NT4(Si)-p53(N15)-antennapedia 诱导人肝癌细胞系细胞死亡。

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line.

机构信息

Department of Radiotherapy Oncology, First Hospital, Medical School of Xi'an Jiao Tong University, Xi'an 710061, Shaanxi Province, China.

出版信息

World J Gastroenterol. 2009 Dec 14;15(46):5813-20. doi: 10.3748/wjg.15.5813.

DOI:10.3748/wjg.15.5813

PMID:19998502

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2791274/

Abstract

AIM

To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53(N15)-antennapedia (Ant), and study its effect on HepG2 cells.

METHODS

Plasmid pLenti6/V5-NT4 p53(N15)-Ant was constructed incorporating the following functional regions, including signal peptide sequence and pro-region of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma (HepG2) cells were used for transfection. 3-[4,5-dimethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, transmission electron microscopy (TEM) and flow cytometric analysis (FCM) were employed to investigate the effects of LV-NT4(Si)-p53(N15)-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4(Si)-p53(N15)-Ant on tumor growth in nude mice.

RESULTS

LV-NT4(Si)-p53(N15)-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4(Si)-p53(N15)-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4(Si)-p53(N15)-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4(Si)-p53(N15)-Ant (P < 0.01). Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, but no significant changes in HepG2 cells infected with LV-EGFP. Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4(Si)-p53(N15)-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4(Si)-p53(N15)-Ant treatment group (682 IU/L) than in control group (45 IU/L, P < 0.01). The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4(Si)-p53(N15)-Ant could induce two different kinds of cell death: necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4(Si)-p53(N15)-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4(Si)-p53(N15)-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight.

CONCLUSION

LV-NT4(Si)-p53(N15)-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

摘要

目的

构建重组慢病毒表达质粒 pLenti6/V5-NT4 p53（N15）-触角（Ant），并研究其对 HepG2 细胞的作用。

方法

将信号肽序列和神经营养因子 4 的前导区、p53 的 N 端 12-26 个残基和 17 个氨基酸的果蝇载体蛋白 Ant 整合到质粒 pLenti6/V5-NT4 p53（N15）-Ant 中。用重组慢病毒感染肝癌细胞 HepG2，采用 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐（MTT）比色法、乳酸脱氢酶（LDH）释放试验、透射电镜（TEM）和流式细胞术（FCM）观察重组慢病毒对 HepG2 细胞的体外作用。体内实验观察重组慢病毒对裸鼠肿瘤生长的抑制作用。

结果

LV-NT4（Si）-p53（N15）-Ant 明显抑制 HepG2 细胞生长。MTT 比色法显示，感染 LV-NT4（Si）-p53（N15）-Ant 的 HepG2 细胞生长抑制率明显高于感染 LV-EGFP 的 HepG2 细胞，48 h 时分别为 46.9%和 94.5%，72 h 时分别为 33.9%和 95.8%（P<0.01）。光镜和 TEM 观察到感染 LV-NT4（Si）-p53（N15）-Ant 的 HepG2 细胞形态发生变化，而感染 LV-EGFP 的 HepG2 细胞无明显变化。感染 LV-NT4（Si）-p53（N15）-Ant 的 HepG2 细胞超微结构发生改变，出现膜降解，导致坏死。感染 LV-NT4（Si）-p53（N15）-Ant 和 LV-EGFP 后 24、48、72 和 96 h 检测 HepG2 细胞 LDH 释放，结果显示，LV-NT4（Si）-p53（N15）-Ant 治疗组 LDH 释放（682 IU/L）明显高于对照组（45 IU/L，P<0.01）。感染后时间越长，LDH 释放差异越大。FCM 分析显示，LV-NT4（Si）-p53（N15）-Ant 可诱导两种不同类型的细胞死亡：坏死和凋亡，以坏死为主，凋亡为辅，提示 LV-NT4（Si）-p53（N15）-Ant 通过诱导坏死发挥其抗癌作用。体内实验表明，LV-NT4（Si）-p53（N15）-Ant 明显抑制肿瘤生长，肿瘤体积和重量抑制率分别为 66.14%。