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人外分泌汗腺细胞分离方法的优化
  • ISSN号:1000-6834
  • 期刊名称:《中国应用生理学杂志》
  • 时间:0
  • 分类:R331[医药卫生—人体生理学;医药卫生—基础医学]
  • 作者机构:[1]天津医科大学,天津301070, [2]解放军总医院第一附属医院,全军创伤修复与组织再生重点实验室,北京100048, [3]浙江大学医学院附属第二医院急诊科,杭州310009
  • 相关基金:国家973计划资助项目(2012CB518105); 国家自然科学基金项目(81121004,81230041,81171798)
中文摘要:

目的:优化人外分泌汗腺细胞的分离方法,以高效地提取外分泌汗腺细胞。方法:新鲜的皮肤样本剪成组织微粒(大小约1 mm3),A组采用胰蛋白酶-乙二胺四乙酸(EDTA)和胶原酶Ⅱ型(2 mg/ml)体积分数1∶1混合的方法;B组采用传统消化方法,即胶原酶Ⅱ型;C组采用胰蛋白酶-EDTA消化的方法,三组同时置于恒温培养箱内,比较三种方法处理后汗腺细胞团的获取情况。将挑取的汗腺细胞团种于培养皿内,观察细胞贴壁和生长情况,并用流式细胞仪测定各组细胞的增殖指数,最后进行免疫细胞化学检测汗腺细胞标志性蛋白的表达。结果:A组和C组在消化30 min后,镜下可见少部分的汗腺细胞团,2 h后A组游离汗腺细胞团明显增多,C组游离汗腺细胞团很少;B组在消化6 h后,才出现部分游离的汗腺细胞团。将汗腺细胞团培养3 d后发现C组汗腺细胞贴壁情况差、成活细胞少;A组和B组的汗腺细胞贴壁情况良好,培养9 d后呈"铺路石样"生长,细胞增殖指数分别为(18±4)%和(17±6)%,无明显差异,免疫细胞化学结果表明:两组细胞癌胚抗原(CEA)和细胞角蛋白7(CK7)表达均为阳性。结论:胰蛋白酶-EDTA和Ⅱ型胶原酶联合消化法能明显缩短汗腺细胞的分离时间,且不影响细胞的活性和增殖特性。

英文摘要:

Objective: To optimize the methods of isolating human eccrine sweat gland cells in vitro so as to get efficiently primary human sweat glands. Methods: The fresh and normal skin tissue was cut into pieces of microskin about lmm3 and the following 3 group digestion buffer was applied to isolated gland cells. The digestion buffer of group A was the eqnivoluminal mixture of Trypsin-Ethylene Diamine Te- tmacetic Acid(EDTA) and collagenase-Ⅱ (2 mg/ml). The digestion buffer of group B was collagenase-Ⅱ (2 mg/ml) traditionally and group C was Trypsin-EDTA. These three groups were placed into an incubator simultaneously and the emerging time of dissociated sweat glands was cal- culated. Sweat glands were sorted out and then placed in culture dish. The adherence and the growth of cells were observed. The proliferation index was detected by tlow cytometry. The identification of cultured cells was performed by immunocytochemical staining. Results. After di- gesting 30 rain in group A and C, a very few of dissociated sweat glands were emerging. But after digesting for 2 h , there were lots of dissoci- ated sweat glands emerging in group A rather than in group C. The emergence of dissociated sweat glands in group B would require at least 6 hours. After seeded in culture dishes, the sweat glands in group C couldn' t adhere to the wall of dish, but the sweat glands in group A and B adhered very well and even grew like paving stones after 9 days. In addition, the proliferation index were ( 18 ± 4) % and ( 17 ± 6) % respec- tively, there was no statistical difference. The results of immunocytochemical staining showed that the cells expressed carcino-embryonic antigen (CEA) and cytokeratin 7(CK7) in group A and B. Conclusion: Trypsin-EDTA combined with collagenase- Ⅱ can shorten the time of isolating sweat gland cells and have no effect on cell activity and proliferation.

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期刊信息
  • 《中国应用生理学杂志》
  • 北大核心期刊(2008版)
  • 主管单位:
  • 主办单位:中国生理学会 卫生学环境医学研究所 军事医学科学院
  • 主编:汪海
  • 地址:天津市大理道1号
  • 邮编:300050
  • 邮箱:tjzgyish@163.com
  • 电话:022-84655184
  • 国际标准刊号:ISSN:1000-6834
  • 国内统一刊号:ISSN:12-1339/R
  • 邮发代号:6-16
  • 获奖情况:
  • 国家印刷质量二等奖,国防科技期刊一等奖
  • 国内外数据库收录:
  • 美国化学文摘(网络版),荷兰文摘与引文数据库,美国生物医学检索系统,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2000版)
  • 被引量:9411