transduction and perception of mechanical signal have become the theoretical front of the research of life science,and how to impose mechanical stimulus on cultured cardiac myocytes is the premise of the study in the field.The aim of this paper is to establish a mechanical stretch model on cardiac myocytes cultured in vitro.METHODS:The experiment was completed in the National Key Laboratory of Trauma Burns and Combined Injury,Third Military Medical University of Chinese PLA during November 2003 to January 2004.Cardiac myocytes were isolated from mice and cultured in vitro with the method of trypsin digestion, and were inoculated to the silicone membrane after purification,then mechanical stimulus was imposed on the cardiac myocytes with 10%of the stretch intensity.The morphology of the cardiac myocytes, changes of content of lactate dehydrogenase(LDH) and propidium iodide(PI) staining positive ratio in the culture medium were observed.RESULTS:During the 10%stretch,the cells were arranged along with the direction of power.After the 10%stretch,the cardiac myocytes spread further,and the number of pseudopodia was increased obviously.There were no obvious changes in the LDH content [12 hours after culture:(710.6±69.5) IU/L,12 hours after stretch:(769.8±66.7) IU/L] and PI staining positive ratio(12 hours after culture:0.53%,12 hours after stretch:0.54%)(P >0.05).CONCLUSION:Better imitating model of stimulating intensity in vivo has been established,and it is verified that 10%stretch is not a damaging factor for cardiac myocytes,and it can up-regulate the cellular function to some certain direction.