ABSTRACT
Objective To explore the effect and mechanism of insulin-like growth factor 2 ( IGF2 ) on the proliferation of human ovarian granulosa cells ( KGN ). Methods KGN cells cultured in vitro and treated with different concentrations of IGF2 were divided into control group and IGF2 group (25 μg/L, 50 μg/L, 100 μg/L), and then cells were divided into control group, 100 μg/L IGF2 group, LY294002 group, and IGF2 +LY294002 group after intervened the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway with LY294002. MTS and 5-ethynyl-2'- deoxyuridine (EdU) method was used to detect the effect of IGF2 on KGN cell proliferation, and enzyme linked immunosorbent assay was used to detect the contents of estrogen and progesterone in cell culture supernatant. The expressions of insulin like growth factor 1 receptor (IGF1R), protein kinase B (Akt), phosphorylated protein kinase B(p-Akt) and CYP19A1 protein in each group were detected by Western blotting. Results With the concentration gradient of IGF2, the proliferation rate of KGN cells and the secretion of estrogen and progesterone gradually increased. The cell proliferation rate and hormone level in the group treated with lOOfig/L IGF2 were the highest (P<0.01), while the PI3K/Akt signaling pathway was inhibited, and the cell proliferation rate and hormone secretion decreased significantly (P<0.01). The protein expression levels of IGF1R, p-Akt and CYP19A1 in different concentration groups increased significantly (P<0.05). While the expression of the above proteins were affected by intervened the PI3K/Akt signaling pathway. Compared with the control group, the protein expression of IGF1R and p-Akt increased significantly in IGF2 group and IGF2 +LY294002 group(P<0.01), CYP19A1 increased significantly in IGF2 group(P<0.01), the protein expression of p-Akt and CYP19A1 decreased significantly in LY294002 group (P<0.05), there was no significant difference in the protein expression of IGF 1R. Compared with the IGF2 group, the protein expression of p-Akt and CYP19A1 decreased in IGF2 +LY294002 group (P<0.01), there was no statistically significant difference in the protein expression of IGF1R, and the expression levels of IGF1R, p-Akt and CYP19A1 were significantly reduced in LY294002 group (P<0.01). Conclusion IGF2 may promote the proliferation and secretion of human ovarian granulosa cells through the PI3K/Akt signaling pathway mediated by IGF1R.
ABSTRACT
Objective To construct the lentiviral expression vector of SOX9 gene and establish a LNcap cell strain with stable expression of SOX9 . Methods SOX9 gene was amplified by PCR and cloned into lentiviral expression vector pLVX-IRES-Puro. pLVX-IRES-FLAG-SOX9 recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. Then we gained recombinant virus particles in packaging cell HEK 293 and infected LNcap cell. The monoclonal LNcap cell strain stably expressed were obtained through puromycin screening. The mRNA level and protein level in the infected LNcap cell were detected by qRT-PCR and Western blot respectively. Results Restriction enzyme digestion and sequencing demonstrated that SOX9 cDNA was successfully cloned into pLVX-IRES-FLAG lentiviral vector. After the transfection to LNCaP cells, the monoclonal cell strain of stably expressed SOX9 were obtained by puromycin screening, which showed expression of SOX9 mRNA detected by qRT-PCR. Western blot analysis revealed that SOX9 protein expressed markedly. Conclusion The recombinant lentiviral vector bearing human SOX9 cDNA has been successfully constructed, and the exogenous expression of SOX9 in LNcap cells was achieved.
