单个结肠平滑肌细胞的制备

The Preparations of Single Intestinal Smooth Muscle Cells

目的 :探讨离体肠道平滑肌细胞分离方法。方法 :采用混合酶法分离豚鼠结肠平滑肌细胞 ,通过台盼蓝染色法检查细胞成活率 ,并应用Ca2 + 荧光指示剂Fura 2 AM测定静息状态下结肠平滑肌细胞内游离钙浓度( [Ca2 + ]i)。结果 :成功地分离出单个结肠平滑肌细胞 ,其存活率为 ( 96.7± 2 .6) % ,在静息状态下且细胞外Ca2 + 浓度为 1.5mmol L时 ,豚鼠结肠平滑肌细胞 [Ca2 + ]i 为 ( 10 8± 9.4 )nmol L (n =6) ;细胞外Ca2 + 浓度为 0时 ,[Ca2 + ]i为( 72± 11.8)nmol L (n =6)。结论 :酶法分离的单个结肠平滑肌细胞活性好 ,可应用于肠道平滑肌细胞电生理、药理学研究。

Objective: To study the dissociation of single smooth muscle cells of the intestine. Methods: Smooth muscle cells were enzymatically dispersed from the isolated longitudinal muscle layer of the guinea pig colon.The cell viability was calculated by Typan Blue staining method. The resting intracellular calcium concentration ([Ca 2+ ] i) was assayed with Fura 2 AM using the freshly isolated colonic smooth muscle cells by biwavelength spectrophotofluorometry. Results: The single colonic smooth muscle cell of guinea pig was successfully isolated and the cell viability was (96.7±2.6)%(n=6). In the resting state, [Ca 2+ ] i in HEPES Ringer solution (CaCl 2 1.5 mmol/L) was (108±9.4)nmol/L(n=6), [Ca 2+ ] i in Ca 2+ free solution was (72±11.8) nmol/L(n=6). Conclusion: The activity of the smooth muscle cells dissociated enzymatically is high,and the smooth muscle cells may be used in electrophysiological and pharmacological experiments of the intestinal smooth muscle cells.