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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 primary isolation, culture and identification of Cajal-like interstitial cells (ICLC) in guinea pig gallbladder. Methods Healthy guinea pigs 5, 4 weeks old, body weight 300 ~ 350 g, male or female. Fasting 12 h, cervical dislocation died. Remove the gallbladder under sterile conditions. Dissection of the gallbladder under a dissecting microscope, strip the gallbladder mucosa and submucosa. The muscle strips were cut, digested, centrifuged and filtered to prepare single cell suspension of gallbladder tissue. Incubate with M199 medium containing stem cell factor (SCF). Cell growth was observed continuously under an inverted microscope, and the cell type was identified by immunofluorescence staining with c-kit specific tyrosine kinase protein kinase receptor. Results After cultured for 1 week, ICLC of gallbladder maintained its inherent features, with many processes and large nuclei. The cells had 2 ~ 3 short processes. At 4 weeks, the cell morphology was clear with protuberances slender. c-kit antibody immunofluorescence staining, fluorescein isothiocyanate (FITC) was positive, 4 ’, 6-diamidino-2-phenylindole (DAPI) counterstain nuclei blue fluorescence. Conclusion The isolation of guinea pig gallbladder ICLC by enzymolysis and its successful culture laid the foundation for the study of the biological characteristics of gallbladder ICLC and its relationship with biliary dyskinetic disorder.