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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(P<0.05);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。
Objective To explore the method of primary culture of vaginal smooth muscle cells (VSMC) in adult large mammals. Methods Vaginal smooth muscle of adult sheep was harvested. The tissue samples were digested with 0.1mm collagenase and digested with 0.1% trypsin-EDTA and 0.1% collagenase I Re-digestion 30 min, the tissue pieces were cultured in culture flasks) VSMC primary culture, and traditional tissue-block method and enzyme digestion method of cell eruption rate and proliferation rate compared to improve VSMC primary culture efficiency; immunofluorescence staining and western blotting method to detect smooth muscle cell markers calponin and a-SMA expression to identify VSMC. Results The primary VSMC obtained by pre-digestion method was faster than the conventional tissue-block method and enzyme digestion method. The cells were erupted on the 3rd day, reached 80% cell fusion on the 9th day, and the characteristic of “Fenggu” was obvious. At 12 days, 80% of the cells were confluent, and the cells were euthanized at about 7 days by conventional tissue-block method and reached 80% confluence on the 14th day. The expressions of calponin and a-SMA in smooth muscle of VSMC were all positive (P <0.05) Amplifiable algebra is limited. After 7 generations of amplification, the cells showed morphological changes, slow proliferation, vacuoles in the nucleus and increased death cells. Conclusion Pre-digested tissue block method can obtain sheep VSMC more rapidly and more.