Gao Yang, Liu Jun-Feng, Zhang Chao, Liu Liang, Liu Yue-Ping, Zhang Sheng-Lei, Zhao Lian-Mei
Department of Thoracic Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China.
Graduate School of Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China.
Exp Ther Med. 2020 Apr;19(4):2933-2948. doi: 10.3892/etm.2020.8560. Epub 2020 Feb 26.
The present study was conducted to examine the feasibility of isolation and primary culture of smooth muscle cells (SMCs) from the esophagogastric junction (EGJ). Smooth muscles of EGJ were harvested from 23 patients with esophageal cancer during esophagostomy from January 2015 to December 2017. Enzymatic dispersion (ED) was performed for isolation. Collagenase II and Trypsin/EDTA were applied by enzyme injection (EI) into tissue fragments or immersion of tissue fragments into enzyme solution. Growth characteristics and proliferation [Cell Counting Kit-8 (CCK-8)] of cells were recorded for both smooth muscle cell medium (SMCM) and DMEM/F12 containing 10% newborn bovine serum (10%-F12). All ED methods could isolate primary cells; EI was the most effective method with low collagenase II concentration (0.5 mg/ml) at 4˚C for 14-24 h. Primary cells demonstrated mainly spindle- and long-spindle-shaped with 'hills and valleys' morphology. The CCK-8 assay in SMCM showed better proliferation results than in 10%-F12. After passaging for 4-8 generations in SMCM or 2-4 generations in 10%-F12, cells enlarged gradually with passages and lost spindle structures. mRNA and proteins of α-smooth muscle actin (α-SMA), smooth muscle 22 α (SM22α), vimentin, desmin, CD90 and proliferating cell nuclear antigen were detected in tissues and cells with different levels of expression. SMCs of esophageal circular muscle, esophageal longitudinal muscle, gastric circular muscle near sling in gastric bottom and gastric circular muscle near clasp in lesser gastric curvature, all cultured in 10%-F12, exhibited superior smooth muscle phenotypes compared with SMCs cultured in SMCM in terms of α-SMA, SM22α and vimentin expression. The EI method of ED at low temperature appears effective for isolation and primary culture of SMCs from human EGJ .
本研究旨在探讨从食管胃交界（EGJ）分离和平滑肌细胞（SMC）原代培养的可行性。2015年1月至2017年12月期间，在食管癌患者行食管造口术时，从23例患者中获取EGJ的平滑肌。采用酶消化法（ED）进行分离。通过将胶原酶II和胰蛋白酶/乙二胺四乙酸（Trypsin/EDTA）注入组织块或把组织块浸入酶溶液的方式进行酶注射（EI）。记录平滑肌细胞培养基（SMCM）和含10%新生牛血清的DMEM/F12（10%-F12）中细胞的生长特性和增殖情况[细胞计数试剂盒-8（CCK-8）]。所有ED方法均可分离出原代细胞；EI是最有效的方法，在4˚C下使用低浓度胶原酶II（0.5 mg/ml）作用14 - 24小时。原代细胞主要呈纺锤形和长纺锤形，具有“峰谷”形态。SMCM中的CCK-8检测显示增殖结果优于10%-F12。在SMCM中传代4 - 8代或在10%-F12中传代2 - 4代后，细胞随传代逐渐增大并失去纺锤形结构。在组织和细胞中检测到α-平滑肌肌动蛋白（α-SMA）、平滑肌22α（SM22α）、波形蛋白、结蛋白、CD90和增殖细胞核抗原的mRNA和蛋白质，其表达水平各异。在10%-F12中培养的食管环行肌、食管纵行肌、胃底部吊带附近的胃环行肌和胃小弯扣附近的胃环行肌的SMC，在α-SMA、SM22α和波形蛋白表达方面，与在SMCM中培养的SMC相比，表现出更优的平滑肌表型。低温下的EI法ED似乎对从人EGJ分离和原代培养SMC有效。
