RESUMO
This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
Assuntos
Animais , Camundongos , Permeabilidade da Membrana Celular , Fisiologia , Sobrevivência Celular , Fisiologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Camundongos Knockout , Podócitos , Fisiologia , Puromicina Aminonucleosídeo , Farmacologia , Canais de Cátion TRPC , Genética , MetabolismoRESUMO
This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
RESUMO
The P85 regulatory subunit protein and gene expression and P110 catalylic subunit activity of phosphatidylinositol 3-kinase (PI-3K) were investigated in adipose tissue of patients with gestational diabetes mellitus (GDM) in order to explore the molecular mechanisms of insulin resistance (IR) of GDM. Samples from patients with GDM (n=50), and controls (n=50) were collected. Fasting insulin (FIN) was determined by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Western blot technique was used to detect the levels of PI-3K P85 subunit in adipose tissues of patients with GDM. The mRNA expression of PI-3K P85 subunit was detected by reverse transcription polymerase chain reaction (RT-PCR) method in the adipose tissue. PI-3K activity was examined by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed statistically. It was found that the levels of FPG, FIN and HOMA-IR in GDM group were significantly higher than those in control group (all P0.05). PI-3K activity was significantly decreased to 82.89% in GDM group as compared with control group (P<0.01) and negatively correlated with HOMA-IR (r=-0.75, P<0.01). It was concluded that PI-3K in GDM patients may be involved in the insulin signaling pathway, resulting in IR of GDM.
RESUMO
The relationship between the expression of resistin in polycystic ovary syndrome (PCOS) and insulin resistance was investigated. The plasma resistin concentrations in 35 patients with PCOS and 40 controls were measured by ELISA. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and fasting insulin (FIN) were tested by radioimmunoassay. Insulin resistance index (HOMA-IR) was calculated. Fasting plasma glucose (FPG) was determined by oxidase test. Western blot and reverse transcriptase PCR (RT-PCR) methods were used to detect the expression of resistin in adipose tissues. The levels of plasma resistin, LH, LH/FSH and FIN and HOMA-IR in patients with PCOS were significantly higher than those in control group (all P<0.05). Plasma resistin was correlated positively with FPG, FIN, HOMA-IR, LH and LH/FSH (r=0.56, 0.60, 0.65, 0.48, and 0.42 respectively). Resistin protein and mRNA expression levels in patients with PCOS were significantly higher than those in normal tissues (all P<0.01). It was concluded that resistin might be involved in the pathogenesis of insulin resistance of PCOS.
RESUMO
The change in serum laminin (LN) level and its clinical significance in epithelial ovarian tumor were investigated. The LN levels in serum and ascites samples from 69 patients with epithelial ovarian tumor and 42 cases as control group before and after operation were analyzed by radioimmunoassay. The results showed that the serum LN levels in the patients with malignant tumors (157.85 +/- 14.37 ng/ml) were significantly higher than that in the control group (125.14 +/- 7.03 ng/ml) and in the patients with benign tumors (128.36 +/- 8.75 ng/ml) (both P < 0.01) before operation. The serum LN levels in the malignant group were decreased significantly after operation as compared with those before operation (P < 0.05). The serum LN levels in low-differentiated tumors was higher than those in moderate-differentiated tumors and high-differentiated tumors (P < 0.05). The LN levels in ascites (172.94 +/- 15.26 ng/ml) was significantly higher than in serum (161.34 +/- 6.59 ng/ml) (P < 0.05) in malignant tumors. The serum LN levels in the patients with lymph node metastasis (165.41 +/- 19.91 ng/ml) was obviously higher than those without lymph node metastasis (152.35 +/- 10.34 ng/ml) (P < 0.05). It was concluded that LN levels in serum and acistis were remarkably increased in malignant epithelial ovarian tumors, suggesting that LN might be one of important diameters reflecting tumor biological characteristics.
Assuntos
Feminino , Humanos , Masculino , Líquido Ascítico , Metabolismo , Biomarcadores Tumorais , Sangue , Metabolismo , Carcinoma , Sangue , Metabolismo , Laminina , Sangue , Metabolismo , Neoplasias Ovarianas , MetabolismoRESUMO
The change in serum laminin (LN) level and its clinical significance in epithelial ovarian tumor were investigated. The LN levels in serum and ascites samples from 69 patients with epithelial ovarian tumor and 42 cases as control group before and after operation were analyzed by radioimmunoassay. The results showed that the serum LN levels in the patients with malignant tumors (157.85 +/- 14.37 ng/ml) were significantly higher than that in the control group (125.14 +/- 7.03 ng/ml) and in the patients with benign tumors (128.36 +/- 8.75 ng/ml) (both P < 0.01) before operation. The serum LN levels in the malignant group were decreased significantly after operation as compared with those before operation (P < 0.05). The serum LN levels in low-differentiated tumors was higher than those in moderate-differentiated tumors and high-differentiated tumors (P < 0.05). The LN levels in ascites (172.94 +/- 15.26 ng/ml) was significantly higher than in serum (161.34 +/- 6.59 ng/ml) (P < 0.05) in malignant tumors. The serum LN levels in the patients with lymph node metastasis (165.41 +/- 19.91 ng/ml) was obviously higher than those without lymph node metastasis (152.35 +/- 10.34 ng/ml) (P < 0.05). It was concluded that LN levels in serum and acistis were remarkably increased in malignant epithelial ovarian tumors, suggesting that LN might be one of important diameters reflecting tumor biological characteristics.