The Study Of Soothing Liver And Regulating Qi On Effects And Mechanism Of The Rat Gastric Interstitial Cells Of Cajal Differentiation And Cytoactive

Objective:To study and analyze the effect of Soothing Liver and Regulating Qi method on promoting the rat gastric interstitial cells of Cajal differentiation and cytoactive and explore its mechanism based on the Ca2+ channel and the SCF/c-kit signal transduction pathway in vitro cell-experiment.Methods:(1) We prepared drug-serums and divided into groups including treatment groups as 5%,10%,20% concentrations of Chaihushugansan, conditional control group as 10% concentration of Motilium and blank control group as 10% concentration of Saline. (2) Interstitial cells of Cajal (ICC) of gastric antrum were isolated by collagenase type Ⅱ digestion method, had a cell primary culture with the M199 complete medium and observed under the inverted microscope. (3) We identified the cultured cells for determination of ICC via Immunofluorescence staining and observed by the upright fluorescence microscope, the ICC logarithmic growth phase curves by MTT method. (4) The cytoactive was detected by CCK-8 method (24h、48h、72h after using the drug-serums), Ca2+ changes of ICC were tested by Fluo-3 fluorescence and observed by the laser scanning confocal microscope, and protein expressions of CaM, SCF and c-kit were detected respectively by Western Blot (72h after using the drug-serums), and then we compared the effect of ICC differentiation and cytoactive of rat gastric antrum with each group.Results:The morphological characteristics of ICC were obvious under the inverted microscope, the logarithmic growth phase curves reflected its growth characteristic, and the c-Kit positive expression of specific membrane binding protein was identified by the immunofluorescence staining and observed by the upright fluorescence microscope, implied that ICC were cultured successfully. The cytoactive on 24h、48h、72h and intracellular Ca2+ fluorescence intensity of ICC both in groups of Chaihushugansan and Motilium, the protein expressions of CaM and c-Kit in the group of Motilium, and the protein expression of CaM, SCF, c-Kit in the groups of 10% and 20% concentrations of Chaihushugansan all increased significantly, which above all compared with the blank control group (P<0.05). The ICC cytoactive on 24h、48h、72h, intracellular Ca2+ fluorescence intensity and the protein expressions of CaM in the groups of 10% and 20% concentrations of Chaihushugansan, and the protein expressions of SCF, c-Kit in the group of 20% concentration of Chaihushugansan all increased significantly, compared with the group of Motilium (P<0.05). The ICC cytoactive both on 48h and 72h and the intracellular Ca2+fluorescence intensity in the groups of 10% and 20% concentrations of Chaihushugansan, and the protein expressions of CaM, SCF, c-Kit in the group of 20% concentration of Chaihushugansan all increased significantly, compared with the group of 5% concentration of Chaihushugansan (P<0.05); The ICC cytoactive on 72h and the protein expressions of c-Kit in the group of 20% concentration of Chaihushugansan all increased significantly, compared with the group of 10% concentration of Chaihushugansan (P<0.05).Conclusions:The method of soothing Liver and regulating Qi had a role to promote the differentiation and cytoactive of the rat gastric ICC. Its mechanism might be related to activate the Ca2+and CaM to raise the ICC intracellular Ca2+and protein expression of CaM by the Ca2+channel, and up-regulate the protein expressions of SCF and c-Kit through the SCF/c-kit signal transduction pathway.