Interstitial Cells Of Cajal Culture, Passage And Expression Of Motilin Receptor

BackgroundInterstitial cells of Cajal(ICC) are a special class of cells dispersed in the muscle and nerve plexuses of the gastrointestinal tract. Nowdays massive studies have comfirmed four major functions for ICCs[1,2]: 1,as pacemaker cells generating slow-wave activity of the gastrointestinal tract; 2,as facilitators of active propagation of electrical events; 3,as mediators of neurotransmission; 4,as stretch sensors coordinating mechanical inputs.There are some evidences of correlations between alterations to ICC and several gastrointestinal motor disorders. Indeed, many GI motor disorders can be related to changes in number, structure, density of ICCs. Therefore, it is significant to treat ICCs as a therapeutic and research target in the gastrointestinal tract. But the means in isolation and culture of ICC still have many difficulties that should be studied and discussed. The feasibility and biological characteristics during long-term culture in vitro have no clear explanation by now. The researches on the culture,passage and recovery of ICC have no been recorded. Acording to records in recently,there are more than ten types of receptprs on ICC,such as Cholecystokinin A[3],G protein-coupled (PKA,PKC)[4],Muscarinic acetylcholine(M2,M3)[5],Neurokinin(NK1, NK3)[6],Purinergic (P2Y1, P2Y4,P2X2, P2X5)[7,8],(5-HT, 5-HT3, 5-HT4)[9,10] and so on. The studies on receptors are benificial for researching function and designing novel medicines.As we know, motilin is a kind of braingut petide composed of 22 amino acid,which is mainly secreted by the endocrine cell in the epithelial mucosa of duodenum and jejunum. Its physiological function is to regulate the composite wave of gastrointestinal transitional movement. The study on motilin receptor is helpful to study themmechanism of gastrointestinal movement regulated by motilin. The receptor for the expression also been demonstrated on the myenteric neurons and smooth muscle cells in different species[11,12,13,14,15,16,17,18]. While as the important part of the gastrointestinal functional unit"nerve ?bers–ICCs-smooth muscle cells",the motilin receptor would be perhaps on ICC .We previously have investigated that the role of interstitial cells of ICC in construction of intestinal tract smooth muscle induced by motilin receptor agonist[19]. Finally we drawn the conclusion that the promotion of motilin receptor agonist to intestinal tract were mediated by ICC and ICC maybe have motilin receptor expressed. ObjectiveWe aim to investigate the culture,subculture,cryopreservation and recovery and to study the biological characteristics of ICC; The other aim of this study was to look for the evidences of motilin receptor on interstitial cells of Cajal (ICC) of the rabbit. MethodPart one : Enzymatic digestion and Ficoll density centrifugation were used to dissociate ICC from the intestinal segment of rabbit offspring. The antibody of c-kit, was used to identify the cultured and recovery ICC under the fluorescence micrographs. Try to find out the conditions of cryopreservation and for ICC and to draw the cell growth curve by MTT method.Part two: Comparing the results of flow cytometry from the freshly cell suspensions of primary culture and from the cells cultured for 15d.,choose the best way to sort ICC by flow cytometry;Cells cultured for a few days were sorted for ICCs with c-kit stained green fluorescent through flow cytometry. The total RNA and proteins extracted from the sorting ICCs were respectively used to verify motilin receptor on the ICCs by Reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The motilin receptor was identified by immunocytochemistry assay with GPR38 antibody,c-Kit antibody and hoechst33342 combined to label ICCs.Results1,We had successfully dissociated and cultured ICCs of rabbit small intestine in vitro.Both observation under light microscope and fluorescent staining with c-Kit antibody confirmed that the culture ICCs were successful. ICCs grew in good condition after several passages.ICC in the logarithmic phase after liquid nitrogen cryopreservation were succeed in recovering identified by cell morphology. We had got the the cell growth curve for 7 days.2,From the cells cultured for 15d, c-kit-positive cell ratio was 19.1% highter than 9.8% with the other method. we finaly chose the cultured cells to get more positive cells. Flow cytometry analyzed that ratio of c-kit positive cell in the cultured cells was 64.3%.The number of sorted ICCs was 6.7×105and 5.6×106 . The results of RT-PCR and Western blot con?rmed the ICCs had motilin receptor expression.Immunocytochemistry also con?rmed the ICCs had motilin receptor expression.ConclusionsIn summary, the culture,passage and recovery of ICC are provided the foundation for establishing the cell lines and given some reliable and scientific pratice experience for the researchers in this field ,which not only save the resources and time but also advance the research more deeply .Basied on the study, we at the first time demonstrate that motilin receptor is present on ICC of the rabbit. The results indicate ICCs may play an important role of gastrointestinal movement induced by motilin.