The Immune Function Of GM-CSF,Thymosin Alpha1 Combined With Docetaxel On The Mice With Lewis Lung Cancer

Background and ObjectiveLung cancer is the world's highest incidence and mortality of malignant tumors.In recent years the incidence was significantly higher,which is the main reason for the death of malignant tumors in China.The National Cancer Center released the latest cancer data in Chinese cities On February of 2017 that about 1 million people are diagnosed with cancer every day,lung cancer is still the incidence of mortality rate double rate "first".The mortality rate of lung cancer was 40.71 for small cities,medium city for 47.79% and the big city is 54.19%.Early lung cancer often have no obvious symptoms,most patients are already in the middle and late stage when they were diagnosised,which have lost the chance of surgical treatment,so it's particularly significant to find an safe and effective chemical treatment measure.Docetaxel is a diterpenoid compound extracted from Taxus,because it can delay the depolymerization of microtubules to forme a stable nonfunctional vascular bundle,so it can inhibit the cell division and cell proliferation to play its unique anti-cancer activity,widely used in lung cancer?gastrointestinal cancer?breast cancer and so on.However,the application of chemotherapy drugs prone to some adverse reactions,such as nausea,vomiting and other gastrointestinal reactions,the common and more serious adverse reactions is neutropenia.Neutrophils as the guardian barrier of the body,its reduction of the number can directly lead to the decreased immune function and poor tolerance to chemotherapy drugs,which can weeken the treatment effect.However,the colony stimulating factor GM-CSF can enhance the major histocompatibility II expression of macrophages,can promote the proliferation and differentiation of hematopoietic stem cells to play a role in stimulating granulocytes.And with the use of chemotherapy drugs,GM-CSF can significantly enhance the latter's anti-tumor effect,with the reduction of adverse reactions and physical tolerance to enhance the body resistance of patients.Thymosin ?1 can promote the maturation of CD₈⁺T cells,and control the development and functioning of immune cells,with an related biological effect on the target cells.MethodsAccording to the experimental need,the experiments can be divided in two parts :cell experiments and animal experiments.The cell experiments include cell lines and cell culture,to detect the inhibitory effect of docetaxel on Lewis lung cancer cell growth by MTT assay.Animal experiments include the establishment of mouse Lewis lung cancer transplanted tumor model,the application of chemotherapy drugs,the detection of chemical indexes in mice after detection and the Lung cancer metastasis when the mice were sacrificed,the histopathological analysis with the tissue sections,to detect the chemical index with the blood,and so on.1?The cell experiments:Lewis cells in logarithmic phase were inoculated on 96-well plates for culture,different concentrations of docetaxel were added after 24 hours,then add MTT solution respectively at 24 h,48h and 72 h.Measure the optical density?OD?of each well at the OD490 nm with the microplate reader to calculate the inhibition,and draw a dose-response curve for DTX.2?The animal experiments:Thirty-five 4-5 weeks old C57BL/6 mice were randomly divided into 5 groups.The Lewis lung cancer cells were injected intraperitoneally for tumor inoculation,and the Lewis lung cancer model was established,then docetaxel 20mg/kg,3times a week for intraperitoneal injection.Thymosin ?1 10mg/kg,twice a week for subcutaneous injection.GM-CSF 20ug/kg,twice a week for subcutaneous injection.3?Detection IndicatorThe survival status of tumor-bearing mice was observed after injection,and measure the body mass,tumor size and the peripheral blood cells for three times a week.To determinate the content of serum cytokines after chemotherapy,to determinate the thymus index,spleen index,the proliferation of T cells,B cell of spleen and the histological examination of spleen.Results 1?The inhibitory effect of DTX on cell growth with Lewis lung cancerThe survival rate of Lewis lung cancer cells decreased with the increase of DTX concentration,the half inhibitory concentration?IC50?of DTX to Lewis lung cancer cells was 5 × 10-10 mol / L.2?The effects of GM-CSF and T?1 on body weight and tumor inhibition rate of the mice with DTX.The body weight of mice with DTX was significantly lower than that in blank control group.The body weight of mice with T?1 or GM-CSF combine with DTX was lower than the control group,but higher than the group only use DTX.The tumor mass of the mice with DTX alone was lower than that of blank control group,the tumor inhibition rate is 34%.The tumor mass of the mice in group with DTX+ T?1,and group with DTX +GM-CSF was significantly lower with the blank control group,the tumor inhibition rate is 46% and 48%.The value of group with DTX + T?1+GM-CSF is 65%,the difference is significant.3?The effects of GM-CSF and T?1 on thymus quality,spleen mass and thymus index,spleen index of DTX chemotherapy mice.The thymus mass,spleen mass and thymus index,spleen index of the mice in the chemotherapy group were significantly lower than those in the blank control group?p<0.01?which the value of the group with DTX +T?1+GM-CSF was obviously higher than the group with DTX only?p<0.05?.4? The effects of GM-CSF and T?1 on the morphology of spleen in DTX Chemotherapy Mice.The structure of white pulp and red bone was less clear on the essence of HE staining,the spleen body structure is also not complete,the splenic sinus expaned,sinus cell proliferated,scattered large nucleus,red cytoplasmic stained macrophages were visible.Macrophages can be seen rich in lysosomes,metastatic tumor tissue disorder,showing a large number of round tumor cells,gathered into cancer-like,visible mitotic image.5?The effects of GM-CSF and T?1 on the proliferation of spleen T cells and B cells in mice treated with DTX.The T cells and B cell stimulation index of DTX drug chemotherapy group were significantly lower than those of blank control group?p<0.01?,which the value og the group with DTX + T?1 + GM-CSF was higher than that of DTX alone?p<0.05?.6? The effects of GM-CSF and T?1 on the peripheral leukocytes in DTX chemotherapy mice.The number of white blood cells in DTX group decreased significantly after treatment,lower than the blank control group.The number of white blood cells was lightly higher in the group with T?1 and DTX than the normal group,which the value is significantly higher in the group with GM-CSF+ DTX than the normal group.The most significant increase in the number of white blood cells is the group with DTX+ T?1+ GM-CSF,indicating that GM-CSF can significantly increase the number of white blood cells,antagonizing the leucopenia caused by DTX.7?The effects of GM-CSF and T?1 on serum cytokines in DTX Chemotherapy MiceThe levels of IL-1,IL-2,IL-4,IL-6 and TNF-? in the chemotherapy group were significantly lower than those in the blank control group?p<0.01?,the content of IFN-? was significantly lower than that of blank control group?p <0.05?.The levels of IL-1,IL-2,IL-4,IL-6,TNF-? and IFN-? in DTX + T?1 group,DTX + GM-CSF group and DTX + T?1 + GM-CSF group were significantly lower than those in blank control group,higher than the simple use of DTX drug group.Especially in DTX + T?1 + GM-CSF group the different was significantly.8?Effects of GM-CSF and T?1 on peripheral blood T cell subsets in mice treated with DTX.CD3+ CD₄⁺,CD3+CD₈⁺ T cells and CD₄⁺ /CD₈⁺ ratio were lower in the groups with DTX than in the blank control group,while the value of DTX+T?1 +GM-CSF group was higher than that of the DTX group alone.The expression of CD3+CD₄⁺,CD3+CD₈⁺ T cells in the body was decreased,while T?1 and GM-CSF could increase the number of CD3 + CD4 +,CD3 + CD8 + T cells ConclusionsThymosin T?1,GM-CSF combined with docetaxel can effectively inhibit tumor growth,while enhancing the body resistance.The weight loss and spleen,thymus quality decline caused by antagonizing docetaxel chemotherapy can be offset,also reduce the damage of docetaxel to T cells and B cells,and antagonize the neutropenia and the decline of IL-1?IL-2?IL-4?IL-6?TNF-??IFN-? produced by chemotherapy to enhance the number and function of T lymphocytes in peripheral blood.Thereby inducing cytotoxic T lymphocyte-mediated cellular immunity to play the role of the body to kill tumor cells immune regulation.Thymosin T?1 and GM-CSF,combined with docetaxel,the effect is better.