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Objective To investigate expression of 7 kinds of immune related human cytokines (IL-2, IL-4, IL-12, IL-13, IFN-γ TNF-α, MIP-1α) in CML patients. Methods Pure monoclonal antibody on the prepared NC membrane glass slides under certain environmental condition to make human eytokines protein microarray were spotted and serum samples (25 patients, 25 the normais) were collected. Cytokines concentration in the serum with the protein microarray were tested by ELISA. Results IL-4 and IL-12 serum concentration in CML patients are lower than that of the normal (P <0.05). However. No statistic difference of MIP-1α was found between CML patients the normal. Conclusion Cell mediated immunity and humoral immunity of CML patients are both inhibited in some extent and the expression of MIP-1α may be inhibited by p210 in CML patients.
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【Objective】 To explore a method for the construction of antitumor suicide-gene therapeutic system. 【Methods】 The thymidine kinase gene of herpes simplex virus type 1 (HSV1-tk) was orientationally cloned into the retroviral vector plasmid (pLXSN) by DNA recombinant technique, and then the recombinant plasmid of pLXSN-tk was identified by restriction endonuclease cutting and DNA sequencing. Introduced by PolyFect Transfection reagent, the recombinant plasmid was transfected into the packaging cell line PT67. Screened by G418, a cell line, which could produce virus stably, was obtained. The virus was transfected into human gastric carcinomatous cell strain SGC-7901 and the transfected SGC-7901 was applied to evaluate an anti-tumor effect. 【Results】 The identification with restriction endonuclease cutting and DNA sequencing showed that HSV1-tk was successfully inserted into the recombined plasmid of pLXSN-tk. The stable cloned PT67/tk was obtained by screening with G418 and then its amount was enlarged by culture, the titer of the PT67/tk solution being 4?10~4 cfu/mL. The anti-G418 cloned cell line SGC-7901/tk was got by infecting SGC-7901 with the virus. Anti-tumor experiment showed that GCV had an obvious toxic effect on SGC-7901/tk but had no effect on SGC-7901, indicating recombinant retroviral HSV1-tk expressed HSV1-tk gene which had biological activity. 【Conclusion】 Cloning HSV1-tk into the retroviral vector is effective in obtaining the recombinant retroviral HSV1-tk which can express HSV1-tk gene, and also effective in establishing an antitumor suicide-gene therapeutic system. This research naturally lays a foundation for further studying of Chinese medicines having synergistic anti-tumor action with suicide gene.
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Suicide - gene therapy is now regarded as a new method for tumor with good prospect. Since bystander effect is the main mechanism and is positively correlated with the immune function, stimulating the immune function of tumor carriers and improving the inflammatory microimmune situation are two important keys to good therapeutic effect. As it is known that Chinese herbal medicine is effective in improving human immune function and has less toxic effect, therefore, suicide - gene therapy combined with Chinese herbal medicine may be a new, safe and economic regimen for tumor.
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Objective To investigate the possibility of establish a combined regimen of Chinese medicine and suicide gene therapy,we observed the killing action of medicated serum of Liuwei Dihuang Bolus (LDB)combined with HSV-tk/GCV suicide gene therapy system on rats hepatoma cell line CBRH7919.Methods The HSV-tk/GCV suicide gene therapy system was constructed and the working solution of ganciclovir (GCV)at 39.2 ?mol/L was detected firstly.Then medicated or non-medicated serum was prepared from SD rats treated with or without LDB by gavage.Meanwhile,the effects of medicated and non-medicated serum on cell proliferation were detected.The control groups without serum added were blank control group,GCV group and suicide gene therapy (SGT)group.Blank serum groups,blank serum + GCV groups,blank serum + SGT groups,medicated serum groups,medicated serum+GCV groups and medicated serum+SGT groups all had two serum concentrations of 5% and 7.5%,respectively.Six double holes were set for each group.CBRH7919/ tk+ and CBRH7919/ tk-were mixed together,and the tk+ cell percentage was adjusted to 0%,5% and 10%,respectively.Then the mixture of CBRH7919/ tk+ and CBRH7919/ tk-was implanted into the 96-hole plates at a density of 3?103 cells/hole,cultured with complete medium for 24 hours,and then treated with medicated or non-medicated serum for 12 hours and sequentially with GCV at 39.2?mol/L for another 60 hours.The number of surrival cells was determined by MTT assay.Q value (the ratio of measured and theoretical pharmacological action)was used to analyze the synergism of Chinese medicine and suicide gene therapy:additive action when 0.85≤Q1.15.Results No cytotoxicity was found when blank serum or medicated serum was below the concentration of 20% (volume fraction).When the concentration of serum was at 5% and 7.5%,the killing action of SGT + medicated serum group was stronger and the cell survival rate was higher those that of medicated serum group and SGT + blank serum group (P1.15).Conclusion HSV-tk/GCV suicide gene therapy system combined with LDB has a synergistic killing action on rats hepatoma cells.
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Aim To explore the proliferation-inhibited,apoptosis-induced and cell cycle-regulated effect of evodiamine on human hepatoma cell line HepG2.Methods MTT,Dapi assay,flow cytometry analysis,comet assay were used.Results Evodiamine could significantly inhibit the growth of human hepatoma cell line HepG2.After 72 hours of treatment with evodiamine at different concentrations(64,16,4,1,0.25 ?mol?L-1),the inhibitory rate of HepG2 was 74.0%,69.0%,60.5%,44.0% and 16.4%,respectively.Meanwhile,HepG2 showed typical apoptosis.After 24 and 36 hours' treatment with evodiamine(1 ?mol?L-1),a typical subdiploid peak before G0/G1 phase was observed by flow cytometry and cell cycle was arrested in the G2/M phase,while the rate of apoptosis was 4.4%,18.0% and 30.3% of treatment with evodiamine for 12,24 and 36 hours respectively.After 24 and 36 hours' treatment with evodiamine(1 ?mol?L-1),the average optical density was lower than that of the control and the length of tail increased compared with the control.Meanwhile the changes were related with time.Conclusion Evodiamine inhibits the proliferation and induces apoptosis of HepG2.
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Aim To investigate the curative effect of Danshen injection combining with HSV-tk/GCV system on rats′ hepatocarcinoma cells and murine transplanted hepatocarcinoma.Methods ① Rats′ hepatocarcinoma cell line CBRH7919(tk~-),CBRH7919/tk(tk~+) and the 5% tk~+ mixed cells were treated with diverse concentrations of Danshen injection,GCV separately,and Danshen injection plus GCV(n=3).The survival rate of each groups was examined using MTT Assay and was analyzed using paired comparison.Q-value analysis method was used to estimate the synergistic effect of Chinese herbal on the suicide gene system.Q-value is a ratio of the actural effect of combination treatment to its theoretical effect.It is thought to be an additive effect when 0.85≤Q