【作者】 赵艳荣；

【导师】 高玉环；

【作者基本信息】 河北医科大学 ， 内科学（专业学位）， 2015， 硕士

【摘要】 目的:通过体外细胞实验明确左旋门冬酰胺酶(L-Asp)抗体是否能够降低L-Asp对NK/T细胞淋巴瘤细胞株SNK-6细胞的杀伤作用及可能机制,并探讨不同浓度抗体对药物抗肿瘤活性的影响。比较NK/T细胞淋巴瘤(NKTCL)患者应用含左旋门冬酰胺酶方案治疗前后血清中L-Asp抗体的阳性率,了解治疗前后患者抗体水平是否发生变化;分析抗体阳性组与阴性组疾病预后及临床特征的差异。最终采取有效措施提高药物疗效,改善疾病预后。方法:1研究对象:NK/T细胞淋巴瘤细胞株SNK-6细胞(郑州大学第一附属医院赠送),于我院科研中心培养。抗体阴性血液标本来自于未使用过L-Asp的自愿者;抗体阳性标本来自于应用L-Asp过程中发生过敏的患者(通过ELISA法证实二者血液中Ig G型L-Asp抗体分别为阴性和阳性)。待测血液来自于2013年10月至2014年10月就诊于河北医科大学第四医院血液内科,病理诊断为NK/T细胞淋巴瘤的患者。共收集血液标本42份(治疗前27份,治疗后15份)。均分离血清,-80℃保存备用。2 CCK法测定左旋门冬酰胺酶及其抗体作用于SNK-6细胞后的细胞生存率。SNK-6细胞培养于含体积分数为10%人AB型血浆、1000U/ml重组人白介素-2的RPMI1640培养液中。取对数生长期细胞配成浓度为106/ml的细胞悬液备用。将细胞分为加抗体组与未加抗体组(抗体为稀释100倍的含Ig G型L-Asp抗体的血清;L-Asp浓度设为1000U/L、100U/L、10U/L、1U/L)及不同浓度抗体组(抗体阳性血清分别稀释100倍、1000倍、10000倍及未加抗体;L-Asp浓度为100U/L),比较各组间SNK-6细胞的生存率,并对其进行统计学分析。3 ELISA法测定NKTCL患者血清中L-Asp抗体水平,并了解应用L-Asp治疗前后患者血清中特异性抗体水平的变化及抗体水平与疾病预后和临床特征的关系。本实验二抗采用羊抗人Ig G(Fc段)抗体,通过预实验得到实验条件后测定抗体水平。当样本OD值(复孔OD值均数)大于抗体阴性血清标本OD值均数+2SD时,抗体为阳性,否则为阴性。比较应用L-Asp治疗前(27份)后(15份)血清中抗体阳性率。另外收集患者相关临床资料,分析抗体阳性组(11例)与阴性组(7例)的无进展(复发)生存率(PFS)及临床体征的差异。结果:1 SNK-6细胞体外实验中随着药物浓度的增加加抗体组与未加抗体组细胞存活率均下降,且未加抗体组细胞生存率低于加抗体组。当药物浓度为100U/L时加抗体组与未加抗体组间细胞生存率差异具有统计学意义(P=0.003)。其他浓度时两组间细胞生存率差异没有统计学意义。2体外细胞实验中,随着抗体浓度降低细胞生存率降低。统计学分析显示抗体稀释100倍与稀释10000倍、未加抗体组间及抗体稀释1000倍与稀释10000倍、未加抗体组间细胞生存率差异存在统计学意义(P值分别为0.03、0.02、0.023、0.021),其他组别间细胞生存率差异没有统计学意义。3比较NK/T细胞淋巴瘤患者应用含左旋门冬酰胺酶方案治疗前后血清中左旋门冬酰胺酶抗体的阳性率。治疗前抗体阳性率为18.5%,治疗后抗体阳性率为53.3%。经统计学(P=0.035)及临床分析,接触L-Asp后患者可产生针对左旋门冬酰胺酶的Ig G型特异性抗体。抗体阳转率为33.3%。4对患者血清中抗体水平与NKTCL预后的关系进行研究。抗体阳性组PFS为72.7%;抗体阴性组PFS为85.7%。统计学分析两组PFS差异无统计学意义(P=0.556)。5血清中左旋门冬酰胺酶抗体水平与NK/T细胞淋巴瘤患者临床特征关系的研究表明抗体产生与发病时鼻腔出血与否及纤维蛋白原(FIB)是否异常有关。其他部分临床特征两组间比例差别较大,但无统计学意义。多因素分析未发现与抗体产生有关的特征。结论:1体外细胞实验证实左旋门冬酰胺酶抗体能够降低L-Asp对SNK-6细胞的杀伤作用,且当二者浓度匹配时抗体可显著降低药物疗效。2体外细胞实验中抗体对左旋门冬酰胺酶疗效的影响存在剂量关系。抗体的中和作用是其降低药物疗效的机制之一。3 NK/T细胞淋巴瘤患者应用含L-Asp的方案治疗后可产生特异性Ig G型抗体。4目前尚不能得出NKTCL患者血清中左旋门冬酰胺酶抗体阳性组PFS低于抗体阴性组。但现有数据提示抗体阳性组PFS有低于阴性组的趋势。5血清中左旋门冬酰胺酶抗体水平与患者临床特征之间的关系目前尚不明确。本实验提示抗体水平与发病时鼻腔出血与否及纤维蛋白原是否异常有关。部分临床特征差异无统计学意义,但抗体阳性组较抗体阴性不良因素比例增高。多因素分析未发现与抗体产生有关的特征。更多还原

【Abstract】 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.更多还原