PLA-CS纳米粒介导Survivin反义寡核苷酸增强阿霉素诱导肝癌细胞凋亡的体外研究
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摘要
目的肝癌(hepatocelular carcinoma，HCC)，是我国最常见的恶性肿瘤之一，恶性程度高、预后差，严重威胁着人类生命与健康。目前，手术切除仍是治疗肝癌首选和较有效的方法。化疗是肝癌一项重要的辅助治疗手段，但对正常细胞的毒性和肿瘤细胞的耐药性是化疗面临的两大难题。研究表明，Survivin是迄今发现的最强的凋亡抑制因子，并具有抑制细胞凋亡和调节细胞周期双重功能，与肿瘤的化疗耐药性密切相关。纳米颗粒基因转运体是近年发展起来的一种新型的安全、有效并具有优良特性的非病毒基因转运载体。本研究：1．制备聚乳酸壳聚糖纳米粒，分析其性质和体外转染效率；2．利用新型的活细胞检测技术分子信标直接检测并证实人肝癌细胞系Bel7402高表达Survivin；3．利用聚乳酸壳聚糖纳米粒介导Survivin反义寡核苷酸研究增强阿霉素诱导肝癌细胞凋亡。
方法1．采用乳化溶剂挥发法，优化工艺条件，成功制备粒径较小、分布均匀的PLA-CS纳米颗粒，用Malvern Zetasizer 3000E(Malvern，UK)检测PLA-CS纳米粒和PLA-CS／DNA复合物的Zeta电位以及粒径分布。使用原子力显微镜(Asylum Research MFP-3D AFMSystems)来检测PLA-CS纳米粒粒径大小和形态。然后利用电泳的方法观察PLA-CS NP与DNA的结合情况和对DNA的保护，MTT实验和流式细胞仪检测PLA-CS NP在体外细胞转染效率和细胞毒性；2．针对Survivin设计的分子信标通过PLA-CS纳米粒介导入正常肝细胞系HL7702细胞和肝癌细胞系Bel7402中，检测Survivin转录产物的差别，台盼兰实验检测分子信标对细胞的生长情况的影响，RT-PCR证实分子信标检测的准确性；3．通过PLA-CS纳米粒转染Survivin反义寡核苷酸进入肝癌细胞，运用RT-PCR和Western blot检测Survivin的变化，MTT检测细胞的增殖抑制，流式细胞仪检测肝癌细胞的凋亡率。用阿霉素处理后，MTT检测细胞的增殖抑制，流式细胞仪和AO／EB染色观测细胞的凋亡指数的变化。
结果1．采用Malvern Zetasizer 3000E激光粒度分析仪进行粒径及分布的检测结果显示，制备PLA-CS NP的平均粒径值为87nm。粒径大小分布呈正态分布，分布均匀。原子力显微镜进行观测。纳米颗粒呈圆球形，表面平滑完整，分散良好，无粘附团聚现象。PLA-CS NP和DNA耦合后，其表面电位为＋10mV±1.5mV。从电泳的结果分析知，纳米粒和质粒DNA能有效的结合起来。MTT显示PLA-CS NP在低于3μg／μl浓度时，细胞生长的抑制率小于10％。流式细胞仪检测转染效率可达43.5％。2．分子信标经PLA-CS纳米粒转染细胞后发现正常的肝细胞系HL7702中几乎没有荧光信号；而肝癌细胞系中Bel7402细胞系内有较强的的绿色荧光信号。台盼兰实验显示细胞的活力基本未受影响。RT-PCR证实Bel7402细胞中Survivin mRNA表达高于HL7702细胞系。3．在转染了Survivin的反义寡核苷酸24小时后，反义寡核苷酸Survivin各组均显示mRNA和蛋白的表达下调，以800ng／ml组最为明显，与对照组比较有显著性差异(P＜0.05)；MTT发现Survivin ASODN处理后各组细胞增殖活性均有不同程度降低；流式细胞仪检测发现Bel7402细胞在400ng／ml，600ng／ml，800ng／ml组的ASODN在作用24小时后凋亡率可分别为7.88±0.15％，14.16±0.35％，32.73±0.78％，与对照空白组0.50±0.06％和SODN转染组0.57±0.09％比较有显著性差异(P＜0.05)。在加用阿霉素后，600ng／mlASODN组的细胞凋亡率可达40.34±0.78％，与单独使用ADM和转染ASODN后的凋亡率16.94±0.54％和15.07±0.35％比较有显著性差异。在AO／EB染色中也可以发现ADM＋ASODN组内的比较多的细胞则发生了核固缩，浓染，破裂，出现了细胞凋亡的核形态变化，而MTT实验显示空白对照组与ADM组和ASODN组在转染细胞24小时后，细胞增殖抑制有明显的差异(P＜0.05)，而ADM＋ASODN组与其余三组比较均有显著性差异(P＜0.05)。
结论1．成功制备平均粒径87nm、分布均匀的PLA-CS纳米颗粒。用壳聚糖修饰该纳米颗粒表面，使其带正电荷从而可逆的结合质粒DNA，组装成PLA-CS纳米颗粒基因转运体系，并对细胞生长毒性低。转染效率较高。2．利用分子信标建立了一种能在单细胞水平上检测活体细胞中基因转录产物的方法，有望发展成为一种能早期检测肿瘤标志物的方法。3．采用PLA-CS纳米基因载体介导转染针对Survivin设计的ASODN，转染效率较高，ASODN发挥了高效的封闭作用，PLA-CS纳米基因载体是一种安全，高效的新型的纳米基因载体。采用PLA-CS纳米基因载体介导转染SurvivinASODN,能有效的封闭Survivin基因，下调Survivin蛋白及mRNA的表达，并促进Bel7402细胞的凋亡。采用ASODN封闭Survivin的表达确实有效地抑制肝癌细胞的生长，增加细胞的凋亡。从而增加ADM的化疗敏感性，改善常规化疗的效果，具有临床应用的潜能。
Objects Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in our country, which is gravely threatening people's health because of its high malignant degree and bad prognosis.Exairesis is still the first-line and most effective treatment to HCC nowadays. Chemotherapy, as an important adjunctive treatment to HCC, has to settle the two tough difficulties of cytotoxicity to normal cells and drug resistance of tumor cells. It is researched that Survivin is the most powerful inhibiting apoptosis factor ever been found with the dual function of inhibiting apoptosis and regulating cellcycle, and it has a close relationship with chemoresistance. Nanoparticle gene transporter, an non-viral gene transporter ,is well developed in recent years because of its safety, efficacy and fine property. In this paper: 1. to prepare polylactic-chitosan(PLA-CS)modified nanoparticles and analyze its properties and vitro transfection efficiency; 2. to test directly using molecular beacon which is a new living cell detection technique and prove that Survivin is high expressed in Be17402; 3.to use Survivin antisense oligodeoxynucleotide (ASODN) loaded PLA-CS nanoparticles to enhance the apoposis of HCC induced by adriamycin(ADM).
Methods 1.to prepare PLA-CS NP with small particle diameter and uniform distribution using solvent emulsionizated volatilixation method and with optimized conditions of technique; to detect Zeta electric potentials and particle diameter distributions of PLA-CS NP and PLA-CS/DNA using Malvern Zetasizer 3000 E; to detect particle diameter and appearance of PLA-CS NP by Atomic force microscope (AFM). Then to observe the combination of PLA-CS NP and DNA and the PLA-CS's protection to DNA by electrophoresis; to detect the transfection efficiency and cytotoxicity of PLA-CS NP in vitro by MTT assay and Flow cytometer (FCM); 2. to transfect molecular beacon designed according to Survivin into HL7702 and Be17402 via PLA-CS NP, then to detect the changes of Survivin transcripts, and to detect the influence of cell growth caused by molecular beacon using Typan blue staining; to confirm the accuracy of molecular beacon detection using RT-PCR;3. to transfect the Survivin ASODN loaded PLA-CS nanoparticles into hepatoma carcinoma cells, then to detect the change of Survivin by methods of RT-PCR and Western blot, to detect cytostasis using MTT assay, and to detect apoptosis rate of HCC using FCM. After acting with ADM, to detect cytostasis using MTT assay and to observe the change of cell apoptotic index using FCM and AO/EB staining.
Results 1.Detection results of the particle diameter and distribution using laser particle size analysor Malvem Zetasizer 3000E showed that prepared PLA-CS NP had small mean diameter of 87 nm and the particle diameter had well Gaussian distribution. It was observed by AFM that NPs were global ones with smooth and complete surface and without adhering and agglomeration. Zeta electric potential was+10mV±1.5mV when PLA-CS NP coupled with DNA. It was analyzed from electrophoresis results that NP and plasmid DNA can be combined together effectively. MTT results showed inhibition ratio of cell growth was less than 10% as the concentration of PLA-CS NP was below 3μg/μl.The transfection efficiency can reach 43.5% detected by FCM. 2. After transfecting molecular beacon loaded PLA-CS NP into HL7702 and Be17402, it was observed that there was hardly any fluorescence in HL7702 when strong green fluorescence signal was found in Be17402. Trypan blue staining essay showed cell vigor was uninfluenced basically. RT-PCR confirmed that the Survivin mRNA in Be17402 was higher expressed than that in HL7702. 3.24 hours after all the groups having been transfected by Survivin ASODN, the mRNA and expression of proteins of them all decreased, especially the one of the group with the concentration of 800ng/ml of Survivin ASODN, and there was significant difference with the control group(P＜0.05); MTT assay results showed that all cells group's proliferation activity degraded disparity after having been transfected by survivn ASODN. FCM detection results showed that 24 hours after the efficient of ASODN the apoptosis rates were 7.88+0.15%, 14.16+0.35%, 32.73+0.78% respectively in the Be17402 groups with concentration of 400ng/ml, 600ng/ml and 800ng/ml, and there was significant difference contrasting to blank group with apoptosis rates of 0.50+0.06% and SODN transfected group with apoptosis rates of 0.57+0.09%(P＜0.05). After the acting of ADM, apoptosis rate of Be17402 was 40.34+0.78% in the group with concentration of 600ng/ml,and there was significant difference contrasting to the apoptosis rates of 16.94+0.54% or 15.07+0.35% respectively in groups using ADM or ASODN transfected only. AO/EB staining results showed more cells in ADM+ASODN group had the change of nucleus morphous including karyopycnosis, thick stain and disruption of nucleus. MTT assay results showed that the cytostasis degree had obvious difference (P＜0.05) between blank control group with ADM group or ASODN group 24 hours after having been transfected.,and there was significant difference between ADM+ASODN group with each of other three groups (P＜0.05).
Conclusions 1.PLA-CS NP with mean diameter of 87 nm and even distribution had been prepared successfully. The NP whose surface had been modified by CS to enable it have positive charge can combine with plasmid DNA reversibly, then PLA-CS NP gene transport system with low cytotoxicity and high transfection efficiency was assembled. 2. That a method of detecting genetic transcript in living monoplast cells by using molecular beacon can be expected to be well developed as a method of detecting tumor markers in the early stage. 3. The transfection induced by Survivin ASODN which has efficient function of blocking loaded PLA-CS NP gene carrier which is safe and efficient has high transfection efficiency. Using PLA-CS NP gene carrier to induce the transfection of Survivin ASODN, it can be more efficient to block survivin and down regulate the expressions of protein and mRNA of surviving which can promote the apoptosis of Be17402 finally. Using ASODN to block expression of survivin can inhibit the growth of HCC efficiently and increase the apoptosis of cell. Also, that can improve the chemosensibility of ADM and the effect of routine chemotherapy. All these make it potential in clinical application.
方法1．采用乳化溶剂挥发法，优化工艺条件，成功制备粒径较小、分布均匀的PLA-CS纳米颗粒，用Malvern Zetasizer 3000E(Malvern，UK)检测PLA-CS纳米粒和PLA-CS／DNA复合物的Zeta电位以及粒径分布。使用原子力显微镜(Asylum Research MFP-3D AFMSystems)来检测PLA-CS纳米粒粒径大小和形态。然后利用电泳的方法观察PLA-CS NP与DNA的结合情况和对DNA的保护，MTT实验和流式细胞仪检测PLA-CS NP在体外细胞转染效率和细胞毒性；2．针对Survivin设计的分子信标通过PLA-CS纳米粒介导入正常肝细胞系HL7702细胞和肝癌细胞系Bel7402中，检测Survivin转录产物的差别，台盼兰实验检测分子信标对细胞的生长情况的影响，RT-PCR证实分子信标检测的准确性；3．通过PLA-CS纳米粒转染Survivin反义寡核苷酸进入肝癌细胞，运用RT-PCR和Western blot检测Survivin的变化，MTT检测细胞的增殖抑制，流式细胞仪检测肝癌细胞的凋亡率。用阿霉素处理后，MTT检测细胞的增殖抑制，流式细胞仪和AO／EB染色观测细胞的凋亡指数的变化。
结果1．采用Malvern Zetasizer 3000E激光粒度分析仪进行粒径及分布的检测结果显示，制备PLA-CS NP的平均粒径值为87nm。粒径大小分布呈正态分布，分布均匀。原子力显微镜进行观测。纳米颗粒呈圆球形，表面平滑完整，分散良好，无粘附团聚现象。PLA-CS NP和DNA耦合后，其表面电位为＋10mV±1.5mV。从电泳的结果分析知，纳米粒和质粒DNA能有效的结合起来。MTT显示PLA-CS NP在低于3μg／μl浓度时，细胞生长的抑制率小于10％。流式细胞仪检测转染效率可达43.5％。2．分子信标经PLA-CS纳米粒转染细胞后发现正常的肝细胞系HL7702中几乎没有荧光信号；而肝癌细胞系中Bel7402细胞系内有较强的的绿色荧光信号。台盼兰实验显示细胞的活力基本未受影响。RT-PCR证实Bel7402细胞中Survivin mRNA表达高于HL7702细胞系。3．在转染了Survivin的反义寡核苷酸24小时后，反义寡核苷酸Survivin各组均显示mRNA和蛋白的表达下调，以800ng／ml组最为明显，与对照组比较有显著性差异(P＜0.05)；MTT发现Survivin ASODN处理后各组细胞增殖活性均有不同程度降低；流式细胞仪检测发现Bel7402细胞在400ng／ml，600ng／ml，800ng／ml组的ASODN在作用24小时后凋亡率可分别为7.88±0.15％，14.16±0.35％，32.73±0.78％，与对照空白组0.50±0.06％和SODN转染组0.57±0.09％比较有显著性差异(P＜0.05)。在加用阿霉素后，600ng／mlASODN组的细胞凋亡率可达40.34±0.78％，与单独使用ADM和转染ASODN后的凋亡率16.94±0.54％和15.07±0.35％比较有显著性差异。在AO／EB染色中也可以发现ADM＋ASODN组内的比较多的细胞则发生了核固缩，浓染，破裂，出现了细胞凋亡的核形态变化，而MTT实验显示空白对照组与ADM组和ASODN组在转染细胞24小时后，细胞增殖抑制有明显的差异(P＜0.05)，而ADM＋ASODN组与其余三组比较均有显著性差异(P＜0.05)。
结论1．成功制备平均粒径87nm、分布均匀的PLA-CS纳米颗粒。用壳聚糖修饰该纳米颗粒表面，使其带正电荷从而可逆的结合质粒DNA，组装成PLA-CS纳米颗粒基因转运体系，并对细胞生长毒性低。转染效率较高。2．利用分子信标建立了一种能在单细胞水平上检测活体细胞中基因转录产物的方法，有望发展成为一种能早期检测肿瘤标志物的方法。3．采用PLA-CS纳米基因载体介导转染针对Survivin设计的ASODN，转染效率较高，ASODN发挥了高效的封闭作用，PLA-CS纳米基因载体是一种安全，高效的新型的纳米基因载体。采用PLA-CS纳米基因载体介导转染SurvivinASODN,能有效的封闭Survivin基因，下调Survivin蛋白及mRNA的表达，并促进Bel7402细胞的凋亡。采用ASODN封闭Survivin的表达确实有效地抑制肝癌细胞的生长，增加细胞的凋亡。从而增加ADM的化疗敏感性，改善常规化疗的效果，具有临床应用的潜能。
Objects Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in our country, which is gravely threatening people's health because of its high malignant degree and bad prognosis.Exairesis is still the first-line and most effective treatment to HCC nowadays. Chemotherapy, as an important adjunctive treatment to HCC, has to settle the two tough difficulties of cytotoxicity to normal cells and drug resistance of tumor cells. It is researched that Survivin is the most powerful inhibiting apoptosis factor ever been found with the dual function of inhibiting apoptosis and regulating cellcycle, and it has a close relationship with chemoresistance. Nanoparticle gene transporter, an non-viral gene transporter ,is well developed in recent years because of its safety, efficacy and fine property. In this paper: 1. to prepare polylactic-chitosan(PLA-CS)modified nanoparticles and analyze its properties and vitro transfection efficiency; 2. to test directly using molecular beacon which is a new living cell detection technique and prove that Survivin is high expressed in Be17402; 3.to use Survivin antisense oligodeoxynucleotide (ASODN) loaded PLA-CS nanoparticles to enhance the apoposis of HCC induced by adriamycin(ADM).
Methods 1.to prepare PLA-CS NP with small particle diameter and uniform distribution using solvent emulsionizated volatilixation method and with optimized conditions of technique; to detect Zeta electric potentials and particle diameter distributions of PLA-CS NP and PLA-CS/DNA using Malvern Zetasizer 3000 E; to detect particle diameter and appearance of PLA-CS NP by Atomic force microscope (AFM). Then to observe the combination of PLA-CS NP and DNA and the PLA-CS's protection to DNA by electrophoresis; to detect the transfection efficiency and cytotoxicity of PLA-CS NP in vitro by MTT assay and Flow cytometer (FCM); 2. to transfect molecular beacon designed according to Survivin into HL7702 and Be17402 via PLA-CS NP, then to detect the changes of Survivin transcripts, and to detect the influence of cell growth caused by molecular beacon using Typan blue staining; to confirm the accuracy of molecular beacon detection using RT-PCR;3. to transfect the Survivin ASODN loaded PLA-CS nanoparticles into hepatoma carcinoma cells, then to detect the change of Survivin by methods of RT-PCR and Western blot, to detect cytostasis using MTT assay, and to detect apoptosis rate of HCC using FCM. After acting with ADM, to detect cytostasis using MTT assay and to observe the change of cell apoptotic index using FCM and AO/EB staining.
Results 1.Detection results of the particle diameter and distribution using laser particle size analysor Malvem Zetasizer 3000E showed that prepared PLA-CS NP had small mean diameter of 87 nm and the particle diameter had well Gaussian distribution. It was observed by AFM that NPs were global ones with smooth and complete surface and without adhering and agglomeration. Zeta electric potential was+10mV±1.5mV when PLA-CS NP coupled with DNA. It was analyzed from electrophoresis results that NP and plasmid DNA can be combined together effectively. MTT results showed inhibition ratio of cell growth was less than 10% as the concentration of PLA-CS NP was below 3μg/μl.The transfection efficiency can reach 43.5% detected by FCM. 2. After transfecting molecular beacon loaded PLA-CS NP into HL7702 and Be17402, it was observed that there was hardly any fluorescence in HL7702 when strong green fluorescence signal was found in Be17402. Trypan blue staining essay showed cell vigor was uninfluenced basically. RT-PCR confirmed that the Survivin mRNA in Be17402 was higher expressed than that in HL7702. 3.24 hours after all the groups having been transfected by Survivin ASODN, the mRNA and expression of proteins of them all decreased, especially the one of the group with the concentration of 800ng/ml of Survivin ASODN, and there was significant difference with the control group(P＜0.05); MTT assay results showed that all cells group's proliferation activity degraded disparity after having been transfected by survivn ASODN. FCM detection results showed that 24 hours after the efficient of ASODN the apoptosis rates were 7.88+0.15%, 14.16+0.35%, 32.73+0.78% respectively in the Be17402 groups with concentration of 400ng/ml, 600ng/ml and 800ng/ml, and there was significant difference contrasting to blank group with apoptosis rates of 0.50+0.06% and SODN transfected group with apoptosis rates of 0.57+0.09%(P＜0.05). After the acting of ADM, apoptosis rate of Be17402 was 40.34+0.78% in the group with concentration of 600ng/ml,and there was significant difference contrasting to the apoptosis rates of 16.94+0.54% or 15.07+0.35% respectively in groups using ADM or ASODN transfected only. AO/EB staining results showed more cells in ADM+ASODN group had the change of nucleus morphous including karyopycnosis, thick stain and disruption of nucleus. MTT assay results showed that the cytostasis degree had obvious difference (P＜0.05) between blank control group with ADM group or ASODN group 24 hours after having been transfected.,and there was significant difference between ADM+ASODN group with each of other three groups (P＜0.05).
Conclusions 1.PLA-CS NP with mean diameter of 87 nm and even distribution had been prepared successfully. The NP whose surface had been modified by CS to enable it have positive charge can combine with plasmid DNA reversibly, then PLA-CS NP gene transport system with low cytotoxicity and high transfection efficiency was assembled. 2. That a method of detecting genetic transcript in living monoplast cells by using molecular beacon can be expected to be well developed as a method of detecting tumor markers in the early stage. 3. The transfection induced by Survivin ASODN which has efficient function of blocking loaded PLA-CS NP gene carrier which is safe and efficient has high transfection efficiency. Using PLA-CS NP gene carrier to induce the transfection of Survivin ASODN, it can be more efficient to block survivin and down regulate the expressions of protein and mRNA of surviving which can promote the apoptosis of Be17402 finally. Using ASODN to block expression of survivin can inhibit the growth of HCC efficiently and increase the apoptosis of cell. Also, that can improve the chemosensibility of ADM and the effect of routine chemotherapy. All these make it potential in clinical application.
引文
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