肿瘤抑素抗肿瘤相关肽对肝癌细胞增殖和凋亡的影响

Effect of Tumstatin Anti-tumor Peptide on Proliferation and Apoptosis of Hepatocarcinoma Cell

摘要:

肿瘤抑素抗肿瘤相关肽-19肽是由肿瘤抑素185~203位氨基酸组成, 具有直接抑制黑色素瘤细胞生长作用, 但其对肝癌细胞增殖和凋亡是否有影响, 对肝癌是否具有治疗作用还需进一步研究。本研究中采用基因工程技术将合成19肽基因与载体pTYB2重组后进行蛋白表达、纯化获得19肽。通过MTT法、生长曲线观察19肽对人肝癌细胞生长抑制作用; TUNEL标记法、流式细胞仪细胞周期检测法、透射电镜观察19肽对肝癌细胞凋亡的影响; 小鼠H22腹水型转移型肝癌实体瘤抑瘤实验证明其体内的抑瘤作用。MTT实验和生长曲线实验表明随着19肽浓度的增加肝癌细胞的存活率下降。在相同19肽浓度下, 随着作用时间延长存活细胞逐渐减少。电镜观察治疗组细胞出现明显凋亡, 流式细胞仪可检测到前G1峰, TUNEL标记法也证实治疗组可见明显的凋亡细胞, 体内19肽作用的小鼠H22腹水型转移型肝癌的抑瘤率达48.46%。可见, 肿瘤抑素19肽可抑制肝癌细胞生长, 促进肝癌细胞凋亡, 对肝癌具有一定的治疗作用。

Abstract:

In this study, the basic sequences of 19peptide were synthesized and inserted into vector pTYB2. After being expressed and purified, the soluble 19peptide was obtained. The inhibiting effect on hepatocarcinoma cell was selected by 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell growth curve. The apoptosis index was observed using TdT-mediated dUTP nick end labeling (TUNEL), flow cytometry and transmission electron microscope (TEM). Its anti-tumor activity in vivo was verified in H22 ascitic fluid transfevent hepatocarcinoma of mice. 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-di- phenyl-2H-tetrazolium bromide (MTT) assay and cell growth curve showed cell viability decrease with the 19peptide.Survival hepatocarcinoma cell decreased with time. In the treatment group, obvious change of apoptosis, the appearance of sub-G1 phase and dyed brown cells were seen by transmission electron microscope (TEM), flow cytometry and TdT-mediated dUTP nick end labeling (TUNEL), The inhibition rate of mouse H22 ascitic fluid transfevent hepatocarcinoma growth was 48.46%. As a whole, 19peptide could directly inhibit hepatocarcinoma cell proliferation and promote hepatocarcinoma cell apoptosis.