Studies On The Expression Of C-erbB₂ Protein In The Periimplantation Mouse Uterus And The Effects On The Implantation Of Mice Blastocyst In Vitro

Objective:To investigate the expression and distribution of C-erbB₂ protein in the periimplantation mouse uterus; to research the separation, cultivation and identification technology of endometrial epithelial cells(EECs) in vitro; to build the mouse implantation model in vitro and find the ideal cultivational system of mouse implantation model; To preliminarily investigate the effection of c-erbB₂ ASODN on the mouse implantation model in vitro.Methods:1,The pro-ovulated method was used to make pregnant animal models; 2,Immunohistochemistry staining was used to examine the localization and expression of the C-erbB₂ protein in the peri-implantation uterus; 3,The EECs was divided from the Day 4 pregnant uterus by enzyme digestion, and the percentage of the EECs expressing CK-19 was more than 90%; 4,We used man-made glass pipes to inhale the mice blastocysts, and cultivated them with the EECs, in order to determine the ideal co-cultivated system then we divided them into 4 groups; 5,We used the contradict method to observe the effects of the c-erbB₂ ASODN on mouse implantation model in vitro, and divided them into 6 groups,after 24 hours'co-culture we recorded the hatching rate of these groups, after 48 hours'co-culture we recorded the adhesion rate and outgrowth rate of these groups; 6,The expression of C-erbB₂ albumen in the EECs was detected by Western Blotting.Results:1,Immunohistochemistry experiments showed that unique uterine cell-specific C-erbB₂ protein distribution. The nagetive control slice and non-pregnant uterus exhibited no signals. On day 1-3 unlike in the stromal cells, the C-erbB₂ protein was detected primarily in the EECs; on day 4-5 besides the EECs, the stromal cells expressed a little C-erbB₂ protein, on day 6-8 the EECs and the decidualized cells around the implanting blastocyst exhibited the accumulation of C-erbB₂ protein. 2,The percentage of the EECs expressing CK-19 was 90% more or less , which were isolated from the mouse day 4 pregnant uterus. 3,As shown in the blank the ratios of embryonic hatchment, attachment and outgrowth of the BSA group and the FBS group were obviously higher than the before two groups (P<0.05), but the difference between the last two groups were no significant (P>0.05);There was no difference in the ratios of embryonic hatchment in six groups; 4,The ratios of embryonic attachment and outgrowth between the c-erbB₂ ODN group and control group were no difference either(P>0.05), the ratio of embryonic attachment and outgrowth in group including 5μmol/L c-erbB₂ ASODN is a little lower than control group(P>0.05), the ratios of 48 hours'embryonic attachment and outgrown in groups including 10,15,20μmol/L c-erbB₂ ASODN were obviously lower than control group (P<0.05,P<0.01); 5,The expression of C-erbB₂ protein in EECs was detected by Western blotting. the level of C-erbB₂ protein in groups including 5 c-erbB₂ ASODN was lower than control group, but the difference has no significance( P>0.05), and the level of C-erbB₂ protein in groups including 10 c-erbB₂ ASODN was lower than control group, the differnence has significance(P<0.05), the level of C-erbB₂ protein in groups including 15,20μmol/L c-erbB₂ ASODN was lower than control group, the difference has big significance (P<0.01); the level of C-erbB₂ protein in groups including 20 c-erbB₂ ASODN acting for 24 hours and 48 hours was lower than control group(P<0.05).Conclusions:1,The expression of C-erbB₂ protein in the periimplantation mouse uterus changes regularly; 2,The method of enzyme digestion is a valid method to separate the EECs from the Day 4 pregnant mouse uterus; 3,The implantation models of blastocysts cultivated with EECs in medium including FBS and BSA are both the ideal implantation models; 4,C-erbB₂ protein could promote mouse embryo implantation in vitro.