豚鼠胆囊Cajal样间质细胞的体外分离、培养和鉴定  

In vitro isolation, culture and identification of interstitial Cajal-like cells from gallbladder of guinea pigs

作　　者：付倍蓓[1] 范莹[2] 谢博[2] 王锴[2] 

机构地区：[1]中国医科大学附属盛京医院信息科,辽宁沈阳110004 [2]中国医科大学附属盛京医院普外二科,辽宁沈阳110004

出　　处：《生物医学工程与临床》2017年第1期8-11,共4页Biomedical Engineering and Clinical Medicine

基　　金：国家自然科学基金资助项目(81000183);辽宁省自然科学基金资助项目(2013021060)

摘　　要：目的探讨豚鼠胆囊Cajal样间质细胞(ICLC)的原代分离、培养及鉴定方法。方法健康豚鼠5只,4周龄,体质量300~350 g,雌雄不限。禁食12 h,颈椎脱臼处死。在无菌条件下取出胆囊。在解剖显微镜下剖开胆囊,剥去胆囊黏膜和黏膜下层。将肌条剪碎,经消化、离心及过滤后制备胆囊组织单细胞悬液。用含有干细胞因子(SCF)的M199培养液培养。于倒置显微镜下连续观察细胞生长情况,用酪氨酸蛋白激酶受体c-kit特异性抗体免疫荧光染色鉴定细胞类型。结果培养1周,胆囊ICLC保持其固有特征,多突起,核大,细胞有2~3个短突起。4周时,细胞形态清晰,突起为细长。c-kit抗体免疫荧光染色,异硫氰酸荧光素(FITC)呈阳性,4’,6-二脒基-2-苯基吲哚(DAPI)复染细胞核呈蓝色荧光。结论酶解法分离豚鼠胆囊ICLC并培养成功,为胆囊ICLC的生物学特性及其与胆道动力障碍性疾病关系的研究奠定了基础。Objective To investigate the method of in vitro isolation,culture and identification of interstitial Cajal-like cells（ICLC） from gallbladder of guinea pigs. Methods Five healthy guinea pigs of either gender, 4-week old with body weight of300-350 g were fasted for 12-hour before sacrificed by cervical dislocation and the gallbladders were removed at sterile condition. Under dissecting microscope, the layers of mucosa and submucosa were removed and smooth muscle layer of gallbladder were cut into small pieces. Single cell suspension was obtained after digestion, centrifugation and filtration. Cells were cultured in M199 medium containing stem cell factor（SCF）. Morphological characteristics were observed with inverted microscope and the cell phenotype was identified by immunofluorescence staining using specific antibody against receptor tyrosine kinase c-kit. Results The ICLC of guinea pig gallbladder was kept its intrinsic features with 2-3 multiple short processes and large nucleus after one-week culture. Cell morphology was clear with thin and long processes after four-week culture. The cells showed fluorescein isothiocyanate（FITC） positive for immunofluorescence staining of c-kit and blue fluorescent staining of nuclei was found after 4＇, 6-diamidino-2-phenylindole（DAPI） re-staining. Conclusion It is demonstrated that the primary culture of ICLC from gallbladder of guinea pig in vitro can be successfully establish, which provide the basis for research into biological function of ICLC and relationship between ICLC and biliary motility disorders.

关 键 词：胆囊 CAJAL样间质细胞 细胞培养 豚鼠 

分 类 号：R575[医药卫生—消化系统]