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AIM: To explore the effect of HBV transfection on apoptosis induced by TWEAK and the regulatory effect of IFN-γ on the apoptosis process.METHODS: BEL-7402-pCDNA3/1.1HBV hepatoma cell line was used as cell model in this experiment, in which BEL-7402 was stably transfected with pCDNA3/1.1HBV and its corresponding empty vector BEL-7402-pCDNA3 was used as control. The effect of HBV transfection on TNF-like weak inducer of apoptosis(TWEAK) induced hepatoma cell apoptosis was determined by CCK-8. The apoptosis before and after HBV transfection induced by TWEAK and the effect of IFN-γ on apoptosis induced by TWEAK in HBV transfected hepatoma cells were detected by TUNEL and caspase 3 activity detection kit.RESULTS: BEL-7402-pCDNA3/1.1HBV and BEL-7402-pCDNA3 hepatoma cell line were successfully established and identified. Cell viability of hepatoma cells was increased after HBV transfection. The apoptosis rate was much higher after HBV transfection compared to the empty vector control and empty cell control. The apoptosis rate induced by TWEAK was much higher in BEL-7402-pCDNA3/1.1HBV cells when exposed to IFN-γ.CONCLUSION: After HBV infection, TWEAK might be involved in the clearance of HBV infected hepatocyte by apoptosis and inflammatory factor such as IFN-γ.
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Objective To explore the role of TRAIL receptors expression on peripheral blood mononuclear cell (PBMC) in the apoptosis of PBMC,and the relation of the expression of TRAIL receptors with the severity of liver damage in chronic hepatitis B patients.Methods The expressions of DR4,DRS,DcR1 and DcR2 in 55 patients and 30 cases of control were assayed by RT-RCR and floweytometery.The relationship of the expression of TRAIL receptors with the severity of liver damage were analized.Results The level of DcR1 in the PBMC of chronic hepatitis B patients was much lower than that of control group (P＜0.05).There was close relation between the expression of DcR1 and liver damage (P＜0.05).The level of DcR1 was negatively correlated with ALT but positively correlated with serum albumin.Conclusion The expression level of DcR1 on PBMC in chronic hepatitis B patients was down-regulated,which may contribute to the increased apoptosis of PBMC in chronic B hepatitis patients.The expression of DcR1 can reflect the degree of liver injury in chronic hepatitis B patients to some degree.
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Objective To study the inhibition effects on hepatoma cells growth by the anti-sense RNA targeting C-MYC binding site on regulation region of hTERT promoter.Methods The rAd virus which express anti-sense RNA complementary to the C-MYC binding site on regulation region of hTERT were constructed using the method of homologous recombination in bacteria cells.The apoptosis of HepG2.2.15 cells infected by rAd-asmycb was detected by the method of Annexin V-FITC/PI labeling,and the morphological changes were observed by electronic microscopy.TRAP-PCR-ELISA and RT-PCR were used to detecte the relative telomerase activity(RTA) and gene transcription at mRNA level of hTERT.Results Cell growth of HepG2.2.15 was retarded and about 40.7% tumor cells were lead to apoptosis.RTA of anti-sense RNA treated cells(1.175) was much lower than the control cells(4.200,P
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<p><b>OBJECTIVES</b>To construct a hepatoma directed gene delivery system which could transfer preS2 antisense RNA to liver cancer cells specifically, and to explore a new therapeutic strategy for hepatocellular carcinoma by blocking hepatitis B virus (HBV) with antisense RNA targeting hepatocellular carcinoma.</p><p><b>METHODS</b>GE7 and HA20 were synthesized and mixed with pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system named AFP-enhancing 4-element complex. Nude mice bearing hepatocelluar carcinoma cells HepG2.2.15 were injected with AFP-enhancing 4-element complex via a tail vein. Total RNA from tissues was extracted, and reversal transcription-ploymerase chain reaction (RT-PCR) was used to detect the expression of preS2. Different doses of AFP-enhancing 4-element complex was injected into nude mice at different time points, and tumor diameter was measured.</p><p><b>RESULTS</b>AFP-enhancing 4-element complex was constructed successfully. RT-PCR showed preS2 antisense RNA delivered by AFP-enhancing 4-element complex only expressed in liver tumor HepG2.2.15 cells of the mice. After the treatment of AFP-enhancing 4-element complex with dose of 0.2 micro g per mouse (once a week for 4 weeks), the mean tumor diameter of nude mice was significantly shorter than that of the control groups (0.995 +/- 0.35 cm vs 2.125 +/- 0.25 cm, P < 0.01).</p><p><b>CONCLUSIONS</b>An HBV antisense RNA gene delivery system targeting hepatocellular carcinoma, AFP-enhancing 4-element complex, was constructed successfully. PreS2 antisense RNA expressed specifically in hepatocelluar carcinoma cells significantly inhibits tumor growth of mice bearing hepatocarcinoma HepG2.2.15 and may have therapeutic potential in HBV related hepatocarcinoma.</p>
Subject(s)
Animals , Male , Mice , Drug Delivery Systems , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Hepatitis B Surface Antigens , Therapeutic Uses , Hepatitis B virus , Genetics , Liver Neoplasms, Experimental , Pathology , Therapeutics , Mice, Nude , Protein Precursors , Therapeutic Uses , RNA, Antisense , Therapeutic UsesABSTRACT
0.05).The C myc and N ras protein expression of 2.2.15 were reduced after ASONs were used 3 days.There was a significant difference between ASON group and control group ( P
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Objective To explore the mechanism of immune tolerance in chronic B hepatitis, the effects of HBV infection on the functions of macrophages and related mechanism of signal transduction by transfection in vitro were studied. Methods Peritoneal macrophages of mice were isolated regularly and cultured, transfected transiently with pcDNA3-HBV or pcDNA3 plasmid DNA and cultured under the stimulation of LPS. After 72 h, RT-PCR was performed to detect the expression of PreS1, TNF-? or IL-1? mRNA. To detect the expression of NF-?B RelA protein by FCM, and the level of nitric oxide in cultural supernatant was measured with Griess reaction. Results After being transfected with pcDNA3-HBV,peritoneal macrophages had the expression of PreS1 mRNA, but have lower level of TNF-?、IL-1? mRNA and transcriptional factor NF-?B, compared with pcDNA3-transfected control group; the level of nitric oxide in pcDNA3-HBV group was also decreased. Conclusions Transient transfection of pcDNA3-HBV could decrease the function of macrophages directly by inhibiting NF-?B activity and effector molecules production, which may be one of the mechanisms of immune tolerance in chronic B hepatitis.
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AIM: To explore the effect of TNF-related apoptosis inducing ligand (TRAIL), a new apoptotic inducing molecule on the biological activity of hepatocarcinoma cell line. METHODS: The expression of membrane binding TRAIL on HepG2 cells was detected by immuno-cytochemistry. Quantity of secretory TRAIL was assayed by ELISA method. The cytotoxicity and apoptosis induced by TRAIL was detected by MTT and TUNEL method, respectively. The telomerase activity of HepG2 cells was detected by TRAP-PCR assay kit. The expression of hTERT, the catalytic subunit of telomerase, was detected by FCM. RESULTS: TRAIL was constitutively expressed on the membrane of HepG2 cell line. Soluble TRAIL was also expressed to a certain degree. Cytotoxicity assay showed that TRAIL significantly inhibited the growth of hepatocarcinoma cells. TUNEL assay indicated that TRAIL induced apoptosis in hepatocarcinoma cells. Detection of telomerase activity showed that TRAIL inhibited telomerase activity and the expression of telomerase catalytic subunit. CONCLUSION: TRAIL is an effective molecule to inhibit the growth of hepatocarcinoma through multiple pathways, such as inducing apoptosis and inhibiting the activity of telomerase.
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AIM: To explore the relationship between caspase activation and the evasion of Legionella from macrophage elimination through a Legionella-infected macrophage model. METHODS: After infected by Legionella, the activity of caspase 3 in macrophages was analyzed by confocal microscopy as well as fluorescence reader. Growth and replication of Legionella in macrophage was assayed. Replication of Legionella was analyzed again to see the effect of caspase 3 inhibition on the growth of Legionella after use of caspase 3 inhibitor. RESULTS: Both confocal microscopy and caspase 3 fluorescent substrate analysis showed that Legionella virulent strain had powerful capability of activating caspase 3 while the mutant non-virulent strain did not have this capability. The virulent strain highly replicated in macrophages and the replication was significantly inhibited by caspase 3 inhibitor. CONCLUSION: Our results indicate that the intracellular caspase 3 is activated shortly after infection by Legionella virulent strain. The evasion of Legionella from the elimination of macrophages may be mediated by caspase 3 activation to a great degree.
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@#本文在“国际残疾分类”的基础上，围绕康复诊断名称的标准化，进行了初步探讨，力求使之适合于临床康复的应用及康复病案管理。
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Objective: To investigate the effects of calcitonin on human breast cancer cell line T47D. Methods: The inhibition rates of salmon calcitonin (sCT) on T47D cells were measured by MTT methods. Then telomerase activity of T47D cells was detected using PCR ELISA methods. Cell apoptosis was observed by transmission electron microscope (TEM). Animal models in vivo were constructed by implanting T47D cells subcutaneously into nude mice. After injection of sCT for 30 days, tumor diameters were measured. The structure of lumbar 3 were separated and compared by a scanning electron microscope (SEM). Results: The inhibitory effects of sCT on T47D cells was observed by MTT method. The PCR ELISA method discovered that sCT could decrease the telomerase activity of T47D cells. TEM found cell apoptosis induced by sCT. Tumor diameters in sCT treatment group showed no statistical difference compared with the control group. SEM of lumbar 3 discovered that sCT could strengthen the bone structure of nude mice. Conclusions: The decrease of telomerase activity and induction of apoptosis are new mechanisms of sCT inhibition on T47D cells. The tumor inhibition in vivo was not observed. This may be attributed to the complicated endocrine response in vivo . sCT is still effective in strengthening the bone structure of those nude mice without osteoporosis.
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Objective:To screen the high performance,specific and nontoxic anti HBV antisense oligonucleotide fragment.Methods:Taking 2.2.15 cells as cell model,the antisense oligodeoxynucleotides(ASON)toward the HBV regulation genesenhancerⅡ(HBV EFⅡ)were designed and synthesized.The role played by ASON in inhibiting the secretion and expession of HBsAg and HBeAg by the host cells was detected by ELISA method.The effect of ASON on prolifezation and metabolism of the cells was detected by MTT method.Results:The inhibitory rate of HBsAg by ASON was 92% and 75%,respectively,indicating that itsdifference from that of noncomplementary sequence control group(inhibitory rate was 11%)had considerable statistical significance( P 0.05).Conclusion:ENⅡ is one of the most important target oriented sequencial selective regions of research on anti HBV nature of antisense oligonucleotide.
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AIM:To explore the effect of HBV transfection on apoptosis induced by TWEAK and the regulatory effect of IFN-? on the apoptosis process.METHODS:BEL-7402-pCDNA3/1.1HBV hepatoma cell line was used as cell model in this experiment,in which BEL-7402 was stably transfected with pCDNA3/1.1HBV and its corresponding empty vector BEL-7402-pCDNA3 was used as control. The effect of HBV transfection on TNF-like weak inducer of apoptosis(TWEAK) induced hepatoma cell apoptosis was determined by CCK-8. The apoptosis before and after HBV transfection induced by TWEAK and the effect of IFN-? on apoptosis induced by TWEAK in HBV transfected hepatoma cells were detected by TUNEL and caspase 3 activity detection kit.RESULTS:BEL-7402-pCDNA3/1.1HBV and BEL-7402-pCDNA3 hepatoma cell line were successfully established and identified. Cell viability of hepatoma cells was increased after HBV transfection. The apoptosis rate was much higher after HBV transfection compared to the empty vector control and empty cell control. The apoptosis rate induced by TWEAK was much higher in BEL-7402-pCDNA3/1.1HBV cells when exposed to IFN-?.CONCLUSION:After HBV infection,TWEAK might be involved in the clearance of HBV infected hepatocyte by apoptosis and inflammatory factor such as IFN-?.
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Objective:To synthesize disubstituted pyridine and I-substituted propynyl carbamates and study their antimicrobial activities, searching for more potent and less toxic antimicrobial agents.Methods: The title compounds were synthesized through the process of electrophilic and nucleophilic substitution. Antimicrobial test in vitro was determined with 13 kinds of common mildews and bacteria(Aspergullus niger, aspergillus flavus, Aspergillus versicolor,Trichoderma viride, Paecilomium varioti Bainier, Chaetomium globsum, Penicillium citrinum, Cladochytrium clodospoium, Escherichia colo, Staphylococcus aureus, Pseudomonas fluorescens Bacillus fluorescens, and Bacillus megatherium). The chemical structures of all compounds were determined by IR, 1 HNMR and elementary analysis.Results: Seven disubstituted pyridine and I-substituted propynyl carbamates obtained were firstly reported. All compounds showed antimicrobial activity, especially compound 3e, who had more potent activity compared with the that of 3-iodo-2-propynyl-butyl- carbamate (IPBC). MIC of the compound 3e was between 2?10 -6 g/ml to 30?10 -6 g/ml . Conclusion: Compound 3e has the best antimicrobial activity and should be futher studied.