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ObjectiveTo investigate the effects of ABI-1 gene knockdown upon the proliferation and migration of human gastric cancer cell NCI-N87 in vitro. MethodsNCI-N87-ABI-I-ShRNA cell model was successfully constructed and validated by Real-time PCR and Western blot. The cellular morphous and skeleton, proliferative and migrative potents, and also AKT expression were compared between NCI-N87-ABI-1-ShRNA and its parents by immunofluorental staining, CCK-8 assay, transwell chamber and Western blotting.ResultsCCK-8 assay showed there was no significant difference in the proliferation rates at different time points between the NCI-N87-Vector and NCI-N87 cells while the proliferation rates at the time points of 36 and 48 hours of the NCI-N87-ABI-1-ShRNA were significantly lower than the NCI-N87( t =2. 85and 4. 166, P ＜ 0. 05 ). Transwell assay showed that migrated cell number were 66 ± 8, 65 ± 8 and 30 ± 4,respectively, and there was significant difference between the NCI-N87-ABI-1-ShRNA and NCI-N87 cells (t =9. 550,P ＜0. 05). Finally, ABI-1- knock-down altered the cellular morphoos and skeleton of 90％NCI-N87 cells and inhibited p-AKT expression.ConclusionABI-1 inhibits proliferation and migration of NCI-N87 cells in vitro probably by PI3K/AKT pathway.
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Objective To investigate the changes of human penile smooth muscle cell contraction and stretch resulting from the integrin-linked kinase (ILK) gene knock-down.Methods Cultured human penile smooth muscle cells were knocked down for ILK using a small interfering RNA (SiRNA).Cellular ILK expression was quantified by Western blot analysis.Cell attachment,spreading and migration were also performed.Furthermore,microfilament dynamics was tested by means of Alexa Fluor 488 phalloidin stain.Results First,blocking the expression of ILK by siRNA significantly inhibited human penile smooth muscle cell attachment,speading and migration; moreover,ILK down-regulation affected actin cytoskeleton reorganization and changed cell morphology in human penile smooth muscle cell.Conclusions Targeting of ILK with a small interfering RNA not only inhibits human penile smooth muscle cell attachment,spreading and migration,but also effectively suppresses microfilament dynamics.This may be of potential therapeutic usefulness in treating erectile dysfunction (ED).
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Objective To study the effects oftacrolimus on the cell proliferation of and secretion of stem cell factors (SCF) by cultured human keratinocytes. Methods Human keratinocyte cell line HaCaT was cultured and treated with various concentrations (0, 10, 102, 103, 104 nmol/L) of tacrolimus. After 48 hours of treatment, cell proliferation was measured with MTT assay, the levels of stem cell factor in the culture supernatant of HaCaT cells were detected with ELISA. Results Inhibited proliferation was observed in HaCaT cells treated with tacrolimus of 103-104 nmol/L. The secretion of SCF by HaCaT cells was enhanced in the presence of 10-102 nmol/L of tacrolimus, was inhibited in the presence of tacrolimus of 104 nmol/L, and remained unchanged with 103 nmol/L. Conclusion Certain concentrations of tacrolimus could enhance the expression Of SCF in HaCaT cells, which may be associated with the efficacy of tacrolimus in the treatment of vitiligo.
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Objective To investigate the inhibitive effect of siRNA(small interfering RNA,siRNA) on the phosphodiesterase type 5(PDE5) in smooth muscle cells of human corpus cavernosum,and provide experimental groundwork for the gene therapy of erectile dysfunction (ED). Methods Small interfering RNAs targeting PDE5 gene were systhesized by using web design software provided by Ambion,there siRNAs and control siRNA were systhesized by Ambion. SiRNAs were transfected into smooth muscle cells of human corpus cavernosum by using siPORTTM Lipid reagent;down-regulation of PDE5 mRNA was detected by RT-PCR;the inhibitive effect of PDE5 was detected by Western Blotting. Results The results of RT-PCR indicated siRNA1、siRNA2 and siRNA3 made down-regulations of PDE5 mRNA expression in the transfected groups 58.2%、14.9% and 11.8%;the PDE5 expression decreased 70.5%、19.8% and 17.3%;however the expression did not have different in control siRNA and frank group. Conclusions The synthesized siRNAs in vitro were able to down-regulate the expression of PDE5.There were different capabilities of the specific siRNAs down-regulation.It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for ED gene therapy.
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Objective Calcineurin-NFAT pathway in the regulation of VSMCs proliferation induced by catecholamines.Methods Primary VSMCs from rat aorta were used as the experimental model.Proliferation of VSMCs was measured by MTT assay and cell count.Calcineurin protein and its activity were assayed with immunoblotting and free inorganic phosphate content analysis respectively.Localization of NFATc1 was detected by immunofluorescence staining.Results Phenylephrine(PE,an ?1-adrenoceptor agonist) increased VSMCs proliferation.Prazosin(an ?1-adrenoceptor antagonist),cyclosporin A(CsA,an inhibitor of calcineurin) and chelerythrine(an inhibitor of PKC) decreased PE-induced absorbance and cell number.Timolol(?-adrenoceptor antagonist) has no effect on absorbance and cell number induced by PE.Additional treatment with CsA further inhibited PE-induced absorbance and cell number compared with the chelerythrine pretreatment group.CsA and chelerythrine alone had no significant effect on either absorbance or cell number.CsA decresed PE-induced alcineurin level and its activity.NFATc1 was translocated from cytoplasm to nucleus upon treatment with PE.This translocation was reversed by CsA.Conclusion CsA partially inhibits PE-induced VSMCs proliferation via inhibiting calcineurin activity and NFATc1 nuclear translocation.Calcineurin-NFATc1 pathway is involved in hyperplastic growth of VSMCs induced by catecholamines.
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0.05) between the two groups diluted by 100-fold and 1 000-fold supernatants.When ASMCs were treated at different concentrations of 0 and 2 ?g/L of pertussis toxin(PTX),the cell number migrated from the test and control groups diluted by 10-fold supernatants,they had statistical significance(74?4 vs 34?3,P0.05).Conclusion:CKLF1 has significant chemotactic effects on ASMCs and such a CKLF1-induced chemotaxis could be inhibiteded by PTX at concentration of 10 ?g/L.
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Objective:To investigate the effect of the integrin-linked kinase(ILK) small interfering RNA(siRNA) on prostate cancer in nude mice by orthotopic injection of human cell line DU145.Methods:The cultured human cell line DU145 was knocked down for ILK using a siRNA.Cellular ILK expression was quantified by RT-PCR and Western blot analysis.Moreover,cell attachment,invasiveness and microfilament dynamics assays were performed.Furthermore,the impact of the ILK siRNA on the prostate cancer was tested using a nude mice model in which prostate cancer was induced by orthotopic injection of human prostate cancer cell line DU145.Gross tumor volume of prostate in nude mice,cell differentiation,the state of apoptosis and proliferation were tested after 5 weeks of injection.Results:The expression of ILK was suppressed significantly by siRNA,cellular mRNA and protein of ILK decreased 87% and 81% separately.The knockdown of ILK also induced the attachment and invasiveness of DU145 cell growing down.The tumor volume,cell differentiation,apoptosis index and proliferation index of prostate in nude mice of ILK siRNA orthotopic injection model were significantly smaller,better,increased and decreased separately than those in control group.Conclusion:Targeting inhibition of ILK not only decreases attachment and invasiveness of human DU145 cells,but also suppresses the growth and development of prostate cancer of orthotopic injection human DU145 cell line model in nude mice.
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Objective:To study the effects of 1?,25-dihydroxyvitamin D 3 and UVB on cell proliferation and melanin synthesis of normal human melanocytes. Methods:Melanocytes of foreskins of healthy men were cultured and treated with various concentration of 1?,25-dihydroxyvitamin D 3 or UVB (55 mJ/cm 2),or both.Cell proliferation was measured with MTT assay .The synthesis of melanin was determined by chromatography. Results: 1?,25-dihydroxyvitamin D 3 and UVB could promote the proliferation of cultured melanocyte,and 1?,25-dihydroxyvitamin D 3 could promote the melanin synthesis.The significant concentration of 1?,25-dihydroxyvitamin D 3 ranging from 10 -7 to 10 -10 mol/L.Conclusion:1?,25-dihydroxyvitamin D 3 and UVB irradiation could promote the proliferation of melanocyte,which indicates that they might be effective in the treatment of vitiligo.
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Objective To explore the expression of high selective A receptor of endothelin-1 (ETA-R) in melanocytes from normal human beings. Methods Normal human melanocytes were cultured and mR-NA expression of ETA-R in melanocytes was measured by reverse transcription-polymerase chain reaction. Results The mRNA expression of ETA-R was seen in melanocytes. Conclusion This study combined with other experiments suggests that endothelin-1 may affect melanocytes by high selective ETA-R which may con-tribute to the pathogenesis of vitiligo.
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Objective To study the effects of endothelin-1 (ET-1) and adrenocorticotropic hormone (ACTH) on the proliferation of cultured normal human melanocytes. Methods Normal human melanocytes were obtained from the foreskins of healthy men, and cultured in minimum essential medium (MEM) with 10% bovine serum. The cultured melanocytes of the 3rd passage were treated with various concentrations of ET-1 and ACTH. Cell proliferation was assessed by the MTT method. Results ET-1 (0.1 ~ 1 000 nmol/L) promoted the proliferation of cultured human melanocytes. ACTH (10-13 ~ 10-9 mol/L) also promoted the proliferation of cultured human melanocytes (P
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Varied TCM constitutions in the patient of acute cerebral infarction were classifyed by using HLA - DQAl allehc gene to analyze hereditary susceptibility of constitution types and relation among constitution, syndrome and treatment. PCR-SSP technique was used for classification of Yin - deficiency Yang - deficiency, Qi - deficiency, phlegm - dampness and blood stasis constitution in 103 cases of acute cerebral infarction and HLA - DQAl allelic gene was used for gent classification in 99 cases of nomal constituion. Results indicated that HLA -DQA * 0501 gene type in Yin -deficiency constitution was significant higher than that of normal constitution CP