摘要
目的:从子宫内膜异位症患者在位子宫内膜中分离、培养子宫内膜干细胞,并鉴定其生物学特性,探索一种简便、高效的分离人子宫内膜异位症子宫内膜干细胞的方法。方法:诊刮获取2012—2014年于天津中医药大学第二附属医院妇产科住院治疗的14例子宫内膜异位症患者的功能层子宫内膜,从中分离出子宫内膜间质细胞,克隆形成法从该细胞中分离出具有强克隆形成能力的克隆形成细胞,对比非克隆形成细胞以噻唑蓝(MTT)比色法测定生长曲线检测两种细胞的增殖能力,连续克隆形成实验检测其克隆形成能力,微球体形成实验检测其微球体形成能力,诱导分化实验检测克隆形成细胞定向分化能力,裸鼠异位病灶形成实验检测其异位病灶形成能力。结果:相较于非克隆形成细胞,生长曲线测定显示克隆形成细胞增殖速度更快,差异有统计学意义(P<0.05)。连续克隆形成实验显示克隆形成细胞5代以内克隆形成率稳定,分别为(12.30±3.75)%、(13.11±3.23)%、(11.86±2.21)%、(12.08±3.01)%、(11.01±3.98)%,非克隆形成细胞随传代次数增加克隆形成率逐渐降低,至第5代已无克隆形成,分别为(5.57±2.13)%、(4.21±2.12)%、(2.23±1.02)%、(0.12±0.07)%、0%,2组细胞对应代数克隆形成率比较差异有统计学意义(P<0.05)。微球体形成实验显示克隆形成细胞3代以内每代成球细胞数量分别为(0.82±0.37)×10~4、(1.45±0.64)×10~4、(3.06±1.73)×10~4,非克隆形成细胞分别为(0.48±0.27)×10~4、(0.37±0.15)×10~4、0,2组对应代数细胞数量比较差异有统计学意义(P<0.05)。诱导分化实验显示克隆形成细胞具有定向分化能力。裸鼠异位病灶形成实验显示克隆形成细胞接种2周后可形成肉眼可见异位病灶,其异位病灶形成率为100%,相较于非克隆形成细胞异位病灶形成率40.62%,差异有统计学意义(χ~2=27.022,P<0.05);克隆形成细胞异位病灶体积为(30.95±12.13)mm^
Objective： To get the endometrium stem cells（ESC） for the eutopic endometrial from women with endometriosis（EMs） by using colony-forming assay and provide the experimental basis for the further study of EMs. Methods：The endometrial samples from women with EMs who treated in Department of Obstetrics and Gynecology, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine in 2012-2014 undergoing fractional curettage respectively.Colony-forming cells（CFCs） were isolated from individual colony-forming units（CFU） which visible macroscopically and contained 4 000 cells. We focused on the characteristics of stem cells between CFCs and N-CFCs, including high proliferative potential（MTT assay）, serial clone formation, mammosphere formation of self-renewal, differentiation potential in vitro, and tissue reconstitution in vivo. Results： The optical densities in cell proliferation were more significantly higher in the CFCs than N-CFCs（P〈0.05）. The cloning efficiency（CE） of CFCs from P1-5 are higher than N-CFCs [CFCs：（12.30±3.75）%,（13.11±3.23）%,（11.86±2.21）%,（12.08±3.01）%,（11.01±3.98）%; N-CFCs：（5.57±2.13）%,（4.21±2.12）%,（2.23±1.02）%,（0.12 ±0.07）%, 0% ]. There are statistical significances between the two groups（P〈0.05）. CFCs originated spheres with a diameter were at least 60 m, spheres were continuously generated at 5-6 day intervals for at least 3 passages with a bigger and bigger size,in contrast, the spheres of N-CFCs cannot be passaged serial [Count the cells of each passage： CFCs：（0.82±0.37）×10~4,（1.45±0.64）×10~4,（3.06±1.73）×10~4, N-CFCs：（0.48±0.27）×10~4,（0.37±0.15）×10~4, 0]. There are statistical significances between the two groups（P〈0.05）. The protein expression of CFCs was negative for CK-19 before induction culture, but were expressed at CFCs, which cultured with the induction medium for 21 days. The endometriotic lesion-forming efficiency（100%）in the CFCs g
出处
《国际妇产科学杂志》
CAS
2017年第1期113-117,125,共6页
Journal of International Obstetrics and Gynecology
基金
中国博士后科学基金(201104309)
国家自然科学基金(81303094)
