【作者】 曾文胜；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2011， 博士

【摘要】 目的：设计与构建Mcl-1 siRNA质粒,明确Mcl-1 siRNA对肝癌细胞的治疗作用和CS-PEG纳米载体介导基因治疗的优势,通过体内外实验探讨奥沙利铂联合Mcl-1 siRNA对肝癌的协同杀伤作用,从而为临床治疗肝癌提供理论依据。方法：1.构建靶向Mcl-1 RNAi片段的pGCsi-H1/Neo/GFP质粒,并进行酶切鉴定和测序分析。2.通过接枝共聚法制备壳聚糖-聚乙二醇(CS-PEG)纳米粒,用基因：纳米粒材料比1：5制备基因纳米复合物,通过其形态、粒径、ζ电位、载药量、包封率、基因保护实验考察载体的理化特性以及基因转染效果。3.利用脂质体或CS-PEG纳米粒为载体将Mcll siRNA质粒转染入肝癌细胞中,对细胞内Mcl-1基因表达进行干扰。4.逆转录多聚酶链反应(RT-PCR)法检测转染Mcl-1 siRNA质粒后肝癌细胞中Mcl-1 mRNA的表达,Western blot法检测转染Mcl-1 siRNA质粒后肝癌细胞中Mcl-1蛋白的表达。5.MTT法检测Mcl-1 siRNA、奥沙利铂或联合用药对人肝癌细胞增殖抑制作用。6.流式细胞仪分析Mcl-1 siRNA、奥沙利铂对人肝癌细胞周期和凋亡的影响。7. Transwell侵袭实验检测Mcl-1 siRNA.奥沙利铂或联合用药对人肝癌细胞的侵袭力变化。8.建立肝癌动物模型,用基因纳米复合物作瘤内多次注射,对比奥沙利铂腹腔注射或联合用药组,绘制肿瘤生长曲线,切取肿瘤检测体积抑制率和瘤重抑制率,免疫组化染色观察肿瘤内部的Mcl-1蛋白的分布情况,Western blot法检测肿瘤组织中Mcl-1蛋白的表达水平。结果：1.重组质粒经酶切鉴定与测序证实构建成功,质粒在HepG2细胞中的转染率为65.80±3.67%。转染48 h后Mcl-1 mRNA水平和蛋白水平均明显低于空白对照组和阴性对照组(p<0.01), Mcl-1 mRNA的抑制率为68.2%,其蛋白水平的抑制率为71.2%。2.空白纳米粒粒径为68.9±12.3nm,粒度分布均匀,ζ电位为32±6.4mV。在基因：纳米粒为1：5(w/w)时制备的基因纳米复合物粒径为111.4±16.9nm,ζ电位为7.5±6.4mV,包封率为86.8±9.7%,载药量为31.2±5.3%,对基因有较好的保护作用。基因纳米复合物组最大转染效率为转染后72h(81.39±3.57%),明显强于脂质体组且持续作用时间长。转染后持续作用时间较脂质体和裸基因均要长,转染72h后对Mcl-1 mRNA抑制率较两对照组明显增强。3.MTT法检测结果显示不同的时间Mcl-1 siRNA对肝癌细胞均有抑制作用,基因纳米复合物组细胞抑制率显著高于纳米粒组和对照组(p<0.01),且与脂质体转染组相比作用时间越长(72h以上)抑制效果越明显(p<0.05)。奥沙利铂对肝癌细胞的抑制作用呈现出剂量、时间依赖性,其联合应用基因纳米复合物时对细胞的抑制作用为(71.46±6.44%),明显大于基因纳米复合物组(26.72±4.69%)和单药(20.30±2.10%)处理后的作用之和。4.流式细胞仪(FCM)分析结果显示纳米粒对肝癌细胞周期的影响不明显,奥沙利铂作用HepG2细胞后S期细胞增加,GO/G1期细胞减少。肝癌细胞经基因纳米复合物转染+奥沙利铂处理后的凋亡率为64.21%,明显高于基因纳米复合物组(28.24%)、奥沙利铂组(21.02%)(p<0.01)。5. Transwell侵袭实验结果显示基因纳米复合物组细胞侵过滤膜数目与CS-PEG纳米粒和对照组相比较差别明显(p<0.01)。基因纳米复合物+奥沙利铂联合作用后穿膜细胞数(53.60±16.64)明显少于对照组(176.70±12.46)、基因纳米复合物组(111.40±9.52)、奥沙利铂组(127.10±9.77)的细胞穿膜数(p<0.01)。6.裸鼠移植瘤动物模型体内实验结果显示联合治疗组、基因纳米复合物治疗组、药物组的肿瘤体积较NC阳性对照组、阴性对照组增长明显缓慢(p<0.01)。联合治疗组、基因纳米复合物治疗组、药物组的抑瘤率分别为69.24%、53.96%、49.47%,明显高于NC阳性对照组的12.16%(p<0.01)。组织病理学检查后发现各治疗组肝癌组织以中重度坏死为主,对照组呈轻度坏死；联合治疗组、基因纳米复合物治疗组中Mcl-1蛋白阳性表达率均有所降低,表达强度较弱。Western blot检测结果显示基因纳米复合物治疗、联合治疗后对肝癌移植瘤组织中Mcl-1的蛋白表达与对照组相比有明显的抑制(p<0.01)。结论：1.成功构建出的靶向Mcl-1的RNA干扰质粒对肝癌细胞内的Mcl-1表达具有明显抑制作用.2.制备出低细胞毒性的CS-PEG纳米载体,载Mcl-1 siRNA质粒后粒径约为111 nm,带正电荷,有很好的基因保护功能、较高的包封率和载药量,能高效率转染至细胞并有效抑制肝癌细胞中Mcl-1的表达,明显降低癌细胞的生存和转移侵袭能力。3.联合应用基因纳米复合物和奥沙利铂在体外可增强肝癌细胞对奥沙利铂的敏感性,抑制肿瘤细胞生长代谢,协同作用效果大大优于单基因或单药作用的效果,能有效的降低肝癌细胞的生长和侵袭力,具有明显的临床应用价值。4.联合应用基因纳米复合物和奥沙利铂在肝癌裸鼠移植瘤模型的体内研究中能明显抑制肿瘤组织的生长,在Mcl-1基因被沉默的情况下奥沙利铂的抑瘤作用更加明显,体积与重量明显减少,组织中癌细胞的异质性减弱,达到治疗的效果。5.证明Mcl-1 siRNA纳米复合物和奥沙利铂联合应用对肝癌具有更好的治疗效果,为临床上对肿瘤进行生物和化学联合治疗的方案提供了理论基础。更多还原

【Abstract】 ObjectiveTo design and construct a plasmid containing Mcl-1 siRNA. Identify the therapeutical effect of Mcl-1 siRNA on human hepatocellular carcinoma cells and the preponderance of gene therapy mediated by CS-PEG nanoparticals. Investigate the synergistic effect of oxaliplatin and Mcl-1 siRNA on hepatocellular carcinoma in vitro and in vivo. And provide theoretical base of clinical treatment of hepatocellular carcinoma.Methods1. The plasmid (pGCsi-Hl/Neo/GFP) containing short hairpin RNA (shRNA) that target at the myeloid cell leukemia-1 (Mcl-1) gene were constructed and identified using restriction enzyme analysis and sequencing analysis.2. Chitosan-polyethylene glycol particle (CS-PEG) was synthesized through stem grafting and copoly-merizing. Gene-nanoparticle compound was prepared at the ratio of 1:5 for gene-nanoparticle. The shape, size, zeta electric potential, envelopment rate and carry gene rate were inspected to evaluate characteristics of the vector and effect of gene transfection was detected.3. Human hepatocellular carcinoma cells were cultured in vitro and transfected with Mcl-1 siRNA by LipofectamineTM 2000 or CS-PEG nanoparticles which interfering the expression of Mcl-1. 4. The expression of Mcl-1 mRNA in human hepatocellular carcinoma cells which transfected with Mcl-1 siRNA was detected by semi-quantitive reverse transcription-polymerase chain reaction (RT-PER). The expression of Mcl-1 protein was detected by Western blot.5. The effects of Mcl-1 siRNA, oxaliplatin or combination treatment on the proliferation inhibit of HEPATOCELLULAR CARCINOMA cells by MTT assay.6. Cell cycle and cell apoptotic rate were assayed by Flow Cytometry (FCM) with the treatment of Mcl-1 siRNA or oxaliplatin.7. The invasion ability was detected through Transwell invasion experiment with the treatment of Mcl-1 siRNA, oxaliplatin or combination treatment.8. Naked mouse hepatocellular carcinoma animal model was established. Gene nanopatical compound was injected into tumor multiple, contrast to intraperitoneal injection of oxaliplatin or gene-drug combination treatment. Drew the growth curve of tumor and took out the tumor to detect volume inhibition ratio and weight inhibition ratio. Distribution of Mcl-1 in transplantation tumor was detected by Strepavidin-peroxidase immunohistochemistry staining. Expression of Mcl-1 protein in tumor tissues was detected by Western blot.Results1. The expression plasmid was confirmed by restriction enzyme analysis and sequencing analysis. The transfection rate of recombinant plasmid in HepG2 cells was 65.80±3.67%.48 hours after transfection the Mcl-1 mRNA and protein levels were significantly lower than that of the blank control group and the negative control group (p<0.01). Inhibition ratio of Mcl-1 mRNA was 68.2%. Inhibition ratio of Mcl-1 protein level was 71.2%.2. The blank nanoparticle size was 68.9±12.3nm and well-distributed. Its zeta electric potential was 32±6.4mV. In conditions of 1:5 for gene/nanoparticle, the size, zeta electric potential, envelopment rate and carry gene rate of gene nanoparticle compound prepared were 111.4±16.9nm,7.5±6.4mV,86.8±9.7% and 31.2±5.3% respectively. It also had very good protection to gene. The highest transfection rate of gene nanoparticle compound was 81.39±3.57%, which appeared after 72h transfection. The persistence time after transfection was longer than both of liposome and bare gene. And the inhibition rate after 72h of transfection was reinforced significantly too.3. MTT assay showed that Mcl-1 siRNA had inhibitory effect on HEPATOCELLULAR CARCINOMA cells by different times. The inhibition of gene nanoparticle compound was higher than nanoparticle group and control group markedly (p<0.01). Contrast to liposome transfect group, the inhibitory rate was increased significantly as the time increased (over 72h) (p< 0.05). The inhibition of oxaliplatin on HEPATOCELLULAR CARCINOMA cells showed a dose-time dependent manner. The inhibitory rate of combination treatment of oxaliplatin and gene nanoparticle compound was 71.46±6.44%, which was higher obviously than the sum of single gene group (26.72±4.69%) and single drug group (20.30±2.10%).4. FCM assay showed that nanopaticals had no obvious effect on cell cycle of hepatocellular carcinoma cells. Oxaliplatin arrested cell cycle in S phase with G0/G1 phase reducing. The apoptotic rate of combination treatment of oxaliplatin and gene nanoparticle compound was 64.21%, which was higher obviously than gene nanoparticle compound group (28.24%) and oxaliplatin group (21.02%) (p<0.01).5. Transwell invasion assay results showed that the invading-membrane cell number of gene nanopartical compound group was fewer than the number of CS-PEG nanoparticles group and the control group markedly (p<0.01). The trans-membrane cell number of combination treatment of oxaliplatin and gene nanoparticle compound was 53.60±16.64, which was lower significantly than the number of the control group (176.70±12.46), gene nanopartical compound group (111.40±9.52) and oxaliplatin group (127.10±9.77) (p<0.01).6. The results of transplantation tumor animal model in vivo showed that tumor volumes of combination treatment group, gene nanoparticle compound group and drug group growed slower than NC positive control group and negative control group significantly (p<0.01). The tumor-inhibition rates of combination treatment group, gene nanoparticle compound group and drug group were 69.24% ,53.96% and 49.47%, which were significantly higher than NC positive control group (12.16%) (p<0.01). Histopathological examination revealed that the treatment groups showed moderate to severe necrosis in hepatocellular carcinoma tissues while the control group showed mild necrosis. Mcl-1 protein positive expression was weaked in combination treatment group and gene nanoparticle compound group. Western blot results showed that Mcl-1 protein expression was inhibited significantly in transplantation tumor tissues of gene nanoparticle compound group and combination treatment group when compared with the control group (p<0.01).Conclusions1. The RNA interference plasmid targeting at Mcl-1 gene is constructed successfully. The Mcl-1 mRNA and protein expressions are suppressed significantly by this given plasmid.2. We prepare CS-PEG nanoparticles with a low cellular toxicity. The nanoparticles have contained Mcl-1 siRNA plasmid with diameter of about 111 nm, a positive charge, a good genetic protection, high encapsulation efficiency and drug loading. The compound can be transfected into hepatocellular carcinoma cells effectively, inhibit Mcl-1 expression and reduce survival and invasive ability of cancer cells significantly.3. Combination of gene nanoparticle compound and oxaliplatin in vitro can enhance the drug sensitivity of hepatocellular carcinoma cells and inhibit cell growth. Synergistic effect is much better than single-gene or single-drug effect and can reduce cell growth and invasion ability of hepatocellular carcinoma cells effectively, which has obvious clinical value.4. Combination treatment of gene nanoparticle compound and oxaliplatin in human hepatocellular carcinoma transplantation tumors in vivo can inhibit the growth of tumor, especially when Mcl-1 gene is silent the antitumor effect of oxaliplatin is more obvious such as the tumor size and weight decrease remarkably, the heterogeneity of cancer cells reduce, which achieves therapeutic effect.5. Combination treatment of Mcl-1 siRNA/nanopartical compound and oxaliplatin has a better effect on hepatocellular carcinoma. It provides a theoretical basis for clinical combination of biological and chemical therapy in cancers.更多还原