| Objective:These experiments are designed to find out whether anti-L-asparaginase(L-Asp) antibodies can reduce the cytotoxicity of L-asparaginase to SNK-6 cell line and its mechanism,as well, to probe the results of different concentrations of antibodies against the cytotoxicity of the drug. Compare the positive rate of the serum anti-L-Asp antibody of NKTCL patients before being trested with L-Asp with that after being treated with in order to investigate the antibody level. Try to find the differences of prognosis and clinical characteristics between the patients with and without the antibodies,which is beneficial for taking corresponding measures to improve the efficacy of L-Asp and the prognosis of this disease.Methods:1 Subject: SNK-6 cell line, a human NK/T cell lymphoma cell line(a generously gift from Prof. Zhang mingzhi in the NO. 1 hospital of Zhenzhou University) was cultured in the research center of our hospital. The antibody negative blood sample was collected from a healthy volunteer and the positive sample(containing Ig G type antibody, confirmed by ELISA method) was from a patient who recently had an allergic reaction. The inspected blood samples came from patients, who were confirmed as NK/T cell lymphoma via pathology in Hebei No. 4 Hospital among Oct. 2013 to Oct. 2014. Totally 42 serum samples were separated and stored at-80℃. 27 samples of them were from patients before treatment and 15 samples from those after treatment.2 The survival of SNK-6 cell line being dealed with L-asparaginase and its antibodies was evaluated by CCK method. SNK-6 cell line was cultivated in the RPMI1640 incubate medium containing 10 % volume fraction of human AB blood type plasma and 1000 u/ml IL-2. Adjust cell concentration into 106/ml and all experiments were done among the ogarithmic phase. Separate SNK-6 cell line into groups with and without antibodies(Antibodis were from serum sample containing Ig G type L-Asp antibodies and were diluted 100 times. L-Asp concentrations were 1000U/L, 100U/L, 10U/L, 1U/L) and groups with different concentrations of antibodies(Antibody positive serum sample was diluted 100 times, 1000 times, 10,000 times and a group without antibody was set up. L-Asp concentration was 100U/L). Assess and analyze the cell survival rate in different groups.3 Use ELISA method to measure the anti-L-Asp antibody levels in serum. Get to know the antibody levels of NKTCL patients before and after being treated with L-Asp, as well, the relationship between the antibody levels and prognosis and clinical characteristics. After preliminary experiments, antibody levels were measured. Goat anti-human Ig G(Fc specific) antibodies were used in this study. When the sample’s OD value was greater than the antibody-negative serum sample’s OD mean value + 2SD, the antibodies were positive, otherwise it was negative.Compare the positive rate of NKTCL patients before and after treatment. And find out the differences of progression free survival(PFS) and clinical characteristics between the patients with and without the antibodies.Results:1 In vitro experiment, the survival rate of SNK-6 cell line decreases with the increase of drug concentration in all groups, and it is higher in the group with the antibodes. There is statistical difference(P=0.003) between the groups with and without antibodies, when the concentration of L-Asp is 100U/L.There isn’t between other groups.2 In vitro experiment, the survival rate of SNK-6 cell line decreases with the decrease of antibody concentration. There is statistical difference between the survival rate in groups treated with antibodies diluted 100 times, as well 1000 times and that in group diluted 100000 times, as well as without antibodies,(Respectively, P were 0.03, 0.02, 0.023, 0.021).There isn’t between other groups.3 Compare the positive rate of NKTCL patients before and after treatment. The positive rates are 53.3% and 18.5%(before vs after). According to statistical(P=0.035) and clinical analysis, we conclude that patients will develop Ig G type anti-L-Asp antibodies when they are treated with L-Asp. And 33.3% of patients will develop antibodies.4 The relationship between the anti-L-asparaginase antibody level and the prognosis of NKTCL patients: The progression free survival(PFS) of antibody-positive group is 72.7%, and the PFS of antibody-negative group is 85.7%. There is no statistical difference(P=0.556) between two groups.5 The relationship between the anti-L-asparaginase antibody level and the clinical characteristics of NKTCL patients: Nasal bleeding at the beginning and the abnormity of FIB connect with the antibody level. Although the proportion of clinical signs vary greatly between the two groups, but there is no statistical significant. Multivariate analysis shows that no characteristic is related to the development of antibody.Conclusions:1 The vitro experiment show that anti-L-asparaginase antibody can reduce the cytotoxicity of L-asparaginase to SNK-6 cell line. The effect will be notable when the concentration of antibody and L-Asp adjusts to each other.2 The vitro experiment confirms that the effect of antibodies on L-asparaginase is influenced by their concentrations. And neutralization is one of the mechanisms.3 The NKTCL patients will produce Ig G type anti-L-Asp antibodies after they are treated with L-Asp.4 We can’t conclude that there is a statistical differenc in PFS between the antibody-positive group and the negative group, but the tendency still exists.5 At present, the relationship between the anti-L-asparaginase antibody level and the clinical characteristics is still unclear. From this study, we know that nasal bleeding and abnormity of FIB connect with the antibody levels. For some characteristics, there is no statistical differences between two groups, but adverse factors proportion increase in antibody-postive group. Multivariate analysis shows that no characteristic is related to the development of antibody. |
PDF Full Text Request