防御素对胃癌细胞系体外的杀伤作用

Anti-tumor activity of defensin on gastric cancer cell line in vitro

目的观察防御素对胃癌细胞系及胃癌耐药细胞系的毒性作用,探讨其毒性作用机制。方法采用液相色谱法制备防御素,即人中性粒细胞多肽(HNP)并鉴定:采用噻唑蓝(MTT)法检测HNP对细胞SGC-7901及SGC-7901/VCR的杀伤活性;应用流式细胞术进行细胞周期分析;采用化学发光技术检测脂质过氧化产物丙二醛(MDA)及一氧化氮(NO)自由基。结果纯化HNP的M_1 3500,具有较高的杀菌活性,HNP对SGC-7901细胞具有显著的杀伤活性,呈剂量及时间依赖性,在100mg·L^1·10h,杀伤率达97％;比较HVP对SGC-7901和SGC-7901/VCR的毒性作用,在80mg·L^1,8h,其杀伤率SGC-7901为84.45％,SGC-7901/VCR为86.75(P<0.01),HNP使SGC-7901细胞S期比例有所减少,实验组(22.2±1.3)％,对照组(19.1±0.6)％,P<0.05,HNP诱导SGC-7901细胞产生MDA及NO自由基,当HNP剂量为10～80mg·L^1时,MDA的检出量为3.21～8.14(10 ^(19)mol·cell^1);NO的检出量为1.73～7.99(10 ^(15)mol·cell^1) 结论防御素对胃癌细胞系和胃癌耐药细胞系具有显著的杀伤作用,特别是对胃癌耐药细胞系的毒性作用更强,这可能与耐药细胞所产生的一系列生物学改变有关,防御素的细胞毒性作用除了其可能的膜损伤作用机制外,还可能为处于DNA合成期的细胞对其更敏感,或是其对细胞S期过程具有延迟或阻抑作用,此外,活性氧及NO等自由基在HNP介导的胃癌细胞损伤过程中可能发挥一定作用。

AIM To testify and observe the cytolytic activity of small peptide molecule termed as defensins generated from polymorphonuclear neutrophils (PMN) on gastric cancer cell line and/or the resistant cell line. METHODS The peripheral blood was collected from healthy blood donors. The PMNs were then homogenized and extracted by centrifugation. The defensin was finally prepared from liquid chromatography. The tetrazolium blue colorimetric assay (MTT) was introduced to evaluate the cytotoxicity of defensin on gastric cancer cell line SGC-7901 and the resistant partner cell line SGC-7901/VCR. The flow cytometry was employed to analyse the cell cycle. The oxygen and reactive nitrogen relevant with lipid peroxidative MDA and NO derived from the cultured gastric cancer cell line exposure to defensin treatment was performed via fluorometric assay. RESULTS The purified defensins of M_r 3500 had good antimicrobiol activity. The anti-tumor experiment of defensins showed that defensins exerted a strong antitumor activity on the cell lines in a concentration and time dependent manner. Flow cytometrical analysis revealed that the proportion of S-phase was decreased among the cells treated. The production of MDA and NO was much higher than that of control group and a lineal dose-dependent correlation was seen. CONCLUSION Our findings laid a new ground that the defensin has confirmed antitumor activity except for these known antimicrobial effect, The data available in this report might extend the conceptual recognization when defensin is concerned. The bio-function of the defensin on the tumors still needs to be explored further.