【Abstract】Objective To investigate the apoptosis induced by TGF-β1 in human hepatic carcinoma cell lines and its relationship with p53 gene and Smad. Methods Three human hepatic carcinoma cell lines which involving in various status of the p53 gene were used in this study. TGF-β1-induced apoptosis in hepatic carcinoma cell lines was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. To study the mechanism of TGF-β1-induced apoptosis, these cell lines were transfected with a TGF-β1-inducible luciferase reporter plasmid containing Smad 4 binding elements (SBE) and luciferase gene using Lipofectamine 2000, then treated with TGF-β1, relative luciferase activity was assayed. Results Of three cell lines studied with TUNEL assay, TGF-β1 induced apoptosis was observed in HepG2 cells (wild type p53). Huh-7 (mutant p53) and Hep3B (deleted p53) cell lines showed less apoptosis. Luciferase activity assay indicated that the response to TGF-β1 induction in HepG2 cells was increased dramatically but was not significant in Huh-7 and Hep3B cell lines. Conclusion HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh7 cell lines. Smad 4 is a central mediator of the TGF-β1 signal transduction pathway.
ObjectiveTo observe the regulation of PTB-associated splicing factor (PSF) exerts on phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway in cultured retinal pigment epithelial (RPE) cells. MethodsARPE-19 RPE cells were divided into five groups including PSF overexpression (0.25, 0.50, 1.00 μg of pEGFP-C2-PSF plasmid DNA), PSF overexpression control (pEGFP-C2 empty vector DNA), PSF inhibition (0.25, 0.50, 1.00 μg of pGenesil-PSF-RNAi plasmid DNA), PSF inhibition control (pGenesil-scramble-siRNA empty vector) and sham transfected group (treated with lipofactamine 2000 reagent, but without adding plasmid DNA) groups. After transfecting with plasmid DNA, the cells were stimulated with insulin-like growth factor-1 (IGF-1). IGF-1-stimulated ARPE-19 cells were also treated with Wortmannin and /or PSF over-expression. WST-1 assay was used to detect the proliferation rates, the VEGF mRNA levels were analyzed using real time polymerase chain reaction (PCR), the levels of phosphorylation Akt and total Akt expression were measured by western blotting. ResultsAfter IGF-1 stimulation, the difference of the cell proliferation rates between PSF overexpression group, PSF overexpression control group and sham transfected group was statistically significant (F=29.728, P<0.05). The difference of the cell proliferation rates between PSF inhibition group, PSF inhibition control group and sham transfected was also statistically significant (F=14.121, P<0.05). Compared with control group, the VEGF mRNA levels was decreased in PSF overexpression group (P=0.000 3), but increased in PSF low expression group (P=0.030 9). Furthermore, overexpression of PSF could down-regulate the activation of pAkt after IGF-1 stimulation. When combined with Wortmannin treatment, the VEGF mRNA levels in PSF overexpression group was significantly lower than the control group (P<0.05). ConclusionsAfter IGF-1 treatment, PSF plays a role in suppressing the proliferation and VEGF expression in RPE cells by inactivating PI3K/Akt signaling pathway.
Objective To investigate the activation and role of signal transduction pathway of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR)-mitogen activated protein kinase (MAPK) in proliferation of human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were stimulated with 0.1%，10% foetal calfserum (FCS) and EGF(0.1, 1, 10, 50 and 100 ng/ml)in 0.1% FCS Dulbeco′s modified Eagle′s medium (DMEM) and in 10% FCS DMEM for 3 days, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and EGFR mRNA，respectively. Activation of MAPK was detected by immunohistochemical method with specific anti-phosphorylated ERK 1/2 antibody. Results The optimal concentrations of EGF were 10 ng/ml in 0.1% FCS DMEM and 1 ng/ml in 10% FCS DMEM. After 3 days of stimulation with EGF, phosphorylated ERK 1/2 staining was detectable in nucleus of RPE cells, whereas cells presented immunostaining for phosphorylated ERK 1/2 in the cytoplasm before stimulation. Conclusions EGF may improve the expression of EGFR protein and EGFR mRNA of RPE cells, and induced MAPK nuclear translocation in a concentration-dependent manner. EGF-EGFR-MAPK signal transduction pathway may play a key role in RPE cells proliferation, and serum exerts an important acceclerating function in the process. (Chin J Ocul Fundus Dis，2004，20：67-132)
ObjectiveTo observe the expressions of p-mTOR, p-S6K1 and p-4EBP1 in the human brain with refractory epilepsy and to explore the role of mTOR signaling pathway in intractable epilepsy. MethodsCollecting the brain tissues of 24 patients with refractory epilepsy for surgical treatment from March 2010 to July 2011 as experimental group in hospitalized Epilepsy Center at the First Hospital of Jilin University, Changchun City. Collecting temporal lobe or frontal lobe brain tissue from 6 autopsy of patients who had emergency surgery for neurosurgery brain tranma during the same period. Using immunohistochemistry to observe the expression of p-mTOR, p-S6K1, p-4EBP1 in the two groups of brain tissues, and analyzed statistically. Results① p-mTOR, p-S6K1 and p-4EBP1 were expressed in both neurons and glial cells of experimental and control groups. P-mTOR, p-S6K1, p-4EBP1 positive cells of experimental group was significantly higher than the control group(P<0.01). The expression level of p-mTOR, p-S6K1, p-4EBP1 in the brain tissues of patients with different seizure frequency and with different duration:the expression level of p-mTOR, p-S6K1, p-4EBP1 in the brain tissues of patients in the group of epilepsy 10 years and more than 10 years were significantly higher than the group of epilepsy fewer than 10 years and the difference was statistically significant (P<0.05). ② The structural changes of brain tissues were observed under the optical microscope and electron microscope. Under the optical microscope:the distribution of nerve cells were uneven, the nucleus was vacuolated, the cytoplasm was less and gliosis. Under the transmission electron microscope:the number of neurons was reduced, nuclear condensation, the heterochromatin was increased, the nucleolus were dissymmetry and the nuclear membrane was breakage, also see neurons became psychotic, cell body became smaller, astrocyte cell membrane became edema, chromatin was dissymmetry, some mitochondrial were swelling and transparent, and others were vacuolated, the mitochondrial crista was in disorder. ③ p-mTOR, p-S6K1 and p-4EBP1 are expressed in the cerebral vascular of the brain in both experimental and control groups.In the experimental group, the expression is high concentration.In the control group, the expression is scattered in a small amount. Conclusionsp-mTOR, p-S6K1 and p-4EBP1 are widely expressed in neurons and glial cells with refractory epilepsy, which was significantly increased compared with control group. The expression of p-mTOR, p-S6K1and p-4EBP1 is related to frequency of epileptic seizures and course.
Objective To probe into the roles of inositol 1, 4, 5-trisphosphate (IP3) and bcl-2 gene expression in inhabiting hepatocellular carcinoma of nude mice by quercetin. Methods Animals with hepatocellular carcinoma in quercetin group were treated with injection peritoneum of quercetin 50 mg/（kg·d ） for 3 weeks, while which in control group were treated with 0.4% DMSO of RPMI 1640 0.05 ml/（g·d）. Then the volume and the weight of tumors were measured, IP3, bcl-2 mRNA and bcl-2 protein were assayed by IP3-［3H］ Birtrak Assay, RT-PCR and Western blot respectively. Results The volume and weight of tumors in quercetin group were lower than those in control group 〔（15.8±10.1） mm3 vs. （52.3±26.5） mm3 in volume, （44.8±10.4） mg vs.（91.3±31.4） mg in weight， P＜0.01〕. Content of IP3 in quercetin group was lower than that in control group 〔（13.4±1.4） pmol/mg prot vs. （35.3±6.6） pmol/mg prot， P＜0.01〕. There was no significant difference in bcl-2 mRNA expression between quercetin group and control group 〔RI （the gray degree multiply area of bcl-2 /the gray degree multiply area of β-actin）： 0.55±0.05 vs. 0.79±0.19， P＞0.05〕， but the expression of bcl-2 protein in quercetin group was lower than that in control group (RI: 1.07±0.12 vs. 6.69±1.80, P＜0.01). Conclusion Quercetin can inhabit the growth of hepatocellular carcinoma tansplanted into liver of nude mice by reducing IP3 production and down-regulating bcl-2 gene expression.
To summarize Notch, basic hel ix-loop-hel ix (bHLH) and Wnt gene signal transduction pathways in the process of differentiation and development of neural stem cells. Methods The l iterature on the gene signal transduction pathway in the process of differentiation and development of neural stem cells was searched and then summarized and analyzed. Results The formation of Nervous System resulted from common actions of multi-signal transduction pathways. There may exist a fixed threshold in the compl icated selective system among Notch, bHLH and Wnt gene signal transduction pathways. Conclusion At present, the specific gene signal transduction pathway of multi pl ication and differentiation of neural stem cells is still unclear.
【Abstract】 Objective To summarize the recent progress in related research on transforming growth factor β1 （TGF-β1）/Smad3 signal transduction pathway and post-traumatic scar formation. Methods Recent related literature at home and abroad on TGF-β1/Smad3 signal transduction pathway and post-traumatic scar formation was reviewed and summarized. Results TGF-β1 is an important influence factor of fibrotic diseases, and it plays biological effects by TGF-β1/Smad3 signal transduction pathway. The pathway is regulated by many factors and has crosstalk with other signal pathways at cellular and molecular levels. The pathway is involved in the early post-traumatic inflammatory response, wound healing, and late pathological scar formation. Intervening the transduction pathway at the molecular level can influence the process of fibrosis and extracellular matrix deposition. Conclusion TGF-β1/Smad3 signal transduction pathway is an important way to affect post-traumatic scar formation and extracellular matrix deposition. The further study on the pathway will provide a theoretical basis for promotion of wound healing, as well as prevention and treatment of pathological scar formation.
Objective To introduce the basic research and cl inical potential of the hair foll icle stem cells related signal transduction in prol iferation and differentiation. Methods The recent original articles about the hair foll icle stem cells were extensively reviewed. Results Many different signal pathways had been involved in the skin development and self-newals.The hair foll icle stem cells could play an important role in the skin self-renewal and regeneration which were modulated by several different signal pathways, which included bone morphogenetic protein/transforming growth factor β, Wnt, Notch and ectodysplasin A genes. Conclusion The hair foll icle stem cells may be a future approach to repair cutaneous wounds as a cell therapy．
OBJECTIVE: To study the effect of overexpression of truncated type II TGF-beta receptor on transforming growth factor-beta 1(TGF-beta 1) autoproduction in normal dermal fibroblasts. METHODS: In vitro cultured dermal fibroblasts were treated with recombinant human TGF-beta 1(rhTGF-beta 1) (5 ng/ml) or recombinant adenovirus containing truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-beta 1 were observed with Northern blotting. RESULTS: rhTGF-beta 1 up-regulated the gene expression of TGF-beta 1 and type I procollagen. Overexpression of truncated receptor II down-regulated the gene expression of TGF-beta 1. CONCLUSION: Overexpression of the truncated TGF-beta receptor II decreases TGF-beta 1 autoproduction via blocking TGF-beta receptor signal. The results may provided a new strategy for scar gene therapy.
Objective To evaluate the effect of integrin-linked kinase (ILK) in the process of retinal neovascularization induced by vascular endothelial growth factor (VEGF). Methods The ILK activities of retinal choriodal endothelial cell line RF/6A were inhibited by LY294002 or siRNA knockdown. VEGF-induced changes of cell adhesion, proliferation, migration and endothelial cell tube-formation were measured then. The in-vivo effects of ILK were also assessed by intraperitoneal injection of LY294002 into an animal model of RNV. Results The cell adhesion measurements of control group, VEGF group, VEGF＋LY294002 group and VEGF＋siRNA group were 0.0726plusmn;0.01961, 0.1137plusmn;0.02631, 0.0837plusmn;0.01503 and 0.0853plusmn;0.02454 , respectively. The difference was statistically significant between VEGF group and control group（t =4.211，Plt;0.01）, and between (VEGF＋LY294002) group or (VEGF＋siRNA) group and control group (t =3.074, 2.91，Plt;0.01). The cell proliferation results of control group, VEGF group and VEGF＋LY294002 group were 0.4162plusmn;0.1392, 0.6412plusmn;0.2420, 0.4476plusmn;0.1834 , respectively. The difference was statistically significant between VEGF group and control group（t=2.608，Plt;0.05）, and between (VEGF＋LY294002) group and VEGF group（t=2.244，Plt;0.05）.The cell migration results of control group, VEGF group and VEGF＋LY294002 group were 83.66plusmn;30.283, 248plusmn;74.748, 138.5plusmn;38.167, respectively. The difference was statistically significant between VEGF group and control group（t=5.436，Plt;0.01）, and between (VEGF＋LY294002) group and VEGF group（t=3.682，Plt;0.01）. There was no obvious tube-formation after ILK activity was inhibited or knocked down. The non-perfusion areas were increased from （62798plusmn;16995.62）mu;m2 to （84722.65plusmn;10435.01）mu;m2 after intraperitoneal injection of LY294002 into animal model of RNV, the difference was statistically significant（t=3.476，Plt;0.01）. Conclusions ILK may play an important role in the process of VEGF-induced retinal neovascularization by regulating the cellular adhesion, proliferation, migration and tube-formation, as all those cellular functions were supressed obviously after the ILK activity was inhibited by LY294002 or the ILK expression was knocked down by siRNA.