摘要
目的:
研究重组腺病毒介导的PTEN基因(第10号染色体同源丢失性磷酸酶张力蛋白基因)表达对肺癌的抑癌和化疗增敏效应及分子机制。
方法:
采用重组腺病毒载体PTEN(简称Ad-PTEN)感染QBI-293A细胞,并用该细胞进行病毒扩增和效价测定。体外将Ad-PTEN感染NCI-H446小细胞肺癌细胞。分为5组:细胞对照PBS组(PBS组)、空载体腺病毒组(Ad组)、Ad-PTEN基因治疗组(简称Ad-PTEN组)、顺铂对照组(DDP组)和Ad-PTEN+DDP组,Western blotting鉴定PTEN基因表达,MTT法和流式细胞仪(FCM)检测Ad-PTEN对NCI-H446小细胞肺癌细胞的生长抑制和诱导凋亡的作用,用RT-PCR检测细胞凋亡相关基因P53、人细胞凋亡相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)、和生存素(Survivin)的转录。采用NCI-H446小细胞肺癌细胞株建立人肺癌裸鼠移植瘤模型,均使用瘤体局部多点注射用药,每次注射量为:PBS组:50μl PBS/只;Ad组:50μl 1x107pfu Ad/只;Ad-PTEN组:50μl 1x107pfu Ad-PTEN /只;DDP组用药浓度为2mg/kg;Ad-PTEN+DDP组:50μl 1x107pfu Ad-PTEN /只+DDP 1mg/kg。隔日一次,共注射6次。动态测量肿瘤体积,并计算瘤重抑瘤率,瘤块HE染色法观察细胞形态,免疫组化染色用以检测NCI-H446小细胞肺癌移植瘤生长相关因子的表达:(1)Caspase3、Bax等促凋亡因子;(2)Bcl-2、Survivin等凋亡抑制因子;(3)血管内皮生长因子(VEGF),为肿瘤血管形成相关因子。
结果:
Ad-PTEN可在QBI-293A细胞中增殖,病毒效价检测为108 pfu/ml。Ad-PTEN感染NCI-H446细胞后,Western-blotting检测到了PTEN基因的表达,Ad-PTEN体外能明显抑制人NCI-H446小细胞肺癌细胞的生长和诱导凋亡,体内同样能明显抑制肺癌裸鼠移植瘤的生长,均优于PBS组和Ad组,具有抑瘤作用;而且Ad-PTEN能提高DDP化疗药物体内外抑制NCI-H446小细胞肺癌细胞及裸鼠移植瘤的生长和诱导凋亡的作用,具有化疗增敏效应。体外实验显示,Ad-PTEN、DDP、Ad-PTEN+DDP能引起NCI-H446细胞P53、Bax及Caspase-3基因表达明显上调,而Bcl-2和Survivin基因表达明显下调;体内实验显示,Ad-PTEN、DDP、Ad-PTEN+DDP能使肿瘤组织中的Caspase-3及Bax基因表达上调,而Bcl-2、Survivin和VEGF基因表达下调,且Ad-PTEN+DDP对上述各基因表达趋势的影响较Ad-PTEN、DDP更为显著。
结论:
1、Ad-PTEN体内外可抑制NCI-H446小细胞肺癌细胞和肺癌裸鼠移植瘤生长和诱导凋亡,呈现明显抑癌作用。
2、Ad-PTEN联合细胞毒药物DDP体内外抑制NCI-H446小细胞肺癌细胞和肺癌裸鼠移植瘤生长和诱导凋亡的作用均较Ad-PTEN组和DDP组明显,具有一定化疗增敏效应。
3、Ad-PTEN+DDP较Ad-PTEN、DDP更具显著上调P53、Caspase3、Bax和下调Bcl-2、Survivin等细胞凋亡相关基因表达的效应,这可能是Ad-PTEN具有抑癌和化疗增敏效应的重要机制之一。
4、Ad-PTEN、DDP和Ad-PTEN+DDP具有下调肿瘤血管形成相关因子VEGF表达的效应,Ad-PTEN+DDP对VEGF表达趋势的影响较Ad-PTEN、DDP更显著,这可能是Ad-PTEN对肺癌裸鼠移植瘤体内生长具有抑瘤和化疗增敏效应的另一机制。
Objective:
To explore the inhibitory and chemosensitivity of recombinant adenovirus PTEN( phosphatase and tensin homologue deleted chromatosome10)on lung cancer and its possible mechanisms.
Methods:
Recombinant adenovirus Ad-PTEN was constructed and transfected into the human embryonic kidney 293 (QBI-293A) cells. The adenovirus completed the amplification in QBI-293A cells and the titration was also detected. The human pulmonary carcinoma cell line NCI-H446 was infected by Ad-PTEN and combined with cisplatin (DDP) in vitro, and were randomly divided into five groups: PBS negative control group(PBS group), idling adenovirus group(Ad group), adenovirus-mediated PTEN group (Ad-PTEN group), DDP positive control group(DDP group) and Ad-PTEN combined with DDP (referred to as joint group). The expression of PTEN in NCI-H446 small cell lung cancer cells was detected by western blotting. The effect of enhanced growth-suppressing and apoptosis-inducing by Ad-PTEN on NCI-H446 small cell lung cancer cells were analyzed by MTT assay and Flow cytometry. The expression of apoptosis-related genes (P53、Bax、Caspase-3、Bcl-2、Survivin) was detected by reverse transcription-PCR (RT-PCR). The human lung cancer mode was established with NCI-H446 cells in athymic nude mouse. Mice of the groups were progressed multi-points injection in tumor, dose of each injection: PBS group: 50μl PBS/only; Ad Group: 50μl 1x107pfu Ad/only; Ad-PTEN Group: 50μl 1x107pfu Ad-PTEN/only; DDP group of drug concentration 2mg/kg, Ad-PTEN+DDP Group: 50μl 1x107pfu Ad-PTEN/only+DDP 1mg/kg. Next day once, a total of 6 injections. The gross tumor volumes were measured dynamically. The ratios of tumor-suppression were calculated. The tumors were observed by HE staining method. Immunohistochemistry for the detection of small cell lung cancer NCI-H446 tumor growth-related factor expression: (1) Caspase3, Bax and other pro-apoptotic factor; (2) Bcl-2, Survivin and other apoptosis inhibitory factor; (3) VEGF and other angiogenesis-related factors.
Results:
After the amplification of Ad-PTEN in QBI-293A cells, the titration was 108pfu/mL. The expressions of PTEN in NCI-H446 cells were detected by western blotting. Adenovirus-mediated PTEN significantly inhibited NCI-H446 cell growth, induced cell apoptosis, and retarded NCI-H446 human lung cell transplanted tumor growth in athymic nude mouse, exhibiting anti-tumor effect. Furthermore, Ad-PTEN significantly enhanced the chemosensitivity to anticancer drug DDP in lung cancer in vitro and vivo. Ad-PTEN group, DDP group, Ad-PTEN+DDP group of NCI-H446 cells in the P53, Bax, and Caspase-3 expression was significantly increased, while Bcl-2 and Survivin gene expression was significantly reduced; and Ad-PTEN+DDP Group effect than Ad-PTEN group, DDP group is more significant. Is the Ad-PTEN with anti-tumor effect of some molecular biological mechanisms.
Conclusion:
1. Ad-PTEN had significant effect in suppressing cell growth and inducing cell apoptosis in NCI-H446 small cell lung cell and small cell lung cancer transplanted tumor growth in athymic nude mouse.
2. Ad-PTEN enhanced chemosensitivity to DDP effect in suppressing cell growth and inducing cell apoptosis in NCI-H446 small cell lung cell and small cell lung cancer transplanted tumor growth in athymic nude mouse.
3. Ad-PTEN+DDP had more potent effect in up-regulating the expression of P53、Caspase3、Bax and down-regulating the expression of Bcl-2、Survivin, which may be responsible for its anti-tumor effect on NCI-H446 small cell lung cell in vitro and in vivo in athymic nude mouse model.
4. Ad-PTEN, DDP and Ad-PTEN+DDP had effect in down-regulating the expression of angiogenesis-related factors VEGF, Ad-PTEN+DDP compared with Ad-PTEN, DDP more significantly, which may be another important mechanism involved in its synergetic effect in suppressing lung cancer transplanted tumor growth in athymic nude mouse model.
引文
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