| Objective: we had established a mouse model of removing ovaries intact uterus toimitate model of postmenopause, and to explore mRNA levels of NOTCH1gene inmice endometrial tissue after high doses of β-E2continuous stimulation, to speculatethe relationship between E2and NOTCH1signaling pathway from the etiology.Methods:（1）The nonpregnant mature female mice(25-30g) were randomly dividedinto four experimental groups,10in each group; group I (Control group) received notreatment, group II(Sham+NS group) only remove of fat next to ovarian tissue and keepintact uterus and ovaries, received intraperitoneal injection of0.9%NaCl after operative;group III(OVX+NS group) included ovariectomized mice on1.5%sodiumpentobarbital anesthesia surgery, removal of the ovaries of mice, and then receivedintraperitoneal injection of0.9%NaCl;groupIV(OVX+β-E2group)includedovariectomized mice, the mice received high dose of β-E2(1mg/kg) every other day for10days.（2）The removed ovaries tissue specimen were preserved with10%(vol/vol)paraformaldehyde, regularly dehydrated, cleared, infiltrated, paraffin-embedded to behardened, sectioned and then stained with HE, examined and photographed under lightmicroscope, to certify the removed tissue specimens were ovaries tissues of mice.（3）Preoperative and postoperative respectively to extract the tail vein blood0.3ml, andstored at-20℃refrigerator.Ten days drug intervention, the bilateral uterine tissues ofall the mice were removed as specimens and weighted, then the uterine index wascalculated; Endometrium tissues were separated, sample preservation solutionimmersion in EP tube at-20℃refrigerator. The levels ofE2in mice serum were detected by ELISA test Preoperative and postoperative and the serum E2of every groupmice under the stimulus of drug intervention.(4)The total RNA was isolated in asingle-step with TRIzol Reagent, the target sequences of target and control genes wereamplified by RT-PCR with special primers, the results of the1.5%agarose gelelectrophoresis were observed and photographed under the FR-200A fully-automaticultraviolet-visible analyzing equipment and Smart view biological electrophoresis photoanalyzing software, the luminous intensity (Adj.Vol.) of the target bands of geneNOTCH1and GAPDH of the four group were semi-quantitatively analyzed andmeasured with the QuantityOne-4.6.2agar gelatine analyzing software.Results:(1) The results of HE staining-paraffin section show that the surgicallyremoved tissue specimenare ovary tissue of mice.(2) The levels of E2in four groupsmice serum preoperative and postoperative were59.57±31.70and70.35±22.38,t=0.393, P=0.70；56.62±24.36and67.90±32.04, t=1.957, P=0.10；60.51±39.39and28.73±5.58, t=2.41, P=0.04；61.21±27.55and36.62±7.73,t=2.90,P=0.02,respectively. The difference between Control and Sham+NS were no statisticallysignificant (P>0.05). The difference between OVX+NS group and OVX+β-E2groupwere statistically significant (P <0.05).(3) Each group statistic F=48.21, underthe stimulus of high doses of β-E2, the level of E2of OVX+β-E2group wassignificantly higher than Control group, Sham+NS group, OVX+NS group (P=0.00, P=0.00, P=0.00), the difference were statistically significant (P <0.05). The level of E2of OVX+NS group was significantly lower than Control group and Sham+NS group (P=0.00, P=0.00), the difference were statistically significant (P <0.05). Control groupcompared with Sham+NS group, P=0.87, the difference between the groups were nostatistically significant（P>0.05）.（4）Under the stimulus of high doses of β-E2, uteruscoefficient（%）of OVX+β-E2group was significantly higher than other groups (P<0.05). The Each group statistic F=9.23, OVX+β-E2group compared with Control group, P=0.00; Compared with Sham+NS group, P=0.02; Compared with OVX+NSgroup, P=0.01.(3) The target bands of NOTCH1gene and GAPDH gene were foundin all of the samples, fragment size were247bp and398bp. The OD values ofNOTCH1in8cases of control group were0.45±0.22;7cases of Sham+NS groupwere0.24±0.12;9cases of OVX+NS group were0.27±0.08, and8cases ofOVX+β-E2group were0.16±0.06.The statistic F in each group was7.26, the mRNAexpression of NOTCH1gene in OVX+β-E2was significantly lower than control groupand OVX+NS group, P=0.03, P=0.01,(P <0.05), the difference were statisticallysignificant (P <0.05).Conclusion:(1) ELISA test can be used to detect the levels of β-E2in mice serumpreoperative and postoperative, the levels of E2in mice serum after ovariectomizedmodel were lower than before ovariectomized Model. Combining with the removedtissue specimens were ovaries tissues, it can be used to determine the ovariectomizedmodel success.(2) Exogenous high dose β-E2stimulation can induce uterine muscle cellhypertrophy and uterine weight gain.(3) The mRNA expression level of NOTCH1genein OVX+β-E2group is significantly lower than Control group and Sham+NS group.These findings suggest that high doses of β-E2promoted endometrial epithelial cellhyperplasia by down-regulating the expression of NOTCH1gene, interaction betweenNOTCH1gene activity and the estrogen play a significance role in the development ofendometrial. |
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