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.
ObjectiveTo investigate the expressions of Patched-1 (Ptch1) and glioma-associated oncogene homologl (Gli1) protein of sonic hedgehog signaling pathway in cholangiocarcinoma tissues, and explore their correlations to the occurrence and development of cholangiocarcinoma. MethodsThe expressions of Ptch1 and Gli1 protein in 62 specimens of cholangiocarcinoma and its bile duct tissues adjacent to cancer were detected by immunohistochemistry, and their positive rate correlated with patients, age, tumor size, differentiation grade, tumor location, lymph node metastasis, TNM stage, operation mode, and postoperative survival time were investigated by statistical analysis. ResultsThe positive rates of Ptch1 and Gli1 protein were significantly higher in cholangiocarcinoma than in tissues adjacent to cancer (74.2% vs. 14.5%, 88.7% vs. 9.7%, P < 0.05). The expressions of Ptch1 and Gli1 protein in cholangiocarcinoma had no correlation to patients age, tumor size, and tumor location (P > 0.05), but were correlated to the operation mode, differentiation grade, lymph node metastasis, TNM stage, and postoperative survival time of patients (P < 0.05). ConclusionsThe elevated expressions of Ptch1 and Gli1 protein of Hh signaling pathway participated in the occurrence and development of cholangiocarcinoma. They may be ideal targets for therapy against cholangiocarcinoma.
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.
Objective To find new ways for wound healing and tissue expansion by reviewing of progress in recent years in functional molecules which are used for signaling channels of mechanical stress perception and mechanotransduction of keratinocyte. Methods The domestic and international articles were reviewed to summarize the functional molecules and signaling channels of mechanical stress perception and mechanotransduction of keratinocytes. Results The mechanism of mechanical stress perception includes mechano-sensitive channels, growth factor receptor-mediated mechanical stress perception, and mechanical stress perception by protein deformation. The mechanism of mechanotransduction includes cell adhesion-mediated signaling, mitogen-activated protein kinase signaling, the cytoskeleton and extracellular matrix, and so on. Conclusion Keratinocytes can response to the mechanical stress and transfer the effective information to undergo shaping, migration, proliferation, differentiation, and other biological behavior in order to adjust itself to adapt to the new environment.
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.
Lipopolysaccharide (LPS), the important component of the outer membrane of Gram-negative bacteria, contributes to the integrity of the outer membrane, and protects the cell against bactericidal agents. LPS, also called endotoxin synonymously, is well known as a potent inducer of the innate immune system that often causes septic shock in the intensive cares. Chemically, the amphiphilic LPS is made up of three parts, i.e. hydrophobic lipid A, hydrophilic core oligosaccharide chain, and hydrophilic O-antigenic polysaccharide side chain. Specially, the lipid A is known to be responsible for a variety of biological effects during Gram-negative sepsis. LPS can elicit a strong response from innate immune system and result in local or systemic adverse reactions. LPS can trigger massive inflammatory responses and may result in immunopathology, for which the molecular basis is mediated by the signal pathway of LPS. In recent years, a tremendous progress has been made in determining the associated proteins and receptors in the LPS signaling that leads to the disease. This review gives a summary of recent progresses of research on LPS and LPS receptors.
Inherited retinal degeneration (IRD) is a group of fundus diseases characterized by a high degree of genetic heterogeneity and clinical heterogeneity, and more than 300 genetic mutations have been identified in association with IRD. Dysregulation of the intracellular second messenger cyclic guanosine monophosphate (cGMP) plays an important role in the development of IRD. cGMP participates in phototransduction process in photoreceptors. Abnormally elevated cGMP over-activate protein kinase G and cyclic nucleotide-gated channel, causing protein phosphorylation and Ca2+ overload, respectively, and these two cGMP-dependent pathways may individually or collectively drive photoreceptor degenerative lesions and death; therefore, reducing cGMP synthesis and blocking downstream signaling can be considered as treatment strategies. Investigating the molecular mechanisms of cGMP dysregulation in photoreceptor degeneration may provide a more comprehensive picture of the pathogenesis of IRD, as well as ideas for finding new therapeutic targets and designing therapeutic programs.
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 construct AWP1 (associated with protein kinase C related kinase 1) recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304. Methods Cloned AWP1 cDNA was inserted into the multiply clone sites (MCS) of plasmid pcDNA3 for adding flag tag, and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV. After identified with restrictional enzymes, plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease PmeⅠ, and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination. After linearized by PacⅠ, the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high-level adenoviruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope (LSCM). Results PCR assay showed that recombinant adenovirus Ad-flag-AWP1 was obtained successfully; and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWP1 recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.
ObjectiveTo explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 (NEP1-40) gene and neurotrophin 3 (NT-3) gene into neural stem cells (NSCs).MethodsNSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups: no-load lentiviral vector transducted NSCs (group A), NEP1-40 transducted NSCs (group B), NT-3 transducted NSCs (group C), NEP1-40 and NT-3 corporately transducted NSCs (group D), and blank control (group E). Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection (MOI; 5, 10, 15) for different time (24, 48, 72 hours). Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium.ResultsThe optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C (P<0.05), but no significant difference was found between groups B and D, and between groups A and C (P>0.05). The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B (P<0.05), but no significant difference was found between groups A and B, and between groups C and D (P>0.05).ConclusionNEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.