The Mechanisms Of Kidney And Liver-Supplementing Chinese Medicine To Prevent Bone Marrow Suppression After Chemotherapy Of Osteosarcoma And Unit CIK Killing Of Osteosarcoma

ObjectiveOsteosarcoma is a primary malignant bone tumor, accounts for 20% about the primary malignant tumor bone. It has high degree of malignancy, easy to occur in young people, easy to recurrence, metastasis and death. With the emergence of new adjuvant chemotherapy, improved surgical techniques, most osteosarcoma patients can treat with limb salvage, the 5-year survival rate can be as high as seventy percent. The methotrexate, doxorubicin, ifosfamide and cisplatin has been recognized and widely used as the main anti-inhibition of osteosarcoma cells for chemotherapy. However, it can lead to decreased immunity, inhibit the hematopoietic system, nausea, vomiting, and the cardiac, liver and kidney function damage and other adverse reactions, also resistance to chemotherapy occurring. As a result, the patients unable to complete chemotherapy, affecting survival quality and life.Therefore, many other treatments of osteosarcoma occuring, including radiation therapy, gene therapy, immunotherapy and molecular target therapy and other new forms of treatment. Tumor immune therapy has become an important method for the treatment of cancer, which stimulating the immune response to antagonize the tumor cells, regulate and balance immune function, differentiation, and inhibit tumor cell growth, and selectively kill the tumor cells. Adoptive immunotherapy with Immunocompetent cells is one of the most Striking means. During the adoptive immunotherapy, the CIK (eytokine-indueed killer) cells are considered the most promising cytokine-induced killer cells. It has highly proliferation ability, highly activity to kill tumor, broad spectrum, and little toxicity to normal bone marrow. It can promote the reconstruction of the immune system of patients, bone marrow purification and the removal of minimal residual disease.It has been used in lung Cancer, liver cancer, renal cell carcinoma, but the relationship between osteosarcoma is unclear. We hope that by studying the role of CIK cells and the effect on osteosarcoma patients, see if it has the function anti-osteosarcoma, provide the basis of using the CIK cells to treat with osteosarcoma.TCM theory holds that the pathogenesis of osteosarcoma is the vir tual standard, due to liver and kidney deficiency, the deficiency of kidney is essential, foreign evils invasion, lead to qi stagnation an d blood stasis. Therefore, Chinese medicine treatment is benefit liv er and kidney and clinical research found that Liu Wei Di Huang Tang is a classic Chinese herbal formula in this righting training. Modern pharmacological studies confirmed that Liu Wei Di Huang Tang can enh ance the body's cellular and humoral immunity. We studied Liuweidihua ngwan serum effect adriamycin-resistant osteosarcoma cells, see if it has the anti-inhibition of adriamycin-resistant osteosarcoma cells.T o provide the experimental basis for clinical application.Clinically, we use antler glue, angelica, astragalus and rhizoma typhonii, With the effection of tonicing kidney Qi. Each one of thes e has numerous reports can relieve the inhibitory effect on the hemat opoietic system when chemotherapy. However, no relevant reports about this prescription reduce the adverse effects of chemotherapy, we hop e that through clinical observation of blood analysis, whether this p rescription can reduce the inhibition of hematopoietic system caused by chemotherapy side effects.MethodsExperimental aspects:By culturing, passaging adriamycin-resistan t osteosarcoma cells, understand the growth regulation of the cells. We uesd the third generation of osteosarcoma cells and make the conce ntration was 1×10^7/mL, injected 1ml under the skin at the back of the neck of nude mice for modeling, observed the success rate in nude mice, and to provide a simple way of making model for osteosarco ma research foundation. We Intragastriced SD rats with Liuweidihuangw an and got the abdominal vein blood, prepared Liuweidihuangwan serum, used this serum effecting osteosarcoma cells. We used 50ml blood fro m the author to Separat human peripheral blood mononuclear cells, sep arated by the lymph liquid separation by centrifugation centrifuge to extract the middle layer of mononuclear cells, by adding the concent ration of day 0 100U/mL of IFN-γ. Into 37℃,5% CO2 incubator for 2 4 hours, adding a concentration of 50 ng/mL CD3, 100U/mL of IL1-αand 300U/mL IL-2, induced the formation of CIK cells, by flow cytomet ry To detect the phenotype. To study PBS solution,10% Liuweidihuangwa n serum and 1640 mediums effect the Adriamycin-resistant osteosarcoma cells. To study PBS solution,10% FBS high glucose DMEM medium, cont aining 400ug/ml ADM 0.9% sodium chloride solution,200ug/ml ADM 0.9% sodium, 100ug/ml ADM 0.9% sodium,50ug/ml ADM 0.9% sodium and 25ug/ml ADM 0.9% sodium effect the Adriamycin-resistant osteosarcoma cells. To study Liuweidihuangwan serum combined with different concentration s of doxorubicin effct Adriamycin-resistant osteosarcoma cells. To st udy CIK cells (1:1 with Adriamycin-resistant osteosarcoma cells), con taining 10% Liuweidihuangwan serum medium, containing doxorubicin 400 ug/ml 0.9% sodium, CIK cells combined with containing 400ug/ml doxoru bicin solution, CIK cells in solution containing 10% Liuweidihuangwan serum, PBS solution effect Adriamycin-resistant osteosarcoma cells. To study Liuweidihuangwan serum combined with different proportion CI K cells with Adriamycin-resistant osteosarcoma cells. The results of the above analysis of variance.Clinical aspects:We selected 10 healthy adults and 10 adult patients with osteosarcoma, got 50ml blood of each one, amplified the CIK cells in vitro.Comparison the FACS phenotype differences by flow cytometry in normal people and osteosarcoma patients with self-induced of CIK cells, to compare the activity of two groups of CIK cells against KH-OS and MG63/ADM. Select the patients (from January 2009 to March 2011) from Joint Orthopaedic in the first affiliated hospital of Guangzhou University of Traditional Chinese Medicine, diagnosed with osteosarcoma. Total of 10 people included,7 men and 3 women, aged from 12 to 21 years.6 people joined the experimental group and 4 patients jinned control group. To observe the effect of reducing the bone marrow suppression of chemotherapy after taking the decoction (antler glue, angelica, astragalus and rhizoma typhonii). Comparing the two groups in WBC, HGB, PLT, LYM%, and NEU%, using t test of its results.ResultThe adriamycin-resistant osteosarcoma cells growth and metabolism faster than normal cells, cell morphology were fusiform, irregular, large nuclei, its proliferated to 7 times only 3 days. The nude modeling which used the concentration of osteosarcoma cells was 1×10-7/mL, injected lml under the skin at the back of the neck of nude mice. The tumor rate was 100%, the pathology report the tumors were the osteosarcoma. Separation of peripheral blood by the lymphatic fluid and centrifuged stratified, divided into 3 layers, the middle layer is white cloudy mononuclear cell layer, by adding the concentration of day 0 100U/mL of IFN-γ. Into 37℃,5% CO2 incubator for 24 hours, adding a concentration of 50 ng/mL CD3, 100U/mL of IL1-αand 300U/mL IL-2, induced the formation of CIK cells, successfully induced CIK cells CIK cells were round translucent cells which high expression of CD3+CD56+by flow cytometry. Through statistical analysis,10% Liuweidihuangwan serum medium without inhibited the growth of osteosarcoma cell proliferation, when compared with the other two groups its p<0.05. The suppression of osteosarcoma cells 400ug/ml,200ug/ml and 100ug/ml 0.9% sodium chloride solution are stronger than the other two groups of doxorubicin (p<0.05), showing enhanced performance with the increase of concentration. When Liu Wei Di Huang Wan serum combined with different concentration of adriamycin effected osteosarcoma cell, there was no significant difference between groups (p=0.618). Liu Wei Di Huang Wan serum associated with different proportions of CIK cells effected the adriamycin-resistant osteosarcoma cells, the result among the groups was statistically significant (p 0.001), the inhibition of the killing effect of osteosarcoma cells showing enhanced performance with the increase of concentration, but When we when the ratio is 50:1(CIK:adriamycin-resistant osteosarcoma cells), it appears less inhibited.We collected peripheral blood from 10 healthy patients and 10 patients with osteosarcoma, inducing CIK cells by the same way. Culturing to 21 days the average number of OS-CIK 35.8-fold increase and ND-CIK increased 81.6 times. CD3+CD56+, respectively, from 6.6%,10.3% in the seventh day to 15.9% (P<0.05),24.8% in the 21th days (P<0.05). Moreover, CD3+ CD56+in the 17th days reached the highest level at that time when CD4 +CD25+-positive rate only 1.31%. OS-CIK, ND-CIK effected on the KH-OS in the target ratio of 40:1 to achieve the inhibition rates were 53.67%, 81.23%, while the effection on the MG63/ADM achieve the suppression tumors were 50.74%,65.70%.10 patients with osteosarcoma drinking tonic the kidney yang medicine during chemotherapy and taking blood analysis the results. The WBC, HGB, PLT, NEU among two groups was no significant difference before chemotherapy.1 week after chemotherapy, two groups of WBC decreased, in the experimental group dropped to 2.73×10^9/L, the control group dropped to 2.048×10^9/L(p= 0.081). HGB both groups decreased, the experimental group dropped 15g/L and the control group was decreased by 19g/L. NEU in both groups decreased.1 month after chemotherapy, both groups continued to decline HGB, the other indexes returned to normal. Throughout the process, PLT are within the normal range.ConclusionOsteosarcoma cells in vitro with the features of fast proliferati on, in line with the clinical features of osteosarcoma fast growth. T he uesd osteosarcoma cells and make the concentration was 1×10^7/mL, injected lml under the skin at the back of the neck of nude mice for modeling, this is a simple way to modeling and high successfull rate s. The killing consequent of adriamycin effected adriamycin-resistant osteosarcoma cells was enhanced with the concentration, showing a co ncentration-dependent relationship.10% Liuweidihuangwan serum medium without inhibited the growth of osteosarcoma cell proliferation, but is conducive to the proliferation of osteosarcoma cells, the specifi c mechanisms still need further research. This reflect the limitation of nature in vitro and inadequate to illustrate the mechanism of ou r Chinese prescription, the mechanism of our Chinese prescription ma ybe by regulating multiple organs of the body to play a role, reflect ing the 1 concept of holism in TCM. This is supply new ideas and new ways for how to better research for prescription drugs. CIK cells ca n be more easily acquisit through the induction, its growth peak at 1 4 to 21 days, OS-CIK and ND-CIK proliferation significantly, but the ND-CIK cells much faster than OS-CIK, were highly expressed in CD3+ CD56+. Both its killing effect of osteosarcoma cells was enhanced as the concentration increased. Our formula can relief bone marrow sup pression caused by chemotherapy, even though no significant differenc e between the two groups statistically, WBC, HGB small decrease in th e control group and PLT no significant drop. The reasons may be relat ed to our limited observation time, less patients and the patient's c ompliance. This clinical study provide a clinical basis for our Tradi tional Chinese Medicine relieve the side effects of chemotherapy, we also need to further study the mechanisms.