| Objective:The expression changes of H1.2,Rp S13,Rp L6 and HSD17B12 genes were detected in the ovaries of POI mice which constructed with triptolian polyglycosides.Furtherly,the proliferation and apoptosis phenotypes of the gene of interest with clearly expressed differently were detected in vitro level by applying overexpression and knockdown techniques.Methods:1.POI modeles were builded and verified,then the expression changes of the target genes were detected:(1)32 C57BL/6 female mice(8-10 weeks old)with normal estrus cycle were selected for enrollment,and randomly divided into control group(n=16)and POI group(n=16).There was no difference in behavioral activity and body weight of each mouse enrolled.The POI group was gavaged with triptolian polyglycoside suspension(80mg/kg/d for 14 days).The control group was given an equal amount of saline.(2)During the modeling,weight changes of the mices were observed daily,and the estrous cycle of mice were detected by vaginal exfoliation cytology.After modeling,the ovarian and uterine organ index were calculated.(2)The concentration changes of AMH in the serum of model mices were measured by ELISA.(3)Pathologic changes and follicle count were observed by the sections of mouse ovaries tissues and HE staining.(4)The m RNA levels expression changes of ER,CYP19A1,H1.2,Rp S13,Rp L6,HSD17B12 gene in the ovaries were detected by q RT-PCR.(5)The protein levels expression changes of H1.2,Rp S13,Rp L6 and HSD17B12 gene in the ovaries were detected by western blot(WB).(6)The statistical analysis of the two groups were carried out by t-test or non-parameter test,and there was a statistical difference of P<0.05.2.The cell modeles of overexpression and knockdown HSD17B12 were constructed,and the changes of cell phenotypic were detected:(1)In the KGN and SKOV3 cell lines,the overexpression HSD17B12(oe-HSD17B12)groups were constructed by transfecting lentivirus vectors of overexpression HSD17B12;the sh RNA HSD17B12(sh-HSD17B12)groups were constructed by transfecting lentivirus vectors of sh RNA HSD17B12;and negative control groups(NC)were constructed by transfecting lentivirus vectors of negative control.The transfection efficiency was verified by fluorescence microscopy,stable strains were screened by puromycin,cell lines were then amplified.(2)The cell models were validated by q RT-PCR and WB methods.(3)After overexpression and knockdown of the HSD17B12 gene,the proliferation phenotype in human ovarian granule cell was detected with CCK-8.(4)In addition,flow cytometry techniques detected the changes of apoptosis phenotype.(5)Two groups were compared with t test or non-parameter test and three groups were analyzed by ANOVA,and P <0.05 is statistically significant.Results:1.The POI model of mices were successfully constructed,and the genes of interest were differentially expressed in the ovaries of POI mice:(1)After modeling,the control group had smooth fur,good mental state and good activity;the POI group had removal hair on the back,average mental state and activity.(2)Compared to the control group,the estrous cycles of the POI mice were prolonged and disordered,indicating that the POI modeles were successfully constructed;the body weight,ovarian index and uterine index of mice decreased(P<0.05).(3)ELISA found that the serum AMH concentration of mice in the POI group decreased significantly(P<0.05).(4)HE staining and follicle count showed that compared with the control group,the ratio of the number of primitive follicles and early growth follicles decreased,and the ratio of the number of atresia follicles increased(P<0.05).(5)q RT-PCR detection found that m RNA expression levels of ER,CYP19A1,Rp L6 and HSD17B12 in the POI group were downregulated markedly(P<0.05),H1.2 and Rp S13 were upregulated obviously(P<0.05).(6)WB showed that protein level expression of H1.2,Rp S13,Rp L6 and HSD17B12 in the POI group were decreased significantly(P<0.05).2.Granule cell proliferation and apoptosis phenotype were affected with the abnormal expression of HSD17B12:(1)The transfection efficiency of KGN and SKOV3 cell lines were above 80% by observation with a fluorescence microscope which indicated that lentiviral transfection were successful.(2)q RT-PCR and WB showed that the m RNA and protein levels expression of HSD17B12 gene in the sh-HSD17B12 group(KGN,SKOV3)decreased(P<0.05)and in the oe-HSD17B12group(KGN,SKOV3)increased(P<0.05)compared with the negative control(NC)group,which confirmed that the cell modeles of knockdown and overexpression were successfully constructed.(3)CCK-8 detection found that compared with the NC group,the cell proliferation capacity of the sh-HSD17B12 group(KGN,SKOV3)decreased after 24 h,48h and 72 h respectively(P<0.05),and the cell proliferation capacity of the oe-HSD17B12 group(KGN,SKOV3)increased after 24 h,48h and 72h(P<0.05).(4)Flow cytometry apoptosis detection showed that compared with the NC group,the apoptosis rate of cells in the sh-HSD17B12 group(KGN,SKOV3)increased(P<0.05),there was no difference in the oe-HSD17B12 group(KGN,SKOV3)(P>0.05);and compared with the sh-HSD17B12 group,the apoptosis rate of cells in the oe-HSD17B12 group(KGN,SKOV3)decreased significantly(P<0.05).Conclusion:(1)POI mouse modeles were successfully constructed with triptolian polyglycosides,and molecular experiments confirmed that the expression of ER and CYP19A1 genes related to estrogen synthesis decreased which further show that POI mouse models can be used for subsequent research.(2)H1.2,Rp S13,Rp L6 and HSD17B12 genes were expressed differently in the ovaries of POI model mice.(3)When the expression of HSD17B12 decreased,the proliferation was inhibited and apoptosis was promoted in human ovarian granule cells;however,the elevated expression of HSD17B12 had the opposite effect.It was suggested that abnormal expression of HSD17B12 affected the proliferation and apoptosis phenotypic changes of human ovarian granule cells. |
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