ObjectiveToobserve the killing effect of herpes simplex virus thymidine kinase/ganciclovir (HSV tk/GCV) suicide gene therapy system on human pancreatic carcinoma cell line. MethdsHSV tk gene was constructed into a retroviral vector pDOR neo. The recombinant plasmid pDOR tk neo was transfected into the retroviral packaging cell PA317. Finally HSV tk gene was transferred into human pancreatic cancer cell line PC 2. The sensitivity to GCV and bystander effect of PC 2/tk cells was tested in vitro. Antitumor effects were also observed after the administration of GCV in nude mice bearing tumor derived from PC 2/tk cells. ResultsHSV tk gene was successfully integrated into host cell. The killing effect of GCV on PC 2/tk cells was in a dose depandent and time depandent manners. The PC 2/tk cells showed a significant "bystander effect". Intraperitoneal injection of GCV postponed the formation of the implanted tumors. ConclusionPancreatic cancer cells expressing HSV tk can be effectively killed by GCV both in vitro and in vivo.

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

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.

Objective To investigate the effect of Rumo Shengji San on promoting tissue regeneration of skin defect after repair with tissue-engineered skin.Methods The dermal scaffolds were reconstructed by collagen sponge in which human skin fibroblasts were transplanted inside and human keratocytes were transplanted on the surface.The tissue-engineered skin was transplanted into the wound surface of defected skin in naked mice.Rumo Shengji San was applied externally once every other day after transplantation for 2 days.The animals were randomly divided into two groups:external application group and control group,4 mice in each group.The differentiation tissue of the tissue-engineered skin was sampled 9 days after transplantation.The epidermis thickness and dermal capillary proliferation in wound of defected skin were observed with HE staining,and double-labeled immunofluorescence method was used to test dermal laminin(Ln) and typeⅠcollagen protein expression.Results Rumo Sheng Jji San promoted the development of epidermis after transplantation for 9 days.The number of capillaries in dermis was increased,and the expression of Ln and typeⅠcollagen protein was promoted in external application group than that in the control group.Conclusion Rumo Shengji San can promote tissue regeneration of skin defect after repair with tissue-engineered skin.

Objective To observe the influence of Lycium Barbarum polysaccharide(LBP) on FasL expression in H22-bearing mice and to explore its anti-tumor mechanism.Methods Kunming mice were randomized into the model group,and low-and high-dose LBP(in the dose of 0.625 and 1.250 g?kg-1?d-1 respectively) groups.H22-bearing mice models were induced through right subaxillary inoculation of H22-ascitic cells.Three days after inoculation,LBP group were given LBP for 14 days.Hematoxylin and eosin(HE) staining method was used to examine the tumor cell density,tumor cell mitotic count,and lymphocyte infiltration in tumor interstitial tissue.FasL expression was observed in the three groups with immunohistochemical method.Results Tumor cell mitotic count was decreased in the two LBP groups(P

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.

【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.

[Objective] To investigate the effect of Lycium barbarum polysaccharide (LBP) on T-lymphocyte subsets levels, dendritic cells (DCs) counting and antigen CD80 expression in peripheral blood and tumor stroma of H22-bearing mice. [Methods] H22-bearing mice models were established. LBP in the dosages of 1.25 and 0.625 g?kg-1?d-1 was orally administered to the mice models for two weeks. The changes of T-lymphocyte subsets levels, DCs counting and antigen CDgo expression in peripheral blood and tumor stroma were detected by flow cytometry (FCM) . [ Results ] LBP increased the numbers of CD4+ and CD8+ T-cells, promoted the proliferation of CD4+ T-cells, and elevated the ratio of CD4+ /CD8+ in peripheral blood. LBP in large dosage also increased the numbers of CD4+ and CD8+ T-cells in the tumor-infiltrating lymphocytes (P

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.

Objective To investigate the potential role of Lycium bararum polysaccharide (LBP) with or without interferon -inducible protein 10 ( CXCL10) in inducing dendritic cells ( DC) functional maturation by monitoring the alteration of cytokines for inducing DC maturation in peripheral blood and by detecting the expression of S-100 protein in tumor tissue, thus to reveal its mechanism of inhibiting experimental liver cancer. Methods H22 bearing mice model was established. The mice were randomized into model group, LBP group (50 mg/kg, ig), CXCL10 (right axillary subcutaneous injection of 15 μg/kg), LBP + CXCL10 group (LBP 50 mg/kg, ig, and right axillary subcutaneous injection of CXCL10 15 μg/kg), 5- fluorouracil (5FU) group ( intraperitoneal injection of 12mg/kg) , 12 mice in each group. The mice were administered the corresponding medicine once a day. After treatment for 2 continuous weeks, blood was sampled from infraorbital vein, and the tumor mass, spleen, thymus were extracted for the calculation of anti-tumor rate, thymus index and spleen index separately . The mRNA expression levels of interleukin 12 (IL-12) and tumor necrosis factor-α (TNF-α) in peripheral blood were detected by fluorescence quantitative PCR, the expression of S-100 protein in tumor tissues was detected by immunohistochemical assay. Results Compared with the model group, tumor growth in LBP group and LBP+CXCL10 group was obviously inhibited, and tumor-inhibitory rate was 55.90%, 50.91%, respectively. Meanwhile, the mRNA expression level of IL-12 was 2.94 folds higher in LBP group and 3.39 folds higher in LBP + CXCL10 group, and TNF-α mRNA expression level was 1.55 folds higher in LBP group and 4.74 folds higher in LBP+CXCL10 group than the model group, the differences being statistical significant ( P<0.05 or P<0.01). Results of immunohistochemical assay showed that S-100+DC number in LBP group and LBP+CXCL10 group was larger than that in the model group (P<0.05 ). Conclusion LBP and LBP+CXCL10 exert significant effect on inhibiting experimental liver cancer. The mechanism may be related with inducing the secretion of IL-12 and TNF-α, which plays a key role in inducing DC maturation, and with the increase of the number of DC in tumor microenvironment.