Study On The Correlativity Of Hormone-Growth Factor-Adhesion Factor Modulator System In Yak Endometrial Epithelium

Yak(Bos grunniens)as an important domestic of the Qinghai-Tibet Plateau,has been challenged by the cold environment for a long time.Yak are seasonal animals with reproductive rates lower than that of other mammals.Yak tends to have one offspring at two years-of-age and two offspring at three-years-of-age.In order to improve the implantation,the study about the hormone-growth factor-adhesion factor regulatory system in yak endometrial epithelial cells.Mainly from the following aspects of the experiment:(1)Isolation and Purified of Endometrial Epithelial Cells: 2/3 segments of yak uterine horn are selected and the epithelial layer are separated,and different digestion methods are used(trypsin digestion,collagenaseI digestion,and two enzyme digestion methods)on endometrial epithelium,and the optimal digestion method was evaluated by the MTT method to calculate the number of digested cells and digestibility.Cells were purified by the methods of differential digestion and differential adherence.The study found that the digestibility of the two enzyme digestion method is the highest,and the best digestion time is first digested with 0.25% trypsin for 10 minutes,and then digested with 0.25% type I collagenase for 3 hours.The highest purity of cells was obtained by a differential adherent method for 30 min to 35 min in cell purification.The high-purity of yak endometrial epithelial cells were obained by the method of the optimized the isolation and purification in this experiment.(2)Identification and Characteristics of Yak Endometrial Epithelial Cells: The cell growth curve was drawn by cell counting method.The cells were identified by cytokeratin 18 and vimentin and PBS.The DNA of the cell were identificated by Feulgen staining and chromosomal analysis.The results showed that the cell growth curve was similar to the "S" growth curve,and the number of cells was logarithmic growth at 3-6 days with an average growth cycle of 1.5-2 days.Cytokeratin 18 was positive,vimentin was positive and PBS was negative,the positive expression rate of cytokeratin 18 was over 99% which can demonstrate that the purity of the cells was very high.The isolated primary epithelial cells had a normal diploid configuration containing 60 chromosomes(29 pairs of autosomes and one pair of sex chromosomes).These are enough to prove that the cells obtained in experiment 1 were yak endometrial epithelial cells.(3)Effects of Different Concentrations of Estradiol and Progesterone on Secretion of Epidermal Growth Factor and Insulin-like Growth Factor in Yak Endometrial Epithelial Cells: The third generation of yak endometrial epithelial cells which were purified was used in this experiment,and add estradiol and progesterone hormones and two hormones mixed together.The optimal growth time of the two hormones for the cells and the best expression time of EGF and IGF-1 were determinated by MTT and Real-time PCR methods.Two kinds of hormones,estradiol and progesterone,were added at different concentrations separate and common.The effect of EGF and IGF-1 were detected by immunofluorescence,Real-time PCR,WB,and ELISA.The results showed that the optimal growth time of cells and the best expression time of growth factors were 24 hours.The expression of two growth factors was positively correlated with hormone concentration when induced by a single hormone but not significant,and the expression of both hormones increased significantly at the same time which were detected by immunofluorescence,Real-time PCR,WB,and ELISA.The best expression concentration was estradiol 10 ng/mL progesterone 10 ng/mL.This experiment found that the combination of progesterone and estradiol could effectively increase the expression of EGF and IGF-1 in endometrial epithelial cells.(4)Effects of Different Concentrations of EGF and IGF-1 and Their Inhibitors on Secretion of Mucin and Placental Growth Factor in Yak Endometrial Epithelial Cells: The third generation of yak endometrial epithelial cells which were purified was used in this experiment,and add EGF and IGF-1 and their respective inhibitors to them.The optimal growth time of the two growth factors for the cells and the best expression time of MUC1 and PGF were detected by MTT and Real-time PCR methods.Two kinds of growth factors and their respective inhibitors were added at different concentrations.The expression of MUC1 and PGF were detected by immunofluorescence,Real-time PCR and WB.The results showed that the optimal growth time of cells and the best expression time of adhesion factors were 24 hours.The expression of MUC1 was a negative correlation the concentrations of EGF and IGF-1.The expression level was the lowest at 100 ng/mL,and positively correlated with the inhibitor concentration of two growth factors,the optimal concentration was 40 ?M/mL.There was a positive correlation between the expression of PGF and the concentrations of EGF and IGF-1.The optimal concentration was 100 ng/mL.There was a negative correlation between the concentration of PGF and the inhibitors of two growth factors.The expression level was the lowest at 40 ?M/mL.This experiment found that growth factors can inhibit the expression of MUC1 on endometrial epithelial cells but can promote the expression of PGF.In this experiment,the regulation system of hormone-growth factor-adhesion factors was studied from yak endometrial epithelial cells which were isolated and purified,and the expression and the induced relationship of estrogen and progesterone and EGF and IGF-1 and MUC1 and PGF were elucidated.The specific conclusions are as follows:1.The best digestion method for yak endometrial epithelium is two-step enzymatic digestion,which is first digested with 0.25% trypsin for 10 minutes and then digested with 0.25% collagenase I for 3 hours.The best purification method is different time to adhere method,the wall by differential adhesion for 30 minutes.2.The cells obtained by digestion and purification were identificated to be yak endometrial epithelial cells by immunohistochemistry,feulgen staining,and karyotype analysis,and the growth curve of the cells was plotted.3.E2 and P4 could promote the expression of EGF and IGF-1 when them used alone.When E2 and P4 were used together,the expression levels of EGF and IGF-1 were significantly increased.The best expression concentration was estradiol 10 ng/mL progesterone 10 ng/mL.4.Both EGF and IGF-1 can inhibit the expression of MUC1 with an optimal inhibition concentration of 100 ng/mL.Both the inhibitor Gefitinib and BMS-536924 promoted the expression of MUC1,and the optimal concentration was 40 ?M/mL.Both EGF and IGF-1 can promote the expression of PGF,and the best promotion concentration is 100 ng/mL.Both inhibitor Gefitinib and BMS-536924 can inhibit the expression of PGF,and the optimal inhibition concentration is 40 ?M/mL.