Effect Of Stem Cell Factor And Insulin On Interstitial Cells Of Cajal Of Mouse Intestins

Interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract, generatingspontaneous electrical depolarizations that organize contractile activity into patternsof phasic contractions. ICC also provide a pathway for active transmission ofelectrical activity in smooth muscles, much like the network of Purkinje fibres in theheart. ICC form synaptic contacts with peripheral nerves, and have been shown tomediate, at a minimum, cholinergic and nitrergic neural inputs to smooth muscle cells.Finally, ICC express mechano-sensitive channels and mechanisms that can transducelength changes to mediate responses to stretch. These cells have important functionsin regulation of mechanical activity, so loss of ICC could certainly result in motordysfunction. Stem cell factor (SCF) is the main ligand of c-Kit, a receptor tyrosinekinase on the surface marker of ICC. SCF-Kit signaling is very important for ICC tomaintain its phenotype, proliferation and differentiation. Smooth muscle cells (SMC )can express SCF and regulate the growth of ICC. Certain concentration of Insulin andInsulin-like growth factor 1(IGF-1) are able to prevent the motor dysfunctionassociated with ICC in diabetes, and also can be helpful to the restoration of SMC. Itis known that there are not insulin receptors on the surfaces of ICC, while SMC hasthe receptors. In the study we investigated the effect of SCF and insulin on mouseintestinal ICC in vitro and discussed the possible mechanisms.Aim: To investigate the effects of SCF and insulin on ICC dysfunction in diabetes andthe possible mechanismMethodsMethods:1. Isolation, culture and identification of mouse intestinal ICC : newborn ICRmouse was sacrificed by breaking down cervical vertebrae. ICC were isolated andcultured from the intestine by enzymolysis. Intestinal ICC were cultured in M199supplemented with 10% fetal bovine serum and recombinant mouseSCF(rmSCF)(5ng/ml ). The cells were identified by c-kit immunofluorescencemethod.2. Detect the content of ICC and SMC with flow cytometer: the cells were culturedfor three days , then were digested into cell suspension and labeled with antibody(c-kit andα-actin),flow cytometer were used to measure the number of ICCand SMC.3. rmSCF intervention: cells were randomly divided into 2 groups, named groupN(without SCF in M199) and group SCF(5ng/ml SCF in M199), the cells werecultured for 3 days , then mRNA gene of c-kit in ICC were analyzed byquantitative Reverse Transcription-Polymerase Chain Reaction, c-kitimmunofluorescence method were used to examine the number and appearance ofICC .4. Bovine insulin intervention: after cultured for 24 hours, cells were randomlydivided into 4 groups and added bovine insulin 0, 10, 30, 50ug/ml seperately inM199 , then the cells were cultured for 2 days , mRNA gene of c-kit and SCF incells were analyzed by quantitative Reverse Transcription-Polymerase ChainReaction, c-kit immunofluorescence method were used to examine the number andappearance of ICC, double immunofluorescence method were used to find thenumber of Ki-67 positive ICC and assess the proliferation of ICC. ResultsResults:1. Culture and identification of intestinal ICC: ICC were attached to the culturevessels by 24h,triangle-shaped or spindle-shaped with big nucleus and shortprocess . 48 to 72 hours later the process became longer and the ICC nearby makenetworks. Around the ICC there were spindle-shaped SMC which has abundantcytoplasm, small nucleus and high diopter. c-kit immunofluorescence methodshowed the membrane and process of ICC were stained with the fluorescentdyes.2. Detect the content of ICC and SMC with flow cytometer: the number of ICC is15.67±3.11% of all cells and SMC number is 64.2±7.35% of all cells.3. rmSCF intervention: compared with group N, the number of ICC in group SCFwas more and more networks of ICC were found in group SCF(P<0.05). Therewas no significant difference between the two groups about the quantity ofmRNA gene of c-kit in ICC (P>0.05).4. Bovine insulin intervention: Compared with N group, the quantity of mRNA geneof c-kit and SCF were enhanced with the administration of Ins (P<0.05), thenumber of ICC also enhanced in group Ins (P<0.05), but the proliferationpercentage of ICC were not different between group N and group Ins.Conclusions:1. Exogenous SCF are helpful to maintain the phenotype of ICC and number of ICC,but it didn't up-regulate the expression of c-kit mRNA, the possible reasons :experiment error; exogenous SCF may cause a negative feedback process todownregulate the expression of c-kit mRNA, single cell RT-PCR may be helpful toexamine the hypothesis.2. In the system where ICC and SMC were cultured together, exogenous Ins arehelpful to increase the number of ICC and can up-regulate the expression of c-kit and SCF mRNA; Exogenous Ins may promote SMC creating more endogenousSCF , and endogenous SCF is helpful to maintain the phenotype of ICC.3. In the system where ICC and SMC were cultured together, exogenous Ins didn'tup-regulate the proliferation percentage of ICC, the reason may be the experimenterror , there are other possibilities: insulin and SCF may decrease the apoptosis ofICC or avoid phenotype change of ICC and maintain the number of ICC in the end.