人子宫内膜异位症在位子宫内膜干细胞的分离和鉴定

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^3,对比非克隆形成细胞异位病灶体积(19.12±10.98)mm^3,差异有统计学意义(t=3.065,P<0.05);HE染色显示:克隆形成细胞及非克隆形成细胞均有间质和腺体样异位病灶形成;进一步行免疫组化染色显示异位病灶组织中,间质样细胞Vimentin表达阳性,腺体样细胞CK-19表达阳性。结论:子宫内膜异位症在位子宫内膜功能层中具有强克隆形成能力的子宫内膜干细胞,应用克隆形成法可以有效收集子宫内膜干细胞,经该法收集的子宫内膜干细胞可以冻存及传代培养,且具有干细胞无限增殖、自我更新、定向分化及异位病灶形成能力。

Isolation and Identification of Stromal Stem Cells from Eutopic Endometrium Functionional Layer of Women with ;Endometriosis

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)× 104, (1.45±0.64)×104, (3.06±1.73)×104, N-CFCs:(0.48±0.27)×104, (0.37±0.15)×104, 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 group was higher than that of N-CFCs group (40.63%), when CFCs and N-CFCs were inoculated into the abdominal subcutaneous of nude mice, respectively. Reconstruction of endometriotic lesions in the CFCs group with an average volume at (30.95 ±12.13)mm3 was bigger than the N-CFCs group (19.12±10.98)mm3. Conclusions:There are stem cells in the function layer of endometrium in EMs patients and it can be isolated from eutopic endometrium using colony-forming assays. The study has given a simple method to get ESC easily for researching EMs. Endometrial stem cells can be cryopreserved and subcultured, and have the ability of unlimited proliferation, self-renewal, directional differentiation and ectopic lesion fomation.