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Objective To observe the initial mechanism investigation in immune escaping correlated T cell proportion of Lewis lung cancer by Fuzheng-Peiyuan formula. To provide a theoretical basis for the application of the Chinese medicine of strengthening the body resistance in clinics. Methods The healthy C57BL/6J mice were divided into six groups by the random number table: the Chinese medicine group, the Chinese medicine plus chemotherapy group, the chemotherapy group, the model group and the normal control group. There were 6 mice in each group. All the groups expect the normal control group were inoculated subcutaneously with Lewis lung cancer cell suspension 0.2 ml. The medicine given to the Chinese medicine group were orally Fuzheng-Peiyuan formula with 8.17 g/kg. The combination group was given intraperitoneal injection of cyclophosphamide 60 mg/kg and orally Fuzheng-Peiyuan formula 8.17 g/kg. The chemotherapy group received the injection of cyclophosphamide 60 mg/kg and intragastric administration of normal saline. The model group and normal control group were administered with saline. After continuous administration for 13 days, the expressions of CD4, Foxp3, CD8 and CD28 in spleen cells of tumor bearing mice were observed by flow cytometry. Results Compared with the model group, the chemotherapy group and the chemotherapy combined with Chinese traditional medicine group showed that tumor weight (1.76 ± 0.42 g, 1.40 ± 0.43 g vs. 4.37 ± 0.59 g) significantly decreased (P<0.01); and the CD4+ Foxp3+ cells of mouse spleen cells in the Chinese medicine group and Chinese medicine plus chemotherapy group (11.25% ± 1.69%, 9.30% ± 2.68% vs. 14.21% ± 1.50 %) significantly decreased (P<0.05 or P<0.01). Conclusions The result initial proved that the Chinese medicine could strengthen the body resistance, adjust the proportion of Treg and CTL in spleen T cell in tumor-bearing mouse.
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Objective To explore changes of the myeloid derived suppressor cell (MDSC), regulatory T cell (Treg), traditional T cell, and their mechanisms in lung tumor mice. Methods Twenty C57BL/6 mice were randomly divided into the experimental and the normal control groups. The experimental group was injected with Lewis lung cancer cells (LLC, 100μL 1 × 106) subcutaneously to prepare the lung tumor model mice, the normal control group was given the same amount of saline (NC). Spleen cells were obtained from LLC and NC groups. Flow cytometry was used to detect the ratio and number changes of MDSC, Treg, CD4+and CD8+T cells in the lung tumor of mice. CD4+and CD8+T cell apoptosis were detected by Annexin-Ⅴstaining, and their proliferation were detected by 5-bromine deoxidization uracil nucleoside (BrdU) incorporation. Results Compared with normal control mice, the ratio and number of MDSC in spleen increased significantly in LLC group (P<0.01), in addition, the ratio of CD4+Foxp3+Treg in CD4+T cells and their number in spleen increased significantly in LLC group. However, the ratio and number of CD4+and CD8+T cells in spleen decreased significantly in LLC group (P<0.05). The proliferation of CD4+and CD8+T cells decreased significantly in LLC group compared with that of NC group (P<0.05), while the apoptosis of CD8+T cells increased significantly (P<0.05). Conclusion MDSC and Treg cells increase in lung tumor model mice, which inhibit proliferation of CD4+and CD8+T cells and promote apoptosis of CD8+T cells.
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Objective:To comparatively observe the effects of 20(R)-ginsenoside Rg3 and PEG-PLGA-Rg3 nanoparticles on the Lewis lung cancer mice and to explore the mechanisms of Rg3 and PEG-PLGA-Rg3 nanoparticle anti-cancer in vivo.Methods:Lewis lung cancer mouse model was established and 60 mice were randomly divided into 5 groups with twelve in each group:PEG-PLGA-Rg3 nanoparticles group (Rg3-N),PEG-PLGA group (PEG),Rg3 group (Rg3 ),normal control group (C),saline control group(NS),and received intragastric administration for 1 4 days.The weights of the mice were measured every 2 days and the weight curves were obtained.At the same time,the color pattern,activity and men-tal status were observed.The mice were sacrificed when the administration was over,and the effects of 20 (R)-ginsenoside Rg3 and PEG-PLGA-Rg3 nanoparticles on tumor weight,and the tumor:weight ratios were analysed.In addition,the tumor microvessel density (MVD)was measured by immunohistochemi-cal staining with anti-CD31 antibody to compare the effects of Rg3 and PEG-PLGA-Rg3 nanoparticles on the tumor angiogenesis in vivo.Furthermore,the levels of such angiogenesis and proliferation factors as MMP-9,HIF-1 α,VEGF,Ki-67 were examined by RT-PCR,Western blot and immunohistochemistry to explore the internal molecular mechanisms of anti-tumor effects in vivo.Results:The trends of variation of the mice weights in NS group and PEG group were rising early but declining later.In contrast,the trends of the other three groups were rising early and became stable later.In comparison with NS group, the mice of Rg3 group and Rg3-N group had better general status:brighter color,more active and better spirit.Compared with NS group,the tumor weight in PEG group,Rg3 group and Rg3-N group showed no significant difference but the tumor:weight ratio and MVD in Rg3 group and Rg3-N group declined signi-ficantly (P <0.01 ).Besides,there was no significant difference between Rg3 group and Rg3-N group. At the same time,the level of VEGF mRNA,the protein expression of MMP-9,HIF-1 α,VEGF in Rg3 group and Rg3-N group decreased compared with NS group.Furthermore,the level of each index above-mentioned in Rg3-N group was lower than that in Rg3 group.The expression of Ki-67 in PEG group,Rg3 group and Rg3-N group showed no significant difference compared with NS group.Conclusion:Rg3 and PEG-PLGA-Rg3 nanoparticle may suppress the expression of VEGF,MMP-9 and HIF-1 αin Lewis lung cancer mice,thereby indirectly contributing to their antitumor effects and alleviating the mice’s general status.In addition,PEG-PLGA nanoparticles embedding can promote Rg3 antitumor effect in vivo.
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Objective To construct human canstatin gene eukaryotic expression vector and investigate the therapeutic effect of intramuscular canstatin gene delivered by electroporation on tumor growth.Methods Canstatin cDNA was amplified from total RNA extracted from fresh fetal liver by reversing transcription polymerase chain reaction (RT-PCR).The canstatin cDNA fragment was in serted into pEGFP-N1 eukaryotic expression vector.The recombination plasmid was delivered to the quadriceps of the mice with Lewis lung carcinomas by electroporation intramuscular.Fluorescence intension measured by fluorescence microscope,reverse-PCR assay,and immunohistochemistry assay were performed to detect the expression of canstatin gene in the muscle and in circulation.The tumor weight and volume were used to detect the biological effects of canstatin gene delivery.Results Recombinant eukaryotic expression vector of recombinant human canstatin was successfully constructed.The canstatin mRNA was significantly increased in the skeletal muscle and intramuscular delivery of canatatin gene by electroporation acquired the expression of enhanced green fluorescent protein (EGFP)/canstatin protein in the circulation and significantly inhibited tumor growth.The percent of the inhibition of tumor weight was 57.7 ％.Conclusions Electroporation mediated gene transfer efficiency in skeletal muscle was compared to simple plasmid injection and lasted for a long time.It was an efficient and safe,convenient and economic,gene transfer methods and might have certain clinical application value.Electroporation mediated canstatin gene transfer in skeletal muscle had obvious inhibitory effect on Lewis lung cancer in mice subcutaneous xenograft tumor growth.
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Objective: To explore the effect of macrophages with different activated phenotypes on tumor growth, lymphangiogenesis and lymp node metastasis in mice bearing Lewis lung carcinoma (LLC). Methods: The xenografted models were established via subcutaneous injection of LLC cells and activated macrophages into mice. They were divided into 4 groups: blank control group, classically activated macrophages (caMphi) group, alternatively activated macrophages (aaMphi) group and RAW264. 7 group with 10 mice in each group. The growth of transplanted tumors was examined dynamically and then the mice were sacrificed on d 28. The transplanted tumors were resected and the lymph nodes and distant metastasis were observed by HE staining. The LYVE-1+ lymphatic vessel densities (LVD) were detected by immunohistochemistry. Furthermore, other 40 mice were randomly divided into the four groups and the survival rates were recorded. Results: Compared with the blank control group, the aaMphi and RAW264. 7 xenografted tumors grew faster and had more lymph nodes and lung metastasis (P 0.05). Conclusion: aaMphi can promote the growth, lymphangiogenesis, lymph nodes and lung metastasis in transplanted LLC tumors, and reduce the survival rate of tumor-bearing mice. In the tumorigenesis of transplanted LLC tumors, RAW264. 7 cells may toward aaMphi.
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Objective To discuss the feasibility on building lewis lung carcinoma mouse models through different methods and improve the methods. Methods The method of culture LLC cells in vitro, trypsin digestion method, Ⅳ collagenase method and homogenate method were compared to make the different dose of cell suspension injected into C57BL/6 mice. The feasibility of the improved method was determined through observing the cell count, the tumor formation ratio, the tumor formation time, tumor volume, weight and life habit. Results The method of culture LLC cells in vitro could get needed cells and its tumor formation ratio was 100 %. Trypsin digestion method and homogenate method could get less cells and its tumor formation ratio was about 80 %～90 % and 60 %～75 %. Whereas 1V collagenase method could get most cell count and its tumor formation ratio was 100 %. Conclusion IV collagenase method is a preferred method which is simple,high efficiency and make a strong base on the cancer experimental study.
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Objective To study the killing action of adenovirus vector carrying HSV-tk(AdCMVtk) on Lewis cells.Methods The recombinant adenovirus carrying HSV-tk was constructed by homologous recombination techniques.The adenovirus transfected efficiency to Lewis cells was measured,the cells were transfected by adenovirus vector with LacZ gene,then stained with X-gal.The effect of AdCMVtk/GCV system on survival rate of Lewis cells was observed.Results The adenovirus transfected efficiency to Lewis cells was increased with the increasing of multiplicity of infection(MOI).100% transfected efficiency needed MOI 500.AdCMVtk/GCV system could kill Lewis cells.When the amount of adenovirus or the concentration of GCV was increased,the cell survival rate was decreased,but AdCMVtk or GCV alone could not kill Lewis cells.The killing action existed bystander effect,74% Lewis cells were died with only 20% Lewis cells transfected AdCMVtk.Conclusion AdCMVtk/GCV system is effective and safe on killing tumor cells.
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0.05).Tumor formation was found in subgroups of positive CXCR4 cells(7 ? 103 ).Negative and positive CXCR cells were inoculated into 3 mice respectively at the concentration of 5 ? 105 and 2 ? 104 cells.No metastasis occurred in the former group.However,lung metastasis was found in 2 mice and ear metastasis was observed in 1mouse of the latter group.Conclusion Subgroups of positive CXCR4 cells in LLC are characterized by certain properties of cancer metastatic stem cells and have the ability to renew themselves and metabolize.