月经血源性子宫内膜干细胞移植治疗重度宫腔粘连的临床前期研究

发布时间：2018-09-03 10:44

【摘要】：近年来的研究认为宫腔粘连(IUA)的发生可能与子宫内膜干细胞减少、缺失或功能障碍有关,所以提出利用干细胞移植来治疗IUA。最新的研究认为月经血源性子宫内膜干细胞(MenSCs)是更适合用作细胞治疗的种子细胞,因为与骨髓间充质干细胞相比,除具有骨髓间充质干细胞的优点外还具有更快的增殖能力、能分化为三个胚层的多种组织和高表达基质金属蛋白等特点,可能与其表达胚胎干细胞(ESCs)抗原OCT-4而具有ESCs的部分特性有关。本研究致力于运用MenSCs移植治疗严重IUA患者的临床前研究,终极目标是恢复IUA患者子宫内膜的生育功能。主要内容分为以下几个部分,第一部分分离、培养和鉴定月经血中子宫内膜干细胞,为干细胞移植治疗宫腔粘连提供细胞来源；第二部分体外诱导MenSCs向子宫内膜细胞分化,为干细胞治疗宫腔粘连提供理论依据；第三部分MenSCs移植于NOD-SCID小鼠肾包膜下或皮下,经雌激素治疗后证实MenSCs在体内可以重建子宫内膜组织；第四部分调查宫腔镜下诊断为重度宫腔粘连者月经血及子宫内膜组织中干细胞,并与有正常生育史的宫腔正常者的比较,确定IUA患者子宫内膜干细胞减少或缺失,说明用干细胞移植治疗IUA是必要的。结果：1)MenSCs在含有10%胎牛血清的低糖DMEM培养基中培养呈克隆性生长,显示了干细胞的生长特性。运用流式细胞分析技术分析培养的经血源性干细胞中OCT-4阳性细胞率为95.13%±0.81%,CD45为0.93%±0.42%,STRO-1为1.80%±0.92%,HLA-DR为1.00%±0.35%,说明培养所得的细胞基本为OCT-4*干细胞,且免疫原性低下,倍增24次细胞染色体核型仍保持正常。2)培养后的MenSCs在条件培养基和17β-戊酸雌二醇共同作用下能够在体外被诱导向子宫内膜细胞方向分化,运用免疫细胞化学检测人子宫内膜上皮细胞角蛋白CK、间质细胞波形蛋白VIM,发现诱导后CK、VIM阳性率明显强于诱导前,并且CK、VIM的mRNA水平和蛋白表达量较诱导前均明显增加,(p0.05),说明MenSCs在体外可以诱导分化为子宫内膜细胞。3)培养的MenSCs移植于垂体降调节去势的雌性NOD/SCID小鼠的腋窝皮下,经雌激素治疗后组织病理检查和免疫组织化学检测CK、VIM、PR、ER和CD31,发现HE染色下可见腺体样结构,免疫组织化学检测CK、VIM和PR有表达,ER和CD31没有表达,证实了MenSCs在体内可以重建子宫内膜组织。CD31不表达说明了重建的过程中子宫内膜组织的血供可能是来自宿主的。4)宫腔镜下诊断为宫腔粘连者较有生育史宫腔正常者MenSCs培养后克隆形成率显著降低((0.74±0.11)×10-6vs(6.8±0.56)×10-6,p0.001),粘连部位的内膜组织干细胞(EnSCs)培养后克隆形成率较宫腔正常者克隆形成率显著降低(0 vs1.21%±0.04%,p0.001),粘连部位旁疑似正常组织的EnSCs培养后较宫腔正常者克隆形成率也显著降低(0.14%±0.03%vs 1.21%±0.04%)。免疫组织化学检测宫腔粘连者OCT-4阳性细胞比例显著低于正常宫腔者(0.1%vs 2%),CD146阳性细胞比例(0.5% vs 1%),正常者子宫内膜功能层CD31阳性细胞数为1%,基底层为2%,而宫腔粘连者功能层0.05%,基底层1%：上皮细胞粘附分子(EpCAM)在宫腔粘连者和正常者子宫内膜表达均0.1%。结论：此临床前研究结果表明MenSCs能够培养扩增获得足够的细胞数量来满足细胞治疗的需要；在条件培养基和适当的雌激素作用下MenSCs能够在体外分化子宫内膜细胞；联合雌激素治疗MenSCs在NOD/SCID小鼠体内能够重建子宫内膜组织；宫腔镜下诊断为宫腔粘连者MenSCs和EnSCs均较宫腔正常者明显减少。总之,运用MenSCs治疗宫腔粘连是可行的和必要的。
[Abstract]:Recent studies suggest that the occurrence of intrauterine adhesions (IUA) may be related to the decrease, deletion or dysfunction of endometrial stem cells, so stem cell transplantation is proposed to treat IUA. In addition to the advantages of bone marrow mesenchymal stem cells (BMSCs), BMSCs have the ability to proliferate faster, differentiate into three embryonic layers and express matrix metalloproteins (MMPs), which may be related to the expression of embryonic stem cell (ESCs) antigen OCT-4 and some characteristics of ESCs. The ultimate goal of this preclinical study is to restore endometrial fertility in patients with IUA. The first part is to isolate, culture and identify endometrial stem cells from menstrual blood to provide cell sources for stem cell transplantation for the treatment of intrauterine adhesions. The second part is to induce MenSCs into endometrial fineness in vitro. Cell differentiation provides a theoretical basis for stem cell therapy of intrauterine adhesions; part three: MenSCs transplanted into NOD-SCID mice renal capsule or subcutaneous, after estrogen treatment confirmed that MenSCs can reconstruct endometrial tissue in vivo; part four: investigation of hysteroscopic diagnosis of severe intrauterine adhesions of menstrual blood and endometrial tissue stem and thin Results: 1) MenSCs grew clonally in low-glucose DMEM medium containing 10% fetal bovine serum, showing the growth characteristics of stem cells. Flow cytometry was used to study the effect of stem cell transplantation on the growth of IUA. The percentage of OCT-4 positive cells, CD45 0.93%+0.42%, STRO-1 1 1.80%+0.92% and HLA-DR 1.00%+0.35% in cultured blood-derived stem cells were 95.13%+0.81%, CD45 0.93%+0.42%, 1.80%+0.92% and 1.00%+0.35% respectively, indicating that the cultured cells were basically OCT-4* stem cells with low immunogenicity, and the chromosome karyotype of the cells doubled 24 times remained normal.2) Conditioned medium and estradiol 17 beta-valerate could induce endometrial cells to differentiate in vitro. The cytokeratin CK and vimentin VIM of human endometrial epithelial cells were detected by immunocytochemistry. The positive rates of CK and vimentin VIM were significantly higher after induction than before induction. The mRNA levels and protein surface of CK and VIM were also detected. The amount of MenSCs was significantly higher than that before induction (p0.05), indicating that MenSCs could be induced to differentiate into endometrial cells in vitro. 3. The cultured MenSCs were transplanted into the axillary subcutaneously of ovariectomized female NOD/SCID mice. After estrogen treatment, histopathological examination and immunohistochemical examination of CK, VIM, PR, ER and CD31 were performed. HE staining was found to be visible. Glandular structure, immunohistochemical detection of CK, VIM and PR were expressed, ER and CD31 were not expressed, confirming that MenSCs can reconstruct endometrial tissue in vivo. CD31 does not show that the blood supply of endometrial tissue in the process of reconstruction may come from the host. 4) Hysteroscopy diagnosis of intrauterine adhesions is more fertile than normal uterine cavity M. The colony formation rate of enSCs cultured in vitro was significantly lower than that of normal uterine cavity ((0.74 65507 The percentage of OCT-4 positive cells in the normal uterine cavity was significantly lower than that in the normal uterine cavity (0.1% vs 2%). The percentage of CD146 positive cells in the normal uterine cavity (0.5% vs 1%) was significantly lower than that in the normal uterine cavity (0.1% vs 2%). Basal layer 1%: EpCAM expression was 0.1% in the endometrium of both intrauterine adhesives and normal subjects. CONCLUSION: Pre-clinical studies have shown that MenSCs can be cultured and amplified to obtain enough cells to meet the needs of cell therapy; MenSCs can differentiate in vitro under conditioned medium and appropriate estrogen. Endometrial cells; MenSCs treated with estrogen can reconstruct endometrial tissue in NOD/SCID mice; MenSCs and EnSCs in hysteroscopically diagnosed intrauterine adhesions were significantly less than those in normal uterine cavity.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R711.74

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