| Objective:Observe the effect of Guizhi Fuling Wan on the function of stromal cells in primary cultured endometriosis and Screen the differential target proteins in stromal cells by differential proteomie technique,then explore the molecular mechanism of Guizhi Fuling Wan in the treatment of endometriosis.Methods:1.Preparation of Guizhi Fuling Wan containing serum:The rats were divided into four groups: blank control group,low concentration group(0.3g / kg),medium concentration group(0.3g / kg)and high concentration group(1.2g / kg).And then use Guizhi Fuling Wan gavage to rats to prepare drug-containing serum2.CCK-8 was used to detect cell proliferation: The primary cultured EMs stromal cells were seeded in 96-well plates,;The cells were divided into four groups: control group,low,medium and high concentration of Guizhi Fuling pill,and CCK-8 method was used to detect the proliferation of cells at 0,24,48,72 and 96 hours.3.Apoptosis was detected by flow cytometry: 10% of different concentrations of Guizhi Fuling pill were treated with interstitial cells for 48 h,and the cells were detected by Annexin V-FITC / PI double staining.4.Transwell detects cell migration and invasion :As the the bottom of the Transwell chamber Similar to biological membrane,Matrigel plastic imitate to physiological invasion barrier.We add the interference factor in the small room and have interstitial cells inoculated in it,fixed and stained the cells 48 hours later,then Calculate cell migration and invasion rates.5.ITRAQ Technique were used to screen differentially expressed proteins in interstitial cells: 10% serum containing interstitial cells for 72 h,the total protein was extracted and the iTRAQ technique was used to screen differentially expressed proteins.6.The expression of typical differential expression protein: 1-3 differentially expressed proteins were selected and the protein expression level was detected by Western blot.Results:1.Cell proliferation results : Compared with the blank control group,the proliferation of cells in the other groups decreased,and the concentration of serum containing different concentration negatively correlated with the serum concentration.The high concentration of serum was the highest in the cell proliferation group,which was statistically significant compared with the other groups.2.Apoptosis results :The apoptotic rate of the other groups was significantly higher than that of the blank serum group(P> 0.05).3.Cell migration and invasion results : In the four groups,the cells in the blank serum group had the highest mobility and attack rate,while the high concentration of drug group have the lowest rate,and the difference was significant(P <0.05).The most important,Cell migration and invasion were negatively correlated with serum concentration.4.Proteomics results: Compared with the blank murine serum group,there were 275 proteins upregulated in the interstitial cells of the serum of Guizhi Fuling Wan and the average expression of kurtosis was more than 1.2 times.There were 97 down regulated proteins with an average expression of kurtosis was more than 0.55 times.5.Western blot results: The expression of VPS53 in stromal cell protein of high concentration serum group was higher than that of blank control group,low concentration drug group and medium concentration group(P <0.05);The expression of FMNL2 in the Endometriosis Interstitial cells protein of the high concentration serum group was the lowest among the four groups.conclusions:1.Guizhi Fuling Wan serum can interfere with endometriosis stromal cell function,inhibit cell proliferation,promote cell apoptosis,and weaken its migration and invasion.2.The differentially expressed proteins were screened out of the interstitial cells of Guizhi Fuling Wan,The results showed that the differentially expressed proteins of VPS53 and FMNL2 were consistent with the results of iTRAQ,but the specific mechanism was to be further studied. |
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