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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。
Objective To observe the toxic effects of defensins on gastric cancer cell lines and gastric cancer drug-resistant cell lines, and to explore its toxic mechanism. METHODS: Defensin (HNP) was prepared by liquid chromatography and identified: MTT assay was used to detect the killing activity of HNP on cells SGC-7901 and SGC-7901/VCR. Flow cytometry was used. Cell cycle analysis was performed using cell culture. Chemiluminescence was used to detect malondialdehyde (MDA) and nitric oxide (NO) free radicals. Results The purified HNP M_1 3500 had high bactericidal activity. HNP had significant killing activity on SGC-7901 cells in dose- and time-dependent manner. The killing rate was 97% at 100 mg·L~1·10 h; HVP was compared. The toxicities of SGC-7901 and SGC-7901/VCR were 84.45% in SGC-7901 and 86.75 in SGC-7901/VCR (P<0.01) at 80 mg·L-1 and 8 h. The proportion of S phase cells in 7901 cells decreased, the experimental group (22.2 ± 1.3)%, control group (19.1 ± 0.6)%, P <0.05, HNP induced SGC-7901 cells to produce MDA and NO free radicals, when the HNP dose was 10 ~ When 80 mg·L~1, the detection amount of MDA was 3.21~8.14 (10 ~(19)mol·cell~1); the detection amount of NO was 1.73~7.99 (10 ~(15)mol·cell~1). Conclusion Defensins have a significant killing effect on gastric cancer cell lines and gastric cancer drug-resistant cell lines, especially on gastric cancer drug-resistant cell lines, which may be related to a series of biological changes in drug-resistant cells. In addition to its possible mechanism of membrane damage, the cytotoxic effect of sulphine may also be more sensitive to cells in the DNA synthesis phase, or it may have a delayed or inhibitory effect on the S phase of cells, in addition to reactive oxygen species and NO. Free radicals Gastric cancer cell-mediated injury HNP may play a role.