Isolation And Culture Of Intestinal Interstitial Cells Of Cajal And The Expression Of P2X₇ In Interstitial Cells Of Cajal In Vitro : A Role Of High Concentration Glucose

Gastrointestinal motility disorder is one of chronic complications of diabets mellitus(DM). It's morbidity is high and the pathogenesis remains unclear. Generally speaking, gastrointestinal motility disorder has relation to autonomic neuropathy and the gastrointinal hormone imblances ,especially the metabolism disorder of glucose may be the base of the disease. Now the treatment is limited to symptoms and the effect is not good. So it is great significance to further explore of the pathogenesis of the disease and more effective treatments. In recent years, more and more experiments have confirmed that interstitial cells of Cajal (ICC) have close relation to it. But it is difficult to isolate and culture ICC in vitro. Purinergic receptors are one kind of non-adrenergic non-cholinergic(NANC)ones. The P2X7 is one of them ,with specific structure and function,and that it have become an important research focus.Objective: To explore the method for isolation and culture of intestinal ICC in Balb/c mice ,observe the expression of P2X7 on ICC, and the effect of high concentrtion gluose on it in vitro, and investigate the cellular mechanism of gastrointestinal dysmotility in diabetic mice.Methods: The intestine segments of mice were dissected in asepsis, then the mechanical and enzymatic dissociation were used to culture ICC. The cells were suspended in M199 medium containing SCF (stem cell factor),their appearance were observed in upside-down light microscope. The cultured ICCs were stained with c-kit immunofluorescence antibody and indentified under the confocal microscopy. The cultured ICCs were double-labeled and indentified with c-kit and P2X7 immunofluorescence antibodies under the confocal microscopy. The ICCs were divided into high glucose and control groups,cell appearance was observed in upside-down light microscopy, and expression of P2X7 and c-Kit mRNA in ICC was showed by RT-PCR.Results: There were large nucleus and more than one primary processes that branched into secondary processes in ICC cells ,and networks formed between them. Fluorescent staining with c- kit antibody confirmed that ICC culture was successful. The immunofluorescence antibodes of P2X7 were also positive in cells in which the immunofluorescence antibodes of c-Kit were positive under the confocal microscopy. After treatment of high glucose,the bodies of ICC were bigger and their processes were shorter ,the network formed from the processes was less.From RT-PCR, we also proved that P2X7 expressed in ICC ,and found the expression of c-Kit was more weaker and the expression of P2X7 was stronger than that of control.Conclusion: It is difficult to isolate and culture ICC. We succeed in finding the method and outlining some question which need to be attached importance to in the study. P2X7 receptors express in ICC. Hyperglycemia may change the appearance , decrease the expression of c-Kit, and enhance the expression of P2X7 in ICC. High glouse may impact the morphology and the function of ICC through P2X7.These may be play roles in the cellular mechanism of gastrointestinal dysmotility in diabetic mice.