| Canine pyometra is one of the most severe obstetric diseases of bitches at the metestrus. According to the previous study, cystic endometrial hyfperplasia(CEH) is the pre-disease of canine pyometra. With the interaction of bacterium and other factors, CEH would turn into canine pyometra. Previous studies indicated that estrogen(E2) and progesterone(P4) are the main inducements for CEH. The main objective of this study was establishment of an ideal procedure of endometrial stromal cells(ESCs) isolation and cultivation; investigation of the progesterone receptor(PR) expression in ESCs; and determination of the effect of E2 and P4 on cell proliferation and PR expression, hoping to clarify the relationship of canine pyometra and E2 & P4, and provide theory support for further study.1. For establishment of an ideal procedure for isolation and cultivation of canine ESCs, exploration of suitable digestion enzymes, cell identification, subcultivation and exploration of suitable fetal bovine serum concentration(FBS) were tried to isolate and culture canine ESCs in vitro. The result indicated that incubating endometrium with 0.125% collagenase at 37℃ for 3~4 h was the best method for canine ESCs isolation. Higher concentration(20%) of FBS was more suitable for the growth of canine ESCs. During the cultivation, the shape of the cells would be gradually showing polygonal and spindle-shaped, while the nuclear was oval-shaped. After 5~6 day’s cultivation, the cells paved into monolayer containing phenomenon of overlapping. Imunocytochemical staining method was used to identify the cells, and the result showed that both the two types of cells were positive for vimentin and negative for cytokeratin. The vimentin positive rate was higher than 90%, so the cells proved to be ESCs.2. Different levels of E2 and P4 were added in the culture medium, and cell proliferative capability was determined by MTT method. After the serum starvation for 24 h, canine ESCs entered logarithmic growth phase on the 2nd day and last for 5 days. The number of cells stopped growing on the 6th day, and then slowly decreased during the 7th day and 8th day. The growth curve of E2(15, 30, 100 pg/m L) group showed no significant difference with control group. The growth rate was improved after adding P4(3, 15, 30 ng/m L), and the cell number was increased in platform phase. In the three phases of the cell cycle(d3: exponential phase, d6: plateau, d8: decline phase), E2 showed no typical effect on the proliferation of ESCs. In each phase, cell growth was not sensitive to E2 concentration(15, 30, 100 pg/mL). P4(15, 30 ng/mL) showed a significant role on the proliferation of ESCs(P < 0.05). In same phase of the cell cycle, ESCs proliferation and P4 concentrations were positively correlated.3. The effect of E2 and P4 on the PR expression in ESCs was evaluated by cell immunohistochemistry. The result showed that canine ESCs was strongly positive to PR antibody. The nuclears were immunostained yellow brown while the cytoplasm couldn’t be stained, indicating that PR was expressed mainly in the nuclears. PR positive rate in control group was always higher than 90%. The positive rate of PR with E2 treatment(15, 30, 100 pg/m L) showed no difference with the control group. P4 treatment(3, 15, 30 ng/m L) showed a significant inhibition from the 6th day(P < 0.05). The PR expression of P4(3 ng / mL) group was gradually decreased during the cultivation, and droped to the lowest value on the 8th day. The PR expression of P4(15, 30 ng/m L) group didn’t decreased significantly after the 6th day. The PR expression of P4(3 ng / mL) group was lower than P4(15, 30 ng/m L) on both the 6th and 8th day.According to the above results, P4 could promote the proliferation of canine ESCs, and inhibit the PR expression canine ESCs cultured in vitro. The effect was related with concentration and the action time indicating that PR expression might be regulated at the cellular level. E2 showed no significant effects on canine ESCs. |
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