Culture Of Interstitial Cells Of Cajal Of Murine Small Intestine And The Effect Of Gene Transfection Of SCL On Its C-kit Expression

BackgroundInterstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and also transduce inputs from the enteric nervous system. In the intestine, slow wave generation has been linked to the presence of a layer of ICC within the Auerbach's plexus region. Interstitial cells of Cajal (ICC) are required for normal gut motility. They generate the electrical slow wave and function as mediators of neuronal input to gastrointestinal smooth muscle. In humans, loss of ICC is associated with a number of motility disorders including slow transit constipation and complications of diabetes. In mice, naturally occurring loss-of-function mutations in the genes for the receptor tyrosine kinase, c-Kit or the c-Kit ligand, stem cell factor result in depletion of ICC and motility disorders. Inhibition of c-Kit signalling with a neutralizing antibody directed against c-Kit (ACK-2) also results in loss of ICC and motility changes. These data clearly indicate that c-Kit signalling is necessary for normal development of ICC and for normal gut motility.Stem cell leukemia (SCL) also called TAL-1 or TCL5, is a basic domain, helix-loop-helix (bHLH) transcription factor mainly required for the development of hematopoietic cells. It has been confirmed that there are SCL binding sites on c-kit gene regulation region and SCL could strongly up regulate c-kit expression in many cells. However there is no clear report on its function in ICC and its expression of c-kit.Although there are many researches on ICC now, the procedure in isolation and culture of ICC still has many difficulties that should be studied and discussed. Their morphologic charactor and Ca2+ oscillation asocciated slow waves during short-term culture in vitro have no clear explanation by now. SCL could strongly enhance many cells to express c-kit, while there was no clear report on that whether it could realize the same action on the expression of ICC.Objective1. Establish the method of ICC cuture in vitro and investigate its morphologic changes and function;2. Construct the SCL expression vector and probe its effect on the expression of c-kit in ICC.MethodEnzymatic digestion and ficoll density centrifugation were used to dissociate ICC from murine small intestine. Factors including contamination, Ca2+, Mg2+and collagenase, and stem cell factor(SCF) etc were investigated. ACK2, the antibody of c-kit, was used to identify the cultured ICC of murine small intestine. Both light microscope and fluorescence microscope were used to observe the changes of ICC of murine small intestine in vitro. Both Ca2+ oscillation and the effect of emodin on it were studied under confocal microscope. SCL expression vector pIRES2-EGFP-SCL was constructed through the strategy of subcolon cDNAs of SCL into medium vector of pLXSN-EGFP. EMSA was used to detect the transcript action of SCL in ICC of murine small intestine, and Western blot was used the investigate up-regulation effect of SCL transfection on the expression of c-kit in ICC.Results1. The method for dissociation and culture of ICC of murine small intestine was successfully established. ICC of murine small intestine were identificted by ACK2, the antibody of c-kit. After 24 hours, cultured ICC exhibited a few axis-cylinders, and longer axis-cylinders were observed to form synapse each other after 3 days. More widespread connections formed within 7 days in vitro. The changes of its morphologic character were obvious within 7 days, however there were no obvious morphological changes after 30 days.2. ICC of murine small intestine cultured in vitro had function of Ca2+ oscillation asocciated slow waves in vitro (about 22 circls/min), which is occodinated with other researches. Emodin could significantly increase the frequency (about 34 circles/min) and amplitude of vibration.3. We have successfully constructed SCL expression vector in that its accodination with the origin sequence by race sequencing.4. Both SCL protein and its DNA binding activity were upregulated by transfection ofthe pIRES2-EGFP-SCL into ICC of murine small intestine (p<0.05).5. Gene transfection of SCL could strongly upregulate c-kit expression in ICC cultured in vitro (p<0.05).Conclution1. We have established the culture method of ICC of murine small intestine, while there were many factors should be discussed: preventing contamination; shortening the time in vitro and minimizing zero Ca2+ exposure; stretching the tissure lightly; appropriate enzyme digestion; changing the culture medium; stem cell factor is essential to maintain ICC in culture.2. ICC of murine small intestine cultured in vitro showed morphologic character. The processes could form secondary and tertiary processes and contact each other and establish multiple networks. Cultured ICC of murine small intestine in vitro had function of Ca 2+ oscillation linked slow waves and emodin could significantly increase its frequency and amplitude of vibration.3. SCL tansfection could significantly upregulate the level of SCL expression and enhance its DNA binding activity in ICC of murine small intestine.4. c-kit expression in ICC of murine small intestine was significantly up-regulated by SCL , which maight be through the increased SCL protein level and enhanced DNA binding activity.