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.

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

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.

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 growth inhibition effect of evodiamine (Evo) on renal carcinoma 786-0 cells and to explore its molecular mechanism. Methods After treated with Evo, methyl thiazolyl tetrazolium ( MTT) assay was used to detect the vitality of 786-0 cells, flow cytometry was employed to examine the cell cycle distribution in 786-0 cells, and immunoblotting was utilized to determine the expression levels of target proteins related to cell cycle progression. Results Evo remarkably inhibited 786-0 cells vitality in dose-dependent manner. Cell cycle analysis indicated that 786-0 cells were arrested in G2/M phase followed by Evo treatment. Furthermore, the results of immunoblotting showed that Evo up-regulated the protein expression levels of P53, P21 and its downstream target gene CyclinB1 in 786-0 cells. Conclusion Evo treatment can induce 786-0 cell cycle G2/M arrest, and its underlying mechanism might be dependent on the P53/P21 signal pathway.

Objective To investigate the inhibitory effect of Lycium barbarum polysaccharide ( LBP) on the tumor growth and metastasis in MMTV?PyMT mouse model of breast cancer. Methods The population of MMTV?PyMT trans?genic mice was expanded and identified. 8?week old MMTV?PyMT?positive female mice were randomly divided into LBP group and control group, 8 mice in each group. The mice of LBP group were given LBP treatment (50 mg/kg, i. p. ), and the control group was given normal saline in the same volume, once every 2 days for 4 weeks. The tumor size was measured every two days. The mice were killed at 4 weeks after treatment, the lungs were removed and fixed in Bouin′s solution to observe the number of metastatic nodules, and tumor tissues were used for immunohistochemical examination of tumor cell proliferation and vascular density. Results The tumor formation rate was 100% in the MMTV?PyMT?positive mice. The tumor weight of LBP group was 4?208 ± 0?4463 g, significantly lower than the 6?477g ± 0?3724 g in the control group (P<0?005). The number of pulmonary nodules of the LBP group was 12 ± 1?155, significantly less than that of the control group (20 ± 2?745) (P<0?05). The immunohistochemical examination using Ki67 and CD31 staining showed that tumor cell proliferation and microvessel density of the LBP group were significantly less than the NS group. Conclusions LBP inhibits breast cancer growth and metastasis through the inhibitory effect on tumor growth and metastasis, inhibition of tumor cell proliferation and angiogenesis in MMTV?PyMT mice. These mice can be used as an ideal model for studies on antitu?mor drug development for the treatment of breast cancer lung metastasis.

Objective To investigate the changes of the immunological factors in subcutaneous exudate and blood components of the rats receiving cutaneous scraping method,and to compare the changes of skin histopathological features before and after cutaneous scraping under microscope.Methods SD rats were randomly divided into two groups,cutaneous-scraping group and non-cutaneous-scraping group.And then each group was divided into three subgroups.The observation indexes included the levels of interleukin (IL)-1β,IL-6 and interferon gamma (IFN-γ) in the blood and the skin,routine blood examination,and skin histopathological features.Results In cutaneous-scraping group,the number of white blood cells in the blood and the levels of IL-1 β and IFN-γ in skin tissues were increased (P ＜ 0.05),the hemolysis rate was increased (P ＜ 0.05).However,the levels of IL-1β,IL-6 and IFN-γin the blood showed no obvious changes.Under the microscope,severe skin edema,vascular congestion and dilatation,and infiltration of inflammatory cells were found in the skin after cutaneous scraping.Conclusion The cutaneous scraping method can activate the immune response rapidly,and the immunological components of the subcutaneous exudate after cutaneous scraping are helpful to the disease treatment.

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.

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.