摘要
目的研究Cajal间质细胞（ICC）上是否有胃动素受体表达。方法以10日龄仔兔小肠为组织来源，采用Ⅱ型胶原酶酶解法并结合密度离心法分离、培养细胞；对培养的ICC细胞，采用免疫细胞化学的方法；应用ICC特异的c．kit抗体进行免疫荧光染色后，用荧光显微镜对其进行鉴定；应用特异性胃动素受体抗体（anti—GPR38）、c—kit蛋白抗体以及细胞核染料（hoechst33342）联合对培养的细胞进行免疫荧光染色；以流式细胞仪分选的ICC为靶细胞，分别采用反转录一聚合酶链反应（RT—PCR）和蛋白免疫印迹（Western blot）技术，将提取的细胞总RNA，经反转录后扩增，行3％琼脂糖凝胶电泳；提取的细胞总蛋白，经SDS聚丙烯酰胺凝胶电泳（SDS—PAGE），转膜，抗体杂交，发光检{贝4；从基因和蛋白水平来验证ICC是否表达胃动素受体。结果特异性的c—kit蛋白抗体荧光染色鉴定成功培养出兔肠道ICC；细胞免疫荧光三标实验显示ICC胞膜上存在胃动素受体表达；流式细胞仪分析培养的细胞中c—kit阳性细胞比率为64．3％，流式细胞仪分选出阳性细胞分别为6．7×10^5和5．6×10^6；对分选出的ICC提取的总RNA和总蛋白分析检测显示胃动素受体mRNA以及受体蛋白定性分析结果均为阳性。结论兔肠道ICC上有胃动素受体的表达，ICC在胃动素促进肠道运动中起到了重要的作用。
Objective To look for the evidences of motilin receptor expression on interstitial cells of Cajal （ICC） of the rabbit. Method Smooth muscle segments with ICC were isolated from the small intestine of 10-day old rabbits. The tissue segments equilibrated in Ca2＋ -free Hanks＇ solution were dispersed with an enzyme solution containing collagenase type Ⅱ and then Ficoll density centrifugation was used to dissociate ICC. The cells were suspended and cultured in the M199 medium. The c-kit antibody was applied to distinguish the cultured ICC. The motilin receptor was identified by immunocytochemical assay with GPR38 antibody, c-kit antibody and hoechst33342 combined to label ICC. Cells cultured for a few days were sorted for ICC with c-kit stained green fluorescent through flow cytometry. The total RNA and proteins extracted from the sorted ICC were respectively used to verify motilin receptor on the ICC by reverse-transcriptase polymerase chain reaction （RT-PCR） and Western blotting. Result We had successfully dissociated and cultured ICC of rabbit small intestine in vitro. Fluorescent staining with c-kit antibody confirmed that the culture ICC was successful. Triple-labeled immunofluorescent staining had detected the motilin receptor on membrane of ICC. Flow cytometry analysis showed that the ratio of c-kit positive cell in the cultured cells was 64. 3%. The number of sorted ICC was 6. 7×10^5 and 5.6×10^6. The results of RT-PCR and Western blot confirmed that the ICC had motilin receptor expression. Conclusion Our study demonstrated presence of motilin receptor on ICC of the rabbit. The present results may suggest that ICC play an important role in gastrointestinal movement induced by motilin.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2011年第4期255-260,共6页
Chinese Journal of Pediatrics
关键词
兔
胃动素受体
流式细胞术
逆转录聚合酶链反应
CAJAL间质细胞
Rabbits
Motilin receptor
Flow cytometry
Reverse transcriptase polymerase chain reaction
Interstitial cells of Cajal
