皖南蝮蛇毒抑瘤组分-Ⅰ对人原代胃癌细胞增殖的影响

doi:10.12092/j.issn.1009-2501.2020.05.004

摘要/Abstract

摘要： 目的:探索皖南蝮蛇毒抑瘤组分-Ⅰ(agkis-trodon halys venom antitumor component Ⅰ, AHVAC-Ⅰ)对人原代胃癌细胞增殖的抑制和诱导发生凋亡的作用。方法:胰酶消化法分离人原代胃癌细胞,无血清培养液培养细胞和差速贴壁法纯化人原代胃癌细胞以及免疫组化法鉴定人原代胃癌细胞。采用细胞增殖和毒性检测(cell counting kit-8, CCK-8)法检测各浓度组的AHVAC-Ⅰ对胃癌原代细胞增殖抑制率。免疫组化法验证AHVAC-Ⅰ诱导人原代胃癌细胞发生凋亡和苏木素-伊红染色(Hematoxylin-Eosin staining, HE)法观察其形态学的变化。采用流式细胞术Annexin V/PI双染色法检测AHVAC-Ⅰ诱导人胃癌原代细胞凋亡率。结果:成功分离纯化人原代胃癌细胞7例,失败11例,免疫组化鉴定癌胚抗原(carcinoembryonic antigen, CEA)和广谱角质蛋白(broad spectrum keratin protein, AE1/AE3)两种抗体均表达阳性。AHVAC-Ⅰ能够抑制人原代胃癌细胞的增殖且呈现剂量效应(P<0.01)。免疫组化检测半胱氨酸天冬氨酸蛋白酶-3(cysteine aspartic protease-3, Caspase-3)表达水平随AHVAC-Ⅰ浓度的升高而升高,HE法观察随着AHVAC-Ⅰ浓度的升高细胞间隙增大、核固缩、凋亡细胞增多。流式细胞术检测人原代胃癌细胞凋亡率随AHVAC-Ⅰ浓度的增加而增加(P<0.05)。结论:AHVAC-Ⅰ能够抑制人原代胃癌细胞的增殖和诱导人原代胃癌细胞发生凋亡,且呈剂量效应。

关键词: 胃癌, 原代细胞培养, 皖南蝮蛇毒抑瘤组分-Ⅰ, 核固缩, 半胱氨酸天冬氨酸蛋白酶-3

Abstract: AIM: To explore the effect of inhibiting proliferation and inducing apoptosis in human primary gastric tumor by the Agkis-trodon halys venom anti-tumor component Ⅰ (AHVAC-Ⅰ). METHODS: Human primary gastric cancer cells were isolated by trypsin digestion, serum-free culture, and purified by differential adherence method, and cells were identified by immunohistochemistry. Cell proliferation and toxicity assay (CCK-8) was used to detect the inhibition rate of AHVAC-Ⅰ in different concentrations of primary gastric cancer cells. Immunohistochemistry was used to verify the apoptosis of human primary gastric cancer cells induced by AHVAC-Ⅰ and the morphological changes were observed by Hematoxylin-Eosin staining (HE staining). AHVAC-Ⅰ-induced primary gastric cancer cell transformation rate was detected by flow cytometry Annexin V/PI double staining. RESULTS: Seven human primary gastric cancer cells were successfully isolated and purified, and 11 cases failed. Immunohistochemical identifications of carcinoembryonic antigen (CEA) and broad-spectrum keratin protein (AE1/AE3) were positive for both antibodies. AHVAC-Ⅰ inhibited the proliferation of human primary gastric cancer cells and showed a dose-dependent effect (P<0.01). Immunohistochemistry showed that the expression level of cysteine aspartic protease-3 (Caspase-3) up-regulated with the increase of AHVAC-Ⅰ concentration. HE staining showed that with the increase of AHVAC-Ⅰ concentration, the cell gap increased, nuclear pyknosis, and apoptosis cells increased. Flow cytometry showed that the apoptosis rate of human primary gastric cancer cells up-regulated with the increase of AHVAC-Ⅰ concentration (P<0.05). CONCLUSION: AHVAC-Ⅰ can inhibit the proliferation and induce apoptosis in human primary gastric cancer cells in a dose-dependent manner.

Key words: gastric cancer, primary cell culture, Agkis-trodon halys venom anti-tumor component Ⅰ, nuclear pyknosis, cysteine aspartic protease-3

中图分类号:

R965.2

田大皓, 卢林明, 支慧, 周珏, 王晓庆, 江玉华. 皖南蝮蛇毒抑瘤组分-Ⅰ对人原代胃癌细胞增殖的影响[J]. 中国临床药理学与治疗学, 2020, 25(5): 505-511.

TIAN Dahao, LU Linming, ZHI Hui, ZHOU Jue, WANG Xiaoqing, JIANG Yuhua. AHVAC-I inhibits the proliferation in human primary gastric cancer cells[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2020, 25(5): 505-511.

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