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Objective:To explore the protective mechanism of phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway in glucagon like peptide-1 (GLP-1) antagonizing the apoptosis of gestational trophoblasts (HTR-8/SVneo) induced by advanced oxidized protein products (AOPP).Methods:Pregnant trophoblast HTR-8/SVneo were cultured in vitro. The cells were divided into control group, AOPP group, GLP-1 group, AOPP + GLP-1 group and AOPP + GLP-1 + LY294002 group. The control group was cultured in 1640 medium; AOPP group was stimulated with 200 μg/ml AOPP; GLP-1 group was stimulated with 50-100 nmol/L GLP-1 for 1 h; AOPP + GLP-1 group was stimulated with 200 μg/ml AOPP for 48 hours, and then GLP-1 (50-100 nmol/L) was added for 1 hour; In AOPP + GLP-1 + LY294002 group, PI3K inhibitor LY294002 was added on the basis of the intervention of AOPP + GLP-1 group. The expression of PI3K/Akt pathway related protein p-Akt was detected by Western blot. Cell viability was detected by cell counting kit (CCK-8). Enzyme linked immunosorbent assay (ELISA) was used to detect the contents of apoptosis promoter protease caspase-9 and caspase-3, and the contents of apoptosis related proteins Bcl-2, Bax and Cyto-c. Results:After AOPP stimulation, the expression of p-Akt in AOPP group was lower than that in control group ( P<0.05); After 50 and 100 nmol/L GLP-1 intervention, the expression of p-Akt in AOPP + GLP-1 group was significantly higher than that in AOPP group (all P<0.05). After 24 and 48 hours of 100 nmol/L GLP-1 intervention, the expression of p-Akt in AOPP + GLP-1 group was significantly higher than that in AOPP group (all P<0.05). After AOPP stimulation, the cell viability of AOPP group was lower than that of control group ( P<0.05); After GLP-1 intervention, the cell viability of AOPP + GLP-1 group was significantly higher than that of AOPP group ( P<0.05). After adding PI3K inhibitor LY294002, the cell viability of AOPP + GLP-1 + LY294002 group was significantly lower than that of AOPP + GLP-1 group ( P<0.05). The results of ELISA showed that the contents of apoptosis promoter protein caspase-3, caspase-9, apoptosis related protein Bax and Cyto-c in AOPP group were higher than those in control group (all P<0.05), and the content of anti-apoptosis protein Bcl-2 was lower than that in control group ( P<0.05); After GLP-1 intervention, the contents of caspase-3, caspase-9, Bax and Cyto-c in AOPP + GLP-1 group were significantly lower than those in AOPP group ( P<0.05), and the content of anti-apoptosis protein Bcl-2 was higher than that in AOPP group ( P<0.05). After treatment with PI3K inhibitor LY294002, the contents of Bcl-2 in AOPP + GLP-1 + LY294002 group were lower than those in AOPP + GLP-1 group, and the contents of Bax and Cyto-c were higher than those in AOPP + GLP-1 group (all P<0.05). Conclusions:GLP-1 may mediate PI3K / Akt pathway to antagonize the apoptosis of HTR-8/SVneo induced by AOPP.
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Objective:To study the effect of miR-18a on expression of estrogen receptor alpha in human normal trophoblast cells(HTR-8).Methods:The synthesized miR-18a (miR-18amimics,miR-18a inhibitor and blank carrier) were transfected into HTR-8 cells.Experiment included experimental group 1,pre-miRNA un-transfection group(NC group) and experimental group 3.RT-PCR detected the expression of miR-18a in the three groups.RT-PCR and Western-blot detected the expression of ERα mRNA and protein in the transfected HTR-8 cells.Results:According to the results of RT-PCR,the expressions of miR-18a in experimental group 1 (0.880 ± 0.060) was significantly higher than that of NC group(0.407 ±0.019) (P ＜0.05),and the expressions of miR-18a in experimental group 3(0.160 ±0.014) was significantly lower than that of NC group(0.407 ±0.019) (P＜0.05);After transfected for 48 hours,there was no significant difference between the expression of ERα mRNA between experimental group 1 (0.249 ±0.003) and NC group(0.313 ±0.010) (P＞0.05),the expression of ERα mRNA in transfected cells in experimental group 3 (0.823 ± 0.023)was significantly higher than that of NC group (P ＜ 0.05);Western-blot results showed that the expression of ERα protein of experimental group 3(1.030 ±0.006) was significantly higher than that of NC group(0.960 ±0.008) (P ＜ 0.05);the expression of ERα protein of experimental group 1 (0.660 ±0.013)was significantly lower than that of NC group (0.960 ± 0.008) (P ＜ 0.05).Conclusions:in human normal trophoblast cells,miR-18a may play regulatory role in the expression of ERα both in mRNA and protein levels.
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Objective To investigate the effect of fatty acid binding protein 4(FABP4)DNA methylation on abnormal lipid metabolism in placental trophoblastic dyslipidemia. Methods Human placental trophoblast cell line(HTR-8)was treated with L-NAME of 100 μmol/L for 48 h. The lipid content in placental trophoblasts was detected by chemical enzyme-colorimetry. The FABP4 DNA methylation level in placenta trophoblasts was detected by nested-touch down methylation specific PCR (NT-MSP). the mRNA and protein expression of DNMT1 and FABP4 were detected by qRT-PCR and Western Blot,respectively,in trophoblast cells. Results The lipid content in trophoblasts significantly increased as compared with the control(P < 0.05). Expression of FABP4 mRNA and protein increased(P < 0.05),while FABP4 methylation level and expression of DNMT1 significantly decreased (P<0.05)after treatment with L-NAME. Conclusions FABP4 DNA methylation is involved in the regulation of lipid metabolism in placental trophoblastic cells of hypertensive disorder complicating pregnancy.
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Objective To construct the plasmid pSilencer4.1/HtrA1 and stably transfect human trophoblastic cell line HPT-8.Methods We detected the expression of HtrA1 in cell line HPT-8 with immunohistochemical SABC,constructed the plasmid pSilencer4.1/HtrA1,transfected human trophoblastic cell line HPT-8 with plasmid pSilencer4.1/HtrA1 and negative plasmid pSilencer4.1, and observed the cell expression after transfection. Results About 90% of cellular cytoplasm of HPT-8 was dyed brown while the nucleus was negatively stained. Selective concentration of G418 was 200 μg/mL for HPT-8 to be transfected pSilencer4.1/HtrA1.With RT-PCR and Western blot methods,the expression of HPT-8 transfected plasmid Psilence4.1/HtrA1 was significantly downregulated compared with that of cells with negative plasmid and normal cells (P 0.05).Conclusion We successfully constructed stable trophoblastic cell line HPT-8/ pSilencer4.1-HtrA1 with silenced expression of HtrA1,which lays foundation for further research.
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Objective:To investigate the effects of IL-2 on the proliferation activity and HCG secretion of first trimester human trophoblastic cells in vitro. Methods:Growing -well 4th generation trophoblastic cells were selected,cultured for 48 h with IL-2 of different concentration. The MTT colorimetric assay was used to analyze the proliferation activity, and radioimmunoassay was used to analyze the secretion of HCG. Results:①Low concentration (100 U/ml,400 U/ml) IL-2 promoted proliferation activity and HCG secretion (P