大型成年哺乳类动物绵羊阴道平滑肌细胞的原代培养改进方法研究

目的探索原代培养成年大型哺乳动物绵羊阴道平滑肌细胞(vaginal smooth muscle cells,VSMC)改良方法。方法成年绵羊阴道平滑肌取材,利用预消化组织块法(将组织剪碎呈1 mm3大小组织块,0.1%Ⅰ型胶原酶消化20 min,0.1%胰酶-EDTA与0.1%Ⅰ型胶原酶混合溶液再次消化30 min,将组织块种于培养瓶)原代培养VSMC,并与传统组织块法和酶消化法进行细胞萌出速度及增殖速度比较,提高VSMC原代培养效率;免疫荧光染色法和western bloting法检测平滑肌细胞标志物calponin及a-SMA表达鉴定VSMC。结果预消化组织块法获得原代VSMC速度快于传统组织块法和酶消化法,3 d可见细胞萌出,9 d可达80%细胞融合,"峰谷"特征明显,而酶消化法至12 d增殖至80%细胞汇合,传统组织块法约7 d可见细胞萌出,14 d可达80%细胞汇合;VSMC平滑肌标志物calponin和a-SMA均呈阳性表达(P0.05);VSMC可扩增代数有限,扩增7代后即表现出细胞形态改变,增殖缓慢,胞核内出现空泡,死亡细胞增加等表现。结论预消化组织块法可较快速多量获得绵羊VSMC。

The study of imprvoved method for the primary culture of sheep vaginal smooth muscle cells(VSMCs) in vitro

Objective Feasibility study of Pre-digested explant culture method for the primary culture of sheep vaginal smooth muscle cells ( VSMC) . Methods Draw material from vaginal smooth muscle of adult sheep,VSMC was isolated by predigested explant culture (cut the tissue into 1mm3 pieces, 0. 1% Type I collagenase digested the tissue for 20min, then the tissue was digested by mixed solution of 0. 1% trypsin-EDTA and 0. 1% type I collagenase for 30min again), the proliferation rate of VSMC derived by predigested explant culture was compared with that of by traditional tissue explant culture or enzymatic digestion to improve the efficiency of primary culture of VSMC. Identification of VSMC by immunofluorescence staining and by western blotting assay to detect the expression of calponin and a-SMA. Results Pre-digested explant culture could get the VSMCs faster than the traditional tissue explant culture and enzyme digestion culture. Eruption of cells can be observed at 3d. For 9d, cell of up to 80% confluence can be observed, and the "peak and valley" pattern can also be observed. Calponin and a - SMA were positive ( P <0. 05 );available amplication generation was limited,cells appeared morphologically changed after 7 generations,proliferation slowed,vacuolus in nucleus and increased dead cells. Conclusion The speed of getting the VSMC by enzymatic digestion culture or traditional explant culture is slower than by pre-digested explant culture.