The Effects Of Disodium Cantharidinate And Vitamin B6 Injection On The Proliferation And Expression Of Some Cytokines Of Cancer Patient Peripheral Blood T Lymph Cells

Objectives: According to differentiation of symptoms and signs of Chinese materia medica, for cancer patient many pathogenesises concomitance, so many therapeutical principles can be used at the same time. The existing studys indicated that Chinese materia medica had clinical efficacy in regulating immune function, inhibiting the generation of tumor new vessels, inducing cell differentiation, promoting the apoptosis of cells, directly killing tumor cells, reversing multidrug resistance of tumor cells, resisting cell mutation and tumor cell metastasis. The anti-tumor mechanisms of Chinese materia medica produce a marked crossed effect through many directions and many ways, so clinicans should combine the determination of treatment based in pathogenesis obtained throu-gh differentiation of symptoms and signs with the treatment based on differentiation of disease.Mylabris sidae is the dry dead body of Mylabris phalerata Pall or Mylabris cichorii Linnaeus being a kind of muscae hispanicae family insect. Mylabris sidae is an important insect medicine. Modern pharmaceutical research indicated that main effective ingredient was cantharides camphor. Cantharidin is hydrolyzed by sodium hydrate then to produce disodium cantharidinate. Disodium cantharidinate and Vitamin B6 compose Disodium Cantharidinate and Vitamin B6 Injection that is a kind of compound preparation. Disodium Cantharidinate and Vitamin B6 Injection retains the anti-tumor effect of cantharidin, meanwhile its anticancer sphere is much broader and its side effects are much less. Disodium Cantharidinate and Vitamin B6 Injection has been generally applied to treat malignant tumor. Previous researches demonstrated that anti-tumor mechanism of Disodium Cantharidinate and Vitamin B6 Injection was mainly cell toxic action, but whether it has other anti-tumor mechanisms is not clear.Through in vitro study, MTT, FCM, RI and RT-PCR were used to reveal possible anti-tumor mechanisms of Disodium Cantharidinate and Vitamin B6 Injection by studying the effects of Disodium Cantharidinate and Vitamin B6 Injection on T lymph cell proliferation and expression of interleukin-2, interleukin-4 and interleukin-8 of T lymph cell from cellular level and molecular level. The results of study will provide more reliable theory and experiment basis for the further development and clinical application of Disodium Cantharidinate and Vitamin B6 Injection.Methods: 1 Separation of peripheral blood mononuclear cells: To draw venous blood of malignant tumor patient using liquemine to anticoagulate. Separate mononuclearcell from peripheral blood with the reference of relevant literature. 2 Using MTT to detect proliferative response of T cells: To add cell suspension to cultivation plate and found a control group and seven test groups. T cells were cultivated for 72 hours. The morphological changes of T lymph cells were observed under inverted microscope. Finally to measure the value of OD. Choose two concentrations (0.010ug/ml and 0.050ug/ml) having the optimal effect of promoting proliferation to do the other tests. 3 The changes of cell cycle of T cell cultured for two days in the control group and two test groups were detected using flow cytometry. 4 The expressions of interleukin-4 and interleukin-8 in supernate were determined using radioimmunity method. The detection procedure referred to the description of kit. 5 The expression of interleukin-2 mRNA in T cells was detected using semi-quantitative RT-PCR: The cells were collected after two days of culture. Total RNA of T cells was extracted. Inverse transcription and amplification of interleukin-2mRNA were done using one-step method. The production was detected using electrophoresis method and then was analyzed using imaging system and analysis software. The relative amount of interleukin-2mRNA was accessed by the ratio of IL-2 absorption factor andβ-actin absorption factor. 6 Using SPSS to analyze data.Results: 1 The morphological changes of T lymph cells were observed under inverted microscope. The result of MTT showed: Disodium Cantharidinate and Vitamin B6 Injection could promote the proliferation of T lymph-cells derived from peripheral blood mononuclear cells. With the concentration being increased, the effect of promoting the cell proliferation enhanced, but when the concentration of Disodium Cantharidinate and Vitamin B6 Injection reached a certain concentration, with the concentration being increased, the effect of promoting the cell proliferation became weak and even could kill T cells. The two optima concentrations of effect of promoting the cell proliferation are 0.010ug/ml and 0.050ug/ml. 2 Debita spissitudine Disodium Cantharidinate and Vitamin B6 Injection could promote the activation of T cells by promoting T lymph-cells from G0/G1 phase to S phase. The percentage of S phase cells between two test groups and a control group had statistical significance (P<0.05). 3 The secretion of IL-8 between two test groups and a control group had statistical significance (P<0.05). 4 The secretion of IL-4 was increased, but the result was not statistically significant (P>0.05). 5 Debita spissitudine Disodium Cantharidinate and Vitamin B6 Injection could promote the synthesis of IL-2 from the gene level. The difference between two test groups and a control group had statistical significance (P>0.05).Conclusions: 1 In vitro, Disodium Cantharidinate and Vitamin B6 Injection could promote the proliferation of T lymph-cells derived from peripheral blood mononuclear cells. The effect of promoting proliferation was implemented by promoting T lymph-cells from G0/G1 phase to S phase. Disodium Cantharidinate and Vitamin B6 Injection may directly enhance the immune function of organism to treat cancer patients by promoting the proliferation of T lymph-cells. 2 The research demonstrated that Disodium Cantharidinate and Vitamin B6 Injection had the most conspicuous effect of promoting the proliferation of T lymph-cells at the concentration of 0.010ug/ml, and with the concentration being increased or decreased the effect of promoting the cell proliferation instead became weak. Furthermore, when Disodium Cantharidinate and Vitamin B6 Injection was increased or decreased to a certain concentration, it could inhibit the proliferation of T lymph-cells. The results give a hint that when clinicians apply Disodium Cantharidinate and Vitamin B6 Injection, the concentration Disodium Cantharidinate and Vitamin B6 Injection should not be too high or too low. 3 One of anti-tumor mechanisms of Disodium Cantharidinate and Vitamin B6 Injection is possibly to decrease the secretion of IL-8 then to inhibit tumor cell migration, growth of tumor vessel and organic selection transfer of tumor cell. 4 Disodium Cantharidinate and Vitamin B6 Injection had the tendency of promoting the secretion of IL-4, but the result was not statistically significant. 5 Disodium Cantharidinate and Vitamin B6 Injection possibly promote the synthesis of IL-2 from the gene level, then to reinforce the immune function to cure tumor indirectly.