黑水虻幼虫抗菌肽粗提物敏感肿瘤细胞株筛选及溶血作用研究

Selection of Tumor Cells Sensitive to Crude Extracts of Antibacterial Peptides from H. illucens Larvae and Study on Related Hemolysis

目的筛选对黑水虻幼虫抗菌肽粗提物敏感的肿瘤细胞株及其溶血作用。方法采用断头取血法收集幼虫血淋巴细胞制作黑水虻幼虫抗菌肽粗提物。体外培养结肠癌细胞株(HCT-8)、肺腺癌细胞株(A549)、宫颈癌细胞株(HeLa)和卵巢癌细胞株(SKOV3) 4株癌细胞,绘制生长曲线。采用CCK-8法检测不同浓度的黑水虻幼虫抗菌肽粗提物对4株癌细胞的增殖抑制率,初步筛选出对黑水虻幼虫抗菌肽粗提物的敏感癌细胞株,并利用CCK-8法研究黑水虻幼虫抗菌肽粗提物对正常人胚肾细胞株(HEK293)的毒性及黑水虻幼虫抗菌肽粗提物对红细胞的溶血作用。结果细胞均在体外培养24 h后进入对数生长期,选取培养24 h的细胞进行传代及后续实验。不同浓度的黑水虻抗菌肽粗提物对HCT-8、A549、SKOV3、HeLa 4种细胞均有一定的抑制作用(P <0.05),且随着浓度的增加,抑制率逐渐升高,其中400 mg/L浓度下的黑水虻抗菌肽粗提物对细胞的抑制率最大。在400 mg/L浓度下,黑水虻抗菌肽粗提物对HCT-8、A549、SKOV3、HeLa以及HEK293 5种细胞的抑制率不同(P <0.05),其中对HCT-8的抑制率最高,对HEK293的抑制率最差;黑水虻抗菌肽粗提物浓度为400 mg/L时对HCT-8细胞的抑制率高于5-氟尿嘧啶(P <0.05);抗菌肽粗提物的浓度分别为31.25、62.5、125、250、500 mg/L时的溶血率分别为(0.16±0.11)%、(0.20±0.12)%、(0.95±0.04)%、(1.09±0.16)%、(1.60±0.18)%,溶血率均低于5%。结论黑水虻幼虫抗菌肽粗提物对HCT-8细胞较为敏感,且对正常细胞无毒、对红细胞不溶血,预示在结肠癌药物的研发中具有良好的前景。

Objective To screen the tumor cell lines sensitive to crude extracts of antibacterial peptides of the H.illucens larvae,and study the related hemolysis.Methods The blood lymphocytes were collected by decapitation to prepare the crude extracts.Four cancer cell lines,including colon cancer cell line(HCT-8),lung adenocarcinoma cell line(A549),cervical cancer cell line(HeLa) and ovarian cancer cell line(SKOV3),were cultured in vitro,and the growth curves were drawn.The proliferation inhibition rate of the crude extracts against the four cancer cells was detected by cell counting kit-8,and the sensitive cancer cell lines were preliminarily screened.Meanwhile,the toxicity on the normal cell HEK293 and the hemolysis activity on the red blood cells of crude extracts of antibacterial peptides of the H.illucens larvae by CCK-8 kit.Results After culturing for 24 h in vitro,it entered the logarithmic growth phase,and the cells were used for passage and subsequent experiments.Studies have shown that different concentrations of the crude extracts had a certain inhibitory effect on HCT-8,A549,SKOV3 and HeLa cells(P < 0.05),and with the increase of the concentration,the inhibitory rate gradually increased,and at the concentration of 400 mg/L the largest inhibitory rate on the cells appeared.At400 mg/L,the inhibitory rates of the crude extracts against HCT-8,A549,SKOV3,HeLa and HEK293 cells were different(P < 0.05),of which the inhibitory rate on HCT-8 was the highest and on HEK293 was the lowest.The inhibition rate on HCT-8 cells was higher than that of 5-fluorouracil(P < 0.05) under the concentration was 400 mg/L.The hemolytic rate was(0.16 ± 0.11) %,(0.20 ± 0.12) %,(0.95 ± 0.04) %,(1.09 ± 0.16) %,(1.60 ± 0.18) % when the concentration of the crude extracts were 31.25,62.5,125,250,500 mg/L respectively,and the hemolysis rates were less than 5%.Conclusion The crude extracts of antibacterial peptides of the H.illucens larvae are sensitive to HCT-8 cells,and non-toxic to normal cells and non-hemolysis to erythrocytes,indicating good prospects in the development of colon cancer drugs.