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2021 Volume 43 Issue 3
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FEI Xiao-li, ZHANG Fan, ZHANG Kai-ji, et al. Expression of IL-32, Cys-C and β2-MG in Peripheral Blood of Multiple Myeloma Patients[J]. Journal of Southwest University Natural Science Edition, 2021, 43(3): 30-35. doi: 10.13718/j.cnki.xdzk.2021.03.005
Citation: FEI Xiao-li, ZHANG Fan, ZHANG Kai-ji, et al. Expression of IL-32, Cys-C and β2-MG in Peripheral Blood of Multiple Myeloma Patients[J]. Journal of Southwest University Natural Science Edition, 2021, 43(3): 30-35. doi: 10.13718/j.cnki.xdzk.2021.03.005
shu

Expression of IL-32, Cys-C and β2-MG in Peripheral Blood of Multiple Myeloma Patients

  • 1.

    Department of Hematology, Chengdu First People's Hospital, Chengdu 610016, China

  • 2.

    Clinical Laboratory, Chengdu First People's Hospital, Chengdu 610016, China

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  • Received Date: 12/05/2020
    Available Online: 20/03/2021

  • MSC: R733.3

  • ObjectiveTo understand the relationship of the changes in serum interleukin-32 (IL-32), cystatin C (Cys-C) and β2-micro-globulin(β2-MG)levels in multiple myeloma (MM) patients with the tumor load, disease stage and clinical efficacy of MM. MethodsSixty-two MM patients (MM group) and 28 healthy subjects (control group) were selected. Fasting blood was collected before and after chemotherapy in the MM group and on the day of physical examination in the control group. The levels of IL-32 were detected by ELISA, and the levels of Cys-C and β2-MG were determined by immunoturbidimetry. ResultsSerum levels of IL-32, Cys-C and β2-MG in the MM group before chemotherapy were significantly higher than those in thecontrol group (p < 0.05). There were significant differences in IL-32, Cys-C and β2-MG levels in MM patients of different ISS stages (p < 0.05): MM patients of stage Ⅲhad significantly higherIL-32, Cys-C and β2-MG levels than patients of stages Ⅰ and Ⅱ (p < 0.05). Serum levels of IL-32, Cys-Cand β2-MG in MM patients before chemotherapy were significantly higher than those after chemotherapy (p < 0.05). After chemotherapy, itseficacy was evaluated. The serum levels of IL-32, Cys-C and β2-MG in MM patients with partial remission and disease progression were all higher than those in MM patients with complete remission (p < 0.05). Spearman correlation analysis indicated thatISS stage of MM patients at initial diagnosis was positively correlated with theirserum levels of IL-32, Cys-C and β2-MG (p < 0.05), while the efficacy after chemotherapy was negatively correlated with theirserum levels of IL-32, Cys-C and β2-MG (p < 0.05). ConclusionsIL-32 may play a role in the development of MM. Changes in serum IL-32, Cys-C and β2-MG levels are correlated with tumor load, disease stage and short-term efficacy of MM patients. Detection of the above-mentioned indexes is simple, rapid and easy to operate, and is recommended for appication in clinical diagnosis and treatment.
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Expression of IL-32, Cys-C and β2-MG in Peripheral Blood of Multiple Myeloma Patients

  • Received Date: 12/05/2020
  • Available Online: 20/03/2021

Abstract: ObjectiveTo understand the relationship of the changes in serum interleukin-32 (IL-32), cystatin C (Cys-C) and β2-micro-globulin(β2-MG)levels in multiple myeloma (MM) patients with the tumor load, disease stage and clinical efficacy of MM. MethodsSixty-two MM patients (MM group) and 28 healthy subjects (control group) were selected. Fasting blood was collected before and after chemotherapy in the MM group and on the day of physical examination in the control group. The levels of IL-32 were detected by ELISA, and the levels of Cys-C and β2-MG were determined by immunoturbidimetry. ResultsSerum levels of IL-32, Cys-C and β2-MG in the MM group before chemotherapy were significantly higher than those in thecontrol group (p < 0.05). There were significant differences in IL-32, Cys-C and β2-MG levels in MM patients of different ISS stages (p < 0.05): MM patients of stage Ⅲhad significantly higherIL-32, Cys-C and β2-MG levels than patients of stages Ⅰ and Ⅱ (p < 0.05). Serum levels of IL-32, Cys-Cand β2-MG in MM patients before chemotherapy were significantly higher than those after chemotherapy (p < 0.05). After chemotherapy, itseficacy was evaluated. The serum levels of IL-32, Cys-C and β2-MG in MM patients with partial remission and disease progression were all higher than those in MM patients with complete remission (p < 0.05). Spearman correlation analysis indicated thatISS stage of MM patients at initial diagnosis was positively correlated with theirserum levels of IL-32, Cys-C and β2-MG (p < 0.05), while the efficacy after chemotherapy was negatively correlated with theirserum levels of IL-32, Cys-C and β2-MG (p < 0.05). ConclusionsIL-32 may play a role in the development of MM. Changes in serum IL-32, Cys-C and β2-MG levels are correlated with tumor load, disease stage and short-term efficacy of MM patients. Detection of the above-mentioned indexes is simple, rapid and easy to operate, and is recommended for appication in clinical diagnosis and treatment.

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  • 多发性骨髓瘤(Multiple myeloma，MM)是一种克隆性浆细胞异常增殖的恶性疾病，发病率仅次于淋巴瘤，约占恶性肿瘤的1%[1]. 目前，各种治疗方案(如免疫疗法、自体造血干细胞移植、靶向药物等)层出不穷为MM患者提供了更多的治疗选择[2-3]. 尽管如此，MM仍无法达到完全治愈，绝大部分患者仍会因疾病进展、复发，最终导致治疗失败[4]. 大量研究证实炎症微环境与肿瘤的发生发展密切相关，炎症微环境中多种免疫细胞分泌大量细胞因子、趋化因子、生长因子等可以促进肿瘤细胞生长和增殖，从而导致肿瘤性疾病进展恶化[5]. 白介素32(interleukin 32，IL-32)呈现出促炎症因子的特性，能够促进MIP-2，TNF-α，IL-6等多种炎症因子分泌增加，从而导致"级联放大效应"[6-7]. 目前，已有文献报道IL-32在多种实体肿瘤如肝癌、肺癌、结肠癌等中呈高表达，被认为是这些疾病的独立不良预后因素[8-9]，但在多发性骨髓瘤中相关研究甚少. 胱抑素-C(Cys-C)是半胱氨酸蛋白酶抑制家族成员，可直接或间接参与肿瘤生长、血管生成、肿瘤细胞转移等过程，有研究表明Cys-C与肿瘤负荷、疾病发展有关[10-11]. 生理代谢活跃的肿瘤细胞可分泌大量β2微球蛋白(β2-MG)，β2-MG能够反映MM细胞增殖活性，研究表明它与MM肿瘤负荷、疾病进展有关[12-13]. 本研究通过比较MM患者不同ISS分期，以及化疗前后血清IL-32，Cys-C，β2-MG水平变化，探讨其与MM发生、发展的关系，旨在为临床工作寻找简单易操作，并且可靠的MM肿瘤负荷、病情分期、近期疗效的评价指标.

1.   资料和方法

    1.1.   病例样本

  • 将2015年1月-2019年7月期间，成都市第一人民医院收治的初诊MM患者62例(MM组)纳入本研究. 其中男性38例、女性24例；年龄36~76岁，中位年龄59岁；IgG型34例，IgA型17例，IgD型2例，轻链型9例，所有病例诊断均符合《2017年中国多发性骨髓瘤诊治指南》的诊断标准[14]. 依据国际分期系统(ISS)进行分期[15]，Ⅰ期11例、Ⅱ期19例、Ⅲ期32例. 纳入MM组患者排除慢性阻塞性肺病、乙型病毒性肝炎、类风湿关节炎等慢性炎性疾病，化疗前或化疗过程中出现感染者. MM组62例患者接受化疗，其中43例接受BD方案化疗：硼替佐米1.3 mg/m2，第1，4，8，11 d；地塞米松20 mg/d，第1~2，4~5，8~9，11~12 d，21 d为1个疗程. 另19例MM患者接受VAD方案化疗：长春新碱0.5 mg/d，第1~4 d；阿霉素10 mg/d，第1~4 d；地塞米松40 mg/d，第1~4，9~12，17~20 d，28 d为1个疗程. 经4个疗程化疗后进行疗效评估，参考2016年国际骨髓瘤工作组(IMWG)疗效标准[16]，其中达完全缓解患者(CR)17例，非常好的部分缓解患者(VGPR)21例，部分缓解患者(PR)20例，疾病进展患者(PD)4例. 此外，选择年龄、性别相匹配的健康体检者28例(对照组)，男性15例、女性13例，年龄38~72岁，中位年龄54岁. 本研究通过成都市第一人民医院医学伦理委员会批准，受试者签署同意书.

    • 1.2.   标本采集和制备

  • MM组患者采集标本时间为化疗前和化疗4个疗程后，对照组为体检当日采集. 分别采集两管空腹静脉外周血各5 mL(肝素抗凝)，其中一管离心提取上层血清置于1.5 mL Eppendorf管中，-70 ℃保存用于IL-32水平测定. 另一管外周血置于全自动生化检测仪中(美国贝克曼库尔特公司)当日完成Cys-C，β2-MG水平测定.

    • 1.3.   检测方法

  • 采用ELISA法检测血清IL-32水平(上海酶联有限公司试剂盒)，严格按照试剂盒说明书进行操作，操作步骤为：对应板孔中加入100 μL标准品工作液或血清标本，37 ℃孵育90 min，弃掉板内液体后，立即加入100 μL生物素化抗体工作液，37 ℃孵育60 min，弃掉板内液体，洗板3次，每孔加入100 μL HRP酶结合物工作液，37 ℃孵育30 min，弃掉板内液体，洗板5次，每孔加入90 μL底物溶液，37 ℃孵育约15 min，每孔加入50 μL终止液，最后在450 nm波长下读取OD值. 使用全自动生化检测仪免疫比浊法测定血清Cys-C，β2-MG水平.

    • 1.4.   统计学处理

  • 所有数据均用SPSS 20.0软件处理. 所有数据呈正态分布，计量资料以x±s表示，两组间均数比较采用t检验，3组及3组以上组间均数比较采用one-way ANOVA，相关性分析采用Spearman进行分析. p＜0.05为差异具有统计学意义.

2.   结果

    2.1.   化疗前MM组和对照组血清IL-32，Cys-C，β2-MG水平比较

  • MM组化疗前血清IL-32，Cys-C，β2-MG水平明显高于正常对照组，差异具有统计学意义(p＜0.05)(表 1).

    • 2.2.   不同ISS分期MM组患者化疗前血清IL-32，Cys-C，β2-MG水平比较

  • Ⅲ期MM患者血清IL-32，Cys-C，β2-MG水平高于Ⅰ期和Ⅱ期患者，差异具有统计学意义(p＜0.05)，Ⅱ期患者血清IL-32，β2-MG水平高于Ⅰ期患者，差异具有统计学意义(p＜0.05)，血清Cys-C水平差异无统计学意义(p＞0.05)(表 2).

    • 2.3.   MM组患者化疗前后血清中IL-32，Cys-C，β2-MG水平变化比较

  • 化疗前MM患者血清IL-32，Cys-C，β2-MG水平显著高于化疗后，差异具有统计学意义(p＜0.05)(表 3).

    • 2.4.   不同疗效MM患者血清IL-32，Cys-C，β2-MG水平比较

  • 部分缓解、疾病进展MM患者血清IL-32，Cys-C，β2-MG水平均高于完全缓解患者(p＜0.05). 非常好的部分缓解患者血清β2-MG水平高于完全缓解患者(p＜0.05)，IL-32，Cys-C水平与完全缓解患者差异无统计学意义(p＞0.05)(表 4).

    • 2.5.   血清IL-32，Cys-C，β2-MG水平与MM患者疾病分期、化疗后疗效的相关性分析

  • 采用Spearman进行相关性分析，发现初诊MM患者ISS分期与血清中IL-32，Cys-C，β2-MG水平均呈正相关(p＜0.05)，患者疾病分期越靠后，血清中IL-32，Cys-C，β2-MG水平越高. MM患者经化疗后疗效评估，疗效与血清IL-32，Cys-C，β2-MG水平均呈负相关(p＜0.05)，患者预后越差，血清中IL-32，Cys-C，β2-MG水平越高(表 5、表 6).

3.   结语

  • 多发性骨髓瘤(Multiple myeloma，MM)是一种克隆性浆细胞异常增殖的恶性疾病，大量单克隆免疫球蛋白产生，导致终末器官损害，老年患者较多[17]. 炎症微环境与肿瘤发生、发展密切相关，在炎症微环境中的免疫细胞分泌大量细胞因子、趋化因子、生长因子，从而促进肿瘤细胞迅速生长和增殖，最终导致肿瘤进展恶化[5]. 约25%的肿瘤性疾病是由慢性炎症进展而成，在炎性微环境中炎性细胞作为重要的支持细胞保护并促进其生长，同时许多肿瘤从慢性炎症的病灶处演变而来，因此炎症和肿瘤有着紧密的联系[18]. IL-32最早在自然杀伤细胞(NK)细胞中发现，最初命名为NK-4 [19]，主要由病原体和促炎细胞因子诱导产生，Monigatti等[20]首次报道了IL-32的促炎症因子特性. IL-32呈现促炎症因子特性，能够促进MIP-2，TNF-α，IL-6，IL-1β等多种炎症因子分泌增加，并起到"级联放大效应". 目前，已有大量文献报道IL-32在多种肿瘤如肝癌[8]、肺癌[9]、慢性淋巴细胞白血病[21]、结肠癌[22]、乳腺癌[23]、骨肉瘤等中高表达，被认为是这些肿瘤性疾病的独立不良预后因素.

    本研究发现MM患者体内高表达IL-32，且经过化疗后血清中IL-32水平明显降低. 采用ELISA法检测初诊未治MM组和对照组血清IL-32水平，结果显示初诊未治MM患者化疗前血清IL-32水平明显高于正常对照组，且ISS分期与IL-32水平呈正相关，提示血清IL-32水平可以反映MM患者肿瘤负荷以及疾病的严重程度，与Wang等[24]、Zahoor等[25]的研究结果一致. 本研究还分别检测了MM组化疗前后IL-32水平变化情况，发现化疗后IL-32水平显著降低，化疗后进行疗效评估，得出化疗后完全缓解MM患者IL-32水平较部分缓解、疾病进展患者明显降低，提示血清IL-32水平可以作为临床监测评估MM患者化疗疗效的指标.

    单一指标的检测很难准确反应疾病病理生理学行为方面的复杂性，因此联合检测现已成为共识. β2-MG是由机体有核细胞合成和分泌的分子量较低的蛋白，生理代谢过程活跃的肿瘤细胞可分泌大量β2-MG，已有大量研究证实它能够反映MM细胞的增殖活性，是MM肿瘤负荷、疾病分期、预后评估公认的可靠指标[26]，也是公认的MM国际分期系统(ISS)的重要指标之一. Cys-C是半胱氨酸蛋白酶抑制家族成员，能够抑制细胞内外半胱氨酸蛋白酶，直接或间接参与肿瘤生长、血管生成、肿瘤细胞转移等过程[27-28]. 因此，本研究与IL-32同期监测了β2-MG，Cys-C血清水平变化，发现血清β2-MG，Cys-C水平变化趋势与IL-32水平变化一致，并进行了相关性分析，发现血清IL-32，Cys-C，β2-MG水平与MM患者肿瘤负荷、疾病分期呈正相关，与化疗后疗效呈负相关，提示IL-32，Cys-C和β2-MG一样，在评估MM肿瘤负荷、疾病分期和化疗疗效中有可靠的临床应用价值，这与张蕴玉等[29]、Gu等[10]的研究结果一致.

    IL-32可能在MM发生、发展中发挥作用，血清IL-32，β2-MG和Cys-C水平不仅能够作为MM肿瘤负荷、疾病分期、进展程度的评价参考指标，还能够预估患者的临床近期疗效，服务于临床工作. 上述指标检测简单、快速、易操作，易于被临床采纳应用.

Table (6) Reference (29)

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