pll-DCIONP介导野生型p53基因转染对耐顺铂肺腺癌靶向治疗和耐药影响的研究
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摘要
研究背景
肺癌是全球恶性肿瘤死亡的主要原因。肺癌将成为21世纪对人类健康威胁最大的恶性肿瘤。虽然化疗药物的发展大大的改善了肺癌的临床疗效，但肺癌患者的5年生存率仍然低于15％。顺铂耐药是导致化疗失败、影响肺癌治愈率和远期生存率的主要原因。因此，寻找有效的方法和制剂逆转肺癌耐药具有十分重要的意义。
p53基因突变是肺癌中发生频率最高的遗传改变。50％的非小细胞肺癌(NSCLC)和70％以上的小细胞肺癌(SCLC)中均可检测到p53基因突变。研究表明：p53基因突变能刺激肿瘤血管生成和肿瘤转移使肿瘤细胞具有更强的侵袭性，还能“序列特异性反式激活”耐药基因启动子，诱导耐药基因表达，对化疗及放疗的敏感性下降，明显降低了放化疗的效果；研究还表明突变型p53蛋白表达率高的患者预后明显较差。因此阻断突变型或功能失活性p53癌基因成为肺癌治疗靶点及逆转多药耐药的最重要策略之一。
有效的基因治疗除了安全有效的治疗基因外，安全高效的基因导入系统也至关重要。目前基因治疗研究面临的严峻挑战之一就是基因治疗的载体系统。基因导入载体包括两大类，即病毒和非病毒载体，病毒载体由于其高转导效率和较好的靶向性而成为肿瘤基因治疗中应用最广泛的方法，但是病毒载体自身具有能诱导宿主免疫反应、潜在的致瘤性、装载容量有限、代价高等缺点；非病毒型载体因具有安全、低毒、装载容量大、操作简单等优势而逐渐引起学者们的关注，但转染效率偏低是其最大缺陷。因此，寻找即安全又有效的基因导入载体非常必要。
纳米技术的出现为解决基因转移载体提供了新的思路。纳米颗粒因为具有小尺寸效应，表面效应，随着颗粒直径变小，比表面积将会显著增大，故具有很高的化学活性，因而纳米成为了最有应用前景的非病毒载体。采用纳米载体转运核苷酸有很多优越性：①有助于核苷酸高效率转染细胞；②能够靶向定位输送核苷酸；③能有效保护核苷酸，防止体内生物酶的降解，逃脱体内网状内皮系统的吞噬作用；④无机纳米粒本身具有杀伤癌细胞的作用，且对正常细胞无损害。磁性纳米除了具有一般纳米颗粒的特性外，还具有超顺磁性，即在磁场中有较强的磁性，没有磁场时，磁性很快消失，从而不会被永久磁化；在外加磁场的作用下，能携带核苷酸实现定向移动，并进行缓释、控制性基因传递，从而实现基因靶向治疗。
本实验对耐顺铂人肺腺癌细胞株A549／CDDP第5～8外显子进行基因测序，检测其突变情况；构建氧化铁磁性纳米颗粒并作为载体介导野生型p53基因(wild-type p53，wt-p53)，进行DNA结合实验、抵抗DNase-Ⅰ消化和血清保护实验，在外加磁场引导下将其转染入耐顺铂人肺腺癌细胞株，检测其转染情况，探讨其对耐顺铂肺腺癌细胞增殖、凋亡及耐药影响，寻求提高肺癌铂类药物化疗疗效的有效途径；并将其转染入耐顺铂肺腺癌动物模型，在外加磁场下，探讨其高效持续的靶向抗肿瘤作用和逆转顺铂耐药作用；并初步探讨氧化铁磁性纳米颗粒转运体系在体内外的毒性情况。
一、氧化铁磁性纳米颗粒作为野生型p53基因载体并转染耐顺铂肺腺癌细胞的可行性研究
目的体外评价氧化铁磁性纳米颗粒(pll-DCIONP)作为体外野生型p53基因(wt-p53)载体并转染耐顺铂人肺癌细胞的可行性方法1．以碱沉淀法制成外包葡聚糖的氧化铁磁性生物纳米颗粒(DCIONP)，在其表面修饰多聚赖氨酸制成多聚赖氨酸-氧化铁纳米颗粒(pll-DCIONP)，扫描电镜观察其形态特征，用激光粒度检测仪测定其粒度分布和Zeta表面电位。2．分光光度计及琼脂糖凝胶电泳分析pll-DCIONP与wt-p53基因的结合力及其复合物抵抗DNase-Ⅰ和血清消化的作用。3．进行纳米颗粒pll-DCIONP携带编码绿色荧光蛋白的EGFP-C2质粒的体外报告基因转染实验，荧光显微镜和流式细胞术观察和分析其转染效率。4．pll-DCIONP作为wt-p53基因载体转染耐顺铂肺腺癌细胞A549／CDDP，RT-PCR以及western blot分析细胞内p53基因的表达。5．MTT法分析纳米颗粒pll-DCIONP转运体系的细胞毒性。结果1．pll-DCIONP的直径在60～80纳米(nanometer，nm)之间，分散状态良好，其Zeta电位无论在酸性、中性或碱性环境里均为正电荷。2．pll-DCIONP无论在酸性、中性及碱性的条件下，均可与wt-p53基因结合，以二者质量比为1：1时结合力最强；pll-DCIONP／wt-p53复合物能抵抗DNase-Ⅰ和血清对wt-p53基因的消化作用。3．pll-DCIONP作为报告基因载体转染肺腺癌细胞，在磁场引导作用下，pll-DCIONP的转染效率明显高于脂质体(P＜0.01)。4．以pll-DCIONP作为wt-p53基因载体转染肺腺癌细胞，随着时间延长细胞内p53基因含量持续增高，而以脂质体作为转染载体时随着时间延长细胞内p53基因含量递减。5．pll-DCIONP转运体系对细胞无明显细胞毒性。结论pll-DCIONP可作为野生型p53基因理想的转染载体之一，并能持续高效地将外源性p53基因转染入耐顺铂肺腺癌细胞中。
二、氧化铁磁性纳米颗粒介导的野生型p53基因转染对耐顺铂人肺腺癌细胞体外持续生长抑制、凋亡诱导作用及耐药的影响
目的1．检测耐顺铂肺腺癌A549／CDDP细胞第5～8外显子基因突变情况。2．探讨pll-DCIONP介导wt-p53基因转染对耐顺铂肺腺癌细胞A549／CDDP体外持续生长抑制、凋亡诱导作用及耐药的影响。方法1．利用DNA测序方法对A549／CDDP细胞第5～8外显子进行基因突变分析。2．MTT法观察pll-DCIONP介导wt-p53基因转染对A549／CDDP细胞持续生长抑制作用。3．应用荧光显微镜、流式细胞仪观察其对A549／CDDP细胞的凋亡作用，RT-PCR检测其对A549／CDDP细胞凋亡促进基因Bax mRNA表达的影响；激酶法检测其对A549／CDDP细胞Caspase-3表达活性的影响。4．MTT法分析其对A549／CDDP细胞耐药性的影响，并计算转染前后的耐药指数的变化。5．RT-PCR以及细胞免疫组化法分析其对A549／CDDP细胞内肺癌耐药蛋白LRP表达的影响。结果1．A549／CDDP细胞p53基因第5外显子第82位核苷酸发生C→A点突变。2．pll-DCIONP介导wt-p53基因转染对A549／CDDP细胞具有持续生长抑制作用，呈时间依赖性和浓度依赖性；联合顺铂时其抑制作用得到进一步放大。而脂质体作为转染载体时，其生长抑制作用短暂；单独使用顺铂干预A549／CDDP细胞，对细胞生长抑制作用不明显，与对照组比较无差异(P＞0.05)。3．pll-DCIONP介导wt-p53基因转染可引起A549／CDDP细胞凋亡改变，表现为细胞变圆变小，细胞核固缩，核边集，核碎裂等，细胞凋亡率明显增高，并能显著上调Bax mRNA的表达和Caspase-3活性，呈时间依赖性和持续性；联合顺铂时其上述作用得到进一步放大。而脂质体作为转染载体时，其上述凋亡诱导作用时间短暂，转染第1天上述其凋亡诱导作用与pll-DCIONP作为转染载体比较无明显差别(P＞0.05)，但第3天后其凋亡诱导作用明显减弱，与pll-DCIONP作为转染载体同一时间点比较存在显著差异性。4．以pll-DCIONP和脂质体作为wt-p53基因转染载体，均可使A549／CDDP细胞对顺铂的IC_(50)及耐药倍数下降；但以pll-DCIONP作为wt-p53基因转染载体时A549／CDDP细胞对顺铂的IC_(50)及耐药倍数下降更为明显(P＜0.01)。5．pll-DCIONP介导wt-p53基因转染A549／CDDP细胞，能持续下调细胞内肺癌耐药蛋白LRP的表达，转染第3天后其作用明显强于脂质体作为基因转染载体。结论1．采用恒定顺铂药物浓度、周期性作用的体外诱导法反复筛选建成的一株耐顺铂的A549细胞系发生了p53基因的突变和p53蛋白构象的改变。2．pll-DCIONP介导wt-p53基因转染对A549／CDDP细胞具有持续生长抑制和凋亡诱导作用。3．pll-DCIONP介导wt-p53基因转染较大程度上逆转了A549／CDDP细胞对顺铂的耐药，通过下调肺癌耐药蛋白(LRP)的表达可能是其逆转耐药的机制。
三、氧化铁磁性纳米颗粒介导的野生型p58基因转染靶向治疗耐顺铂人肺腺癌裸鼠移植瘤的实验研究
目的1．建立耐顺铂人肺腺癌细胞A549／CDDP移植瘤裸鼠模型。2．体内探讨pll-DCIONP介导的wt-p53基因靶向治疗耐顺铂人肺腺癌移植瘤的可行性。方法1．常规体外培养A549／CDDP细胞，采用皮下注射法建立移植瘤裸鼠动物模型。2．在外加磁场作用下，pll-DCIONP介导的wt-p53基因转染尾静脉注射裸鼠，观察肿瘤生长情况，称瘤重，计算肿瘤生长指数。3病理组织切片和HE染色观察pll-DCIONP介导的wt-p53基因转染对肝脾肾的损伤作用。4．通过流式细胞术检测细胞凋亡率。5．实时荧光定量PCR以及免疫组化法分析其对细胞内肺癌耐药蛋白LRP表达的影响。结果1．pll-DCIONP介导的wt-p53基因转染尾静脉注射裸鼠无明显毒副作用，HE染色表明肝脾肾均无明显的损伤。2．pll-DCIONP-wtp53组移植瘤不但生长缓慢，部分瘤体体积出现缩小，瘤重和生长指数分别为0.698±0.226g和1.153±0.657，明显低于对照组和顺铂组(P＜0.001)，与脂质体-wtp53组也存在差异(P＜0.01)，pll-DCIONP-wtp53组移植瘤联合顺铂时其作用更为明显；单独使用顺铂组与对照组比较无明显差别(P＞0.05)。3．pll-DCIONP-wtp53组移植瘤细胞凋亡率为42.49±11.76％，明显低于对照组(10.62±3.89％)和顺铂组(10.47±4.12％)(P＜0.001)，与脂质体-wtp53组(18.63±5.26％)也存在差异(P＜0.01)，pll-DCIONP-wtp53组移植瘤联合顺铂时其凋亡率更为明显；单独使用顺铂组与对照组凋亡率比较无明显差别(P＞0.05)。4．pll-DCIONP-wtp53组移植瘤组织LRP表达明显减弱，明显低于对照组和顺铂组(P＜0.001)，与脂质体-wtp53组也存在差异(P＜0.01)，pll-DCIONP-wtp53组移植瘤联合顺铂时其减弱程度更为明显。结论1．纳米颗粒pll-DCIONP能携带wt-p53基因在磁场引导作用下体内靶向发挥抗肿瘤作用，并增强肿瘤细胞对顺铂的化疗敏感性，其作用明显强于脂质体作为wt-p53基因载体。2．纳米颗粒pll-DCIONP介导wt-p53基因转染能明显下调耐顺铂肺腺癌裸鼠肿瘤组织LRP的表达，从机理上解释了其逆转耐药的可能机制；其作用也明显强于脂质体作为wt-p53基因载体。3．纳米颗粒pll-DCIONP在体内具有良好的生物安全性。
Background
As one of the leading causes of death among all the malignant tumorsin the world, lung cancer will become the biggest health-killer in the 21 stcentury. Great improvements have made in the efficacy of treating lungcancer with chemotherapy. However, the overall 5-year survival rate ofpatients is less than 15％. A majority of patients with lung cancer rapidlydevelop resistance to cisplatin causing therapy failure. For this reason, itis of importance to search for an appropriate and effective way to reversecisplatin resistance in lung cancer.
p53 mutations are the most frequently found of genetic change in thelung cancer.Mutations in the p53 gene have been identified with a highfrequency of 50％and 70％in NSCLC and SCLC respectively. It isthought that p53 mutations could stimulate angiogenesis and metastasisof neoplasm, make neoplasm more metastatic, transactivate promoter ofdrug resistance gene in a sequence-specific way and induce expression ofdrug resistance gene so as to show a poor treatment response tochemotherapy and radiation. Furthermore patients with mutated p53presented a poor survival. For this reason, replacing mutated p53 orfunctionally inactivating p53 will be one of the most important strategiesof therapeutic gene targeting and reversal of multidrug resistance.
Efficient and nontoxic gene delivery and expression of deliveredexogenous gene are important determinant of gene therapy. It is also adraconic task for research of gene therapy. Nowadays gene deliverysystems are mainly divided into viral vectors and non-viral vectors. Theviral vectors have very strong capabilities of delivering genes so that it isused most extensively in gene therapy of tumor. But viral vectors areassociated with immunogenicity, toxicity, lack of tissue specificity,difficulty in large-scale production and potential risk of inducing tumorigenic mutations. Non-viral vectors have become an attractivealternative because it can be easily scaled up, produced at relatively lowcosts and with less toxicity. But most non-viral vectors are limited in lowefficiency of transfection, especially in vivo. Therefore it is verynecessary to search for efficient and non-toxic targeted gene deliverysystems.
The development of nanotechnology has provided us new ways toovercome barrier of gene delivery vectors. The nanoparticles can beeffectively endocytosed by the cells resulting in a high cellular uptake ofthe entrapped DNA because of its subcellular size. There are manyadvantages of nanoparticles as gene delivery vectors: (1) They have ahigh efficiency for nucleotide delivery. (2) They can deliver nucleotide tothe target size. (3) They can protect nucleotide from degradation bydigestive enzyme and phagocytosis by reticuloendothelial system. (4)They have the abilities of killing cancer cells, but not normal cells.Magnetic nanoparticles have character of superparamagnetism besidesthose mentioned above. Magnetic nanoparticles can deliver the focusednucleotide to the target site/cells via high-field/high-gradient magnets.Thus the nucleotide is released slowly from the nanoparticles resulting insustained intracellular gene delivery and it plays a role of targeting genetherapy.
The authors examined p53 mutations in cisplatin-resistant humanlung adenocarcinoma cells A549/CDDP. Poly-L-Lysine modified ironoxide magnetic nanoparticles(pll-DCIONP) was synthesized as wt-p53gene carriers, The potential adsorbing wt-p53 gene and resistingDNase-Ⅰand blood serum digestion of pll-DCIONP/wt-p53 complexswas analyzed, magnetic field used to direct movement of pll-DCIONPand A549/CDDP cells was transfected with wt-p53 DNA-loadednanoparticles. The efficiency of transfection was analyzed to study effectof magnetic iron oxide nanoparticle-mediated wild-type p53 genedelivery on sustained antiproliferative activity and apoptosis in human lung adeno-carcinoma cell lines A549/CDDE The authors further treatedA549/CDDP xenograft in nude mice with an intravenous injection ofwt-p53 DNA-loaded nanoparticles so as to study effect of highly efficientand targeted tumor-killing, reversal of cisplatin resistance and in vitro andvivo toxicity of pll-DCIONP delivery systems.
PartⅠUse of magnetic iron oxide nanoparticles as wt-p53 genecarrier and transfeetion with it in cisplatin-resistant lungadenocarcinoma cells
Objective To evaluate the feasibility of using iron oxidenanoparticles as gene carder and transfecting with it in cisplatin-resistantlung adenocarcinoma cells. Methods 1.The dextran coated iron oxidenanoparticles (DCIONP) was synthesized with deposition. The surface ofDCIONP was modified by self-assembled poly-L-lysine to form particlecomplexes (pll-DCIONP).The configuration of pll-DCIONP was detectedby SEM. The diameter and Zeta surface potential of pll-DCIONP weredetected by Zetasizer. 2. The potential adsorbing wt-p53 gene andresisting DNase-Ⅰand blood serum digestion of pll-DCIONP wasanalyzed by spectrophotometer and agarose gel electrophoresis. 3. To testthe ability of pll-DCIONP to transfer gene in vitro, assays of delivery ofreporter plasmid DNA into A549/CDDP cells, The efficiency oftransfection was analyzed by fluorescent microscope and flow cytometry.4. The pll-DCIONP was evaluated as a kind of wt-p53 gene carrier bytransfecting human lung adenocarcinoma cell line A549/DDP in vitro.The expression of intracellular p53 gene was analyzed by RT-PCR andWestern blot. 5. The toxicity of delivery systems of pll-DCIONP in vitrowas measured by MTT. Results 1. The diameter of the pll-DCIONP is60～80nm with a satisfactory dispersed status, the Zeta surface potentialof pll-DCIONP is positively charged at different pH. 2. Under thedifferent condition of pH, the pll-DCIONP have the potential to adsorbwt-p53 gene and the potential of adsorbing was the strongest aspll-DCIONP/wt-p53 complexes prepared at w/w ratio of 1:1, pll-DCIONP/wt-p53 complex have the potential to resist DNase-Ⅰandblood serum digestion. 3. Magnetic field was used to direct movement ofpll-DCIONP capable of transferring reporter plasmid DNA into cells evenmore efficiently than the lipids examined in vitro(P＜0.01). 4. Cellstransfected with wt-p53 DNA-loaded nanoparticles pll-DCIONPdemonstrated a sustained and increased p53 gene levels with incubationtime as opposed to a decrease in gene levels in the cells transfected withthe wt-p53 DNA-lipofectamine complex. 5. There was no significanttoxicity of delivery systems of pll-DCIONP. Conclusion pll-DCIONPwill become one of favored gene carriers for wt-p53 gene delivery and ithave the potential of transfecting the wt-p53 gene in cisplatin-resistantlung adenocarcinoma cells with a sustained and significantly efficientgene expression.
PartⅡMagnetic iron oxide nanoparticle-mediated wild-type p53gene delivery resulted in sustained antiproliferative activity,apoptosis and its efficacy of reversing resistance in cisplatin-resistanthuman lung adenocarcinoma cells
Objective 1. To examine p53 mutations in cisplatin resistant humanlung adeno-carcinoma cells lines A549/CDDP. 2. To study effect ofmagnetic iron oxide nanoparticle-mediated wild-type p53 gene deliveryon sustained antiproliferative activity, apoptosis and its efficacy ofreversing resistance in cisplatin-resistant human lung adenocarcinomacell lines A549/CDDP. Methods 1. Mutation of p53 gene was screenedby DNA direct sequencing in cell lines A549 and A549/CDDP. 2.Theanti-proliferative effect of magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery against A549/CDDP cells was tested by MTT.3. The apoptosis of cells transfected with wt-p53 DNA-loadedpll-DCIONP was detected by fluorescent microscope. The apoptosis ratioof tumor cells transfected with wt-p53 DNA-loaded pll-DCIONP wasmeasured with flow cytometry. The variation of expression ofpro-apoptosis gene Bax mRNA of cells transfected with wt-p53 DNA-loaded pll-DCIONP was determined by RT-PCR. The activity ofCaspase-3 of cells transfected with wt-p53 DNA-loaded pll-DCIONP wasmeasured by colorimetric assay. 4. The resistance to cisplatin of cellsA549/CDDP was evaluated by MTT and the resistance index of cellscalculated respectively before and after transfection with wt-p53DNA-loaded pll-DCIONP. 5. The expression of mRNA and protein oflung resistance protein (LRP) of cells transfected with wt-p53DNA-loaded pll-DCIONP was measured by RT-PCR and IHC SP assayrespectively. Results 1. There was C to A transversion in 82nd nucleotideof 5th exon of cells A549/CDDP. 2. The anti-proliferative effect was moresustained and greater in cells transfected with wt-p53 DNA-loadedpll-DCIONP than the DNA-lipofectamine complex. The anti-proliferativeeffects became stronger with incubation time and with an increase in thecase of pll-DCIONP. The anti-proliferative effect became stronger withwt-p53 DNA-loaded pll-DCIONP and low-does cisplatin together,whereas the effect was transient and lasted for only 1 day when the cellswere transfected with DNA-lipofectamine complex and the inhibitoryeffect with DNA-lipofectamine complex did not extend beyond 3 dayspost-transfection. There was no significant difference in theanti-proliferative effect of cells cultivated with low-does cisplatin withcontrols(P＞0.05). 3. As compared with the controls, cells transfected withwt-p53 DNA-loaded pll-DCIONP caused typical apoptotic changes,including distinct morphological changes characterized by chromatincondensation, nuclear shrinkage and nuclear cleavage. And the cells grewmore rounded, smaller and some floated. The apoptosis ratio and activityof Caspase-3 were more sustained and higher in cells transfected withwt-p53 DNA-loaded pll-DCIONP than the DNA-lipofectamine complex.The apoptosis ratio grew higher with incubation time in the case ofpll-DCIONP, The apoptosis ratio and activity of Caspase-3 became higherwith wt-p53 DNA-loaded pll-DCIONP and low-does cisplatin together,whereas the effect was transient and lasted for only 1 day when the cells were transfected with DNA-lipofectamine complex and the apoptosiseffect and activity of Caspase-3 with DNA-lipofectamine complex didnot extend beyond 3 days post-transfection. There was no significantdifference in the apoptosis effect and activity of Caspase-3 of cellscultivated with low-does cisplatin with control(P＞0.05).pll-DCIONP-mediated wild-type p53 gene delivery could up-regulatelevels of expression of Bax mRNA of A549/CDDP cell. Meanwhile itdemonstrated a higher Bax mRNA level compared to cells transfectedwith wt-p53 DNA-lipofectamine complex(P＜0.05). 4. The IC_(50) andresistance index to cisplatin were down-regulated in cells transfected withwt-p53 DNA-loaded pll-DCIONP and the DNA-lipofectamine complex,but the effect of down-regulate was more significant in cells transfectedwith wt-p53 DNA-loaded pll-DCIONP than the DNA-lipofectaminecomplex(P＜0.01). 5. Cells transfected with wt-p53 DNA-loadedpll-DCIONP demonstrated a relatively lower level of LRP mRNA andprotein than the wt-p53 DNA-lipofectamine complex on 3rd and 5th dayin this study. Conclusion 1. Genetic and protein pattern changes werefound in p53 mutations. 2. Magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery resulted in sustained antiproliferative activityand apoptotic effects on cisplatin-resistant human lung adenocarcinomacell lines A549/CDDP. 3. Magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery resulted in a reversal effect of cisplatinresistance in cisplatin-resistant human lung adenocarcinoma cell linesA549/CDDP, Down-regulating the levels of cellular expression of LRPmay be one of mechanism of resistance reversal.
PartⅢMagnetic iron oxide nanoparticle-mediated wild-type p53gene delivery resulted in targeting gene treatment with cisplatin-resistant human lung adenocarcinoma xenograft in nude mice
Objective To explore the feasibility of magnetic iron oxidenanoparticle-mediated wild-type p53 gene delivery results in targetinggene treatment with cisplatin-resistant human lung adeno-carcinoma xenograff in nude mice. Methods 1. Cisplatin-resistant cell linesA549/CDDP were cultured routinely and A549/CDDP cells implanted tosubcutaneous in nude mice to establish cisplatin-resistant xenograftanimal model. 2. Magnetic field was used to direct movement ofpll-DCIONP. The wt-p53 DNA-loaded pll-DCIONP was injectedintravenously and the tumor growth was then observed. Volumes andweight of tumor mass were detected respectively. Then tumor growthindex was thereby calculated. 3. Then specimens were fixed byparaformaldehyde and then paraffin section, Hematoxylin and eosin (HE)staining and histological examination performed. 4. Apoptotic index intumor tissues was measured by flow cytomety. 5. Expression of lungresistance protein (LRP) mRNA was measured through real-time PCRand expression of LRP protein detected by IHC SP assay. Results 1.There was no obvious side-effect for treatment following the wt-p53DNA-loaded pll-DCIONP to be injected intravenously and liver, kidneyand spleen tissues was proved no distinct injured by HE staining assay. 2.Treatment with single wt-p53 DNA-loaded pU-DCIONP, The growth ofxenograft was slow even part of them shrank. The weight of tumor massand growth index of tumor were 0.698±0.226g and 1.153±0.657respectively and it was significantly less than control groups andCDDP groups treated with low-does cisplatin(P＜0.001) and also lessthan Lip groups treated with the wt-p53 DNA-lipofectaminecomplex(P＜0.01). The effect became stronger with wt-p53 DNA-loadedpll-DCIONP and low-does cisplatin to be co-administrated. 3. Treatmentwith single wt-p53 DNA-loaded pll-DCIONP, The apoptotic index oftumor tissues was 42.49±11.76％and it was significantly more thancontrol groups (10.62±3.89％)and CDDP groups (10.47±4.12％)treated with low-does cisplatin(P＜0.001). And it was also more thanLip groups(18.63±5.26％) treated with the wt-p53 DNA-lipofectaminecomplex(P＜0.01). The effect became stronger with wt-p53 DNA-loadedpll-DCIONP and low-does cisplatin to be co-administrated. 4. Treatment with single wt-p53 DNA-loaded pll-DCIONP, The expression of LRP oftumor tissues was down-regulated and it was significantly less thancontrol groups and CDDP groups treated with low-doescisplatin(P＜0.001). And it was also less than Lip groups treated withthe wt-p53 DNA-lipofectamine complex(P＜0.01). The levels ofexpression of LRP dropped with wt-p53 DNA-loaded pll-DCIONP andlow-does cisplatin to be co-administrated. Conclusion 1. Magnetic ironoxide nanoparticle-mediated wild-type p53 gene delivery resulted intargeting anti-tumor with cisplatin resistant human lung adeno-carcinomaxenograft in vivo and reversed the resistance in cisplatin resistant humanlung adenocarcinoma xenograft in vivo. The effect grew stronger thanxenograft treated with the wt-p53 DNA-lipofectamine complex. 2.Down-regulating the expression levels of of LRP in tumor tissues may beone of mechanism of resistance reversal. 3. There was no significant invivo toxicity of delivery systems of pll-DCIONP.
肺癌是全球恶性肿瘤死亡的主要原因。肺癌将成为21世纪对人类健康威胁最大的恶性肿瘤。虽然化疗药物的发展大大的改善了肺癌的临床疗效，但肺癌患者的5年生存率仍然低于15％。顺铂耐药是导致化疗失败、影响肺癌治愈率和远期生存率的主要原因。因此，寻找有效的方法和制剂逆转肺癌耐药具有十分重要的意义。
p53基因突变是肺癌中发生频率最高的遗传改变。50％的非小细胞肺癌(NSCLC)和70％以上的小细胞肺癌(SCLC)中均可检测到p53基因突变。研究表明：p53基因突变能刺激肿瘤血管生成和肿瘤转移使肿瘤细胞具有更强的侵袭性，还能“序列特异性反式激活”耐药基因启动子，诱导耐药基因表达，对化疗及放疗的敏感性下降，明显降低了放化疗的效果；研究还表明突变型p53蛋白表达率高的患者预后明显较差。因此阻断突变型或功能失活性p53癌基因成为肺癌治疗靶点及逆转多药耐药的最重要策略之一。
有效的基因治疗除了安全有效的治疗基因外，安全高效的基因导入系统也至关重要。目前基因治疗研究面临的严峻挑战之一就是基因治疗的载体系统。基因导入载体包括两大类，即病毒和非病毒载体，病毒载体由于其高转导效率和较好的靶向性而成为肿瘤基因治疗中应用最广泛的方法，但是病毒载体自身具有能诱导宿主免疫反应、潜在的致瘤性、装载容量有限、代价高等缺点；非病毒型载体因具有安全、低毒、装载容量大、操作简单等优势而逐渐引起学者们的关注，但转染效率偏低是其最大缺陷。因此，寻找即安全又有效的基因导入载体非常必要。
纳米技术的出现为解决基因转移载体提供了新的思路。纳米颗粒因为具有小尺寸效应，表面效应，随着颗粒直径变小，比表面积将会显著增大，故具有很高的化学活性，因而纳米成为了最有应用前景的非病毒载体。采用纳米载体转运核苷酸有很多优越性：①有助于核苷酸高效率转染细胞；②能够靶向定位输送核苷酸；③能有效保护核苷酸，防止体内生物酶的降解，逃脱体内网状内皮系统的吞噬作用；④无机纳米粒本身具有杀伤癌细胞的作用，且对正常细胞无损害。磁性纳米除了具有一般纳米颗粒的特性外，还具有超顺磁性，即在磁场中有较强的磁性，没有磁场时，磁性很快消失，从而不会被永久磁化；在外加磁场的作用下，能携带核苷酸实现定向移动，并进行缓释、控制性基因传递，从而实现基因靶向治疗。
本实验对耐顺铂人肺腺癌细胞株A549／CDDP第5～8外显子进行基因测序，检测其突变情况；构建氧化铁磁性纳米颗粒并作为载体介导野生型p53基因(wild-type p53，wt-p53)，进行DNA结合实验、抵抗DNase-Ⅰ消化和血清保护实验，在外加磁场引导下将其转染入耐顺铂人肺腺癌细胞株，检测其转染情况，探讨其对耐顺铂肺腺癌细胞增殖、凋亡及耐药影响，寻求提高肺癌铂类药物化疗疗效的有效途径；并将其转染入耐顺铂肺腺癌动物模型，在外加磁场下，探讨其高效持续的靶向抗肿瘤作用和逆转顺铂耐药作用；并初步探讨氧化铁磁性纳米颗粒转运体系在体内外的毒性情况。
一、氧化铁磁性纳米颗粒作为野生型p53基因载体并转染耐顺铂肺腺癌细胞的可行性研究
目的体外评价氧化铁磁性纳米颗粒(pll-DCIONP)作为体外野生型p53基因(wt-p53)载体并转染耐顺铂人肺癌细胞的可行性方法1．以碱沉淀法制成外包葡聚糖的氧化铁磁性生物纳米颗粒(DCIONP)，在其表面修饰多聚赖氨酸制成多聚赖氨酸-氧化铁纳米颗粒(pll-DCIONP)，扫描电镜观察其形态特征，用激光粒度检测仪测定其粒度分布和Zeta表面电位。2．分光光度计及琼脂糖凝胶电泳分析pll-DCIONP与wt-p53基因的结合力及其复合物抵抗DNase-Ⅰ和血清消化的作用。3．进行纳米颗粒pll-DCIONP携带编码绿色荧光蛋白的EGFP-C2质粒的体外报告基因转染实验，荧光显微镜和流式细胞术观察和分析其转染效率。4．pll-DCIONP作为wt-p53基因载体转染耐顺铂肺腺癌细胞A549／CDDP，RT-PCR以及western blot分析细胞内p53基因的表达。5．MTT法分析纳米颗粒pll-DCIONP转运体系的细胞毒性。结果1．pll-DCIONP的直径在60～80纳米(nanometer，nm)之间，分散状态良好，其Zeta电位无论在酸性、中性或碱性环境里均为正电荷。2．pll-DCIONP无论在酸性、中性及碱性的条件下，均可与wt-p53基因结合，以二者质量比为1：1时结合力最强；pll-DCIONP／wt-p53复合物能抵抗DNase-Ⅰ和血清对wt-p53基因的消化作用。3．pll-DCIONP作为报告基因载体转染肺腺癌细胞，在磁场引导作用下，pll-DCIONP的转染效率明显高于脂质体(P＜0.01)。4．以pll-DCIONP作为wt-p53基因载体转染肺腺癌细胞，随着时间延长细胞内p53基因含量持续增高，而以脂质体作为转染载体时随着时间延长细胞内p53基因含量递减。5．pll-DCIONP转运体系对细胞无明显细胞毒性。结论pll-DCIONP可作为野生型p53基因理想的转染载体之一，并能持续高效地将外源性p53基因转染入耐顺铂肺腺癌细胞中。
二、氧化铁磁性纳米颗粒介导的野生型p53基因转染对耐顺铂人肺腺癌细胞体外持续生长抑制、凋亡诱导作用及耐药的影响
目的1．检测耐顺铂肺腺癌A549／CDDP细胞第5～8外显子基因突变情况。2．探讨pll-DCIONP介导wt-p53基因转染对耐顺铂肺腺癌细胞A549／CDDP体外持续生长抑制、凋亡诱导作用及耐药的影响。方法1．利用DNA测序方法对A549／CDDP细胞第5～8外显子进行基因突变分析。2．MTT法观察pll-DCIONP介导wt-p53基因转染对A549／CDDP细胞持续生长抑制作用。3．应用荧光显微镜、流式细胞仪观察其对A549／CDDP细胞的凋亡作用，RT-PCR检测其对A549／CDDP细胞凋亡促进基因Bax mRNA表达的影响；激酶法检测其对A549／CDDP细胞Caspase-3表达活性的影响。4．MTT法分析其对A549／CDDP细胞耐药性的影响，并计算转染前后的耐药指数的变化。5．RT-PCR以及细胞免疫组化法分析其对A549／CDDP细胞内肺癌耐药蛋白LRP表达的影响。结果1．A549／CDDP细胞p53基因第5外显子第82位核苷酸发生C→A点突变。2．pll-DCIONP介导wt-p53基因转染对A549／CDDP细胞具有持续生长抑制作用，呈时间依赖性和浓度依赖性；联合顺铂时其抑制作用得到进一步放大。而脂质体作为转染载体时，其生长抑制作用短暂；单独使用顺铂干预A549／CDDP细胞，对细胞生长抑制作用不明显，与对照组比较无差异(P＞0.05)。3．pll-DCIONP介导wt-p53基因转染可引起A549／CDDP细胞凋亡改变，表现为细胞变圆变小，细胞核固缩，核边集，核碎裂等，细胞凋亡率明显增高，并能显著上调Bax mRNA的表达和Caspase-3活性，呈时间依赖性和持续性；联合顺铂时其上述作用得到进一步放大。而脂质体作为转染载体时，其上述凋亡诱导作用时间短暂，转染第1天上述其凋亡诱导作用与pll-DCIONP作为转染载体比较无明显差别(P＞0.05)，但第3天后其凋亡诱导作用明显减弱，与pll-DCIONP作为转染载体同一时间点比较存在显著差异性。4．以pll-DCIONP和脂质体作为wt-p53基因转染载体，均可使A549／CDDP细胞对顺铂的IC_(50)及耐药倍数下降；但以pll-DCIONP作为wt-p53基因转染载体时A549／CDDP细胞对顺铂的IC_(50)及耐药倍数下降更为明显(P＜0.01)。5．pll-DCIONP介导wt-p53基因转染A549／CDDP细胞，能持续下调细胞内肺癌耐药蛋白LRP的表达，转染第3天后其作用明显强于脂质体作为基因转染载体。结论1．采用恒定顺铂药物浓度、周期性作用的体外诱导法反复筛选建成的一株耐顺铂的A549细胞系发生了p53基因的突变和p53蛋白构象的改变。2．pll-DCIONP介导wt-p53基因转染对A549／CDDP细胞具有持续生长抑制和凋亡诱导作用。3．pll-DCIONP介导wt-p53基因转染较大程度上逆转了A549／CDDP细胞对顺铂的耐药，通过下调肺癌耐药蛋白(LRP)的表达可能是其逆转耐药的机制。
三、氧化铁磁性纳米颗粒介导的野生型p58基因转染靶向治疗耐顺铂人肺腺癌裸鼠移植瘤的实验研究
目的1．建立耐顺铂人肺腺癌细胞A549／CDDP移植瘤裸鼠模型。2．体内探讨pll-DCIONP介导的wt-p53基因靶向治疗耐顺铂人肺腺癌移植瘤的可行性。方法1．常规体外培养A549／CDDP细胞，采用皮下注射法建立移植瘤裸鼠动物模型。2．在外加磁场作用下，pll-DCIONP介导的wt-p53基因转染尾静脉注射裸鼠，观察肿瘤生长情况，称瘤重，计算肿瘤生长指数。3病理组织切片和HE染色观察pll-DCIONP介导的wt-p53基因转染对肝脾肾的损伤作用。4．通过流式细胞术检测细胞凋亡率。5．实时荧光定量PCR以及免疫组化法分析其对细胞内肺癌耐药蛋白LRP表达的影响。结果1．pll-DCIONP介导的wt-p53基因转染尾静脉注射裸鼠无明显毒副作用，HE染色表明肝脾肾均无明显的损伤。2．pll-DCIONP-wtp53组移植瘤不但生长缓慢，部分瘤体体积出现缩小，瘤重和生长指数分别为0.698±0.226g和1.153±0.657，明显低于对照组和顺铂组(P＜0.001)，与脂质体-wtp53组也存在差异(P＜0.01)，pll-DCIONP-wtp53组移植瘤联合顺铂时其作用更为明显；单独使用顺铂组与对照组比较无明显差别(P＞0.05)。3．pll-DCIONP-wtp53组移植瘤细胞凋亡率为42.49±11.76％，明显低于对照组(10.62±3.89％)和顺铂组(10.47±4.12％)(P＜0.001)，与脂质体-wtp53组(18.63±5.26％)也存在差异(P＜0.01)，pll-DCIONP-wtp53组移植瘤联合顺铂时其凋亡率更为明显；单独使用顺铂组与对照组凋亡率比较无明显差别(P＞0.05)。4．pll-DCIONP-wtp53组移植瘤组织LRP表达明显减弱，明显低于对照组和顺铂组(P＜0.001)，与脂质体-wtp53组也存在差异(P＜0.01)，pll-DCIONP-wtp53组移植瘤联合顺铂时其减弱程度更为明显。结论1．纳米颗粒pll-DCIONP能携带wt-p53基因在磁场引导作用下体内靶向发挥抗肿瘤作用，并增强肿瘤细胞对顺铂的化疗敏感性，其作用明显强于脂质体作为wt-p53基因载体。2．纳米颗粒pll-DCIONP介导wt-p53基因转染能明显下调耐顺铂肺腺癌裸鼠肿瘤组织LRP的表达，从机理上解释了其逆转耐药的可能机制；其作用也明显强于脂质体作为wt-p53基因载体。3．纳米颗粒pll-DCIONP在体内具有良好的生物安全性。
Background
As one of the leading causes of death among all the malignant tumorsin the world, lung cancer will become the biggest health-killer in the 21 stcentury. Great improvements have made in the efficacy of treating lungcancer with chemotherapy. However, the overall 5-year survival rate ofpatients is less than 15％. A majority of patients with lung cancer rapidlydevelop resistance to cisplatin causing therapy failure. For this reason, itis of importance to search for an appropriate and effective way to reversecisplatin resistance in lung cancer.
p53 mutations are the most frequently found of genetic change in thelung cancer.Mutations in the p53 gene have been identified with a highfrequency of 50％and 70％in NSCLC and SCLC respectively. It isthought that p53 mutations could stimulate angiogenesis and metastasisof neoplasm, make neoplasm more metastatic, transactivate promoter ofdrug resistance gene in a sequence-specific way and induce expression ofdrug resistance gene so as to show a poor treatment response tochemotherapy and radiation. Furthermore patients with mutated p53presented a poor survival. For this reason, replacing mutated p53 orfunctionally inactivating p53 will be one of the most important strategiesof therapeutic gene targeting and reversal of multidrug resistance.
Efficient and nontoxic gene delivery and expression of deliveredexogenous gene are important determinant of gene therapy. It is also adraconic task for research of gene therapy. Nowadays gene deliverysystems are mainly divided into viral vectors and non-viral vectors. Theviral vectors have very strong capabilities of delivering genes so that it isused most extensively in gene therapy of tumor. But viral vectors areassociated with immunogenicity, toxicity, lack of tissue specificity,difficulty in large-scale production and potential risk of inducing tumorigenic mutations. Non-viral vectors have become an attractivealternative because it can be easily scaled up, produced at relatively lowcosts and with less toxicity. But most non-viral vectors are limited in lowefficiency of transfection, especially in vivo. Therefore it is verynecessary to search for efficient and non-toxic targeted gene deliverysystems.
The development of nanotechnology has provided us new ways toovercome barrier of gene delivery vectors. The nanoparticles can beeffectively endocytosed by the cells resulting in a high cellular uptake ofthe entrapped DNA because of its subcellular size. There are manyadvantages of nanoparticles as gene delivery vectors: (1) They have ahigh efficiency for nucleotide delivery. (2) They can deliver nucleotide tothe target size. (3) They can protect nucleotide from degradation bydigestive enzyme and phagocytosis by reticuloendothelial system. (4)They have the abilities of killing cancer cells, but not normal cells.Magnetic nanoparticles have character of superparamagnetism besidesthose mentioned above. Magnetic nanoparticles can deliver the focusednucleotide to the target site/cells via high-field/high-gradient magnets.Thus the nucleotide is released slowly from the nanoparticles resulting insustained intracellular gene delivery and it plays a role of targeting genetherapy.
The authors examined p53 mutations in cisplatin-resistant humanlung adenocarcinoma cells A549/CDDP. Poly-L-Lysine modified ironoxide magnetic nanoparticles(pll-DCIONP) was synthesized as wt-p53gene carriers, The potential adsorbing wt-p53 gene and resistingDNase-Ⅰand blood serum digestion of pll-DCIONP/wt-p53 complexswas analyzed, magnetic field used to direct movement of pll-DCIONPand A549/CDDP cells was transfected with wt-p53 DNA-loadednanoparticles. The efficiency of transfection was analyzed to study effectof magnetic iron oxide nanoparticle-mediated wild-type p53 genedelivery on sustained antiproliferative activity and apoptosis in human lung adeno-carcinoma cell lines A549/CDDE The authors further treatedA549/CDDP xenograft in nude mice with an intravenous injection ofwt-p53 DNA-loaded nanoparticles so as to study effect of highly efficientand targeted tumor-killing, reversal of cisplatin resistance and in vitro andvivo toxicity of pll-DCIONP delivery systems.
PartⅠUse of magnetic iron oxide nanoparticles as wt-p53 genecarrier and transfeetion with it in cisplatin-resistant lungadenocarcinoma cells
Objective To evaluate the feasibility of using iron oxidenanoparticles as gene carder and transfecting with it in cisplatin-resistantlung adenocarcinoma cells. Methods 1.The dextran coated iron oxidenanoparticles (DCIONP) was synthesized with deposition. The surface ofDCIONP was modified by self-assembled poly-L-lysine to form particlecomplexes (pll-DCIONP).The configuration of pll-DCIONP was detectedby SEM. The diameter and Zeta surface potential of pll-DCIONP weredetected by Zetasizer. 2. The potential adsorbing wt-p53 gene andresisting DNase-Ⅰand blood serum digestion of pll-DCIONP wasanalyzed by spectrophotometer and agarose gel electrophoresis. 3. To testthe ability of pll-DCIONP to transfer gene in vitro, assays of delivery ofreporter plasmid DNA into A549/CDDP cells, The efficiency oftransfection was analyzed by fluorescent microscope and flow cytometry.4. The pll-DCIONP was evaluated as a kind of wt-p53 gene carrier bytransfecting human lung adenocarcinoma cell line A549/DDP in vitro.The expression of intracellular p53 gene was analyzed by RT-PCR andWestern blot. 5. The toxicity of delivery systems of pll-DCIONP in vitrowas measured by MTT. Results 1. The diameter of the pll-DCIONP is60～80nm with a satisfactory dispersed status, the Zeta surface potentialof pll-DCIONP is positively charged at different pH. 2. Under thedifferent condition of pH, the pll-DCIONP have the potential to adsorbwt-p53 gene and the potential of adsorbing was the strongest aspll-DCIONP/wt-p53 complexes prepared at w/w ratio of 1:1, pll-DCIONP/wt-p53 complex have the potential to resist DNase-Ⅰandblood serum digestion. 3. Magnetic field was used to direct movement ofpll-DCIONP capable of transferring reporter plasmid DNA into cells evenmore efficiently than the lipids examined in vitro(P＜0.01). 4. Cellstransfected with wt-p53 DNA-loaded nanoparticles pll-DCIONPdemonstrated a sustained and increased p53 gene levels with incubationtime as opposed to a decrease in gene levels in the cells transfected withthe wt-p53 DNA-lipofectamine complex. 5. There was no significanttoxicity of delivery systems of pll-DCIONP. Conclusion pll-DCIONPwill become one of favored gene carriers for wt-p53 gene delivery and ithave the potential of transfecting the wt-p53 gene in cisplatin-resistantlung adenocarcinoma cells with a sustained and significantly efficientgene expression.
PartⅡMagnetic iron oxide nanoparticle-mediated wild-type p53gene delivery resulted in sustained antiproliferative activity,apoptosis and its efficacy of reversing resistance in cisplatin-resistanthuman lung adenocarcinoma cells
Objective 1. To examine p53 mutations in cisplatin resistant humanlung adeno-carcinoma cells lines A549/CDDP. 2. To study effect ofmagnetic iron oxide nanoparticle-mediated wild-type p53 gene deliveryon sustained antiproliferative activity, apoptosis and its efficacy ofreversing resistance in cisplatin-resistant human lung adenocarcinomacell lines A549/CDDP. Methods 1. Mutation of p53 gene was screenedby DNA direct sequencing in cell lines A549 and A549/CDDP. 2.Theanti-proliferative effect of magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery against A549/CDDP cells was tested by MTT.3. The apoptosis of cells transfected with wt-p53 DNA-loadedpll-DCIONP was detected by fluorescent microscope. The apoptosis ratioof tumor cells transfected with wt-p53 DNA-loaded pll-DCIONP wasmeasured with flow cytometry. The variation of expression ofpro-apoptosis gene Bax mRNA of cells transfected with wt-p53 DNA-loaded pll-DCIONP was determined by RT-PCR. The activity ofCaspase-3 of cells transfected with wt-p53 DNA-loaded pll-DCIONP wasmeasured by colorimetric assay. 4. The resistance to cisplatin of cellsA549/CDDP was evaluated by MTT and the resistance index of cellscalculated respectively before and after transfection with wt-p53DNA-loaded pll-DCIONP. 5. The expression of mRNA and protein oflung resistance protein (LRP) of cells transfected with wt-p53DNA-loaded pll-DCIONP was measured by RT-PCR and IHC SP assayrespectively. Results 1. There was C to A transversion in 82nd nucleotideof 5th exon of cells A549/CDDP. 2. The anti-proliferative effect was moresustained and greater in cells transfected with wt-p53 DNA-loadedpll-DCIONP than the DNA-lipofectamine complex. The anti-proliferativeeffects became stronger with incubation time and with an increase in thecase of pll-DCIONP. The anti-proliferative effect became stronger withwt-p53 DNA-loaded pll-DCIONP and low-does cisplatin together,whereas the effect was transient and lasted for only 1 day when the cellswere transfected with DNA-lipofectamine complex and the inhibitoryeffect with DNA-lipofectamine complex did not extend beyond 3 dayspost-transfection. There was no significant difference in theanti-proliferative effect of cells cultivated with low-does cisplatin withcontrols(P＞0.05). 3. As compared with the controls, cells transfected withwt-p53 DNA-loaded pll-DCIONP caused typical apoptotic changes,including distinct morphological changes characterized by chromatincondensation, nuclear shrinkage and nuclear cleavage. And the cells grewmore rounded, smaller and some floated. The apoptosis ratio and activityof Caspase-3 were more sustained and higher in cells transfected withwt-p53 DNA-loaded pll-DCIONP than the DNA-lipofectamine complex.The apoptosis ratio grew higher with incubation time in the case ofpll-DCIONP, The apoptosis ratio and activity of Caspase-3 became higherwith wt-p53 DNA-loaded pll-DCIONP and low-does cisplatin together,whereas the effect was transient and lasted for only 1 day when the cells were transfected with DNA-lipofectamine complex and the apoptosiseffect and activity of Caspase-3 with DNA-lipofectamine complex didnot extend beyond 3 days post-transfection. There was no significantdifference in the apoptosis effect and activity of Caspase-3 of cellscultivated with low-does cisplatin with control(P＞0.05).pll-DCIONP-mediated wild-type p53 gene delivery could up-regulatelevels of expression of Bax mRNA of A549/CDDP cell. Meanwhile itdemonstrated a higher Bax mRNA level compared to cells transfectedwith wt-p53 DNA-lipofectamine complex(P＜0.05). 4. The IC_(50) andresistance index to cisplatin were down-regulated in cells transfected withwt-p53 DNA-loaded pll-DCIONP and the DNA-lipofectamine complex,but the effect of down-regulate was more significant in cells transfectedwith wt-p53 DNA-loaded pll-DCIONP than the DNA-lipofectaminecomplex(P＜0.01). 5. Cells transfected with wt-p53 DNA-loadedpll-DCIONP demonstrated a relatively lower level of LRP mRNA andprotein than the wt-p53 DNA-lipofectamine complex on 3rd and 5th dayin this study. Conclusion 1. Genetic and protein pattern changes werefound in p53 mutations. 2. Magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery resulted in sustained antiproliferative activityand apoptotic effects on cisplatin-resistant human lung adenocarcinomacell lines A549/CDDP. 3. Magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery resulted in a reversal effect of cisplatinresistance in cisplatin-resistant human lung adenocarcinoma cell linesA549/CDDP, Down-regulating the levels of cellular expression of LRPmay be one of mechanism of resistance reversal.
PartⅢMagnetic iron oxide nanoparticle-mediated wild-type p53gene delivery resulted in targeting gene treatment with cisplatin-resistant human lung adenocarcinoma xenograft in nude mice
Objective To explore the feasibility of magnetic iron oxidenanoparticle-mediated wild-type p53 gene delivery results in targetinggene treatment with cisplatin-resistant human lung adeno-carcinoma xenograff in nude mice. Methods 1. Cisplatin-resistant cell linesA549/CDDP were cultured routinely and A549/CDDP cells implanted tosubcutaneous in nude mice to establish cisplatin-resistant xenograftanimal model. 2. Magnetic field was used to direct movement ofpll-DCIONP. The wt-p53 DNA-loaded pll-DCIONP was injectedintravenously and the tumor growth was then observed. Volumes andweight of tumor mass were detected respectively. Then tumor growthindex was thereby calculated. 3. Then specimens were fixed byparaformaldehyde and then paraffin section, Hematoxylin and eosin (HE)staining and histological examination performed. 4. Apoptotic index intumor tissues was measured by flow cytomety. 5. Expression of lungresistance protein (LRP) mRNA was measured through real-time PCRand expression of LRP protein detected by IHC SP assay. Results 1.There was no obvious side-effect for treatment following the wt-p53DNA-loaded pll-DCIONP to be injected intravenously and liver, kidneyand spleen tissues was proved no distinct injured by HE staining assay. 2.Treatment with single wt-p53 DNA-loaded pU-DCIONP, The growth ofxenograft was slow even part of them shrank. The weight of tumor massand growth index of tumor were 0.698±0.226g and 1.153±0.657respectively and it was significantly less than control groups andCDDP groups treated with low-does cisplatin(P＜0.001) and also lessthan Lip groups treated with the wt-p53 DNA-lipofectaminecomplex(P＜0.01). The effect became stronger with wt-p53 DNA-loadedpll-DCIONP and low-does cisplatin to be co-administrated. 3. Treatmentwith single wt-p53 DNA-loaded pll-DCIONP, The apoptotic index oftumor tissues was 42.49±11.76％and it was significantly more thancontrol groups (10.62±3.89％)and CDDP groups (10.47±4.12％)treated with low-does cisplatin(P＜0.001). And it was also more thanLip groups(18.63±5.26％) treated with the wt-p53 DNA-lipofectaminecomplex(P＜0.01). The effect became stronger with wt-p53 DNA-loadedpll-DCIONP and low-does cisplatin to be co-administrated. 4. Treatment with single wt-p53 DNA-loaded pll-DCIONP, The expression of LRP oftumor tissues was down-regulated and it was significantly less thancontrol groups and CDDP groups treated with low-doescisplatin(P＜0.001). And it was also less than Lip groups treated withthe wt-p53 DNA-lipofectamine complex(P＜0.01). The levels ofexpression of LRP dropped with wt-p53 DNA-loaded pll-DCIONP andlow-does cisplatin to be co-administrated. Conclusion 1. Magnetic ironoxide nanoparticle-mediated wild-type p53 gene delivery resulted intargeting anti-tumor with cisplatin resistant human lung adeno-carcinomaxenograft in vivo and reversed the resistance in cisplatin resistant humanlung adenocarcinoma xenograft in vivo. The effect grew stronger thanxenograft treated with the wt-p53 DNA-lipofectamine complex. 2.Down-regulating the expression levels of of LRP in tumor tissues may beone of mechanism of resistance reversal. 3. There was no significant invivo toxicity of delivery systems of pll-DCIONP.
引文
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