Effect Of Cholesterol On Interstitial Cajal-Like Cells In Guinea Pig Gallbladder That Was Isolated And Cultured In Vitro

Objective: Gallbladder cholesterol stones are a common digestive system disease,and their etiology is complex,which is the result of a combination of various factors.Common causes such as dietary factors,genetic factors,changes in bile composition,gallbladder dysmotility,etc.are hot topics at home and abroad.In recent years,with the improvement of people's living standards,the incidence of cholesterol gallstones has also increased year by year,seriously endangering human health.Gallbladder interstitial Cajal-like cells are the pacemaker cells of the gallbladder.They participate in the spontaneous rhythmic electrical activity and auto biological contractile activity of the gallbladder,mediate excitatory and inhibitory neurotransmitter effects from the nerve,and are important for maintaining gallbladder motility.effect.Both the gallbladder specimens and the CG animal models of CG patients have a reduction in the number of gallbladder ICLCs,and the mechanism of the reduction in the number of ICLCs remains unclear.Some scholars have suggested that the reason for the decrease in ICLCs in the gallbladder of CG patients may be caused by the excessive absorption of cholesterol by the gallbladder.Whether this statement is correct or not remains to be confirmed.Most previous studies on gallbladder ICLCs have been limited to histology,which limits our further study of the pathogenesis of CG.In this study,the primary culture of guinea pig gallbladder ICLCs and the addition of water-soluble cholesterol in cell culture medium were used to simulate the high cholesterol environment faced by ICLCs in patients with CG,and to explore the effect of cholesterol on the proliferation and apoptosis of ICLCs.And preliminary study on the mechanism of cholesterol affecting the proliferation and apoptosis of ICLCs.Method: Digestion of guinea pig gallbladder tissue by enzymatic digestion,isolation and culture of guinea pig gallbladder ICLCs,identification of ICLCs by c-kit immunofluorescence staining,addition of water-soluble cholesterol-containing culture solution to ICLCs culture medium to simulate the environment with high cholesterol concentration in bile,cck-8 The proliferation rate of ICLCs was detected by flow cytometry.The apoptosis rate of ICLCs was detected by flow cytometry.The expression of ICLC c-kit protein was detected by Western blot,and the expression levels of ICLCs c-kit mRNA were detected by Real-time PCR.Results : The guinea pig gallbladder ICLCs was successfully isolated by enzymatic hydrolysis and subcultured.After 1 week of cell culture,it can be seen under the microscope that there are many spindle-shaped cells,a few of which are triangular,round,elliptical,and star-shaped.The nucleus is larger,the cytoplasm is less,and there are 2 to 3 protrusions.connection.As the culture time increases,the cell processes are elongated and interconnected to form a network,forming typical ICLCs morphological features.The cultured cells were passaged 3 times and immunofluorescence staining(ie c-kit monoclonal antibody and FITC-labeled secondary antibody treatment)was c-kit positive,showing FITC green fluorescence,nuclear DAPI staining,and blue fluorescence.It was confirmed that the cultured cells were ICLCs.After incubation with ICLCs containing 25 mg/L,50 mg/L,and 100 mg/L cholesterol,the proliferation rate of ICLCs was significantly decreased compared with the control group(P<0.05);the apoptosis rate of ICLCs was significantly increased(P<0.05).<0.05);ICLCs c-kit protein and c-kit mRNA expression were significantly decreased(P<0.05).Conclusions :(1)Cholesterol has the effect of inhibiting the proliferation of ICLCs and promoting apoptosis of ICLCs,and the higher the cholesterol concentration,the more obvious the effect of inhibiting the proliferation of ICLCs,and the higher the apoptosis rate of ICLCs.(2)Cholesterol can inhibit the expression of ICLCs c-kit proteins,and inhibit the expression of ICLCs c-kit mRNA.(3)The reason for the decrease of ICLCs in gallbladder of CG animals may be that cholesterol inhibits the expression of c-kit/SCF signaling pathway,slows the growth and development of ICLCs,and increases the apoptosis of ICLCs,which ultimately leads to a decrease in the number of ICLCs.