Clinical And Experimental Study On Effect Of The Principle Of Reinforcing The Vital Energy And Consolidating The Constitution On Immune Function After Chemotherapy Of Osteosarcoma

Osteosarcoma is a common bone tumor which has very high malignant.The occurrence and development of osteosarcoma correlates closely with immune system of the body.Patients with malignant bone tumor frequently have immune inhibition or damage.Immune condition of body has improtant role to development and improvement of bone tumor.With remarkable improvement of surgery,application of synthesis chemotherapy schemes and increasing of chemotherapy drugs' bid,effect of osteosarcoma has promoted evidently.However,Osteosarcoma succumbs to relapse and durg-resistance after surgery and chemotherapy.Meanwhile,chemotherapy drugs damage to immune ability greatly besides skilling tumor cells,so influence curious effect seriously and threat human life.Therefore,increasing immune functions of osteosarcoma patients is very improtant to therapy of osteosarcoma.Traditional Chinese Medicines have unique roles on enhanced immune functions of tumor patients.The principle of reinforcing the vital energy and consolidating the constitution is one of improtant rules of TCM curing malignant tumor,in particular,the body regulatory roles of immune functions significantly.This study undestood changes on immune indexs among chemotherapy of osteosarcoma patients by using Chinese herbs(Liuwei Dihuang (LW) Decoction),and analyzed immune condition of patients.Here the paper studied cytokines-induced killer(CIK) against osteosarcoma cells in vitro,so as to obtain efftective evident of immunotherapy on osteosarcoma patients.Objects:1.Assessed change of many immune indexes from peripherial blood lymphocytes before and after chemotherapy of osteosarcoma patients;2.Observed immune regulatory roles of Liuwei Dihuang(LW) Decoction before and after chemotherapy of osteosarcoma patients;3.Observed amplification and phenotype of CIK cells from different sources and the CIK cells after co-culture with dendritic cells(DC);4.Studied the cytotoxic activity against osteosarcoma cells and osteosarcoma durg-resistance cells by CIKs from different sources;5.Defined the induce and proliferation of CIK cells using Liuwei Dihuang(LW) Decoction;6.Studied the cytotoxic activity against osteosarcoma cells by CIKs in vitro that were induced by using drug serum of Liuwei Dihuang(LW) Decoction.Methods:1.The study detected T lymphocytes antigens(CD3,CD4,CD8),B lymphocytes antigens(CD19,CD20) and NK cells(CD16+CD56) deriving from peripherial blood of 104 osteosarcoma patients by flow cytometry;2.104 osteosarcoma patients divided into TCM therapy group,blank group and immune therapy control.It detected changes of immune indexes before and after therapy by using flow cytometry,and defined influence of immune functions of osteosarcoma patients and immune regulatory roles of Chinese herbs(Liuwei Dihuang(LW) Decoction) to osteosarcoma patients;3.The study expanded CIK cells from osteosarcoma patients and normal donors,and pulsed dendritic cells(DCs) by using tumor lysis from osteosarcoma patients.CIK cells co-cultured with DCs.The proliferation of expanded CIK cells was detected by Flow Cytometry.The cytotoxic activity of expanded CIK cells was tested with ⁵¹Cr cytotoxic method;4.The study observed proliferation and cell phenotypes of CIK cells by using drug resum of Liuwei Dihuang(LW) Decoction.The cytotoxic activity of expanded CIK cells was tested with MTT method.Results:1.The level of CD₃⁺,CD₄⁺,CD₈⁺and CD₄⁺/CD₈⁺ after chemotherapy of osteosarcoma patients were evidently lower than before chemotherapy(P＜0.01).The chemotherapy of patients made T cells reduce.The inhibitive T cells increased evidently.The cell immune functions of osteosarcoma patients reduced evidently.The level of CD19 and CD20 reduced evidently before and after chemotherapy of osteosarcoma patients(P＜0.05 or 0.01).The humour immune also were inhibited in osteosarcoma patients by T10 chemotherapy scheme.2.The level of CD₃⁺ and CD₈⁺ of osteosarcoma patients increased evidently after therapy of Liuwei Dihuang(LW) Decoction(P＜0.01).The level of CD₄⁺ and CD₄⁺/CD₈⁺ increased but didn't evident(P＞0.05).Chinese herbs can increase T cells and sub-population,but immune globins didn't evidently improve,and promote immune functions of osteosarcoma patients.The level of CD19 and CD20 increased evidently by therapy of chinese herbs(P＜0.05 or 0.01).It is showed that chinese herbs can not only improve the levelof immune cells of osteosarcoma patients,but also increase the level of humour immune.3.The level of CD₄⁺ and CD₃⁺ increased but didn't evident(P＞0.05) by using IL-2 during the period of chemotherapy.The ratio of CD₄⁺/CD₈⁺ increased evidently(P＜0.01).It is showed that IL-2 can improve immune functions of osteosarcoma patients.The level of CD19 and CD20 also increased by therapy of IL-2 and didn't statistical significant(P＞0.05).It is showed that IL-2 was limited to humour immune.4.Both groups were successfully induced to CIK cells.The proliferation rate and cytotoxicity of the normal donors-CIK (ND-CIK) were slightly stronger than the osteosarcoma patients-CIK(OS-CIK). Cytotoxicity of CIK cells from each group increased progressively along with increasing of the effect target density ratio.The proliferation and cytotoxicity of CIK cells co-cultured with tumor lysate-puling DCs were evidently higher than CIK solely in two groups regardless of normal and patients(P＜0.05).The CD3⁺CD56⁺ co-expression of all CIK cells increased greatly among total culture.Furthermore the cytotoxicity of CIK against osteosarcoma drug-resistance cell was very strong.5.Drug resum of Liuwei Dihuang(LW) Decoction didn't induce CIK cells sololy.The proliferation folds of CIK cells by using drug resum of Liuwei Dihuang(LW) Decoction were higher evidently than by using non-drug resum and non resum(P＜0.05).The proliferation rates of CIK cells by using drug resum at middle bid were higher than low bid and high bid.The CIK cells phenotype was the best of CD3⁺CD56⁺ co-expression in drug resum at low bid(2%) which evidently higher than CIK cells of other groups.CIK cells didn't promote coexpression of CD3⁺CD56⁺ in drug resum at middle bid.The cytotoxicity of CIK against osteosarcoma cell line OS-732 in drug resum at low bid(2%) was stronger than other groups(P＜0.005).Conclusion:1.Chemotherapy could reduce immune ability of osteosarcoma patients;2.Liuwei Dihuang(LW) Decoction and immune therapy(IL-2) can active T lymphocytes and increase immune function of patients.Liuwei Dihuang(LW) Decoction can improve the levelof immune cells of osteosarcoma patients and the level of humour immune.IL-2 can improve immune functions of osteosarcoma patients and be limited to humour immune 3.It indicated that the proliferation and cytotoxicity of CIK cells are related to organism condition in vitro.CIK should exist effectiveness in clinical therapy of osteosarcoma.4.Drug resum of Liuwei Dihuang(LW) Decoction didn't induce normal CIK cells sololy.Liuwei Dihuang(LW) Decoction can promote proliferation of CIK cells,especifically drug resum at middle bid.The phenotype in drug resum at low bid were most steady.The cytotoxicity of CIK against osteosarcoma cell line in drug resum at low bid was the strongest.CIK cells could apply for clinical therapy of osteosarcoma effectively.