中文摘要：

目的：探讨一种高效、稳定的从小鼠心肌组织中分离培养心肌微血管内皮细胞的方法并观察细胞的生物学特性。方法i以多聚赖氨酸对培养皿作预包被，采用Ⅱ型胶原酶消化、差速贴壁分离法，结合内皮细胞专用培养基，分离培养微血管内皮细胞并进行扩增。利用倒置显微镜观察细胞生长情况；台盼蓝法、绘制生长曲线和MTT法分别测定心肌微血管内皮细胞传代成活率、不同代生长及增殖特征；以Dil．ac，LDL和FITC．UEA．1荧光双标，结合CD31、vwF和CD34免疫荧光鉴定细胞表面特异性抗原；体外血管形成实验检测心肌微血管内皮细胞的功能。结果：酶消化、差速贴壁法分离培养2d后，细胞呈散在的、小簇状聚集性生长，4～5d迅速生长呈单层排列，细胞为梭形、三角形或多角形，7—8d后呈铺路石样即可传代；传代细胞经台盼蓝法检测成活率均为95％以上；第1、3代细胞生长曲线近似“s”形，MTF法显示第1、3代细胞在第2—4d吸光度变化较明显（P〈0．05）；随传代次数的增加，第5代细胞生长曲线近似平缓，吸光度无明显变化，细胞逐渐衰老；DiI．ac．LDL和FITC．UEA．1荧光双标阳性率为（89．2±3．5）％，相关特异性抗原CD31、vwF和CD34免疫荧光鉴定阳性率为（56．7±3．7）％、（78．5±2．6）％和（67．8±4．2）％；体外血管形成实验显示，6—12h后出现明显的管腔结构。结论：以多聚赖氨酸作预包被，采用Ⅱ型胶原酶消化、差速贴壁法并结合内皮细胞专用培养基，可获得纯度较高的小鼠心肌微血管内皮细胞，为心脏疾病的研究提供种子细胞。

英文摘要：

AIM： To investigate an effective and stable method to isolate myocardial microvascular endothelial cells （MMVECs） from the mouse heart and to observe their biological characteristics. METHODS： The cells from the mouse heart were isolated by collagenase II digestion followed by different speed adhesion. Then the cells were cultured on the dish coated with polylysine in endothelial cell-specific culture medium. The biological characteristics were observed by trypan blue staining. The cell growth curve and the cell proliferation were also evaluated by MTT assay at different passa- ges. The expression of DiI-ac-LDL, FITC-UEA-1, specific markers of endothelial cells and the expression of CD31, vWF and CD34 were determined by immunofluoresence staining. To evaluate the function of the MMVECs, in vitro tube formation was evaluated under microscope. RESULTS： Two days after the enzyme digestion, the MMVECs formed small and isolated clusters. The MMVECs grew quickly in monolayer with the characteristics of endothelial cell shape at 4 - 5 d. The cells became confluent and cobblestone-like which were ready for passage at 7 - 8 d. After passage, the viability of the cells was more than 95%. The first and the third generation of the cells presented an S-shape growth curve. The cell prolif- eration of the first to the third generation was quick and slowed down after the fifth passage. MMVECs were highly positive for DiI-ac-LDL and FITC-UEA-I [ （89. 2 ± 3.5 ） % ], indicating the cells were MMVECs. The other relative antigen ex- pression （CD31, vWF and CD34） on the MMVECs was （56. 7 ±3.7）%, （78.5 ±2.6）% and （67.8 ±4. 2）%, respec- tively. The MMVECs formed the tubes in vitro after cultured for 6 ±12 h. CONCLUSION： We can obtain high-purity MMVECs using the combination of collagenase II digestion, the different speed adhesion process and the endothelial cell- specific culture medium for effective and reliable MMVECs isolation and culture.