室温酶解结合差速离心法分离子宫内膜腺上皮细胞及活性评估  

The Isolation and Evaluation of Endometrial Epithelial Cells using the Hydrolysis at Room Temperature Combined with Differential Centrifugation

作　　者：刘玉锋[1] 哈春芳[2,3] 马远[1] 杨眉[1] 年妍[1] 

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学总医院妇科,银川750004 [3]宁夏医科大学生育力保持教育部重点实验室,宁夏回族自治区生殖与遗传重点实验室,银川750004

出　　处：《宁夏医科大学学报》2014年第2期133-136,F0003,共5页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81160078);宁夏自然科学基金(NZ11283)

摘　　要：目的探讨室温酶解结合改良式差速离心法分离培养人子宫内膜腺上皮细胞的效果及细胞活性评估。方法选择子宫腺肌症患者在位增殖晚期及分泌期内膜10例,所得内膜分为两份,一份采用室温酶解结合改良式差速离心法(改良组)分离,一份采用37℃水浴消化结合筛网法(对照组)分离,通过细胞计数、细胞免疫化学及MTT法分别比较两种方法分离培养子宫内膜腺上皮细胞的数目、纯度及细胞生长活性。结果两组中10份标本均获得成功;改良组每g内膜组织可得到(9±1.7)×107个腺上皮细胞,免疫细胞化学角蛋白表达阳性,纯度达(97.8±0.002)%;对照组可得到(3.9±0.78)×107个腺上皮细胞,角蛋白表达阳性,纯度达(88.6±0.006)%,改良组分离的腺上皮细胞数量和纯度均明显高于对照组(P<0.05)。MTT结果显示改良组细胞在对数生长期生长活性明显高于对照组(P<0.05)。结论采用室温酶解结合改良式差速离心法可高纯度分离子宫内膜腺上皮细胞,并且细胞数量多,活性好,可用于子宫内膜疾病的药物干预及机制学的研究。Objective To explore the effect of room temperature enzymolysis combined with improved differential centrifugation on the isolation and culture of human endometrial glandular epithelial cells and the evaluation of cell viability.Methods Endometrium specimens were collected at late proliferative and secretory phase from 10 patients with endometriosis.Each sample was divided into two for cell isolation via room temperature enzymolysis combined with improved differential centrifugation （improved group） or 37 ℃ water bath digestion combined with mesh method （control group）.The number,purity,and viability of the isolated cells were compared by immunocytochemical staining and MTT assay.Results The cell isolation and culture was successful for all specimens.We obtained （9 ± 1.7） × 10^7 gland epithelial cells per 1 g endometrial tissue in the improved group,as compared with （3.9 ±0.78） × 10^7 cells in the control group.The immunohistochemistry revealed that the expression of cytokeratin was positive for both groups and the purity for the improved and control group was （97.8 ±--0.002） % and （88.6 ± 0.006） %,respectively （P 〈 0.05）.The MTT assay showed significant difference in cell viability between two groups during logarithmic phase （P 〈 0.05）.Conclusion The room temperature enzymolysis combined with improved differential centrifugation can separate high purity endometrial glandular epithelial cells with a large quantity,good viability,and long growth cycle so as to be applied in the research of reproductive physiology of endometrial disease intervention.

关 键 词：子宫内膜 腺上皮细胞 细胞分离培养 生长活性 

分 类 号：R711.71[医药卫生—妇产科学]