KCNN3/SK3基因修饰的自体脂肪干细胞移植治疗薄型子宫内膜的研究

The implantation and development of zygote would be prevented if the endometrial thickness was too thin. Thin endometrium has become one of the serious disease that does harm to female reproductive health. Recently, the therapies to promote growth of endometrium are still confined to administration of drugs , which has unsatisfactory curative effect and fails to solve problems of endometrium regeneration radically. Therefore, seeking a novel effective target seems to be of great importance. Based on previous research results, stem cells and small conductance calcium-activated potassium channels type 3(KCNN3/SK3) had multiple biologic functions in endometrium regeneration and endometrial function recovery, including not only the proliferation and migration of endometrial cells, but also local angiogenesis. Our previous studies showed that adipose-derived stem cells(ADSCs) also had induced differentiation capacity of endometrial epithelial cells, adipocytes, myocytes, endothelial cells and neuroblasts. Furthermore, The endometrial thickness of animal model with thin endometrium significantly increased after intrauterine infusion of autologous ADSCs. Additionally, decrease of KCNN3/SK3 gene expression level was positively correlated with the thickness in patients who suffered from thin endometrium. These results suggest that ADSCs and KCNN3/SK3 gene could be the available target stem cells and gene for endometrium regeneration and repair. In this project study, KCNN3/SK3 gene and ADSCs will be used as a target gene and stem cell carrier in gene modified stem cells for endometrium regeneration and repair. Additionally, cell proliferation, apoptosis, migration, implantation and differentiation of stem cells, local vascular remodeling and regional blood flow control will be involved to make clear the effects and mechanisms of KCNN3/SK3 gene modified ADSCs on endometrium repair and endometrial receptivity, which not only will contribute to its effectiveness, timeliness and feasibility for the treatment of thin endometrium, but also lay a foundation of further study on gene modified stem cell therapy for intractable thin endometrium from gene and tissue level.

薄型子宫内膜已成为严重影响成年女性生殖健康的顽疾之一，目前现有方法疗效有限且尚难以解决子宫内膜再生难题。研究发现，干细胞和小电导型钙激活钾离子通道3（KCNN3/SK3）通过发挥促进子宫内膜细胞增殖、迁移和局部血管生成等效应促使子宫内膜再生和功能恢复。课题组前期研究发现薄型子宫内膜模型大鼠自体脂肪干细胞（ADSCs）宫腔内灌注可使子宫内膜厚度显著增加，且人薄型子宫内膜中KCNN3/SK3基因表达水平显著降低且与内膜厚度呈正相关性。本项目将ADSCs和KCNN3/SK3作为基因修饰干细胞促进子宫内膜再生修复的干细胞载体和分子靶点，拟从细胞增殖、迁移、干细胞种植分化、组织血管重塑及血流量调控等角度探索KCNN3/SK3基因修饰的ADSCs在组织学和基因水平对子宫内膜再生修复和容受性的有效性、时效性、可行性及作用机制，从而为子宫内膜再生修复和基因修饰干细胞治疗难治性薄型子宫内膜提供新的研究思路。

薄型子宫内膜阻碍受精卵在子宫内膜着床和发育，已成为严重影响成年女性生殖健康和困扰临床医师的顽疾之一。目前，临床上治疗薄型子宫内膜的方法较多，但疗效欠佳。干细胞具有无限制自我更新和增殖分化为多种细胞类型的能力，对治疗薄型子宫内膜具有巨大的应用潜能。目前主要应用于薄型子宫内膜治疗的干细胞具有分离纯化困难、体外扩增受限、安全风险较大、伦理学受限等方面的缺点，使其虽然在动物模型和体外水平中发挥一定作用,但临床应用受到极大限制。研究提示脂肪干细胞(adipose-derived stem cells ，ADSCs)易获取、易培养、性状稳定且扩增能力强，能成为干细胞治疗薄型子宫内膜的理想细胞来源。随着基因工程技术和干细胞技术的长足发展，将两者相结合的基因修饰干细胞移植术为单纯干细胞治疗开辟了新的研究方向并显示出较好的临床应用前景。已有研究初步发现，子宫内膜中KCNN3/SK3表达异常可能与薄型子宫内膜有关。课题组前期研究发现，ADSCs和KCNN3/SK3可作为基因修饰干细胞移植治疗薄型子宫内膜最理想和最具潜力的干细胞载体和基因靶点。本项目在前期工作基础上，将KCNN3/SK3基因修饰的ADSCs尾静脉注射治疗大鼠薄型子宫内膜模型内，并从细胞增殖、迁移、干细胞种植分化、组织血管重塑及血流量调控等角度探索KCNN3/SK3基因修饰的ADSCs在组织学和基因水平对子宫内膜再生修复和容受性的有效性、时效性、可行性及作用机制。本研究结果显示，ADSCs 将充分发挥其多向分化潜能特性，有效改善子宫内膜厚度和容受性，达到从组织学水平治疗薄型子宫内膜的目的；另一方面，在尾静脉注射KCNN3/SK3基因修饰的ADSCs治疗大鼠薄型子宫内膜后，可增加薄型子宫内膜厚度、改善纤维化面积和容受性、抑制子宫内膜细胞凋亡并增加其微血管密度，同时可能通过Raf-MEK1/2-ERK1/2、PI3K-Akt-mTOR和PI3K/Akt/GSK-3β/VEGF通路促进而调控子宫内膜的增殖、分化、生长，达到从基因水平治疗薄型子宫内膜的目的，从而为基因修饰干细胞自体移植治疗薄型子宫内膜寻找到良好的基因靶点和更优良的干细胞载体，为临床应用基因修饰干细胞技术治疗薄型子宫内膜奠定夯实基础。