Lamprey serum can kill HeLa and NB4 tumor cells.
Lamprey serum can kill HeLa and NB4 tumor cells.
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DOI：
10.1093/abbs/gmu039
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发表时间：
2014-07
影响因子：
3.7
通讯作者：
Yinglun Han;Y. Pang;Tao Yu;R. Xiao;Biyue Shi;Peng Su;Xin Liu;Qingwei Li
中科院分区：
文献类型：
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作者：
Yinglun Han;Y. Pang;Tao Yu;R. Xiao;Biyue Shi;Peng Su;Xin Liu;Qingwei Li
自然界主要由生物组成，其中许多是病原微生物，能够杀死其他生物并将其转化为自身的复制品[1，2]。为了自卫，真核生物逐渐进化出免疫系统，包括先天和适应性免疫系统[3-7]。天然免疫已在植物、真菌和后生动物中发现;而有颌脊椎动物除了天然免疫防御外，还具有进化的适应性免疫系统[8]。以七鳃鳗[9]和盲鳗为代表的无颌类动物是最古老的具有适应性免疫防御的脊椎动物，尽管TCR和BCR是否存在于无颌脊椎动物中仍然未知。这种适应性免疫系统经历不同LRR插入的生殖系基因组重排以产生各种淋巴受体（VLR）。在七鳃鳗中已经鉴定出三种类型的VLRs，VLRA、VLRB和VLRC。VLRA和VLRB分别在类似于有颌脊椎动物的T细胞和B细胞的淋巴细胞中表达。在被特定病原体感染后，B细胞样淋巴细胞增加VLRB的表达，并且VLRB以类似于B细胞分泌免疫球蛋白的方式分泌[10，11]。在这项研究中，我们报告七鳃鳗血清可以在体外杀死HeLa和NB 4细胞。七鳃鳗的细胞杀伤现象不同于传统的有颌脊椎动物的细胞毒效应，这一结果可能有助于人类肿瘤性疾病的早期诊断和治疗研究。从黑龙江同江段（中国同江）采集成年七鳃鳗，并在208 ℃下饲养在砂衬水族箱中。HeLa和NB 4细胞由辽宁师范大学生命科学学院（中国大连）提供。HeLa细胞维持在Dulbecco改良的Eagle培养基（Sigma-Aldrich，圣刘易斯，美国）中，NB 4细胞维持在RPMI 1640培养基（Sigma-Aldrich）中。两种培养基均补充有10%胎牛血清（Sigma-Aldrich）、100 U/ml青霉素（Sigma-Aldrich）和100 mg/ml链霉素（Sigma-Aldrich）。将细胞在378 ℃下在用5%CO2和95%空气加湿的培养箱中培养。将正常成年七鳃鳗（体重200- 220 g）的尾部切断，将血液抽入10 ml塑料离心管中，并使其在48 ℃下凝结过夜。通过在48 ℃离心（1680 g）10 min分离血清，并在220 ℃储存在1.5 ml离心管中。使用前，将5 ml七鳃鳗血清用磷酸盐缓冲盐水（PBS，三次更换，总共2 l）透析（分子量截止，10 kDa）24小时，并调节至最终蛋白质浓度为20 mg/ml。使用碘化丙锭（PI; Sigma-Aldrich）染色进行细胞死亡分析，随后进行荧光激活细胞分选（FACS）分析。将HeLa和NB 4细胞（5x 106）与20 mg/ml七鳃鳗血清透析液在室温下孵育15分钟，并将PBS用作阴性对照。之后，将细胞培养物以150 g离心5 min，收集细胞，洗涤并重悬于1 ml冷PBS中。将细胞与100 ml PI在室温下进一步孵育15 min，然后加入400 ml 1x膜联蛋白结合缓冲液用于流式细胞术（FACSAria II; BD Corporation，纽约，美国）分析。结果显示，将七鳃鳗血清与HeLa和NB 4细胞孵育15分钟导致外膜破裂并导致水泡形成（图1A）。此时，通过监测膜完整性来评估膜质量。HeLa细胞的死亡率更高（98...
Nature consists primarily of living materials, many of which are pathogenic microorganisms capable of killing and converting other organisms into copies of themselves [1, 2]. For self-defense, eukaryotes have gradually evolved immune systems, including the innate and adaptive immune systems [3–7]. Innate immunity has been found in plants, fungi, and metazoans; while jawed vertebrates have an evolved adaptive immune system in addition to the innate immunity defense [8]. Agnathans, represented by lamprey [9] and hagfish, are the oldest vertebrates that possess the adaptive immune defenses, although whether TCR and BCR exist in jawless vertebrates remains unknown. This adaptive immune system undergoes germline genomic rearrangements of insertion of diverse LRRs to generate various lymphatic receptors (VLRs). Three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. VLRA and VLRB are expressed in lymphocytes that resemble T cells and B cells of jawed vertebrates, respectively. After being infected by a specific pathogen, B-cell-like lymphocytes increased the expression of VLRB, and VLRB was secreted in a manner analogous to the secretion of immunoglobulins by B cells [10, 11]. In this study, we report that lamprey serum can kill HeLa and NB4 cells in vitro. The phenomenon of cell killing in lamprey is different from the traditional cytotoxic effect in jawed vertebrates, and the results might be helpful for the research on the early diagnosis and therapy of neoplastic diseases of human beings. Adult lampreys were collected from the Tongjiang Section of the Heilongjiang River (Tongjiang, China) and housed in sand-lined aquariums at 208C. HeLa and NB4 cells were supplied by College of Life Science, Liaoning Normal University (Dalian, China). HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St Louis, USA) and NB4 cells were maintained in RPMI 1640 medium (Sigma-Aldrich). Both media were supplemented with 10% fetus bovine serum (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), and 100 mg/ml streptomycin (Sigma-Aldrich). Cells were cultured in an incubator humidified with 5% CO2 and 95% air at 378C. Normal adult lamprey (200–220g in weight) was tailsevered, and blood was drawn into a 10-ml plastic centrifuge tube and allowed to clot at 48C overnight. Serum was separated by centrifugation (1680 g) for 10 min at 48C and was stored in a 1.5-ml centrifuge tube at 2208C. Before use, 5ml of lamprey serum was dialyzed (molecular weight cut-off, 10 kDa) against phosphate buffered saline (PBS, three changes, a total of 2 l) for 24 h and adjusted to a final protein concentration of 20 mg/ml. Cell death analyses were performed using propidiumiodide (PI; Sigma-Aldrich) staining with subsequent fluorescence-activated cell sorting (FACS) analysis. HeLa and NB4 cells (5 x 106) were incubated with 20mg/ml lamprey serum dialyzate for 15 min at room temperature, and PBS was used as a negative control. After that, the cell cultures were centrifuged at 150 g for 5 min, and the cells were collected, washed, and resuspended in 1 ml of cold PBS. The cells were further incubated with 100 ml of PI for 15 min at room temperature, and then 400 ml of 1x annexinbinding buffer was added for flow cytometry (FACSAria II; BD Corporation, New York, USA) analysis. The results showed that incubation of lamprey serum with HeLa and NB4 cells for 15 min caused outer-membrane disruption and led to the formation of blebs (Fig. 1 A). At this time, the membrane quality was assessed by monitoring the membrane integrity. There was a higher percentage of cell death for HeLa (98 …