脐静脉内皮细胞来源的微囊泡促进腹膜样组织血管化的初步研究

目的探讨人脐静脉内皮细胞(hUVECs)来源的微囊泡(MVs)促进大鼠腹膜间皮细胞(rPMC)增殖及促进组织工程化腹膜样组织血管化的作用。方法采用胶原酶消化法获得hUVECs,无血清培养法刺激释放MVs,并对MVs行电镜检测。rPMC与hUVECs来源的MVs体外共培养,对细胞及上清液分别行CCK、ELISA和RT-PCR检测。将rPMC种植在小肠粘膜下层(SIS)表面,分别与加入MVs的培养基和单独培养基预培养1周后,植入裸鼠皮下;2周后取出移植物行HE染色,镜下观察。结果透射电镜下可以看到有完整的膜结构的hUVECs分泌的MVs。加入MVs组与对照组相比,能明显促进rPMC增殖(P<0.05),同时在上清液中加入MVs组VEGF浓度明显高于对照组(P<0.05)。分别对两组rPMC行VEGF的RT-PCR检测,加入MVs组比对照组表达明显增高。对取出的移植物行HE染色,加入MVs组和对照组相比,可见新生的微血管密度明显增高(P<0.05)。结论 hUVECs来源的MVs能够明显促进rPMC的增殖,诱导rPMC产生VEGF高表达,刺激组织工程化腹膜样组织血管化,为以腹膜间皮细胞作为尿道组织工程种子细胞和大范围尿道缺损修复提供了理论基础。

A Preliminary Study of the Microvesicles Derived from Human Umbilical Vein Endothelial Cells Promote Tissue-Engineered Peritoneum-Like Tissue Vascularization

Objective To investigate the possibility of microvesicles （MVs） derived from human umbilical vein endothelial cells （hUVECs） promote the proliferation of rat peritoneal mesothefial cells （rPMC） and enhance tissue-engineered peritoneum-llke tissue vascularization. Methods hUVECs were isolated from human umbilical cord by collagenase digestion, serum-free culture method to stimulate the release of MVs and for electron microscopy examination. In vitro, rPMC co-cultured with MVs derived from hUVECs and supernatant were detected by CCK, ELISA and RT-PCR. In vivo, rPMC grown in the small intestinal submucosa （SIS） surface,respectively with the added MVs medium or medium pre-cuhured for one week,and implanted subcutaneously into nude mice. Two weeks after the operation, the grafts were harvested and detected by HE staining. Results Under a transmission electron microscope, the complete membrane structure of MVs derived from hUVECs was observed. MVs group can significantly promote the proliferation of rPMC compared with the control group （P〈0.05） and the VEGF concentration was significantly higher in the supernatant compared with the control group （P〈0.05）. Meanwhile, RT-PCR revealed the expression of VEGF on the rPMC of MVs group was significantly higher compared to the control group. After two weeks, HE staining showed the newborn microvessel density was significantly higher in the MVs group compared with the control group （P〈0.05）. Conclusion MVs derived from HUVECs can significantly promote the proliferation of rPMC, increased expression of VEGF, stimulated tissue-engineered peritoneum-like tissue vascularization and provide a good theoretical basis for the application in the future that peritoneal mesothelial cells as the urethra tissue engineering seed cells in a wide range of urethral defect repair.