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Mol Med Rep ; 16(1): 764-772, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28560395


Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non­toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1­pAFP­TK, carrying the suicide gene driven by the alpha­fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription­polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1­pAFP­TK plasmid, whereas HSVtk gene expression was not detected in normal HL­7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1­pAFP­TK decreased in a dose­dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1­pAFP­TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase­3 staining demonstrated that activated caspase­3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase­3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.

Efeito Espectador , Ganciclovir/metabolismo , Plasmídeos/genética , Pró-Fármacos , Simplexvirus/genética , Timidina Quinase/genética , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ganciclovir/farmacologia , Regulação Viral da Expressão Gênica , Humanos , Neoplasias Hepáticas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , alfa-Fetoproteínas/genética
Int J Clin Exp Med ; 8(1): 1051-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25785092


Lung cancer is one of the most common cancers in the world, especially in China. It is believed that genetic polymorphisms played a role in cancer susceptibility. Here we investigated the association of interleukin-6 (IL-6) and interleukin-10 (IL-10) gene polymorphisms with the susceptibility of lung cancer in never-smoking Chinese Han population. In this study, we performed a case-control study including 330 cases of never-smoking lung cancer patients and 336 cancer-free never-smoking controls in Chinese Han population. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to identify gene polymorphisms, and then verified by sequencing method. The results indicated that the four single nucleotide polymorphisms (IL-6 -1363T/G and -572G/C, IL-10 -819T/C and -592A/C) were genotyped by PCR-RFLP and confirmed by sequencing, and we found that the allelic frequencies of G in IL-6 -1363T/G, C in IL-10 -819T/C and C in IL-10 -592A/C were significantly increased in lung cancer patients, by comparing with the control group. However, there was no significant difference in the distribution of the IL-6 572G/C polymorphisms between patients and controls. In conclusion, the IL-6 -1363T/G, IL-10 -819T/C and IL-10 -592A/C polymorphisms are closely related to genetic susceptibility to lung cancer in never-smoking Chinese Han population, and these genetic variants might be used as molecular markers for detecting lung cancer susceptibility.

FASEB J ; 20(7): 916-25, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16675849


Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors,and not on gene deletion. Here we used ROCK1knockout (ROCK1-/-) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1-/- mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1-/- mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFbeta2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of Gq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli.

Cardiomegalia/metabolismo , Fibrose/metabolismo , Fibrose/prevenção & controle , Coração/fisiopatologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Fibrose/patologia , Regulação Enzimológica da Expressão Gênica , Genótipo , Coração/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Fenótipo , Pressão , Proteínas Serina-Treonina Quinases/genética , Remodelação Ventricular/fisiologia , Quinases Associadas a rho
J Mol Cell Cardiol ; 38(4): 685-91, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15808845


Conditional transgene expression in the heart is a useful approach to explore the physiological basis of the cardiac phenotype. The present study describes the development of a binary transgenic system in which transgene expression in the mouse heart can be turned on/off by administration/withdrawal of an exogenous compound. We generated a transgenic line (alphaMHC-Glp 65) harboring a mifepristone (RU486)-controlled chimeric transcription factor (Glp 65) under the regulatory control of the cardiac-specific alpha-myosin heavy chain (alphaMHC) promoter. In the presence of RU486, Glp 65 expressed in the heart is able to bind to a target gene promoter containing four copies of the 17-mer GAL4 binding site, resulting in ligand-inducible transactivation of the target gene. We tested this system by crossing the transgenic mice, alphaMHC-Glp 65, with a transgenic line harboring human growth hormone (hGH) target gene. We observed that expression of hGH could be induced in adults as well as in the embryonic hearts of bigenic mice by RU486. The basal hGH expression was very low and the inducible level in the heart was estimated over 800-fold higher versus the basal level after 4 days of administration of RU486 at 500 microg/kg body weight per day at 2-5 months of age. The level of the transgene returned to the basal level within 7 days after withdrawal of RU486. This system can be used to control cardiac-specific expression of transgene in a time- and dose-dependent manner.

Regulação da Expressão Gênica , Camundongos Transgênicos/genética , Miosinas Ventriculares/genética , Animais , Marcação de Genes , Hormônio do Crescimento/análise , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Ventrículos do Coração/química , Ventrículos do Coração/citologia , Humanos , Ligantes , Camundongos , Camundongos Transgênicos/metabolismo , Mifepristona/farmacologia , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transgenes , Regulação para Cima
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue ; 16(8): 458-9, 2004 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-15298798


OBJECTIVE: To investigate the effects of reconstructive human acidic fibroblast growth factor (aFGF) and wild type aFGF on skin cell proliferation in rat. METHODS: Neonatal rat skin (area of 2 mmx2 mm) was cultured in Dulbecco's modification of Eagle's medium containing reconstructive human aFGF and wild type aFGF, respectively. The concentrations of aFGF were 1 microg/L, 10 microg/L, and 100 microg/L. After being cultured for 4 days, the area of skin was measured. RESULTS: After treatment with two different growth factors in three different concentrations (1 microg/L, 10 microg/L and 100 microg/L) for 4 days, the areas of skin in three reconstructive human aFGF groups were 1.4, 1.5 and 1.3 fold of that of control, respectively and the areas of three wild type aFGF groups were 1.5, 3.2 and 1.6 fold of that of control, respectively, while the area of skin in the control group was (2.96+/-1.12) mm(2). In comparison with those of other groups, the skin area of 10 microg/L wild type aFGF group was significantly increased (P<0.05). CONCLUSION: Reconstructive human aFGF confers less impact on cutaneous cell growth. The capability of wild type aFGF to induce cutaneous cell proliferation is much greater than that of reconstructive human aFGF.

Fator 1 de Crescimento de Fibroblastos/farmacologia , Pele/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Ratos , Ratos Wistar , Pele/citologia , Técnicas de Cultura de Tecidos
Acta Pharmacol Sin ; 24(6): 563-8, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12791183


AIM: To investigate whether endothelin-1 (ET-1) can promote human umbilical artery smooth muscle cell (HUASMC) proliferation through pathway of extracellular signal-regulated kinase (ERK) and cyclin D1. METHODS: The effects of ET-1 and PD98059 on HUASMC were evaluated by MTT assay. The content of DNA was defined by [3H]TdR assay and cell cycle was analyzed by flow cytomerty. Western blot analysis was employed to detect the active phosphorylated state of ERK and the expression of cylin D1. RESULTS: Firstly, ET-1 (100 nmol/L) stimulated HUASMC proliferation compared with the group without ET-1 (P<0.05) and PD98059 group (P<0.05). PD98059 inhibited the HUASMC proliferation stimulated by ET-1 (P<0.05). Secondly, ET-1 stimulated DNA synthesis of HUASMC compared with the group without ET-1 (P<0.05). Thirdly, ET-1 promoted the cell cycle transition from G0/G1 phase to S phase. G0/G1 phase cell percentage was obviously decreased compared with the group without ET-1 (P<0.05). S phase cell percentage was increased compared with the group without ET-1 (P<0.05). Fourthly, ET-1 increased the phosphorylated level of ERK and the expression of cylin D1, an inhibitor of ERK blocked phosphorylated level of ERK and cyclin D1 expression. ERK phosphorylated level of ET-1 group was evidently increased compared with PD98059 group (P<0.05), Cyclin D1 protein expression also was increased compared with PD98059 group (P<0.05). While nonphosphorylated ERK expression remained unchanged. CONCLUSION: Endothelin-1 promoted vascular smooth muscle cell proliferation through pathway of ERK and cyclin D1.

Ciclina D1/metabolismo , Endotelina-1/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Flavonoides/farmacologia , Humanos , Músculo Liso Vascular/citologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais , Artérias Umbilicais/citologia