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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
Objective To study the effect and mechanism of antisense oligodeoxynucleotides (ASONs) inhibiting hepatitis B virus (HBV) expression. Methods We designed and synthesized antisense oligodeoxynucleotides directly against HBV PreS 2 gene and noncomplementary sequence control. 2.2.15 cells were chosen as cell model. Inhibitory effect of ASONs on HBV gene expression were assayed by ELISA. Cell apoptosis and proliferation were detected by Fascan Flow Cell Cytometer. Effect of ASONs on cell metabolism was detected by radioimmunoassay (RIA) and MTT assay. Results ASONs were able to effectively inhibit HBV expression. Their inhibitory rates of HBsAg and HBeAg were 66% and 91%, respectively. Noncomplementary sequence control group (both inhibitory rates were 11%) was not able to inhibit HBV expression. ASON might induce host cell apoptosis. Cell apoptosis rates on 3rd and 6th day were 6.10% and 6.43%, respectively. Proliferation index were 37% and 36%, respectively. Results of RIA and MTT showed that ASON had not cytoxicity on host cell. Conclusions Not only are ASON able to inhibit HBV gene expression with sequence specific but also clear HBV in the way of apoptosis.
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Objective:To investigate the effect of arterial baroreflex(ABR)on survival rate of rats with cecal ligation and puncture(CLP)-induced sepsis.Methods:Male Sprague-Dawley rats were divided into 2 groups:sham-operated rats(n=22)and sinoaortic denervated(SAD)rats(n=22).Four weeks after SAD rats were subjected to CLP-induced sepsis,the blood pressure and heart period(HP)were monitored for 12 hours in conscious state and the survival of rats was observed.Results:Both the diastolic and systolic blood pressue gradually decreased after CLP;the HP shortened first and then drastically prolonged until the death of rats.At 12 h after CLP the survival rate of SAD rats was lower than that of the sham-operated rats(59% vs 86%).Significant differences were found between the Kaplan-Meier survival curves of the rats in 2 groups(P
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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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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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The peroxisome prolifrator-activated receptors(PPARs)? and ? constitute a subfamily of nuclear receptors. PPAR? has been shown to be activat ed by the hypolipidemic drugs of the fibrate class; While the antidiabetic TZD a re synthetic ligands for PPAR?. Upon binding and activation by their ligands, t hey regulate the transcription of numerous genes involving lipid metabolism and insulin resistance. The research indicated that PPAR? also plays a key role in lipid metabolism. PPARs therefore constitute interesting targets for the develop ment of single and dual agonists useful in the treatment of obesity and type 2 d iabetes.
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Aim To investigate the effects of the novel compound C333H on reduce blood glucose and lipid in vivo.Methods Normal KM mice,hyperlipidaemia mice and type 2 diabetic mice by intragastric gavage were used and total RNA from liver,adipose and skeletal muscle were isolated for RT-PCR.Results C333H reduced blood lipid level and improved glucose metabolism.In addition,C333H increased expressions of LPL,aP2 and GluT4 at transcriptional level.Conclusion C333H is a novel PPAR?/? agonist,signivicantly reducing blood lipid and glucose,which had potential as a therapy for type 2 diabetes.