| Background:Lymphoma is currently one of the fastest growing incidences of malignant tumors, B cell lymphoma is on the rise. Hepatitis B virus(HBV) infection is common throughout Asia and Africa. China is a high HBV infection epidemic area. Epidemiological studies performed over the last decade have demonstrated a positive association between persist-ent, hepatitis B surface antigen(HBs Ag)-positive HBV infection and B-cell non-Hodgkin lymphoma(NHL), with HBV-infected patients having a 2-3-fold higher risk to develop NHL than non-infected patients. It has been reported that patients suffering from chronic HBV infection is significantly increased risk of NHL, but the mechanism is not explained clearly. Many research shows that the HBV has infected not only liver cell but also lymphocyte. Foreign scholars put forward a multi-factor model that basing on HBV infection, integration, chronic antigen stimulation(CAS) and lymphoma causality, of which HBV plays a necessary but not sufficient etiology. In this study, we using recombinant HBs Ag antigen stimulation the human peripheral lymphoblastoid cell line IM-9, simulation CAS, to explore the relationship between chronic HBV infection and subsequent development of NHL.Methods:1. The human peripheral lymphoblastoid cell line IM-9 is a result of human B lymphocytes without tumor features and immortalized with the Epstein Barr virus, it could be used for genetic or functional researchs.2. IM-9 incubates with different concentration of HBs Ag antigen( 100ug/m L,200ug/m L,400ug/m L),the nonradioactive cell proliferation MTS assay assesses Cell viability.3. Under Annexin V staining, the fluorescence-activated cells sorting analysis was carried out after treating with HBs Ag antigen(100ug/m L,200 ug/m L,400ug/m L).The* etermine was done using ACScan flow cytometer by Annexin V-FITC Apoptosis Detection Kit.4. Western Blot analysis was performed in IM – 9 cells induced by different concentration of HBs Ag antigen(100ug/m L, 200ug/m L, 400ug/m L),key molecular of SIRT1-NF-κB signaling pathways histone H3 acetylation, the expression level of SIRT1,NF-κB,P21 and apoptosis related proteins Caspase 3, Caspase 8, Caspase 9, the Bcl,Bcl-x L and Bax were determined.5. Enzyme-linked immunosorbent assay(ELISA) to detect cytokine IL-4, IL-10,IL-12, chemokines IP-10, RANTES level.6. SIRT1 activator SRT1720(SRT), niacinamide SIRT1 inhibitors(Nicotinamide,NAM) was used to respectively complicated with 200ug/m L HBs Ag in cell culture, after incubation for 48 h cell viability and cell apoptosis were detected.Western Blot analysis of histone H3 acetylation, P21, SIRT1 and NF-κB were performed.Results:1.IM–9cells with different concentrations(100ug/m L,200ug/m L,400ug/m L)of recombinant HBs Ag antigen after incubation for24 h,48h,72 h, cell prolife-ration percentage gradually raised with the increase of HBs Ag antigen concentration and time of 24 h cell proliferation percentage(79.87±2.39,85.92±3.70,92.02±1.52),48 h cell proliferation percentage(74.26±1.34,82.15±1.02,93.47±3.25),72 h cell prolife-ration percentage(55.60±2.38,72.89±3.20,94.15±2.32), respectively, and 24 h in the control group(PBS)(72.34±1.88),48 h control(PBS)(61.23±2.41), and 72 h control group(PBS)(32.71±1.57) compared with statistically significant differences(* P < 0.05,P < 0.01). * *2.IM – 9 cells with different concentrations(100 ug/m L, 200 ug/m L, 400 ug/m L) ofVII recombinant HBs Ag antigen after incubation for 24 h, 48 h, 72 h,cell apoptosis was determined by Annexin-V/7-AAD staining and FACS analysis, which revealed anti-apoptosis activity to rely on dosage and time.24 h Annexin V+/ 7AAD+ apoptosis rate( 9.34±1.15,7.57 ±0.46,5.97 ±1.02), 48 h Annexin V+/7AAD+ apoptosis rate( 8.06±0.44,5.89 ±1.02,4.37 ±0.33), 72 h Annexin V+/ 7AAD+ apoptosis rate(7.27±0.19,3.92 ±0.86,2.17 ±1.03), respectively, and 24 h in the control group PBS)(13.27±2.60),48 h control(PBS)(12.43±3.01),72 h control(PBS)(14.51±1.11)ompared to treatment group compared with control group, the difference statistically significant(* P< 0.05, P < 0.01) * *.3. IM – 9 cells with different concentrations(100 ug/m L, 200 ug/m L, 400 ug/m L) of recombinant HBs Ag antigen after incubation for 48 h incubation for Western blot analysis,compared with the control group(PBS). The results showed that HBs Ag antigen decreases the expression of p21 and histone H3 acetylation in a dose-dependent manner.4. IM-9 cells with different concentrations(100 ug/m L, 200 ug/m L, 400 ug/m L) of recombinant HBs Ag antigen after incubation for 48 h incubation for Western blot analysis,compared with the control group(PBS). Along with the HBs Ag antigen concentrations increased, the expression of the Bcl-2,the Bcl-x L,SIRT1 and NF-κB were elevated, but the expression of Bax was decreased, caspase 9 level has not changed.5. Cytokine IL-4, IL-10, IL-12 and chemokines IP-10, RANET level are detected with a Human Thirty-Plex Antibody bead kit in IM-9 cells treated with different concentrations(100 ug/m L,200ug/m L,400ug/m L) of recombinant HBs Ag antigen. HBs Ag antigen elevated levels of IL-4, and IL-10, but reduced levels of IP-10, IL-12, and RANTES.(100ug/m L, 200 ug/m L,400ug/m L) of IL-4(25.29±1.15,34.57±0.51,43.88±1.49), respectively,and the control group(PBS)(16.34±2.19) is statistically significant(* P < 0.05, * *P <0.01).(100ug/m L,200ug/m L,400ug/m L) of IL-10( 749.06±2.31, 806.25±0.93,1010.19±1.75), respectively, and the control group(PBS)(601.24±1.52)is statistically significant(* P<0.05,* *P<0.01).(100 ug/m L,200ug/m L,400ug/m L) of IL-12(36.14±1.27,32.53 ±2.79, 23.77 ±3.10), respectively, and the control group(PBS)(47.82±1.31)is statistically significant(* P < 0.05, * *P < 0.01).(100 ug/m L, 200 ug/m L, 400 ug/m L) ofthe IP-10(705.06±2.91、629.18±1.02、478.59±2.14)respectively, and the control group(PBS)(812.59±3.57)is statistically significant(* P < 0.05, * *P < 0.01).(100 ug/m L, 200ug/m L, 400 ug/m L) of RANET( 1788.62±3.14 、 1495.23 ±1.13 、 1345.70 ±1.09)respectively, and the control group(PBS)(2312.56±2.97)is statistically significant(* P <0.05, * * P < 0.01).6. The IM – 9 cell proliferative activity induced by HBs Ag antigen could be promoted by SRT.(200 ug/m L +10 u M) HBs Ag+SRT( 94.38±2.16),respectively, and(200 ug/m L)HBs Ag(83.27±1.21)and the control group(PBS)(62.35±2.31)is statistically signify-cant(* P < 0.05, * *P < 0.01). IM-9 cell proliferation induced by HBs Ag antigen couldbe inhibited by NAM.(200 ug/m L+10 u M) HBs Ag +NAM(37.48±2.02), respectively, with(200 ug/m L)HBs Ag(83.27±1.21),the control group(PBS)(62.35±2.31)is statistically significant(* P < 0.05, * *P < 0.01). SRT promote HBs Ag induce anti-apoptotic activity,(200 ug/m L+10 u M) HBs Ag+SRT( 4.71±2.03),respectively, and(200 ug/m L) HBs Ag(6.53±1.61), the control group(PBS)(12.35±3.51) is statis-tically significant(* P <0.05, * *P < 0.01). Anti-apoptosis activity induced by HBs Ag anti-gen could be inhibited by NAM.(200 ug/m L + 10 u M) HBs Ag + NAM(24.43±1.48),respectively,and(200ug/m L) HBs Ag(6.53±1.61), the control group(PBS)(12.35±3.51) is statistically significant(* P < 0.05, * *P < 0.01). The down-regulation level of histone H3 acetylation and p21 induced by HBs Ag antigen could be emphasize by SRT and inhibited by NAM.Moreover, the up-regulation of SIRT1 and NF-κB induced by HBs Ag antigen could be emphasized by SRT and inhibited by NAM.Conclusion:1. HBs Ag antigen shows promotes proliferative activity in a dose- and time-dependent man-ner in the human peripheral blood stem cell lines from B lymphoid IM-9 cells.2. HBs Ag antigen shows anti-apoptosis activity in a dose- and time-dependent manner in IM-9 cells.3. HBs Ag antigen downregulates the expression of p21 and histone H3 acetylation ina dose-dependent manner at the molecular level.4. HBs Ag antigen up-regulated the expression of anti-apoptotic Bcl-x L and Bcl-2proteins, and inactivated the intrinsic apoptosis pathway by down-regulating Bax and increased the expression of SIRT1 and NF-κB in dose-dependent manner.5. HBs Ag antigen elevated levels of IL-10, and IL-4, but reduced levels of IP-10,IL-12, and RANTES. As did IL-4, a necessary for Th2 cell differentiation. IL-10, which is pivotal growth factor of NHL cancer cells. Cytokines IL-12 is a necessary for Th1 cells differentiation. Enhanced Th2 immune response, weakened the Thl immune response,while the latter plays an important role in antitumor immunity, which helps the immune escape of tumor cells.6. These effect induced by HBs Ag antigen was suppressed by NAM,but promoted by SRT.In conclusion,HBs Ag antigen has anti-apoptotic activity in IM-9 cell lines including some mechanisms: promotion of proliferation, inhibition of apoptosis, regulation of chemokines and cytokines, acetylation of histone H3, SIRT1 and NF-κB. Simulating chronic antigen stimulation,the consecutive stimulation of HBs Ag antigen promoted proliferation of human B lymphoblastoid cell line IM-9 through regulating SIRT1-NF-κB pathway. It may be the potential mechanism of HBV-related NHL. |
PDF Full Text Request