差速贴壁法体外分离培养小型猪脂肪间质干细胞的实验研究  

Cultivation of adipose-derived mesenchymal stem cells by differential adhesion

作　　者：焦国华[1] 高洪丽[2] 吴贽 张志光[3] 

机构地区：[1]深圳市宝安区松岗人民医院口腔科,518105 [2]深圳市龙岗区人民医院检验科,518172 [3]中山大学光华口腔医学院广东省口腔医学重点实验室,广州510055

出　　处：《中华细胞与干细胞杂志（电子版）》2016年第3期160-166,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家自然科学基金(81302550);广东省科技计划项目(2014A020212390);广东省自然科学基金(2015A030311039)

摘　　要：目的探讨差速贴壁法对小型猪脂肪间质干细胞（ADSC）提取和纯化的可行性。方法从两成软骨10月龄的中国实验用I系小型猪皮下脂肪组织获取ADSC，培养4 ~ 6 h倒置显微镜下观察，见少量细胞贴壁，立刻将含有未贴壁细胞的培养基接种到新培养瓶内继续培养，传统培养法不换培养瓶，以后均每3 d换液1次。完全培养基传至第3代MTT法绘制生长曲线，并测定两种培养法获得的细胞不同代数的贴壁时间；诱导培养基向脂肪细胞、成骨细胞、成软骨细胞诱导并鉴定。贴壁时间、表面抗原阳性率、分化诱导染色阳性率实验数据比较采用单因素方差分析及t检验。结果采用差速贴壁培养法可分离培养出小型猪ADSC，并可除去部分贴壁的成纤维细胞、上皮细胞和脂滴，传代后培养瓶中脂滴明显减少；差速贴壁培养法获得的ADSC在贴壁时间方面较传统方法获得的ADSC短，前者传代后的贴壁时间（3.5±0.2）h较后者（5.8±0.3）h短，差异具有统计学意义（t = 12.55，P = 0.01）；两种方法获得的ADSC中CD34阳性率：差速法为（1.17±0.01）﹪，传统法为（0.99±0.03）﹪；CD44阳性率：差速法为（88.90±0.03）﹪，传统法为（89.23±0.10）﹪；CD106阳性率：差速法为（1.18±0.05）﹪，传统法为（1.56±0.06）﹪；差异均统计学意义（t = 0.12，1.23，0.37，P = 0.06，0.07，0.06）。油红O、苏丹黑B、Von kossa、茜素红、阿尔新蓝、II型胶原染色结果表明差速贴壁法获得的ADSC可分化为脂肪细胞、成骨细胞和成软骨细胞，且阳性染色率分别为（78±2）﹪、（82±6）﹪、（85±1）﹪、（83±3）﹪、（76±9）﹪、（74±1）﹪，较传统法的（68±1）﹪、（69±2）﹪、（73±0）﹪、（75±1）﹪、（69±3）﹪、（65±4）﹪高，差异具有统计学意义（t = 5.21，12.56，16.27，10.33，17.01，23.97；P = 0.02，0.03，0.01，0.01，0.00，0.00）。结ObjectiveTo investigate the method of purification of adipose-derived stem cell（ADSC）in vitro and to discuss whether the method of differential adhesion have more advantages than the conventional one in harvesting ratio and purification. MethodsAbout 10 g subcutaneous fatty tissue was obtained from the cervical part or back of the type I Chinese experimental minipig, then the substance was cultivated on the underlayer after the step of enzymatic digestion by collagenase type I for 4 hours, change a new culture flask when observed some adherent cells under the inverted microscope. The conventional one does not deal with this step. Change the medium 3 days a time. Change the complete medium with inductive medium（induce into adipocyte and osteoblast）for 2 ~ 4 weeks after the cells passage to 3, then the efficiency of differentiation was assessed. Simultaneously, draw a growth curve by the method of MTT. The experimental data were compared with one factor analysis of variance and t test. ResultsThe ADSC can be obtained successfully from the fatty tissue of minipig by the method of differential adhesion and the observation by microscope and growth curve by MTT indicated that the cell morphology and bionomics of ADSC which were obtained by differential adhesion are superior to those of conventional one. Differential velocity adherent culture method to obtain the ADSC in adherent time aspects compared with the traditional method to obtain the ADSC is shorter, after the former passage affixed to the wall time（3.5 ± 0.2）h shorter than the latter（5.8 ± 0.3）h, the difference has statistical significance（t =12.55, P = 0.01）. Two methods to obtain the ADSC in CD34 positive rate of differential speed method for（1.17 ± 0.01）﹪, the traditional method for（0.99 ± 0.03）﹪; CD44 positive rate of differential speed method for（889 ± 0.03）﹪, the traditional method for（89.23 ± 0.10）﹪; CD106 positive rate of differential speed method for（1.18 ± 0.05）﹪, the traditional method for（

关 键 词：脂肪细胞 间质干细胞 成骨细胞 软骨细胞 细胞培养技术 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]