Application Of Protein Chip Technology Hbv Chronic Liver Disease Tcm Syndromes And Cell Factor

Object: The small molecule protein substances with biological activity were secreted by the cells and referred as cytokine.As well, the significant correlation between cytokine and the evolvement of chronic liver disease has been proved.Based on gene microarray, protein microarray has developed as a new biological detection technology.It's great promoting effect on the development of multiple disciplines, such as Biology, Clinical Laboratory Medicine, Genetics, Oncology and Pharmacology, has changed the Status of biomedical research and diagnosis fundamentally. Currently, ELISA method has always been used to detect cytokine, although some defects like low-sensitivity, low-repeatability, narrow measurement range were exist, so protein microarray, with the advantages of rapid, concurrent, automation and high throughput, was been chosed to avoided the defects above.Set seven kinds of cytokine (IL-2, IFN-γ, IL-4, TNF-α, sICAM-1, TGF-β₁, TIMP-1) as the research targets and constructed protein microarray, compared the difference in sensitivity and specificity between protein microarray technology and ELISA method.Explored the change regulations of cytokine in different chronic liver diseases by protein microarray technology, then found the correlation between cytokines and different chinese medicine syndrome types of Chronic Hepatitis B patients and patients with HBV-liver Cirrhosis, expected to lays the foundation for the diagnosis, treatment and Dialectical Thinking of Traditional Chinese Medicine on chronic liver disease.Method: Stayed the slides at APES which has been diluted by acetone in the proportion of one to fifty for about 20～30seconds, then flushed the unconjugated APES with pure acetone solution, afer that dried them in room temperature.Diluted the monoclonal antibodies of the seven kinds of serum markers with PBS(0.01 mol/L, pH7.0)to 500mg/L, the Cartisian 5500 robot would print the sample diluent on APES- slides, set four copies for every sample and eight same microarray per slide.Incubated the chips for 4 hours at 37℃, then washed them by PBST for three times about 15 seconds every time, after be sealed for 2 hours at 37℃, repeated the operation above, so dried and stored the chips. Assumed the BSA which were marked by biotin as positive contrast groups, unmarked ones were negative control groups. Dissolved the serum samples stored at -80℃, the first centrifugation was done at 4℃, 5000g for 5 minutes.Diluted 10μl serum by sample diluent, and shocked in ice bath for 30 minutes at 400～600r/min.Added 100μl sample to every well, incubated for one hour and washed PBST for three times, after add the monoclonal antibodies of the seven kinds of cytokine 20μl (concemtration 1μg/ml)and the avdin marked by Cy3, incubated and washed again.Formed the images by Scan Array 4000-confocal scanner, then used the Imagene software to analyse the fluorescence intensity, gave all raw data the standardization and minus background processing according to the reference values of positive and negative, found the average of four wells' fluorescence intensities, finally calculated and analysed the results with standard curve.Compared the results between protein microarray and ELISA method, discussed the change regulations of cytokine in different chronic liver diseases and the correlation between cytokine and different chinese medicine syndrome types of chronic liver disease.Results: (1) After constructed the serum cytokine protein microarray successfully.In the prediction test we detected the cytokines (IL-2, IL-4, and TNF-α) of thirty four samples by protein microarray and ELISA method and compared the sensitivity of two methods, the result showed that IL-2 and IL-4 were not detected, we found TNF-αin serum samples of eight severe chronic viral hepatitis B patients by protein microarray while the number was five by ELISA method. (2) The detection rates of cytokines between chronic liver disease group and the normal group were notable different (P<0.05), Th1 cytokines (IL-2, IFN-γ) was normal in mild chronic viral hepatitis B patients, the concentration of chronic severe viral hepatitis patients was minimum (P<0.01); Th2 cytokines (IL-4, 258.56±36.72 pg/ml) was maximum in chronic severe viral hepatitis patients (P<0.01), the change regulations were mild < class A < moderate < severe = class B < class C < chronic severe viral hepatitis; the change regulations of TNF-αwere mild = class A < moderate = class B < class C < severe < chronic severe viral hepatitis; the change regulations of TGF-β₁ were mild < moderate < severe < class A < chronic severe viral hepatitis < class B < class C; the change regulations of sICAM-lwere mild < class A < class B = moderate < severe < class C = chronic severe viral hepatitis; the change regulations of TIMP-lwere mild < moderate < class A < severe < class B = chronic severe viral hepatitis < class C. (3) Compared with the normal group the content of IL-2 was notable drop (P<0.05～0.01); the content of IL-4 was 86.43±19.24 pg/ml in the patients of damp-heat endogenous retention type, there is no difference between damp-heal endogenous retention type and the normal group, the content in another types were notable increase; the content of TNF-α, TGF-β₁, sICAM-1 and TIMP-1 in different types were notable increase compared with the normal group (P<0.01), the content change extent of TNF-αin damp-heat endogenous retention type was maximum, which is 433.58±24.44 pg/ml. the content of TGF-β₁, sICAM-1 and TIMP-1 increased significantly in blood stagnation endogenous retention type; the content of IFN-γin stagnation of liver qi and spleen deficiency type, damp-heat endogenous retention type and the normal group were similar, while the content in other three groups were notable lower than the normal group (P<0.01).(4) the content of IFN-γin damp-heat endogenous retention type of patients with HBV-liver Cirrhosis was 62.68±8.56 pg/ml, which was similar with the normal group, and serum cytokines in anther types were different significantly (P<0.01); the content of IL-2 in damp abundance due to splenic asthenia type, deficiency of spleen and kidney yang type and blood stagnation type were lower significantly than stagnation of liver qi type (P<0.01), and the contents of IL-4 and TNF-αwere higher then stagnation of liver qi type (P<0.01); the contents of TGF-β₁ and sICAM-1 were minimum in damp-heat endogenous retention type (P<0.05～0.01); the content of TIMP-1 in stagnation of liver qi type was 167.88±14.21 ng/ml, which was minimum (P<0.05～0.01); the contents of IFN-γin all types were lower than stagnation of liver qi type (58.37±9.75 pg/ml) except the damp-heat endogenous retention type group, which was 62.68±8.56 pg/ml (P<0.01).Conclusion: The sensitivity and repetition of the detection of serum cytokines by protein microarray were higher than ELISA method; the contents of serum cytokines (IL-2, IFN-γ, IL-4, TNF-α, sICAM-1, TGF-β₁ and TIMP-1) in chronic liver disease patients might reflect the degree of liver inflammation and fibrosis; the different chinese medicine syndrome types of Chronic hepatitis B patients and patients with HBV-liver Cirrhosis were relevant to serum cytokines, they could be considered to be new indexes to identity chinese medicine syndrome types of patients with chronic HBV infection.