Establishment Of Murine Interstitial Cells Of Cajal Loss Model And Study On SCF Promoting Its Restoration

Recent studies have indicated that the interstitial cells of Cajal (ICC) are gastroenteric pacemakers that generate and propagate electrical slow waves. They also mediate excitatory and inhibitory motor neurotransmission. Slow waves, also called basic electrical rhythms, are of great functional importance because they determine the rate and timing of activity of the muscles of the gastrointestinal system, and their impairment is likely to cause various motility disorders. If ICC is essential to gastrointestinal function, it can be expected that ICC abnormalities will lead to motility disturbances. Research in the last few years has indeed demonstrated that loss or defects in ICC are associated with disease including infantile hypertrophic pyloric stenosis, Hirschsprung's disease, idiopathic slow transit constipation and chronic idiopathic intestinal pseudoobstruction, etc. The most important advance in ICC study came when it was recognized that ICC express the gene product of c-kit, a protooncogene that encodes a receptor tyrosine kinase. Another important advance was the realization that cell signaling via c-kit and its ligand stem cell factor (SCF) is essential for the occurrence, development and maintenance of ICC networks. And the disruption of SCF/c-kit signaling passway will cause breakdown in ICC networks and absence of slow waves. ICC has the characteristic of plasticity so that lost ICC will likely regenerate. Thus that if SCF/c-kit signaling passway is restored will promote to restore ICC. But no descriptions have elucidated those. Therefore, the present study was primarily designed to establish a model of ICC loss. We also examined whether SCF could promote the restoration of ICC. Objective: 1. To observe the normal distribution of interstitial cells of Cajal and the slow waves activities in the murine small intestine. 2. To establish the model of ICC loss by blocking the c-kit receptors and inquire into its mechanisms in principium. 3. To study the effect of SCF promoting ICC restoration by the way of muscular strips culture in vitro. 4. To study the effect of SCF promoting ICC restoration in vivo. Methods: 1. Firstly, the adult murine were enrolled. The activities of slow waves in vivo were determined in small intestine, and the distribution and configuration of ICC were observed with immunohistochemistry. 2. Secondly, new-born murine were injected intraperitoneally antibody of c-kit (ACK2) 100μg every second day, and control groups were injected saline. The activities of slow waves and interstitial cells of Cajal were investigated when the murine were 10 days postpartum. RT-PCR and Western blotting technique were employed to determine the mRNA and protein expression of c-kit. Apoptosis was detected with TUNEL. And the ultrastructure of ICC was observed by an electron microscope. 3. Thirdly, the muscular strips of small intestine obtained from ICC loss model murine were cultured for different time (1 day, 3 days and 5 days) with various concentrations of SCF (0ng/ml, 50ng/ml and 100ng/ml) to examine the dose-dependent and time-dependent alteration of the slow waves activities in response to SCF. Control groups were cultured without SCF. Based on the above results, the muscular strips of small intestine were enrolled in the most effective concentration of SCF and culture time. The distribution of ICC and the mRNA and protein expression of c-kit were measured. 4. Fourthly, ICC loss model murine were injected intraperitoneally SCF 30μg/kg·d for 5 days continuously, and control groups were injected saline. The activities of slow waves in vivo and the distribution of ICC and the mRNA and protein expression of c-kit were determined in small intestine. Results: 1. The slow waves of small intestine in vivo were proximal cosinal waves in the adult Balb/c murine. The frequency were 17.78 ±1.52 cycles/min and the amplitude were 0.16 mV ±0.02 mV. 2. ICC were located mainly in myenteric plexus region (called ICC-MY) and between the internal thin layer and the thick outer layer of the circular muscle (called ICC-DMP) in the small intestine. The distribution of ICC-MY were continuous.3. The slow waves and the distribution of interstitial cells of Cajal in control groups resembled in adult murine. But the slow waves were arrhythmic and the activities were lower when c-kit receptors were blocked (ACK2 groups). The frequency were 2.38 ±0.89 cycles/min and the amplitude were 0.045 mV ±0.012 mV (p<0.05 vs control). And the distribution of ICC were lost and the mRNA and protein expression of c-kit could not be detected in ACK2 groups. 4. Apoptosis were revealed obviously within the mucosal and submucosal layers, while no apoptosis were found in the myenteric plexus region in ACK2 groups. 5. Electron microscope showed that ICC was few observed and no obvious apoptosis bodies and death cells were found in ACK2 groups. ICC-resembling cells which contained mitochondria less could be observed by chance. 6. The frequency of the slow waves of the muscular strips of small intestine in culture from ICC loss model increased in response to SCF50ng/ml for 5 days or SCF100ng/ml for 3 days and 5 days. But the frequency increased more significantly in SCF100ng/ml for 5 days and the amplitude also increased (p<0.05 vs SCF 0ng/ml for 5 days). 7. The distribution of ICC in the myenteric plexus region could be observed, while discontinuous, when the muscular strips of small intestine from ICC loss model were cultured in the presence of SCF100ng/ml for 5 days. And both the mRNA and protein expression of c-kit also increased (p<0.05 vs SCF 0ng/ml for 5 days), while both were markly lower compared with normal control groups. 8. The activities of slow waves in vivo in ICC loss murine with SCF injected intraperitoneally increased significantly, especially the frequency (p<0.01 vs NS injected groups). And the discontinuous distribution of ICC in the myenteric plexus region could also be observed. Both the mRNA and protein expression of c-kit also increased. Conclusions: 1. Two types of ICC including ICC-MY and ICC-DMP are observed in the Balb/c murine small intestine which are bipolar cells or multipolar cells. ICC-MY are interconnected into a network and have close anatomic relationships with smooth muscle cells and neurons. 2. When the c-kit signal is blocked, ICC loss and slow waves absent are found while ICC appears to differentiate toward a smooth muscle-like cell but no apoptosis or death.3. SCF can promote ICC restoration partially whether is used in vivo or in vitro, and the slow waves are improved as well. ICC and the slow waves show a dose-dependent and time-dependent restoration in response to SCF. 4. The mechanisms of SCF action on ICC may relate with c-kit upregulated.