大鼠脂肪来源血管内皮细胞的改良式培养与观察

Modified culture of rat adipose-derived vascular endothelial cells andits observation

目的对目前常用的大鼠脂肪血管内皮细胞(VECs)的原代培养方法进行改良,建立一种操作更为简单、更经济的VECs的原代培养方法。方法取SD大鼠睾丸囊脂肪组织,采用胶原酶消化法,在保留血管及脂肪组织、缩短消化时间的条件下分离、获取细胞,普通高糖培养基培养,光镜下观察细胞形态及生长情况,CD29、CD31与Ⅷ因子相关抗原流式鉴定及Ⅷ因子相关抗原免疫荧光鉴定,MTT比色法检测细胞冻存前后增生率,并讨论改良培养法较传统培养法的优越性。结果体外培养的原代VECs在光镜下初始呈偏圆不规则多角形,细胞融合后呈铺路石样生长;流式鉴定CD31阳性和CD29、Ⅷ因子相关抗原弱阳性,Ⅷ因子相关抗原荧光染色阳性;细胞传至第20代后,仍未见贴壁困难、生长停滞等迹象,平均传代时间为5～7d;液氮中冻存12个月复苏后细胞形态及生长情况无明显改变。结论改良的大鼠脂肪来源VECs的分离和培养方法,较传统方法更简单、经济,培养时间更短,可长期传代,是一种较为理想的培养方法。

Objective To modify primary culture of rat adipose vascular endothelial cells （VECs） so as to establish a more simple and economical method of culturing VECs. Methods Digest SD rats testicular capsule adipose tissues were digested by collagenase,the vascular and fat tissue were retentioned and shortened the digestion time to obtain and culture the cells with the ordinary high glucose culture medium. Then cells morphology and growth were observed with microscopy,the cells were identified with flow cytometry analysis and immunofluorescence. Methylthiazol tetrazolium （MTT） assay was used to detect the proliferation rate before and after cryopreservation and compared the superiority of the traditional culture method. Results The primary VECs vitro observed with microscopy were partial round and irregular polygonal,and changed to cobble stone-like when cells became fusion. CD31 antigen was positive,CD29 and factor Ⅷ related antigen were weakly positive,factor Ⅷ related antigen showed fluorescent signs of positive staining. No difficulty of cell adherent and growth was found in the 20th generation of cells （average generation time was 5-7 days） and no significant changes were found in cell morphology and growth after 12 months of cryopreservation. Conclusions The modified culture method of rat adipose-derived VECs was much simpler than the traditional method. It was an ideal culture method which had the advantages of economical,shortening culturing time and long-term passage.