The Influence Of17β-estradiol On Gene D111Expression In The Uterine Tissue Of Ovariectomized Mouse

Objective: To study the influence of17β-estradiol interference on the gene Dll1expression level in uterine tissue of the ovariectomized mouse.Methods:①40adult female mice were divided into four groups (eight mice died inthe experiment process), the normal control group, Sham+NS group, OVX+NS groupand OVX+E2group, the normal control group was free of any interference, a little ofgonadal adipose tissue of the mice in Sham+NS group was removed and this group wasinterfered with certain volumes of NS, the OVX+NS group was interfered with certainvolumes of E2solvent after bilateral ovaries tissues were surgically removed and theOVX+E2group was interfered in a dosage of1mg E2/kg·W/d in every two days for10d after bilateral ovaries were surgically removed.②The removed ovaries tissuespecimen from surgical modeling were preserved with10%neutral buffered formalin,regularly dehydrated, cleared, infiltrated with paraffin, paraffin-embedded to behardened, continuously sectioned and then stained with HE, examined andphotographed under light microscope, to certify the removed tissue specimens wereovaries tissues of mice; and then test the changes of serum estrogen levels of the fourgroups via the method of ELISA before and after modeling and interference with NSand E2, to confirm that the surgical modeling of ovariectomization succeed andwhether the exogenous estrogen had entered the blood circulation and took anbiological effect.③After the ovariectomization modeling and NS/E2interference werefinished, the bilateral uterine tissues of all the mice were removed as specimens and weighted wet, then the uterine index of all mice was calculated. The total RNA of theuterine specimen was isolated in a single-step method with TRIzol Reagent, the targetsequences of target gene Dll1and control genes Gapdh were amplified by RT-PCR withcorresponding special primers, all the productions of the RT-PCR were electrophoresedwith the1.5%agarose gel, and the results of the electrophoresis were examined andphotographed under the FR-200A fully-automatic ultraviolet-visible analyzingequipment and Smart view biological electrophoresis photo analyzing software. Theluminous intensity (Adj.Vol.) of the target bands of gene Dll1and Gapdh of the groupof Normal, Sham+NS, OVX+NS and OVX+E2were semi-quantitatively analyzed andmeasured with the QuantityOne-4.6.2agar gelatine analyzing software; the ratios ofAdj.Vol（. x±s）of the target gene Dll1and control gene Gapdh of the group of Normal,Sham+NS, OVX+NS and OVX+E2were analyzed with the method of one wayanalysis of variance, and average values of each group were compared with the methodof multiple comparison, the data differences among the group of Normal, Sham+NS,OVX+NS and OVX+E2were analyzed by the software SPSS18.0to see whether thedata differences had sense statistically.Results:①The results of HE staining-paraffin section examination show that theremoved tissue specimen of the surgical ovariectomization are ovary and oviduct tissueof mouse；the serum estrogen level of the seventeen mice from the OVX+NS group andOVX+E2group declines significantly after modeling of surgical ovariectomizationcompared to that before modeling of surgical ovariectomization (Z＝-3.479，P=0.001，P<0.05), which demonstrates that the modeling succeeds. After ten dates of E2and NSinterference was finished, the serum estrogen level of the OVX+E2group comparingwith that of the group of Normal, Sham+NS, OVX+NS significantly increases, and thedifferences have sense statistically(P<0.05). The uterine index of the OVX+E2group issignificantly greater than that of the rest groups of Normal, Sham+NS,OVX+NS(P<0.05).③The target bands of target gene Dll1and control gene Gapdh of all the samples of the four groups of Normal, Sham+NS, OVX+NS and OVX+E2couldbe detected under the biological electrophoresis image analyzing system, and theluminous intensity (Adj.Vol.) ratio of Dll1/Gapdh (x±s）of the Normal, Sham+NS,OVX+NS and OVX+E2group are0.33±0.12、0.36±0.08、0.30±0.12�'�0.78±0.11respectively. The data differences of the gene Dll1expression level among the fourgroups have sense statistically（F＝35.580，P＝0.000，P<0.05）with the method of oneway analysis of variance using the SPSS18.0software. And then the method ofmultiple comparison was taken, the result of the multiple comparison show that thegene Dll1expression level of the OVX+E2group is significantly higher than thegroups of Normal, Sham+NS and OVX+NS（P<0.05）.Conclusion: The constant high-dose estrogen interference could increase the uterineindex, induce abnormal hyperplasia and increase gene Dll1expression level in theuterine tissue of mouse, which further leads to hyper-activation of Notch signalingpathway, which possibly is the molecular pathological basis for the estrogen-dependentabnormal hyperplasia even cancers of the uterine tissue, its mechanism and meaningneed to be further studied.