| Objective: To investigate the effect of hepatitis B virus (HBV) antigen on normal human lymphocyte apoptosis and the function of thymic peptideα1(Tα1) as an inhibitor of lymphocyte apoptosis induced by HBV antigen.Mothods:1 Isolating the lymphocytes: The venous blood from healthy donors, anti- coagulated by heparin and diluted to its half concentration by RMPI 1640 culture medium, was added slowly to the upper layer of lymphoctye separation medium and then centrifuged to isolate the lymphocytes which were then cleaned and diluted with 1640 culture medium to make a cell suspension of 2×106 cells per milliliter.2 The experiment of lymphocyte apoptosis induced by HBV positive serum (HBV(+)S): The lymphocytes were divided into four groups, each of which included six parallel wells:①FCS control group: 0.9ml cell suspension +0.1ml FCS;②Normal human serum (NHS) control group: 0.9ml cell suspension +0.1ml NHS;③HBV(+)S group: 0.9ml cell suspension +0.1ml HBV(+)S(induced by HBV DNA of different concentration: 2.33×107copy/ml, 3.47×108copy/ml and 4.70×109 copy/ml);④Dexamethasone (DEX) positive control group: 0.9ml cell suspension +0.1ml FCS + DEX(final concentration: 1×10-5mol/L). The cell suspensions mentioned above were added into the 12-well plates and cultured in incubator at 37℃,5% CO2, saturated humidity. The cultured lymphocytes were collected at different time points. The morphological feature of lymphocytes was observed with Wright staining and TdT-mediated dUTP-biotin nick end labeling(TUNEL), and their hypodiploid peaks were detected with flow cytometry(FCM) to calculate apoptosis ratio.At the same time, the fresh lymphocyte control group, which wasn't cultured, was observed with above methods.3 The experiment of effect of Tα1 on lymphocyte apoptosis: The experimental groups were the same as the above. The Tα1 of different concentration (final concentration: 0μg/ml, 10μg/ml, 50μg/ml and 100μg/ml) was added into every group. The cultured lymphocytes were collected after 72 hours of inoculation to detect their apoptosis ratio with FCM.Results:1 The characteristics of apoptotic lymphocyte: The morphological feature of apoptotic lymphocytes was observed by light microscope. The nuclear chromatin condensed, gathered to nuclear membrane and formed apoptotic bodies under Wright staining; the nucleus was stained in dark brown with TUNEL; the special hypodiploid peaks were seen by FCM.2 The results of lymphocyte apoptosis induced by HBV antigen: The apoptosis ratio of HBV(+)S group was obviously higher than that of the NHS control group and FCS control group (P<0.01), and rose with the increase of the concentration of HBV DNA. The difference of apoptosis ratio among low dose group(2.33×107 copy/ml) , middle dose group(3.47×108 copy/ml ) and high dose group(4.70×109 copy/ml) had significance (P<0.01). The lymphocyte apoptosis could hardly be detected in uncultued group, but increased with the culture time. The apoptosis ratio of different culture time(24h,48h,72h) was different between every two groups (P<0.01). The apoptosis ratio of the lymphocytes was positively correlated with the dose of HBV DNA(r=0.813, P<0.01) and the cultured time(r=0.846, P<0.01).3 The results of Tα1 inhibiting the lymphocyte apoptosis induced by HBV antigen: Tα1 of middle and high dose (50μg/ml, 100μg/ml) respectively was added into the FCS control group, NHS group and HBV(+)S group, and the lymphocyte apoptosis of those groups was obviously inhibited compared with the homologous groups without Tα1 (P<0.01). Only the HBV(+)S group with Tα1 of low dose(10μg/ml) showed lymphocyte apoptosis inhibition in contrast to the group without Tα1(P<0.01), but the FCS control group and NHS group with Tα1 of low dose did not (P>0.05). There was significant difference of the inhibition among those groups of different doses of Tα1 , and the inhibition was dependent upon the dose of Tα1. The lymphocyte apoptosis ratio was negatively correlated with the dose of Tα1(r=-0.683, P<0.01).Conclusions:1 The methods to observe lymphocyte apoptosis were successfully established including Wright staining, TUNEL and FCM (PI staining).2 HBV antigen's effect on the apoptosis of normal lymphocytes in vitro was proved systematically with various methods detecting apoptosis. The effect of HBV antigen on normal human lymphocyte apoptosis in vitro, which depended on the culture time and the dose of HBV DNA, was proved through many indexes such as morphology and apoptosis peak.3 It was observed and confirmed by FCM that not only the apoptosis of normal lymphocyte but also the lymphocyte apoptosis induced by HBV was inhibited by Tα1 which had a greater inhibiting effect upon the lymphocyte apoptosis induced by HBV than upon the apoptosis of normal lymphocytes. |
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