| Background: Anovulatory dysfunctional uterine bleeding(ADUB) is related to defect of self-controlled endometrial hemostasia, mainly due to retarded endometrium repair. The process of endometrium repair includes re-epithelialization and vessels regeneration. There has been no evidence to show if endometrial re-epithelialization of ADUB is the same as that of menstruation. The microenvironment involved in endometrial repair consists of growth factors and their receptors, cytokines and their receptors, vascoactivators, extracellular matrix, estrogen receptor and progesterone receptor interacting with complex signal cross-talk. Many study results suggested that the expression of these regulators in endometrium around menstruation is identical with that in other tissue during wound healing. Therefor, the mechanism of endometrial repair of menstruation is likely to share features with wound healing which is regulated by a wealth of regulators. Among these regulators, keratinocyte growth factor(KGF) specifically stimulates the proliferation and differentiation of epithelial cells. Due to these properties, KGF maybe play an important role in epithelial repair process. However, it has not been demonstrated yet that effects of KGF on endometrium epithelial cell ( EEC) of ADUB and menstruation.The aim of present study is to investigate the role of KGF in process of ADUB endometrial repair via observing the effects of KGF on growth, secretion and expression of ER and PR of EEC from ADUB cultured successfully by optimizing the EEC primary culture methods in taking sample, cultural condition and purification.Method:1. EEC of ADUB is digested with collagenase, purified with metal sieves and cultured in DMEM/F12 medium with 5% fetal bovine serum(FBS).2. Characterizing morphology and growth process of EEC from ADUB by light microscope, electron microscope; detecting its purity by immunocytochemistry of cytokeratin-19(CK19).3. Compared with normal EEC from endometrium of proliferative phase and withendometrium of ADUB, biological property of ADUB EEC was investigated. Drawing growth curve by MTT, detecting concentration of carcinoma antigen(CA125) by magnetic antibody immuno assay (MAIA), and analyzing gray degree of proliferating cell nuclear antigen (PCNA), estrogen receptor(ER), progesterone receptor (PR) by immunocytochemistry and OD ratio of ER and PR by RT-PCR.4. Four groups were designed in this study: EEC from normal proliferative phase endometrium(N), EEC of ADUB(A), N+KGF, A+KGF. Contrast their OD value measured by MTT, concentration of CA125 detected by MAIA, and gray degree of PCNA, ER and PR by immunocytochemistry and OD ratio of ER and PR by RT-PCR.Result:1. The yield of EEC from ADUB could be significantly increased by modification of the primary procedure without any contamination and the purity of EEC is 95%. The best time for experiment is on the fifth to the sixth day after inoculation.2. The survival rate of ADUB EEC is similar to that of normal EEC; and the survival rate of EEC from simple hyperplasia is higher than that of EEC from proliferative phase of endometrium(P<0.05).3. The appearance of ADUB EEC is the same asthat of normal EEC, they have same cytoskeleton marker and both of them excrete CA125. EEC of ADUB keep the characteristic ultrastructure of endometrium of ADUB, the pathological changes were found in both cell membrane and organell.4. In vitro, the proliferation of EEC from ADUB is lower than that of normal EEC(P<0.05), but both of them have same growth period and initiate to decay from the seventh day.5. The concentration of CA125 of EEC from ADUB is higher than that of normal EEC(P<0.05). Till the seventh day, it has been increasing.6. The expression of ER and PR is descent significantly in normal endometrium of follicular phase, proliferative phase of endometrium and simple hyperplasia endometrium, while the expression of PCNA is increased. In vitro, the expression of ER,PR and PCNA in EEC from ADUB is lower than that in normal EEC.7. The OD value and CA125 concentration in both N+KGF and A+KGF groups are significantly increased at 50ng/ml level compared to the control group(P<0.05). Increased concentration of CA125 is related to enhancive OD value(N group r=0.681,P<0.05;ADUBgroup r=0.885,P<0.01).8. The gray degree of PCNA,ER and PR of EEC in both N+KGF and A+KGF groups were upregulated significantly at 50ng/ml level compared to the control group(P<0.05).9. The enhanced range of OD value, CA125 concentration and gray degree of PCNA in A+KGF group was significantly lower than that in N+KGF group.Conclusions:1. Depending on improved methods of primary culture of EEC in getting samples, purifying cells and culture condition, we isolated and cultured EEC from endometrium of ADUB firstly, moreover, provided a fundamental tools for researching the function of EEC inADUB.2. Finding out that EEC of ADUB in vitro retained pathologic configuration and function the same asm vivo, alternatively, which were responsible for the retarded growth and the abnormal secretion.3. Both expression and activity of ER were decreased in endometrium and that of EEC in vitro of ADUB, implying abnormal steroids function.4. KGF stimulated cell proliferation and secretion of both ADUB and normal EEC in dosage dependence and the most efficient concentration is 50ng/ml.5. The reactivity of ECC in ADUB to KGF was significantly duller than that in normal ones. This maybe due to its abnormal structure and function or culture condition in vitro.6. Effects on EEC of ADUB by KGF that activated EEC growth and secretion and up-regulated the expression of ER and PR indicate that KGF maybe play an important role in endometrial repair of ADUB.
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