重组高效抗肿瘤抗病毒蛋白对胰腺癌细胞BxPC-3的体内外实验研究
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摘要
胰腺癌是常见的消化系统恶性肿瘤,也是世界范围内恶性程度最高的癌症之一,目前的治疗方法疗效均不佳,胰腺癌患者基本无法治愈,死亡率极高；5年相对生存率仅为5%。目前应用的胰腺癌化疗药物疗效有限,且容易引发多种药物不良反应；寻找到更为安全有效的药物或者治疗方案,将为胰腺癌的治疗带来新的希望。
干扰素联合其他化疗药物治疗能够明显提高胰腺癌患者的5年生存率,缓解胰腺癌发展进程,但其应用具有局限性。重组高效抗肿瘤抗病毒蛋白注射液即乐复能是一种来源于干扰素的蛋白质工程药,对皮肤癌、前列腺癌、肝癌细胞的研究表明,重组高效抗肿瘤抗病毒蛋白的抗肿瘤活性是天然人干扰素的200多倍,抗病毒活性则达10倍以上；但其对胰腺癌细胞的作用及机制尚不清楚,研究重组高效抗肿瘤抗病毒蛋白对胰腺癌细胞的影响及作用机制,将为重组高效抗肿瘤抗病毒蛋白应用于胰腺癌临床治疗提供切实的理论与实验依据。
本文第一章采用胰腺癌BxPC-3细胞作为研究对象,应用MTT法、流式细胞术、荧光染色、Transwell侵袭实验分别观察了不同浓度、不同时间重组高效抗肿瘤抗病毒蛋白对胰腺癌细胞增殖、凋亡及侵袭能力的影响,结果发现5ng/ml浓度的重组高效抗肿瘤抗病毒蛋白处理BxPC-3细胞24h和48h后对细胞生长有明显的抑制作用,随着重组高效抗肿瘤抗病毒蛋白浓度的增加,抑制作用越来越明显,胰腺癌细胞停滞在G1期,细胞凋亡率也随之升高,且细胞的侵袭能力也随着重组高效抗肿瘤抗病毒蛋白浓度的增加而减弱；重组高效抗肿瘤抗病毒蛋白的作用效果呈剂量依赖方式。
为探讨重组高效抗肿瘤抗病毒蛋白抑制胰腺癌细胞增殖与侵袭、促进胰腺癌细胞凋亡的分子机制,本文第二章选取细胞凋亡、血管生成、细胞侵袭过程中的几个关键基因,采用RT-PCR和Western blot分别于mRNA和蛋白质水平检测重组高效抗肿瘤抗病毒蛋白对其表达的影响。结果表明,重组高效抗肿瘤抗病毒蛋白促进胰腺癌BxPC-3细胞caspase-3、caspase-8的表达,抑制VEGF-A和MMP-2的表达,作用呈剂量依赖方式；对caspase-9和MMP-9的表达无影响。
前二章从体外细胞实验角度明确了重组高效抗肿瘤抗病毒蛋白抗胰腺癌细胞的作用,为了进一步明确其在体内的作用,本文第三章采用胰腺癌BxPC-3细胞异位种植瘤模型为研究对象,观察了重组高效抗肿瘤抗病毒蛋白对异位种植瘤瘤体生长的影响,结果表明10μg/kg治疗剂量的重组高效抗肿瘤抗病毒蛋白能够显著抑制胰腺癌细胞异位种植瘤的生长,组织切片与TUNEL实验观察到重组高效抗肿瘤抗病毒蛋白治疗组的瘤体内细胞发生退变,凋亡明显；采用Western blot检测cleaved caspase-3、caspase-8、VEGF-A、MMP-2的表达,结果与前述体外细胞实验一致,重组高效抗肿瘤抗病毒蛋白促进瘤体细胞cleaved caspase-3、caspase-8的表达,抑制VEGF-A(?)(?)MMP-2的表达；与阳性对照药物吉西他滨相比,重组高效抗肿瘤抗病毒蛋白对抗胰腺癌的效果相近,但剂量较吉西他滨明显减小。
综上所述,重组高效抗肿瘤抗病毒蛋白能够通过促进死亡受体途径关键凋亡基因caspase-8的表达激活下游效应型caspase-3,从而促进胰腺癌细胞的凋亡；重组高效抗肿瘤抗病毒蛋白能够抑制血管生成因子VEGF-A的表达,从而抑制肿瘤内血管新生；重组高效抗肿瘤抗病毒蛋白能够抑制蛋白水解酶关键基因MMP-2的表达,从而抑制胰腺癌细胞的侵袭与转移；体外实验与体内实验的结果相符。重组高效抗肿瘤抗病毒蛋白的临床应用目前处于Ⅱ/Ⅲ临床试验阶段,本研究从体内和体外实验的角度明确其高效对抗胰腺癌的作用,可为药物的临床应用提供理论与实验依据,更可为新药物的开发、胰腺癌化疗方案的优化提供新的线索。
Pancreatic cancer is the common digestive carcinoma, with the high malignancy. Current treatment for pancreatic cancer is unsatisfactory, which the patients hold the high mobility, and the relative survival rate in5years is only5%. The chemotherapeutics will induce the multiple side effects and the curative effect is limited. Therefore, it will bring the new potential for pancreatic cancer patients to find out one new chemotherapeutic drug or scheme.
The treatment of interferon combining with other chemotherapeutics can improve the5-year survival rate of pancreatic cancer patients, and alleviate the progression of pancreatic cancer, but the application of interferon is limited. Recombinant antitumor antivirus protein (RAAP; Trade name:Novaferon) is a kind of protein engineering drug derived from interferon, its anti-tumor efficacy has been established in skin, liver, lung, and prostate cancers. The antitumor activity of RAAP is the200times of natural human interferon, and the antivirus activity is above the10times. However, the effect of RAAP on pancreatic cancer cells and the related mechanism remain unknown. To investigate the effect of RAAP on pancreatic cancer cells will supply the confirmative evidences for the clinical application of RAAP in the treatment of pancreatic cancer.
The first chapter used pancreatic cancer cell line BxPC-3, to observe the effect of RAAP in different concentrations and different time periods on proliferation, apoptosis and invasion of BxPC-3cells by MTT assay, flow cytometry, fluorescence staining, and Transwell assay. The results showed that RAAP in5ng/ml concentration could inhibit the proliferation of BxPC-3cells obviously, the time period of24hours was a positive time point. Moreover, with the increment of concentration of RAAP, the inhibitive effect was more significant, as well as the cell cycle arrest in G1stage, and the apoptotic rates of cells. On the contrary, the cell invasion was alleviated with the increment of RAAP concentration. All effects of RAAP on BxPC-3cells were dose dependent.
In order to explore the molecular mechanism of RAAP inhibiting proliferation and invasion of BxPC-3cells, and promoting apoptosis of BxPC-3cells, the second chapter applied RT-PCR and Western blot to determine the expressions of some key genes during cell apoptosis, angiogenesis and cell invasion. The results showed that RAAP could increase the expressions of caspase-3and caspase-8, decrease the expressions of VEGF-A and MMP-2, while had no effect on the expressions of caspase-9and MMP-9.
The first two chapters determined the effect of RAAP in vitro, and furthermore, in order to confirm the effect of RAAP in vivo, the third chapter used xenografts of nude mice by pancreatic cancer BxPC-3cells, to investigate the effect of RAAP on the development of xenografts. The results showed that RAAP (10μg/kg) could inhibit the growth of xenografts significantly; by histological sections and TUNEL assay, the tumor cells in the RAAP group were apoptotic obviously; meanwhile, RAAP could promote the expressions of caspase-3and caspase-8, and reduce the expressions of VEGF-A and MMP-2, which were consistent with the results in vitro. Compared with the positive drug Gemcitabine, the efficacy of RAAP is similar, but the effective dose is significantly reduced.
Taken together, RAAP could promote the apoptosis of pancreatic cancer cells by activating caspase-8and further caspase-3; RAAP could inhibit the angiogenesis by decreasing the expression of VEGF-A; RAAP could reduce the cell invasion by down-regulate the expression of MMP-2, which was the key gene for proteolysis of extracellular matrix; the results in vitro and in vivo were consistent. Currently, the clinical application of RAAP is still under phase II/III clinical trial, the investigation on RAAP anti-pancreatic cancer can supply the theoretic and laboratory evidences for the clinical application, also the new clues for the development of new drugs and the optimization of chemotherapy for pancreatic cancer.
干扰素联合其他化疗药物治疗能够明显提高胰腺癌患者的5年生存率,缓解胰腺癌发展进程,但其应用具有局限性。重组高效抗肿瘤抗病毒蛋白注射液即乐复能是一种来源于干扰素的蛋白质工程药,对皮肤癌、前列腺癌、肝癌细胞的研究表明,重组高效抗肿瘤抗病毒蛋白的抗肿瘤活性是天然人干扰素的200多倍,抗病毒活性则达10倍以上；但其对胰腺癌细胞的作用及机制尚不清楚,研究重组高效抗肿瘤抗病毒蛋白对胰腺癌细胞的影响及作用机制,将为重组高效抗肿瘤抗病毒蛋白应用于胰腺癌临床治疗提供切实的理论与实验依据。
本文第一章采用胰腺癌BxPC-3细胞作为研究对象,应用MTT法、流式细胞术、荧光染色、Transwell侵袭实验分别观察了不同浓度、不同时间重组高效抗肿瘤抗病毒蛋白对胰腺癌细胞增殖、凋亡及侵袭能力的影响,结果发现5ng/ml浓度的重组高效抗肿瘤抗病毒蛋白处理BxPC-3细胞24h和48h后对细胞生长有明显的抑制作用,随着重组高效抗肿瘤抗病毒蛋白浓度的增加,抑制作用越来越明显,胰腺癌细胞停滞在G1期,细胞凋亡率也随之升高,且细胞的侵袭能力也随着重组高效抗肿瘤抗病毒蛋白浓度的增加而减弱；重组高效抗肿瘤抗病毒蛋白的作用效果呈剂量依赖方式。
为探讨重组高效抗肿瘤抗病毒蛋白抑制胰腺癌细胞增殖与侵袭、促进胰腺癌细胞凋亡的分子机制,本文第二章选取细胞凋亡、血管生成、细胞侵袭过程中的几个关键基因,采用RT-PCR和Western blot分别于mRNA和蛋白质水平检测重组高效抗肿瘤抗病毒蛋白对其表达的影响。结果表明,重组高效抗肿瘤抗病毒蛋白促进胰腺癌BxPC-3细胞caspase-3、caspase-8的表达,抑制VEGF-A和MMP-2的表达,作用呈剂量依赖方式；对caspase-9和MMP-9的表达无影响。
前二章从体外细胞实验角度明确了重组高效抗肿瘤抗病毒蛋白抗胰腺癌细胞的作用,为了进一步明确其在体内的作用,本文第三章采用胰腺癌BxPC-3细胞异位种植瘤模型为研究对象,观察了重组高效抗肿瘤抗病毒蛋白对异位种植瘤瘤体生长的影响,结果表明10μg/kg治疗剂量的重组高效抗肿瘤抗病毒蛋白能够显著抑制胰腺癌细胞异位种植瘤的生长,组织切片与TUNEL实验观察到重组高效抗肿瘤抗病毒蛋白治疗组的瘤体内细胞发生退变,凋亡明显；采用Western blot检测cleaved caspase-3、caspase-8、VEGF-A、MMP-2的表达,结果与前述体外细胞实验一致,重组高效抗肿瘤抗病毒蛋白促进瘤体细胞cleaved caspase-3、caspase-8的表达,抑制VEGF-A(?)(?)MMP-2的表达；与阳性对照药物吉西他滨相比,重组高效抗肿瘤抗病毒蛋白对抗胰腺癌的效果相近,但剂量较吉西他滨明显减小。
综上所述,重组高效抗肿瘤抗病毒蛋白能够通过促进死亡受体途径关键凋亡基因caspase-8的表达激活下游效应型caspase-3,从而促进胰腺癌细胞的凋亡；重组高效抗肿瘤抗病毒蛋白能够抑制血管生成因子VEGF-A的表达,从而抑制肿瘤内血管新生；重组高效抗肿瘤抗病毒蛋白能够抑制蛋白水解酶关键基因MMP-2的表达,从而抑制胰腺癌细胞的侵袭与转移；体外实验与体内实验的结果相符。重组高效抗肿瘤抗病毒蛋白的临床应用目前处于Ⅱ/Ⅲ临床试验阶段,本研究从体内和体外实验的角度明确其高效对抗胰腺癌的作用,可为药物的临床应用提供理论与实验依据,更可为新药物的开发、胰腺癌化疗方案的优化提供新的线索。
Pancreatic cancer is the common digestive carcinoma, with the high malignancy. Current treatment for pancreatic cancer is unsatisfactory, which the patients hold the high mobility, and the relative survival rate in5years is only5%. The chemotherapeutics will induce the multiple side effects and the curative effect is limited. Therefore, it will bring the new potential for pancreatic cancer patients to find out one new chemotherapeutic drug or scheme.
The treatment of interferon combining with other chemotherapeutics can improve the5-year survival rate of pancreatic cancer patients, and alleviate the progression of pancreatic cancer, but the application of interferon is limited. Recombinant antitumor antivirus protein (RAAP; Trade name:Novaferon) is a kind of protein engineering drug derived from interferon, its anti-tumor efficacy has been established in skin, liver, lung, and prostate cancers. The antitumor activity of RAAP is the200times of natural human interferon, and the antivirus activity is above the10times. However, the effect of RAAP on pancreatic cancer cells and the related mechanism remain unknown. To investigate the effect of RAAP on pancreatic cancer cells will supply the confirmative evidences for the clinical application of RAAP in the treatment of pancreatic cancer.
The first chapter used pancreatic cancer cell line BxPC-3, to observe the effect of RAAP in different concentrations and different time periods on proliferation, apoptosis and invasion of BxPC-3cells by MTT assay, flow cytometry, fluorescence staining, and Transwell assay. The results showed that RAAP in5ng/ml concentration could inhibit the proliferation of BxPC-3cells obviously, the time period of24hours was a positive time point. Moreover, with the increment of concentration of RAAP, the inhibitive effect was more significant, as well as the cell cycle arrest in G1stage, and the apoptotic rates of cells. On the contrary, the cell invasion was alleviated with the increment of RAAP concentration. All effects of RAAP on BxPC-3cells were dose dependent.
In order to explore the molecular mechanism of RAAP inhibiting proliferation and invasion of BxPC-3cells, and promoting apoptosis of BxPC-3cells, the second chapter applied RT-PCR and Western blot to determine the expressions of some key genes during cell apoptosis, angiogenesis and cell invasion. The results showed that RAAP could increase the expressions of caspase-3and caspase-8, decrease the expressions of VEGF-A and MMP-2, while had no effect on the expressions of caspase-9and MMP-9.
The first two chapters determined the effect of RAAP in vitro, and furthermore, in order to confirm the effect of RAAP in vivo, the third chapter used xenografts of nude mice by pancreatic cancer BxPC-3cells, to investigate the effect of RAAP on the development of xenografts. The results showed that RAAP (10μg/kg) could inhibit the growth of xenografts significantly; by histological sections and TUNEL assay, the tumor cells in the RAAP group were apoptotic obviously; meanwhile, RAAP could promote the expressions of caspase-3and caspase-8, and reduce the expressions of VEGF-A and MMP-2, which were consistent with the results in vitro. Compared with the positive drug Gemcitabine, the efficacy of RAAP is similar, but the effective dose is significantly reduced.
Taken together, RAAP could promote the apoptosis of pancreatic cancer cells by activating caspase-8and further caspase-3; RAAP could inhibit the angiogenesis by decreasing the expression of VEGF-A; RAAP could reduce the cell invasion by down-regulate the expression of MMP-2, which was the key gene for proteolysis of extracellular matrix; the results in vitro and in vivo were consistent. Currently, the clinical application of RAAP is still under phase II/III clinical trial, the investigation on RAAP anti-pancreatic cancer can supply the theoretic and laboratory evidences for the clinical application, also the new clues for the development of new drugs and the optimization of chemotherapy for pancreatic cancer.
引文
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