Study On Specific Biomarkers For Graft-Versus-Host-Disease

Objectives:Hematopoietic stem cell transplantation(HSCT)is one of the best methods for treating hematological malignancies.Graft-Versus-Host Disease(GVHD)is the main complication after transplantation,which seriously affects the success rate of transplantation and patient safety.Graft-versus-host disease is divided into acute and chronic subtypes.Acute Graft-Versus-Host Disease(aGVHD)progresses rapidly and is critically serious,leading to early death of patients after transplantation.Chronic Graft-Versus-Host Disease(cGVHD)persists for several years,with a long course,seriously affecting the quality of life of patients.At present,the diagnosis of GVHD is mainly based on clinical symptoms and pathological biopsy,but the lack of specificity of clinical symptoms and the invasiveness of pathological biopsy seriously affect the early diagnosis and treatment of GVHD.Screeni ng of serum biomarkers is simple and rapid,non-invasive,and changes in the expression levels of biomarkers can reflect the pathological immune status of the body,which is conducive to the early warning of GVHD and the study of mechanisms.Therefore,detecting serum specific biomarkers of GVHD has important clinical reference value for early diagnosis and treatment.Methods:1.Establishment of GVHD mice model: SPF grade male C57BL/6(H-2b)mice were selected as donor mice,femur,tibia bone marrow and spleen were takenand SPF grade female BALB/C(H-2d)mice were used as recipient mice.The whole body was irradiated by 650 cGy 60 Co gamma ray.Then they were divided into two groups: control group and aGVHD group(experimental group).In the control group,5x10^6 bone marrow cells were injected into the tail vein of the donor mice.In the aGVHD group,additional 1x10^6 spleen cells were added and then injected.2.Dynamic changes of 10 biomarkers were detected in aGVHD animal models,and specific biomarkers related to aGVHD were screened.20 patients with aGVHD and 20 patients without aGVHD in our center were selected to detect and verify the clinical significance of 10 biomarkers in patients with GVHD by protein chip technology,and to explore the value of biomarkers in the pathogenesis of aGVHD.3.The expression of 12 biomarkers in 30 patients with cGVHD and 60 patients without cGVHD were detected by liquid suspension chip technology.Specific biomarkers associated with cGVHD were screened and their significance in the occurrence and development of GVHD,disease severity and different target organs was evaluated.Results:1.After whole body irradiation with 650 cGy 60 Co gamma rays,recipient mice in the experimental group were injected with donor bone marrow cells and spleen cells.On the 14 th day after transplantation,FISH technique was used to detect the successful implantation of donor stem cells.GVHD manifestations such as hair loss,weight loss,bow back and diarrhea were observed in the experimental group.2.Through the detection of 10 specific biomarkers,compared with the control group,the expression of IL-2 and TLR4 in aGVHD mice and aGVHD patients increased significantly(P<0.05),while the expression of TGF-β in aGVHD mice and patients decreased significantly(P<0.05).3.The expression levels of CXCL9,CXCL13,CCL11,CCL17,MPIF-1/CCL23 were significantly increased in patients with cGVHD compared with the control group(P<0.05).The expression levels of CXCL13 and CCL17 in patients with severe cGVHD were significantly higher than those in the control group(P<0.05).The expression of CXCL9 in skin and CCL17 in liver was significantly increased(P<0.05).Conclusions:1.The aGVHD mouse model can be successfully constructed by whole body irradiation of recipient mice with 650 cGy 60 Co gamma rays and transfusion of donor mouse bone marrow cells and spleen cells.2.IL-2,TLR4,TGF-β can be used as a biomarker for early prediction and diagnosis of aGVHD,and are vulable for clinical application reference.3.CXCL9,CXCL13,CCL11,CCL17,MPIF-1/CCL23 can be used as specific biomarkers for early diagnosis and evaluation of cGVHD.CXCL13 and CCL17 can be used as specific biomarkers for predicting the occurrence of severe cGVHD.CXCL9 and CCL17 can provide auxiliary reference for different target organ damage.