Research On ICC And SP In Gastric Antrum Of Diabetic Gastroparesis And Isolation, Culture And Identification Of ICC

Objective To observe the changes of interstitial cells of Cajal(ICC)and substance P (SP) in gastric antrum in diabetic gastroparesis rat model,then to investigate the related mechanism of gastric motility changes in diabetic gastroparesis.And to study the mothod of isolating,culturing and identifying the KM mice gastric interstitial cells of Cajal,then offer the base for investigating the transplantation,replacement therapy and drugs experiment of ICC.Method(一) To observe the changes of interstitial cells of Cajal(ICC)and substance P(SP) in gastric antrum in diabetic gastroparesis rat model1.Thirty SD rats were selected and randomly divided into diabetic model group(n=20) and normal control group(n=10).2.Rats in the diabetic model group were intraperitoneal injected with strotozotocin(STZ),and were observed the general condition and monitorde blood glucose during this period.The rats with random blood glucose more than 16.7mmol/L served as diabetic rats.3.Using gastrogavage with radioactive ^99mTC to quantify liguid food emptying rate after three months.The tissue specimens were obtained from the rats of control group and the diabetic gastroparesis model group.Immunohistochemical methods were used to investigate changes of the C-kit positive interstitial cells of Cajal and substance P in rat gastric antrum.Using quantitative image analysis software for statistical analysis.4.To observe the ultrastructure of ICC by electron microscope.(二) Isolation,culture and identification of mice gastric interstitial cells of Cajal1.Enzymatic digestion was used to dissociate ICC from KM murine stomach.2.The ICC were cultured and subcultured in M199 culture medium,then were observed with invert microscope.3.The antibody of c-kit was used to identify the cultured ICC under immunofluorescent microscope and confocal microscopy.Results Duplicating successful models of diabetic gastroparesis.Compared with the control group,gastric transmission rate of diabetic gastroparesis rats was significantly reduced(P＜0.01).C-kit positive interstitial cells of Cajal was reduced (P＜0.01),the expression of substance P decreased(P＜0.01).Electron microscopy exhibite that the junctions between interstitial cells of Cajal and other cells were decreased and the structures were damaged,and organelles decreased significantly,mitochondria swelled vacuoles formed,myelin changed,endoplasmatic reticulum dilated,cytoplasm dissolved and the pernuclear space broadened;the cell basilemma and the epicyte were partly separated.ICC exhibit a few triangle or spindle shaped axiscylinders after attachment,and the longer processes could be obsered to build synapse together after 3days.After immunohistochemical stains,the ICC showed C-Kit posotive expression,the nucleus and synapse was significantly pigmentated.Conclusion The gastric motility of rats with diabetic was markedly delayed, which may be associated with the changes of interstitial cells of Cajal number and ultrastructure in gastric antrum;This also may be associated with the decreased expression of substance P.The enzymolysis mehtod for isolating cells was sucessful established.ICC were adherent after 24hours and built the network after 72hours then were stably cultured over 3w.The normal cells morphology was gradually changing,and could apoptosis after 20d.The topics laid a experimental basis for the study of the pathogenesis of delayed gastric motility in diabetic,even provide a new direction for clinical treatment,offer the base for investigating the transplantation,replacement therapy and drugs experiment of ICC.