Effects of the ITPR1 gene overexpression on Ca²⁺ concentration, lipid content and calcium transport-related genes in duck uterine epithelial cells / 生物工程学报

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an important intracellular channel for releasing Ca²⁺. In order to investigate the effects of the ITPR1 overexpression on Ca²⁺ concentration and lipid content in duck uterine epithelial cells and its effects on calcium transport-related genes, the structural domain of ITPR1 gene of duck was cloned into an eukaryotic expression vector and transfected into duck uterine epithelial cells. The overexpression of the ITPR1 gene, the concentration of Ca²⁺, the lipid content, and the expression of other 6 calcium transport-related genes was determined. The results showed that the concentration of Ca²⁺ in uterine epithelial cells was significantly reduced after transfection (P<0.05), the triglyceride content was significantly increased (P<0.01), and the high-density lipoprotein content was significantly decreased (P<0.01). The correlation analysis results showed that the overexpression of the C-terminal half of the ITPR1 gene was significantly positively correlated with the total cholesterol content (P<0.01), which was significantly positively correlated with the low-density lipoprotein content (P<0.05). The overexpression of the N-terminal half of the ITPR1 gene was significantly positively correlated with the triglyceride content (P<0.01), which was significantly negatively correlated with the concentration of Ca²⁺ (P<0.05). RT-qPCR results of 6 calcium transport-related genes showed that the overexpression of the C-terminal half of the ITPR1 gene significantly inhibited the expression of the IP3R2, VDAC2 and CAV1 genes, and the overexpression of the N-terminal half of the ITPR1 gene significantly promoted the expression of the IP3R3 and CACNA2D1 genes. In conclusion, the ITPR1 gene overexpression can promote Ca²⁺ release in duck uterus epithelial cells, promote the synthesis of triglyceride, low-density lipoprotein and cholesterol, and inhibit the production of high-density lipoprotein, and the ITPR1 gene overexpression affected the expression of all 6 calcium transport-related genes.

Effects of SCD-1 gene overexpression on the content of calcium ion and lipids in duck uterine epithelial cells / 生物工程学报

Stearoyl-CoAdesaturase-1 (SCD-1) is a key regulator of monounsaturated fatty acid synthesis. It plays a vital role in lipid synthesis and metabolism. Ca²⁺ is an important cation in the body and plays an important role in the organism. The aims of this study were to investigate the correlation of SCD-1 gene overexpression with lipid indexes and calcium ion level. The pcDNA3.1 (+) + SCD-1 +Flag eukaryotic expression vector and cultured duck uterine epithelial cells were co-transfected. The overexpression of SCD-1 gene was measured using the Flag Label Detection Kit. Ca ions and lipid contents were detected through Fluo-3/AM Calcium Ion Fluorescence Labeling method and Lipid Measuring Kit, respectively. SCD-1 gene overexpression was negatively correlated with triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C), and positively correlated with Ca ion, total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels. Meanwhile, Ca ion was positively correlated with TG, LDL-C and HDL-C contents, and negatively correlated with TC and VLDL-C levels. Overexpression of SCD-1 gene could regulate Ca ion secretion, as well as lipid synthesis and transport in duck uterine epithelial cells.

Effect of CD82 on the expression of integrin αV,β3, E-cadherin andβ-catenin in uterine epithelial cells in pregnant mice / 中国实验动物学报

Objective Uterine epithelial cells were isolated from pregnant mice and cultured in vitro , and exam-ined the effect of CD82 on the expression of integrin αV,β3, E-cadherin and β-catenin in the cells.Methods The uter-ine epithelial cells were primarily isolated from pregnant mouse uterus .The recombinant adenovirus containing mouse CD 82 gene which had been constructed in our lab infected the uterine epithelial cells .Immunocytochemistry was used to detect the protein expressions of integrin αV,β3, E-cadherin and β-catenin in the uterine epithelial cells , which were infected with CD82 adenovirus or not .Results 1.The purity of primary cultured cells was (93.2 ±0.6)%.2.The transfection efficiency of CD82 recombinant adenovirus was ( 92 ±4.5 )%.The adenoviral particles carrying CD 82 gene indeed ex-pressed CD82 gene and protein in the primary uterine epithelial cells after 24 hours or 48 hours.3.The uterine epithelial cells of pregnant mice on d4 expressed integrin αV, β3, E-cadherin and β-catenin.4.In contrast to the control group, when CD82 adenovirus infected cells , the uterine epithelial cells of pregnant mice on d 4 increased the expression of integrinαV,β3 and β-catenin protein, had no significant changes of E-cadherin.Conclusions CD82 may have effect on the ex-pression of integrin αV,β3 and β-catenin in mouse uterine epithelial cells before implantation .

Estrogen up-regulates MMP2/9 expression in endometrial epithelial cell via VEGF-ERK1/2 pathway

OBJECTIVE@#To study the effect of estrogen on anovulatory dysfunctional uterine bleeding (ADUB).@*METHODS@#Primary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolytic activity of gelatinase was determined by gelatin zymography analysis. Cellular mRNA and protein synthesis was blocked respectively to determine whether the increased expression of MMP-2/9 was induced by estrogen. The expression of VEGF was blocked by siRNA. After treatment with various factors, MMP-9, VEGF, total Erk and phosphorylated Erk expression in primary uterine epithelial cells was detected by Western blotting analysis. Cell MMP-2/9mRNA levels was measured by real-time RT-PCR.@*RESULTS@#The activity and expression of MMP2/9 was increased in the endometrium of patients with ADUB. Estrogen could up-regulate the expression of VEGF and activate Erk 1/2-Elk1 signal path. After interference by siRNA, ERK1/2 pathway was blocked in cells, and the expression of MMP-2/9 was down-regulated. ERK1/2 specific blocker U0126 blocked ERK phosphorylation, and it could down-regulate the expression of MMP-2/9.@*CONCLUSIONS@#The results showed that the estrogen can increase the expression of VEGF, and thus activate ERK1/2 pathway to induce MMP-2/9 expression.