【作者】 彭程；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 生物医学工程， 2007， 博士

【摘要】 目的:研究表明p53基因在绝大部分肿瘤中存在高突变率,这种突变与肿瘤细胞的多药耐药有着密切关系。本研究拟探讨采用磁性纳米颗粒介导wtp53基因转染人肝癌耐药细胞株HepG2/5-Fu诱导其发生凋亡及其耐药的研究。研究方法:(1)人肝癌耐药细胞株HepG2/5-Fu多药耐药性分析,检测HepG2/5-Fu细胞株对不同抗肿瘤药物的敏感性。(2)流式细胞仪分析HepG2/5-Fu细胞在5-Fu作用的凋亡情况以及对5-Fu的耐受分析。(3)RT-PCR分析HepG2/5-Fu细胞株中凋亡相关基因p53、bcl-2、bcl-xl、bax等基因的表达情况。(4)SSCP方法分析HepG2/5-Fu细胞株中p53基因的突变情况。(5)制备IONP-PLL纳米颗粒,构建氧化铁磁性纳米转运体系,分析其生物学效应。(6)以IONP-PLL纳米颗粒为基因转运载体,将wtp53基因转入HepG2/5-Fu耐药性细胞株,观察5-Fu对转染有wtp53基因的HepG2/5-Fu细胞的杀伤效应。结果:(1)HepG2/5-Fu细胞与HepG2细胞相比除对5-Fu耐药外,对其它几种常用化疗药均有不同程度的耐药性,其中对5-氟尿嘧啶耐药倍数达107.0倍,对紫杉醇的耐药倍数达49.0倍。(2)HepG2细胞中未经5-Fu处理的对照组、30μmol/L 5-Fu组及150μmol/L 5-Fu组的凋亡指数分别为(6.5±1.0)%、(14.0±4.0)%和(20.0±5.0)%;30μmol/L 5-Fu组和150μmol/L5-Fu组与对照组相比,凋亡分别增加了1.2倍和2.1倍,差异具有显著性(P<0.05)。而经同样浓度5-Fu处理的HepG2/5-Fu细胞中对照组、30μmol/L 5-Fu组及150μmol/L 5-Fu组的凋亡指数分别为(6.7±0.7)%、(7.1±2.2)%和(6.5±2.0)%:30μmol/L5-Fu组150μmol/L 5-Fu组与其对照组相比,细胞凋亡变化不明显。(3)经30μmol/L和150μmol/L 5-Fu处理后,HepG2细胞与其对照组相比,呈现明显的G0/G1期增加,S期、G2/M减少,而HepG2/5-Fu细胞与其对照组相比细胞周期未见改变。(4)PCR分析发现与HepG2细胞相比,HepG2/5-Fu细胞耐药株bcl-xl、bcl-xs和bax的表达增高,而p53和cpp32的表达减少,特别是p53基因,表达明显减少。在人肝癌细胞HepG2和HepG2/5-Fu细胞mRNA水平均没有检测到bcl-2的表达。(5)应用碱沉淀法制成的氧化铁纳米颗粒经葡聚糖修饰后形成的DCIONP-PLL纳米颗粒粒径小,直径47±15nm,分布均匀,悬浮性好。在酸性的条件下,IONP具有结合DNA的能力,但是在中性和碱性的条件下,DNA结合能力差。在IONP和DNA的质量比为1∶1时,pH=3时,IONP和DNA结合力强。IONP与DNA的质量比为2:1时,结合率达98%。在酸性的条件下,氧化铁纳米颗粒可保护DNA不被降解。(6)在中性环境下,IONP-PLL有很强的DNA结合力,且随着IONP-PLL和DNA质量比的增加,IONP-PLL的DNA结合力不断增强。IONP-PLL能保护DNA抵御核酸酶的降解。IONP-PLL和IONP-PLL/DNA复合物对于细胞几乎没有毒性,,但随着IONP-PLL的增加,对细胞的毒性略有增加。(7)转染wtp53基因的HepG2/5-Fu细胞对5-Fu呈剂量依赖性杀伤效应和时间依赖性杀伤效应,在5-Fu作用下,能发生明显的凋亡效应。结论:(1)wtp53基因的突变与肝癌细胞的细胞周期的改变、耐药性的产生相关,是肝癌细胞HepG2/5-Fu产生多药耐药性的机制之一。(2)在氧化铁纳米颗粒表面修饰多聚赖氨酸,使氧化铁纳米颗粒表面能可逆结合生物治疗用的核酸分子,该纳米颗粒能作为高效的基因转运载体,且毒性小,安全可靠。(3)以wtp53基因为靶点的肝癌基因治疗有望成为治疗晚期肝癌的一种新的治疗策略。更多还原

【Abstract】 Object:The research indicated the p53 gene has the high rate of mutation in the major tumor, this kind of mutuation change and the tumor cell multi-medicines drug resistant to have the close relation. This research plans to discuss that the wtp53 gene was transfected into cell line HepG2/5-Fu of the human liver cancer using magnetic nano particles t to analyses the apoptosis and drug resisitant induced by 5-Fu.Methods:(1) Analyze multi-medicine drug resistance the of human liver cancer cell line HepG2/5-Fu in order to examines the HepG2/5-Fu cell line to the different anti-tumor medicine sensitivity.(2) Analyze the to apoptosiss and the 5-Fu tolerance of HepG2/5-Fu cell induced by 5-Fu through FAS.(3) Analyzes apoptosiss correlation genes expression on the HepG2/5-Fu cell line such as p53, bcl-2, bcl-xl, bax et al.by RT-PCR.(4) Analyzes the p53 gene mutaion in the HepG2/5-Fu cell line through the SSCP method.(5) Prepares IONP-PLL nano particles to construct the oxidation ferromagnetism nanometer transportation system, and analyzes its biology effect.(6) The wtp53 gene was transfected into the HepG2/5-Fu by IONP-PLL nano particles as the gene transportation carrier, observes the lethal effect that the cell line HepG2/5-Fu was treated with 5-Fu.Result:(1) The HepG2/5-Fu cell has the varying degree drug resistance to 5-Fu and other several kind of commonly used chemotherapy medicine compared with HepG2 , among of total, the drug resistance to 5-Fu reach 107.0 times, to Paclitaxel reach 49.0 times compared with the drug resistanct of HepG2.(2) When treated the HepG2 with the concent ration of 5-Fu (0μmo/L , 30μmo/L or 150μmol/L), the AI was (6.5±1.0) %、(14.0±4.0) % and (20.0±5.0) %, inspectively.The apoptosis rateincreased 1.2 time and 2.1 time compared with not treated with 5-Fu, inspectively, which wrer significantly ( P< 0.05) .But treated the HepG2/5-Fu with the same concent ration of 5-Fu (30μmo/L or 150μmol/L), the AI was (6.7±0.7) %、(7.1±2.2)% and (6.5±2.0)(3) When treated with the same concent ration of 5-Fu (30μmo/L or 150μmol/L) , the proportion of apoptosis was significantly increased and G0/G1 phase was increased and S and G2/M phases were reduced in HepG2 cells , but the proportion of apoptosis and cell cycle was not changed in HepG2/5-Fu cells. (4) The expression of bcl-xl , bcl-xs , and bax mRNA were significantly increased and the expression of p53 and cpp32 mRNA were significantly decreased in resistant HepG2/5-Fu cells , as compared with HepG2 cells by RT-PCR.(5) IONP was prepared by alkaline precipitation of divalent and trivalent iron chloride in the presence of high concentration of dextran. In this system, iron oxide nanoparticles was equably dispersed in surfactant in suspend state.The diameter of IONP-PLLIDNA was 47nm±15nm. Negative staining showed DNA bourid on the surface of IONP-PLL. The DNA binding assay showed that at acid pH, IONP can bind DNA, but at pH 7 and 9, IONP has no the potential to bind DNA. The binding is achieved by interaction between the negatively charged DNA phosphate groups and positively charged gene vectors. When titrated to pH 3, the IONP showed an increase binding of plasmid DNA by this type of particle. The electronstatistic interaction can also protect DNA against the digestion of nuclease at acid pH.(6) Whether IONP-PLL and IONP-PLL/DNA complexes influenced cell viability was investigated in several cell lines by MTT assays. IONP-PLL and IONP-PLL/DNA complexes showed no obvious negative effect on cell viability.(7) The cell line HepG2/5-Fu tansfected wtp53 displayed the dosage dependence effect and the time dependence effect to 5-Fu. The cell line HepG2/5-Fu tansfected wtp53 appeared bviously apoptosis induced by 5-Fu. Conclusion:(1) wtp53 gene mutation was relative with cell cycle change and the drug resistance of liver cancer, which is one of multi-medicine drug resistance mechanisms of the liver cancer cell HepG2/5-Fu.(2)Poly-L-Lysine modified Iron Oxide Nanoparticles (IONP-PLL) system was optimized to enhance the gene delivery efficiency. IONP-PLL/DNA complexes showed no obvious negative effect on cell viability and safty.(3) Take the wtp53 gene as the target to treat later period liver cancer shall be hopefully one kind of new treatment strategy.更多还原