Based On PD-1/PD-L1 Signal Pathway Research The Mechanism Of Action Of The Kidney Detoxification Method In The Treatment Of CHB

ObjectiveThrough systematically study the main steps of HBV infection affect the immune tolerance:PD-1/PD-L1 signal pathway, to study the effect of different stages of HBV immune tolerance and kidney detoxification method of targets and mechanisms of the effect of anti HBV may, illustrate the kidney immune activation mechanism of detoxification method in the treatment of CHB, and HBV persistent infection immune escape and fu qi theory of TCM "kidney evil v liver blood" the relationship between the pathogenesis, reveals the essence of chronic hepatitis b v gas pathogenesis, for kidney detoxification method to provide comprehensive scientific basis in the treatment of CHBMethods1.39 patients included in the standard of chronic hepatitis b patients were randomly divided into Chinese and western medicine therapy and western medicine treatment of two groups.20 cases of Chinese and western medicine treatment group, western medicine treatment group (19 cases). Another 10 cases of healthy volunteers as normal group. Chinese and western medicine treatment group is taking kidney detoxification and the formulas of nucleotides analogues (lamivudine) for treatment, western medicine group with oral nucleotide analogues (lamivudine) is a treatment plan. Respectively before and 6 months after treatment to observe peripheral blood CTL (CD8+T) surface molecular PD 1, the expression of PD-L1.2. By flow cytometry detection technique health blood donors and patients with CHB peripheral blood CTL (CD8+T) surface molecular PD 1, the expression of PD-L1.3. of the tumor cells by CCK8 method (CEMX174) cytotoxicity test, determine a kidney detoxification agent for the highest non-toxic concentration.4. CHB patients and healthy volunteers collected 20 ml peripheral venous blood, separation of PBMC, and then at 6 holes in the cell culture plate to R10 medium, rhGM-CSF, rhIL-4, rhTNF alpha and beta IL - 1 stimulation induced PBMC be DC, mature, to be detected in kidney detoxification method of mixed culture liquid form the highest non-toxic concentration 48 h. By flow cytometry instrument respectively to detect the DC cell surface molecules CD80, CD86, PD-L1 and PD-changes in the L2.5. CHB patients and hel.39 patients included in the standard of chronic hepatitis b patients were randomly divided into Chinese and western medicine therapy and western medicine treatment of two groups.20 cases of Chinese and western medicine treatment group, western medicine treatment group (19 cases). Another 10 cases of healthy volunteers as normal group. Chinese and western medicine treatment group is taking kidney detoxification and the formulas of nucleotides analogues (lamivudine) for treatment, western medicine group with oral nucleotide analogues (lamivudine) is a treatment plan. Respectively before and 6 months after treatment to observe peripheral blood CTL (CD8+ T) surface molecular PD 1, the expression of PD-L1.2. By flow cytometry detection technique health blood donors and patients with CHB peripheral blood CTL (CD8+T) surface molecular PD 1, the expression of PD-L1.(3) of the tumor cells by CCK8 method (CEMX174) cytotoxicity test, determine a kidney detoxification agent for the highest non-toxic concentration. 4. CHB patients and healthy volunteers collected 20 ml peripheral venous blood, separation of PBMC, and then at 6 holes in the cell culture plate to R10 medium, rhGM-CSF, rhIL-4, rhTNF alpha and beta IL-1 stimulation induced PBMC be DC, mature, to be detected in kidney detoxification method of mixed culture liquid form the highest non-toxic concentration 48 h. By flow cytometry instrument respectively to detect the DC cell surface molecules CD80, CD86, PD-L1 and PD-changes in the L2.5. CHB patients and healthy volunteers collected 10 ml peripheral venous blood, separation of PBMC, and then by the method of magnetic bead separation of CD8 +T cells and CD8 T cells, and then in the 24 hole cell culture plate to the R20 medium, by PHA, IL-2 training amplification, join the kidney detoxification method of mixed culture liquid form the highest non-toxic concentration 48 h. By flow cytometry instrument CD8+T cells was detected in surface molecular CD28, CTLA4, PD, PD-1-L1 and PD-changes in the L2. althy volunteers collected 10 ml peripheral venous blood, separation of PBMC, and then by the method of magnetic bead separation of CD8+T cells and CD8 T cells, and then in the 24 hole cell culture plate to the R20 medium, by PHA, IL 2 training amplification, join the kidney detoxification method of mixed culture liquid form the highest non-toxic concentration 48 h. By flow cytometry instrument CD8+T cells was detected in surface molecular CD28, CTLA4, PD, PD-1-L1 and PD-changes in the L2.Results1. PBMC Peripheral blood cells of PD-1 and PD-the expression of L1 in the treatment of chronic hepatitis b patients before compared with normal people, patients with chronic hepatitis b in the T express of PD on CD8+T-1 is lower than normal, the rest in T cells to CD8-, CDS+T cells, T CD8-are higher than normal, but these differences have no statistically significant (P> 0.05). CHB patients with PD-LI are higher than normal, but only in the CD8+T cells, T CD8+is higher than normal person have statistically significant (P< 0.05.24 weeks after treatment, Chinese and western medicine (kidney detoxification method+nucleotide analogue) group and western medicine (nucleotide analogue) he would heal rise, PD-1 set of CHB patients before and the rise and heal in contrast there are statistically significant (P< 0.05; Western medicine treatment of CHB patients after 24 weeks of PD-1 rise less than 24 weeks after the Chinese and western medicine treatment of CHB patients with PD-1 rise and have statistical significance. 24 weeks after treatment, Chinese and western medicine (kidney detoxification method+nucleotide analogue) treatment group and western medicine (nucleotide analogue) treating CHB patients with PD-L1, compared to heal before by statistical calculation has significance (P< 0.05).2. Use CEMX174 tumor cells to drug toxicity experiment method of CCK 8 and inhibition of the 10%, the results showed that kidney detoxification highest non-toxic concentration of the formulas is 1:20 is 0.125 mg/ml.3. In vitro induced to develop healthy people and patients with CHB peripheral blood mononuclear source DC, herb tea and kidney drug money supreme non-toxic concentration of mixed culture:under inverted microscope to see before the cell body is bigger, a lot of things like hair on the outside of the cell body, the development is in the form of DC half suspension form of cell growth, no matter from shape change or growth state is in line with the typical dendritic cells morphological characteristics. Mature DC after dosing mixed culture, CD80, CD86, PD-L1 and PD-L2 are raised, but only CD86 and PD-a rise in L2 has statistically significant (P< 0.05. CHB patients and healthy people mature DC after dosing mixed culture, CD80, CD86, PD-L1 and PD-L2 are raised, but only CD80, CD86 and PD-L2 increase was statistically significant (P< 0.05. PD-there is no statistically significant rise in L1.4. Induced in vitro cultivation of healthy people and patients with CHB peripheral blood CD8+T and CD8 T cells, and CD8+T cells with kidney detoxification liquid form the highest non-toxic concentration after mixed culture, current detection CD28, CD152 rise, PD-1, PD-L1 and PD-L2 cut, but only the CD152 raised is statistically significant, more have no statistical significance. CHB patients and normal CD8+T cells and kidney detoxification liquid form the highest non-toxic concentration after mixed culture, current detection CD28, CD152 rise, PD-1, PD-L1 and PD-L2 cut, but there is no statistical significance.Conclusion1. The body after infected with hepatitis b virus (HBV), flow detection of peripheral blood lymphocytes, PD-1, the PD-L1 expression is higher than normal. PD-1 the expression of closely related and the differentiation of CTL, PD-1 a up, top, down to the natural development process of rebirth. Experimental results showed that the treatment of 6 months after rise in PD1 May be related to this reason. Rise under Chinese and western medicine group and western medicine have experimental research shows that antiviral drugs can cut PD-1 expression in the short term, this could explain the results. After 6 months after treatment, PD-L1 is cut. But comparing Chinese and western medicine and western medicine treatment group was not statistically significant, so it is difficult to determine the kidney detoxification method in change PD-because western medicine treatment of L1 expression quantity to achieve.2. Induction training DC and CD8+T cells in vitro, herb tea and kidney drug money again the highest non-toxic concentration mixed culture, make DC cell phenotype CD80, CD86 and PD-L2 rise, explain kidney detoxification method in the aspect of immune cell can seek to its therapeutic effect. But CD8+T cells in detection of CD28, CD152 and PD, PD-1-L1 and PD-L2 basically do not have statistical significance.With "kidney drug money" for the concept of traditional Chinese medicine combination of effective prescriptions treating patients with CHB is through a lot of curative effect observation and experimental study confirmed that from molecular immunology aspect also shows that it has the role of immune regulation, the regulation of many cytokines have impact. Programmed according to our entire death receptor 1 PD-and its ligand L1 combined inhibition of channel of the analysis of experimental results, can speculate that kidney detoxification may of peripheral blood in patients with CHB PD-1/PD-L1 expression cut and blocking PD-1/PD-L1 inhibition pathway has certain effect, so as to achieve the purpose of the treatment of CHB.