Cre/LoxP系统联合SV40 LTag诱导可复性永生化大鼠皮肤成纤维细胞系的建立  

The Establishment of the Reversed Immortalized Rat Dermatic Fibroblast Cell Line Induced by Cre/LoxP and SV40 LTag

作　　者：李晓航[1] 张佳林[1] 康铁利[1] 李乐[1] 程颖[1] 石蕊[1] 赵宁[1] 刘永锋[1] 

机构地区：[1]中国医科大学附属第一医院普通外科教研室肝胆外科暨器官移植科,沈阳110001

出　　处：《中国医科大学学报》2011年第11期979-982,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30371359;30870644;31140059)

摘　　要：目的探索可复性永生化大鼠皮肤成纤维细胞的建立方法。方法采用胰酶消化法分离培养大鼠皮肤成纤维细胞;逆转录病毒载体(SSR69)转染成纤维细胞,筛选稳定表达SV40 LTag的永生化成纤维细胞;用表达Cre重组酶的腺病毒感染永生化成纤维细胞,筛选获得回复后成纤维细胞。对原代、永生化及回复后成纤维细胞,倒置显微镜下观察细胞生长状态;采用细胞生长曲线法测定细胞增殖能力。免疫荧光染色检测Vimentin在原代及永生化成纤维细胞中的表达情况,平板克隆实验评价回复后成纤维细胞的致瘤性。结果提取了大鼠皮肤成纤维细胞,并筛选出表达SV40 LTag的永生化成纤维细胞,二者形态无明显差异,均贴壁生长,呈长梭形或多角形,但永生化成纤维细胞体外增殖能力明显高于原代成纤维细胞(P<0.05),在体外培养传代>25代。细胞免疫荧光提示两种细胞均在细胞质内表达Vimentin。回复后成纤维细胞不表达SV40 LTag,其体外增殖能力与原代细胞无明显差异(P>0.05),无致瘤性。结论应用Cre/LoxP系统联合SV40 LTag可以建立可复性永生化大鼠皮肤成纤维细胞系。Objective To explore the establishment of the reversed immortalized rat dermatic fibreblast cells. Methods The primary fibmblast cells,isolated from the newly born rat skin by collagenase digestion, were transfected with the retroviral vector SSR69 and the immortalized cells stably expressing SV40 LTag were screened. Then the immortalized cells were infected by adenovirus expressing the Cre-recombinase, and the reversed cells not expressing SV40 LTag were screened. Compared molphology, proliferation in vitro and expression characteristics between primary and immortalized fibroblast by means of inverted nficroscope, making growth curve and cell immunofluorescence. Tu- morigenicity of reversed cells were examined by plate cloning experiment. Results The primary fibroblast ceils were successfully isolated from the rat skin, and the immortalized cells were obtained by transfecting the primary fibroblast cells with the retroviral vector SSR69. Mar- phology of the two cells were similar, showing elongated spindle or polygonal, and grew with adherent characteristic. However, the proliferation of immortalized fibroblast was significantly superior than that of primary fibroblast （P 〈 0.05 ）, and the immortalized cells were cultured more than passage 25. Cell Immunofluorescence confirmed that vimentin were expressed in the cytoplasm of two cells. The reversed cells were successfully obtained after the excision of SV40 LTag gene. The proliferation of reversed cells was similar to that of primary fibroblast cells （P 〉 0.05 ）, and were not tumorigenic. Conclusion The reversed immortalized rat dermatic fibroblast cell line could be established by means of Cre/LoxP and SV40 LTag.

关 键 词：永生化 猿猴病毒大T抗原基因 Cre/LoxP位点特异性重组酶系统 

分 类 号：R587.1[医药卫生—内分泌]