An Experimental Study Of Regulative Mechanisms Underlying The Plasticity Of Interstitial Cells Of Cajal In Adult Mammals

Interstitial cells of Cajal (ICCs) are a kind of speical interstial cells, and distribute throughout the entire gastrointestinal tract. In recent two decades, a lot of researches have been carried out to investigate the morphology, ultrastructure, origination and physiology of these cells, and it have been identified that ICCs are the pacemaker cells that generate and propagate the slow waves in the alimentary tract, and act as the mediators of inputs from the enteric nervous system to GI smooth muscles; they also serve as a stretch sensor of the GI tract.Clinical studies have indicated that quite a number of GI motility disorders, such as diabetic gastroparesis, slow transit constipation, Crohn's disease and post-operational gut dysmotilities, are characterized by a reduction of ICC numbers and impairment of the cellular network. However, the underlying reasons for ICCs loss and impairment are not yet well-known. Clarification of the regulatory factors that control survival and proliferation of ICCs will further our understanding and treatment of GI motility disorders.ICCs express the gene product of c-kit, a proto-oncogene that encodes the receptor tyrosine kinase (Kit). Its ligand, stem cell factor (SCF), is produced by smooth muscle cells (SMCs) and neurons. Inactivation of Kit with neutralizing antibodies or Kit blocker in fetal and neonatal animals, or non-lethal mutations of Kit or SCF in rats and mice, leads to defects in ICC networks and pacemaker activity of GI movement. Therefore, the Kit/SCF signal pathway is considered essential for the development, proliferation, differentiation and survival of ICCs during the fetal and neonatal periods.ICC precusors begin to express Kit protein at E12, and day-2 post-partum these cells further develop in morphogy, ultrastructure and function almost same with the mature ones. In vitro experiments have also shown that only cultured ICCs from the mice before day-6 post-partum can proliferate in response to SCF, indicating that the proliferation of ICCs is time-limited and SCF-dependent under culture conditions. Our previous experiments have shown that after semi-transection and anastomosis, ICCs at the anastomotic site are lost and then ICCs adjacent could proliferate and gradually recover to normal distribution. This finding suggests that ICCs in adults still possess a proliferative ability. Besides that, we also have found that the new ICCs always emerge with SCF-expressing smooth muscle cells, which hinted that Kit/SCF signal pathway might play an important role in the regulation of the proliferation of ICCs in adults.Although ICCs keep expressing the c-kit gene and its product, Kit protein, the effects of Kit signal pathway on mature ICCs are still unknown. Besides that, serveal issues are still needed to be clarified, such as how mature ICCs response to the inhibition of Kit signal pathway? Apoptosis? Death? Whether ICCs could recover to normal after loss? Which method ICCs are dependent on during the recovery? What role is the Kit/SCF signal pathway plays? Furthermore, it is also unclear that how ICCs response to disease, and whether the alterations of ICCs is under the regulation of Kit/SCF signal pathway. Therefore, to investigate the effects of Kit/SCF signal pathway on mature ICCs would benefit the understanding of the underlying mechanism of gastrointestinal motility disorders and exploring possible therapy strategies.Supposing Kit/SCF signal pathway regulate the plasticity of ICCs in adult, this would benefit the understanding of gastrointestinal disorders, and provide possible therapic method. Therefore, this investigate is divided into two part:Part 1:Imatinib is a potent inhibitor of Kit and can used in vivo. We treated adult guinea pigs and mice with Imatinib, and observed the alterations of the cell numbers and process length of ICCs by immunoflourosent staining with anti-Kit and Vimentin antibodies after a peroid of drug treatment. Also Kit/a-SMA double labeling were carried out to investigate whether ICCs transdifferentiate toward a phenotype of smooth muscle cells after drug treatment, and the mechanical contractions of small intestine were measured under the condition of in vitro organ bath. After drug withdrawal, except the observation of ICCs numbers and process length, and the measurement of mechanical contraction, Kit/PCNA and Kit/BrdU double staining were used to detect the proliferative events of ICCs. All the methods were used to clarify the association between the plasticity of ICCs and Kit/SCF signal pathway.The results are the followings:1 After Imatinib treatment, ICCs reduced in cell number and process length, meanwhile the mechanical contractions of small intestine were inhibited.2 Both Kit/a-SMA double labeled cells and Kit(-)/a-SMA(+) ICC-like cells were observed, which suggest a possibility that ICCs might transdifferentiate toward a phenotype of smooth muscle cells.3 After Imatinib withdrawal, ICCs recovered in cell numbers and process length, along with a regain of mechanical contractions.4. At the same time, a number of Kit/PCNA and Kit/BrdU double stained cells were found out, which hinted a proliferative events of ICCs.The above result have indicated that Kit singal pathway is essential for the maintanence, survival and proliferation, under the condition of Kit inactivation ICCs might transdifferntiate towards a smooth muscle cell phenotype, and ICCs in adults could proliferate after lost.Part 2:Mice with mechanical intestinal obstruction were used as animal models. During the intestinal obstruction, the changes of ICCs, including cell numbers and transdifferentiation, were investigate by Kit and Kit/a-SMA single and double immunoflouresence, and the alterations of SCF and p-Kit were assayed by western blot, beside that intestinal mechanical contrations were measured in in vitro organ bath. After removal of the obstruction, besides the ICCs number and expression of SCF and p-Kit protein, Kit/BrdU double labeling were applied to detect the proliferative events of ICCs. The methods were used to investigate the alterations of ICCs during intestitnal obstruction and after removal of obstrution, meanwhile analysized the expression of SCF and associated activation/inactivation of Kit. These results might clarify the regulative effects of Kit/SCF signal pathway on the plasticity of ICCs, and provide possible therapic strategies.The results were the followings:,1. The mechanical intestinal obstruction led to a reduction of the cell numbers of ICCs which was most obvious adjacent to the obstructive site, meanwhile Kit/a-SMA double positive cells were observed, and intestinal motitlity were inhibited obviously.2. After removal of obstruction, ICCs increased in amounts, and BrdU/Kit double labeled cells were observed accompanying with the recovery of intestinal motility.,3. The expression of SCF and p-Kit were apperantly down-regulated to about 1/2 or 1/3 during obstruction, which suggested the Kit/SCF signal pathway was inhibited.4. After removal of obstruction, SCF and p-Kit restored to normal level, which indicated that the activation of Kit/SCF signaling was closely associated with the revovery and proliferation of ICCs.The second part revealed that the expressive alterations of SCF was closely associated with the activation/inactivation of Kit receptor which tightly linked with the proliferation and transdifferentiation of ICCs. These finding suggested Kit/SCF signal pathway regulated the plasticity of ICCs in adult mice.To sum up, the results above proved that ICCs in adults still possess vivid plasticity, in other word, these cells could transdifferentiate towards a smooth muscle cell phenotype and reduced in numbers, also could proliferate and restore normal amounts, distribution and function. The smooth muscle cells could regulate the the plasticity of ICCs by up/down regulation of SCF expression. Therefore the present study suggested that one of the mechanisms underlying the gastrointestinal motility was insufficience which would lead to inactivation of Kit receptor and reduction of ICCs, meanwhile up-regulating the expression of SCF or directly activating the Kit signaling might be one of the potential therapic strategies.