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Objective To study CYP11B2 mRNA and aldosterone secretion alteration in human adrenocortical carcinoma H295R cell after angiotensin Ⅱ (AT-Ⅱ) and potassium chloride stimulation,and to investigate the effect of adrenocorticotropic hormone receptor (ACTHR) on them.Methods Lentiviral vector was used to increase ACTHR expression.It was transfected into the H295R cells.Similarly,another H295R cells,without ACTHR vector,was used as the control group.ACTHR alteration was measured by Western blot and real-time polymerase chain reaction (RT-PCR).CYP11B2 mRNA was detected at 24 hours after 100 nmol/L AT-Ⅱ/16 mmol/L KCL stimulation,and the amplification of the two groups was compared.Aldosterone was measured by ELISA kit.Results Compared with those in control ceils,the protein and mRNA level of ACTHR in experimental cells were increased 2.4 times and 18 times respectively (P＜0.05).CYP11B2 mRNA of experimental cells was 1.7 times higher than control cells after 24 h stimulation of AT-Ⅱ.Aldosterone production was 121.98+8.31 and 104.05+6.88 ng/L respectively.The former amplification was 2.06 times higher than that of the latter (P＜0.05).Similarly,CYP11B2 mRNA of experimental cells was 19.2 times higher than control cells after 24 h stimulation of KCL.Aldosterone production was 137.67±10.35 and 104.05 ± 6.88 ng/L respectively.The former amplification was 3.13 times higher than that of the latter (P＜0.05).Conclusion Overexpression of ACTHR increases the sensitivity and response of CYP11B2 mRNA and aldosterone to AT-Ⅱ and KCL stimulation,and ACTHR is expected to become a key protein in understanding primary aldosteronism.
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Objective To study the influence of inhibited steroidogenic factor-1 on human adrenocortical H295R cells, and explore its role in the pathogenesis of adrenal tumors. Methods The plasmids pGenesil1-SF-1-shRNA which containing U6 promoter and SF-1-specific short hairpin RNA (shRNA) and pGenesil1-negative-shRNA containing unspecific shRNA were transfected into H295R cell. The expression of SF-1 was measured by Western blot and real-time polymerase chain reaction(RT-PCR). Cell proliferation was analyzed by WST-1 assay and cell count. Ki-67 expression was detected by immunohistochemistry and cell apoptosis was examined by TUNEL assay. Results Compared with those in control cells, the protein and mRNA level of SF-1- transfected cells were reduced by 69.7% and 71.2% (P＜0. 01). WST-1 and cell count method showed that SF-1 gene silencing obviously inhibited cell proliferation(P＜0. 01). By contrast, there was a 3. 7-fold increase in the percentage of apoptotic H295R cells in SF-1-inhibited group than that of control group (P＜0. 01). Immunohistochemistry showed that Ki-67 positive cells in SF-1-inhibited cells were lower than the negative control cells (16.90±2.17) % and (33. 48±3.16)%,(P＜0. 01). Conclusion SF-1 gene silencing can inhibit the proliferation of adrenocortical cells, and it is expected to become a key protein in understanding pathogenesis of adrenal tumors or treating them.
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Objective To explore the role of CD4+ CDhigh25 regulatory T cells in the pathogenesis of chronic abacterial prostatitis/chronic pelvic pain syndrome (CAP/CPPS).Methods The percentage of CD4+ CD+25 and CD4+ CDhigh25 regulatory T cells was detected by flow cytometry from 45 CAP/CPPS pa-tients and 18 normal controls.The levels of interleukin-6(IL-6),IL-10,tumor necrosis factor-α(TNF-α) and transforming growth factor-β1 (TGF-β1) in serum and seminal plasma were measured by ELISA in the same cohort.Results There was no significant difference in the percentage of peripheral blood CD4+CD+25 and CD4+Cdhigh+ cells between CAP/CPPS patients and normal control (P>0.05).The ser-CD4+CD+25 and CD4+Cdhigh+ cells between CAP/CPPS patients and normal control (P>0.05>.The ser-um levels of TGF-β1 in patients with CAP/CPPS were markedly lower than those in controls (P<0.05),serum TNF-α and seminal plasma IL-6,TGF-β1 and TNF-α in CAP/CPPS patients were markedly higher than those in controls (P<0.05).There was a positive correlation between the IL-6 and the NIH-CPSI.There was also a positive correlation between the IL-10 and the pain index.In ad-dition,the percentage of peripheral blood CD+4CDhigh25 cells was positively correlated with serum TGF-β1.But the percentage of CD+4CDhigh25 cells had no correlation with ages,duration of CAP/CPPS pa-tients,NIH-CPSI and the other cytokines.Conclusions The defective function of peripheral blood CD+4CD+25 regulatory T cells may be related with the pathogenesis of CAP/CPPS.The cytokines may also play an important role in the process of pathogenesis of CAP/CPPS.