微载体辅助的干细胞疗法对盆腔脏器脱垂阴道筋膜再生的研究

Pelvic organ prolapse has seriously affected the quality of life in women. Weak vaginal fascia is one reason that pelvic organ prolapse occurs. Currently, the pelvic reconstructive surgery aims at supporting the prolapsed organ but has significant complications. Thus, how to repair and regenerate the vaginal fascia is the urgent clinical question to be solved. New treatment modalities such as stem cell-based therapy have showed great potential in tissue regeneration, however, the study of mesenchymal stem cells used in the vaginal fascia regeneration is still in an early exploration stage. The possible reason may be that the loss and low survival rate of transplanted MSCs due to unfavorable microenvironment for growth at lesion sites; another reason may be that lack of enough effective evidence for the therapeutic effect of transplanted MSCs. Therefore, there has great value to investigate the method that benefits MSCs survival and verify the therapeutic effect of MSCs in the vaginal fascia. In this study, we will apply injectable 3D microcarriers, which were innovatively developed as stem cell carriers for in vivo delivery by our previous work, to provide protection during injection and microenvironment that benefit MSCs survival. We will pre-culture human umbilical cord MSCs in the microcarriers to form microtissues, and then inject the microtissues into vaginal wall in SD rat models. We will observe the in vivo survival of GFP-Luciferase labelled MSCs in different time by bioluminescence imaging and evaluate the difference in histomorphology, host response on the molecular level, biomechanical property and the change of fibroblast and smooth muscle cells. Consequently, it will determine the repair and regenerative effect of microcarrier-assisted MSC therapy and provide basic data for the clinical application of MSCs in the treatment of pelvic organ prolapse.

阴道筋膜薄弱是造成盆腔器官脱垂的病因之一，目前临床广泛采用的盆腔脱垂修补手术是针对盆底解剖修复，如何实现阴道筋膜结构功能的修复和再生成为临床亟待解决的问题。干细胞治疗成为新兴疗法，但间充质干细胞对阴道筋膜的再生还处于早期探索阶段，其原因可能是注射后MSC暴露在不利于生长的微环境导致存活率低和流失以及注射后的有效性研究不足，因此，进一步研究有利于MSC存活的条件和验证其对阴道筋膜的再生疗效具有重要价值。本研究拟采用我们前期研究开发的细胞体内运输的微载体为工具，为MSC提供注射保护和利于存活的微环境。预先将人脐带MSC在微载体内体外培养形成微组织；在大鼠模型的阴道前壁注射微组织，小动物活体成像观察MSC不同时段的体内活性，评估治疗前后组织形态学、生物力学，成纤维细胞和平滑肌细胞等的差异，从而明确微载体辅助的MSC对阴道筋膜的修复和再生效果，为MSC在盆腔器官脱垂治疗的临床应用提供理论基础。

盆底支持组织功能异常尤其是阴道薄弱是造成盆腔器官脱垂的病因之一，目前临床广泛采用的盆腔脱垂修补手术是针对盆底解剖修复，如何实现阴道结构功能的修复和再生是临床亟待解决的问题。干细胞治疗成为新兴疗法，但间充质干细胞对阴道筋膜的再生还处于早期探索阶段，其原因可能是注射后MSC暴露在不利于生长的微环境导致存活率低和流失以及注射后的有效性研究不足，因此，进一步研究有利于MSC存活的条件和验证其对阴道筋膜的再生疗效具有重要价值。本项目应用我们前期开发的细胞体内运输工具微载体支架，辅助MSC在阴道内的移植和治疗。首先分离培养并通过表面标志物鉴定了人脐带间充质干细胞，并成功构建了共表达绿色荧光蛋白和荧光素酶的间充质干细胞以用于体内细胞活性的追踪和评价。进一步将MSC在微载体内体外培养形成微组织，定量评价MSC在微载体内的活性和增殖能力，结果表明细胞均匀生长在微载体内，增殖能力良好；此外，微载体培养促进MSC分泌细胞外基质、生长因子和炎症调节相关因子。进而，建立大鼠卵巢切除模型，在阴道前壁注射微组织后，通过小动物活体成像观察MSC不同时段的体内活性，植入7天后细胞仍具有活性，表明微载体对植入到阴道壁的细胞有较好的保护作用。治疗12周后组织形态学评价显示微载体已降解、各组炎症反应不明显，微载体和细胞组的肌层明显增厚；α-SMA免疫组化染色结果也表明微载体辅助细胞对阴道平滑肌有较好的修复作用。而且，相对对照组，微载体和细胞组的弹力纤维表达明显增多。本项目的结果表明微载体辅助的细胞疗法有利于平滑肌和弹力纤维的生长，促进阴道的修复，为盆腔器官脱垂的临床治疗提供了新思路。