Experimental Research On IL-21 Inhibiting The Growth Of Colorectal Carcinoma By Regulating Immune Balance

Objective:Colorectal carcinoma is one of the common malignant tumors which threatens human health seriously.The combination of surgery, radiotherapy and chemotherapy is the main treatment method currently.Although there has been a considerable progress compared with the previous treatment, the effects are not optimistic.Because the occurrence and development of the malignant tumor are related with the immune function of the body, immune treatment is more and more valued.The immune balance(the balance of Th1and Th2 cell, Th1/Th2 balance)theories provided an powerful tool for the immunity research of malignant tumor.Research discovered that compared with normal the level of immunity in malignant tumor patient is lower, and the Th1/Th2 balance is inclined to Th2 type.It is thinked currently that the adjustment of Th1/Th2 balance is beneficial to the body.We purpose to observe the level of Th1/Th2 balance in colorectal carcinoma patient and Colon26 mice which was transplanted with Colon26 cells in clawpad by flow cytometry and ELISA method.At the same time we purpose to develop Colon26/IL-21 mice model by transplanting Colon26/IL-21 cells in clawpad.In the experiment we will observe how Colon26/IL-21cells effect the Thl/Th2 ballance of mice by secreting IL-21 cytokine and how the adjustment of Th1/Th2 balance effect the tumorgrowth of the mice.Methods:Part 1 We collected randomly 30 cases of colorectal carcinoma patients who were operated in the Surgery Department of No.4 Hospital of Hebei Medical University.We confirmed the diagnosis by pathologic examination before operation. Meanwhile we collected randomly 30 cases of healthy adults as control group.We collected their vein blood on an empty stomach before operation.①The vein blood was collected with anticoagulation tub and was used to experiment immediately.we activate T cell with stimulant PMA before it was detected by flow cytometery.CD4+IFN-γ+ cell(Thl)and CD4+IL-4+ cell(Th2) content was indicated by accounting for CD4+ cell percentage.The ratio of CD4+IFN-γ+ cell and CD4+IL-4+ cell was Th1/Th2.②Obtain serum by centrifugating vein blood. IFN-γand IL-4 levels were measured by enzyme-linked immunosorbent assay techniques. Analyze and statistice data by T-test.Part 2 We divided 90 Balb/c-mice randomly into three groups.The first group of mice were transplanted with Colon26/IL-21cells in clawpad.The second group of mice were transplanted with Colon26cells in clawpad.The third group of mice were injected with normal sodium in clawpad.Obtain serum by centrifugating vein blood after 24 days. IFN-γand IL-4 levels in the serum were measured by enzyme-linked immunosorbent assay techniques. The ratio of IFN-γand IL-4 was Th1/Th2.Analyze and statistice data by ANOVA.Measure the weight of the tumors of two groups.The tumor organization of two groups was observed and contrast with HEstainning.Results:1 The level of Th1 cell in colorectal carcinoma patients and healthy adults was 11.78±5.04% and 11.39±3.62%.The difference of the data between two groups wasn't significant(P>0.05).The level of Th2 cell in colorectal carcinoma patients and healthy adults was 16.0±7.02% and 10.63±4.55%.The difference of the data between two groups was significant(P<0.01).The level of Th1/Th2 in colorectal carcinoma patients and healthy adults was 0.74±0.27 and 1.26±0.9,The difference of the data between two groups was significant(P<0.05).The level of IFN-γin the serum of colorectal carcinoma patients and healthy adults was 6.26±1.43 pg/ml and 9.18±2.31 pg/ml,The difference of the data between two groups was significant(P<0.05).The level of IL-4 in the serum of colorectal carcinoma patients and healthy adults was 8.88±1.30pg/ml and 5.20±1.71pg/ml,The difference of the data between two groups was significan(tP<0.01).The level of IFN-γ/IL-4 in the serum of colorectal carcinoma patients and healthy adults was 0.70±0.10和1.82±0.35,The difference of the data between two groups was significant(P<0.01). 2①The tumor of the mice which was transplanted with Colon26cells in clawpad continued to grow.The value of tumor of the mice which was transplanted with Colon26/IL-21cells in clawpad arrived at 1.3mm3 after 16 days,then diminuted gradually.The weight of Colon26-mice and Colon26/IL-21-mice was 2.48±0.31g and 0.27±0.10g.The difference of the data between two groups was significant(P<0.01).②HE staining confirmed on the 24th day that the tumor in clawpad of Colon26 mice and Colon26/IL-21 mice are all malignant.③The level of IFN-γin the serum of Colon26/IL-21-mices, Colon26-mice and normal mice group was 44.80±8.07 pg/ml,26.40±2.31 pg/ml and 31.21±3.36 pg/ml.The difference of the data between each group was significant.The level of IL-4 in the serum of Colon26/IL-21-mice, Colon26-mice and normal group was 134.61±16.41pg/ml,170.42±19.77pg/ml and 120.60±16.38pg/ml.The difference of the data between Colon26/IL-21-mice and Colon26-mice groups was significant(P<0.01).The difference of the data between Colon26-mice and normal mice groups was significant ( P<0.01 ) .The difference of the data between Colon26/IL-21-mice and normal mice groups wasn't significant(P>0.05).The level of IFN-γ/IL-4 in the serum of Colon26/IL-21-mices, Colon26-mices and normal group was 0.33±0.06,0.16±0.01 and 0.26±0.03.The difference of the data between each group was significant(P<0.01).Conclusions: 1 Compared with normal immune balanceTh1/Th2 balance)of colorectal carcinoma patients was inclined to Th2 type.2 IL-21 can enhance the function of Th1 cells and regulate the ratio of Th1/Th2 cells,thus correct the immune imbalance.3 The effect of anti-tumor can be enhanced by correcting the Th1/Th2 imbalance in individual.4 Regulating the immune balance can be expected to become a major means of colorectal carcinoma immunotherapy.