【作者】 李高鹏；

【导师】 陈孝平；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分羟基磷灰石纳米颗粒作为基因载体可行性的研究目的对羟基磷灰石纳米材料颗粒（nHAP颗粒）的物理属性进行检验看其是否可能作为基因载体。方法利用了Ca²⁺及左旋多聚赖氨酸（PLL）对其进行表面修饰；应用原子力学显微镜，透射电镜，Zeta电位分析仪分析羟基磷灰石纳米材料颗粒的颗粒大小，分布情况及颗粒表面的带电情况；应用琼脂糖凝胶电泳，兔血清消化法观察nHAP颗粒对质粒DNA的结合情况；利用了以上结果与目前基因转移到了最新理论要求比较，判断此材料是否可能作为基因转移载体并为基因转移提供科学依据。结果nHAP颗粒本身大小小于100nm，但由于颗粒表面本身带负电，不能包括有效地结合及保护DNA；经Ca²⁺修饰后，颗粒可带正电，可一定程度地结合和保护DNA，但聚合物本身的中性电与带负电的细胞膜结合，而且本身的颗粒则变地容易聚集而成为很大的颗粒，也不利于细胞的吞噬；经PLL修饰后，颗粒溶解性提高，直径变小，表面带正电，电泳结果显示PLL-nHAP颗粒可包括有效地结合和保护DNA免受兔血清的破坏，而且复合物表面的正电也包括有利于与细胞膜的结合与吞噬。无论哪种修饰方法，nHAP颗粒与DNA质量比必须达到了15才能完全结合和保护DNA。结论nHAP颗粒本身不可作为基因转移到了载体。经过PLL修饰后可作为基因转移载体，nHAP颗粒与DNA质量比必须达到了15才能完全结合和保护DNA。第二部分野生型P53，Rb抑癌基因真核细胞表达载体的构建目的：利用了分子生物学的基本原理构建野生型P53，Rb抑癌基因真核细胞表达载体。方法：（1）：Trizol法提取正常胎肝细胞L02 mRNA后，利用了RT-PCR克隆野生型P53基因的全长CDNA。利用了Xho和BamHⅠ分别酶切CDNA的两端后，与相同酶切的PEGFP-C2真核细胞表达载体连接后转化，提取重组质粒后进行酶切，PCR鉴定正确后，进行测序验证。（2）：Trizol法提取正常足月胎盘组织mRNA后，利用了巢式RT-PCR克隆野生型Rb基因的全长CDNA。利用了Xho和BamHⅠ分别酶切CDNA的两端后，与相同酶切的PBK-CMV真核细胞表达载体连接后转化，提取重组质粒后进行酶切，PCR鉴定正确后，进行测序验证。结果：（1）：成功克隆1.2kb野生型p53基因，并亚克隆到了4.7 kb真核细胞表达载体PEGFP-C2，p53基因与EGFP在一个阅读框内形成一个融合基因，其重组载体称为PEGFP-C2-p53。（2）：成功克隆2.9 kb野生型Rb基因，并亚克隆到了4.5kb真核细胞表达载体PBK-CMV内，其重组载体称为PBK-CMV-Rb。结论：成功构建人携带野生型P53，Rb基因真核表达质粒PEGFP-C2-P53，PBK-CMV-Rb。第三部分羟基磷灰石纳米在体外基因转染的研究目的研究新型非病毒载体羟基磷灰石（hydroxyapatite，HAP）纳米颗粒在体外对肝细胞癌细胞基因治疗中的作用及机制。方法（1） MTT法评价各个浓度HAP纳米颗粒对肝细胞癌细胞HePG2及正常肝细胞L02生长的影响，从而选择合适的剂量使用范围用于后续实验。（2）应用氯化钙修饰HAP纳米颗粒，以增强型绿色荧光蛋白基因（PEGFP-N1）为报告基因，联合脂质体共同作为基因载体转染肝细胞癌细胞HePG2，荧光显微镜下观察转染成功后，流式细胞术测定转染效率和平均荧光值，并与传统脂质体方法进行比较。（3）应用PLL修饰HAP纳米颗粒，以增强型绿色荧光蛋白基因（PEGFP-N1）为报告基因，作为基因载体转染肝细胞癌细胞HePG2，荧光显微镜下观察转染成功后，流式细胞术测定转染效率和平均荧光值，并与传统脂质体方法进行比较。（4）应用PLL修饰HAP纳米颗粒，以p53基因（PEGFP-C2-p53）目的基因，作为基因载体转染肝细胞癌细胞HePG2，荧光显微镜下观察转染成功后，流式细胞术测定癌细胞的凋亡率和死亡率，并与传统脂质体方法进行比较。结果（1）体外应用nHAP颗粒不能超过终浓度20ug／ml，联合脂质体应用nHAP颗粒不能超过终浓度5ug／ml。由于第一部分研究结果提示nHAP颗粒与DNA质量比必须达到了15才能完全结合和保护DNA。故后续浓度选用nHAP颗粒与DNA各为15ug／ml及1ug／ml。（2）氯化钙修饰的HAP纳米颗粒单独应用无基因转移功能。联合HAP纳米颗粒与脂质体作为基因转染的载体成功将绿色荧光蛋白基因导入HePG2细胞，其转染效率和平均荧光强度显著高于单用脂质体组(P^12h效率=0.002，P^36h效率=0.000，P^72h效率=0.000，P^36h强度=0.000，P^72h强度=0.000，P^all＜0.05)。（3） PLL修饰的nHAP颗粒可作为基因转移的载体。（4） PLL-nHAP颗粒可介导p53抑癌基因入进入癌细胞并促进其抑癌作用，由于纳米材料颗粒本身具包括有抗癌作用，其总体作用甚至高于传统脂质体法介导的基因治疗。结论（1） Ca²⁺修饰后HAP具包括有结合和保护质粒DNA的作用，单纯应用无基因转移能力。可以作为脂质体的辅助载体，提高转染效率，但联合脂质体增加对正常肝细胞的毒性。（2）单纯PLL修饰的nHAP颗粒可在安全剂量范围内介导基因转染肝细胞癌细胞，最适合后续体内基因治疗。（3）在合适的量，nHAP颗粒（PLL）可以引起肝细胞癌HePG2凋亡和死亡而对正常细胞影响较小，nHAP颗粒（PLL）可以介导抑癌基因P53入胞，加上其本身的抗癌作用，其抑癌效比脂质体包括有延迟性但更好。nHAP颗粒（PLL）适合作为体外肝细胞癌基因治疗的载体第四部分超液化碘油羟基磷灰石纳米制作抗癌特异性基因转移载体在动物体内的研究目的研究新型非病毒载体羟基磷灰石（hydroxyapatite．，HAP）纳米颗粒在动物体对肝细胞癌模型的基因治疗中的作用及机制。方法（1）肝功能法评价各个剂量的nHAP-PLL-PLL颗粒纳米颗粒对兔正常肝脏的的影响，从而选择合适的剂量使用范围用于后续实验。（2）为了判断各组剂型组合，给药方式及脂质体对转染效率的影响。分别设立以下各组比较转染效率。每组3只兔子。A组：NS+PEGFPC2组（插管），B组：nHAP颗粒+PEGFPC2组（插管），C组：nHAP颗粒+PEGFPC2组（局部注射），D组：超液化碘油nHAP颗粒+PEGFPC2组（插管），E组：超液化碘油nHAP颗粒+PEGFPC2组（局部注射），F组：脂质体+PEGFPC2组（插管），G组：脂质体+PEGFPC2组（局部注射），H组：脂质体+超液化碘油nHAP颗粒+PEGFPC2组（插管）。灌注后三天后处死兔子消化法提取肿瘤细胞，流式细胞术检测转染效率。确定最高转染效率组为后续实验。（3）观察肝动脉灌注超液化碘油羟基磷灰石纳米粒（nHAP颗粒）与P53基因，Rb基因对兔VX2肝肿瘤生长，瘤兔生存期及肝功能的影响，同时观察其对p53，Rb，BCRP，G-pg，CD34，VEGF，TWIST及表达的影响。实验设生理盐水插管组26只（A组），nHAP颗粒超液化碘油插管组35只（B组），超液化碘油nHAP颗粒-P53复合物插管组40只（C组），超液化碘油nHAP颗粒-Rb复合物插管组35只（D组），超液化碘油nHAP颗粒+（Rb+P53）插管组35只（E组）。共171只兔子。结果（1） nHAP-PLL颗粒剂量在5-50mg／kg时，经肝动脉给药对兔肝VX2肿瘤的生长具包括有抑制作用，并能提高瘤兔的生存期，肝功能损害是可逆的。为了保证安全性，我们选择5mg／kg的nHAP颗粒与0.33mg／kg DNA用于后续实验（2）肝动脉插管时，单纯纳米材料颗粒没包括有肝脏肿瘤基因转移效应，必须配合超液化碘油发挥其阻滞作用，才能转移基因；局部注射无须超液化碘油即可发挥转移效应，联合超液化碘油效率可增加，但效率均极低（荧光显微镜下看不到）；肝动脉插管效率明显高于局部注射，插管和超液化碘油包括有协同提高转染效率；联合脂质体不能提高超液化碘油nHAP颗粒的基因转染效率。（3）超液化碘油nHAP颗粒联合基因短期内可能影响肝功能，但一周内可以恢复。超液化碘油nHAP颗粒可携带抑癌基因入胞，短期内抑制肿瘤生长，两者包括有协同作用。长期作用不明显。超液化碘油nHAP颗粒联合抑癌基因可以提高瘤兔生存时间。联合双基因治疗效果更好。结论经肝动脉灌注超液化碘油nHAP颗粒可介导的p53和Rb可联合用于治疗兔VX2肝细胞癌，从而提高抗癌作用，延长瘤兔生存时间。其机制包括如利用下调多种肿瘤恶性行为相关基因来实现。更多还原

【Abstract】 PartⅠThe study of the feasibility for the hydroxyapatite nanoparticleas the gene transfer vectorObjective: To evaluate the physical characters of hydroxyapatite nanoparticle that arerelated to the gene delivery of sythenic material.Methods: The diameter, the surface zeta-pontential of the nanoparticle was investigatedby the atomic force microscope （AFM）, transmission electron microscopy （TEM） andzeta-potential analyzer. The DNA combination and protection assay was evaluated by theagarose gel electrophoresis method.Results: The diameter of the nHAP is less than 100nm. However, the negative pontentialof its surface can’t bind the DNA effectively. After the surface modification of nHAP byCa2+, the nanoparticle can indeed combine and protect the DNA, whereas, the morebigger diameter may also hinder the effective gene transfer through the cell membrane.PLL modification changed the very negative zeta-potential of nHAP into a positivelycharged nHAP-PLL nanoplex. The nanoplex were smaller than the unmodified nHAP; thelipiodol/nanoplex emulsion, with the mean diameter at 75±11.8nm can disperse moreuniformly and be more soluble and stable, contrary to the unmodified nHAP and nanoplex Conclusion: Nanoplex/pDNA mass ratios greater than 15 （w/w） had exhibited completepDNA absorption onto positive charged polyplex and pDNA protection from thedestruction of nucleinase in rabbit serum. Unmodified nHAP may can’t induce effectivegene delivery.PartⅡThe construction of the recombint eukaryotic expressive vector ofwild-type human p53 and RbObjective: To construct the recombint eukaryotic expressive vector of wild-type humanp53 and Rb by utilizing the theory of molecular medicine.Methods: （1） The p53 gene, cloned from normal L02 cells after the mRNA is extracted byTrizol, was subcloned into the C-terminal of EGFP in PEGFP-C2 （CMV promoter, BDBiosciences, USA） by using BamHI and XhoI restriction enzymes. Subsequent sequenceanalysis was done following the PCR and enzyme cutting indentification. （2） The Rb gene,cloned from the tissue of normal placenta by nest PCR after the mRNA is extracted byTrizol, was subcloned into the PBK-CMV by using BamHI and XhoI restriction enzymes.Subsequent sequence analysis was done following the PCR and enzyme cuttingindentification.Results: （1）: The 1.2 kb wild-type p53 was cloned and subcloned to the 4.7 kbPEGFP-C2 vector, with the sequence reported in gene bank （No. AF307851）, with theEGFP reading frame linked in frame to the p53 gene by intervening amino acids"QISSSSFEF". The recombinant vector was called PEGFP-C2-P53. （2）: The 2.9Kb Rbgene, cloned from human placenta tissue successfully, was subcloned into the multiclonesite of PBK-CMV vector by using the same restriction enzyme followed by sequenceanalysis（Invitrogen, USA）, which is completely same to the Pubmed. The recombinantvector is called PBK-CMV-Rb. Conclusion: The recombint eukaryotic expressive vector of wild-type human p53 and Rbwere constructed successfully.PartⅢThe in vitro study of gene delivery of hydroxyapatitenanoparticle to the HepG2 cellObjective （1）: The effect of different concentration of nHAP, lipid and thecombination of the two above to the growth of the cells were studied by the method ofMTT. （2）: Combining liposome and Calcium chloride modified hydroxyapatite （HAP）nanoparticles, the PEGFP-N1 plasmids were transfected into HepG2 cells. The genetransfering rate and the mean fluorescence intensity were observed by flow cytometry afterthe green fluorescence of the cells was seen under fluorescence microscope. （3）: By usingliposome and poly-lysine （PLL） modified hydroxyapatite （HAP） nanoparticles, thePEGFP-N1 plasmids were transfected into HepG2 cells. The gene transfering rate and themean fluorescence intensity were observed by flow cytometry after the green fluorescenceof the cells was seen under fluorescence microscope. The two was compared after 12hs,36hs and 72hs incubation. （4）: By using liposome and poly-lysine （PLL） modifiedhydroxyapatite （HAP） nanoparticles, the PEGFP-C2-P53 plasmids were transfected intoHepG2 cells. The apoptosis and necrosis rate of the cells were observed by flow cytometryafter. The two was compared after 36hs and 72hs incubation.Results （1） The maximal safe dose of nHAP is 20ug/ml while it is 5ug/ml while theliposme is used together. As the result of part 1 indacate, the weight ratio of nHAP andDNA must be more than 15, can the nHAP can complete combine and protect the DNA.So, the subsequent concentrations for the application in vitro are 15ug/ml and 1ug/ml fornHAP and DNA respectively. （2） The gene can’t be transferred by the Ca2+ modified HAPnanoparticles alone. Combining the nanoparticles and liposome, the gene can be transferred very efficiently and the transfering rates were significantly higher than the lipid48 hours later (P^12hR=0.002, P^36hR=0.000, P^72hR=0.002, P^36hI=0.000, P^72hI=0.000,P^all＜0.05). （3） The PLL modified nHAP can diliver the gene transfer to the HepG2 cells.（4） The PLL-nHAP nanoplex can diliver the p53 gene expression in the hepatoma cell lineand enhance the antitumor activity, even more effective than the traditional nonviralvector liposome due to the antirumoral effect of the nanoparticle itself.Conclusion: （1） The Ca2+ modified nHAP can’t induce the gene delivery alone while canenhance the gene delivery of liposome when the combination of the nHAP and liposme.However, the combination can make more cytoxity to the nomal liver cell L02. （2） ThePLL modified nHAP can transfer the gene to the hepatoma cells within safe dosage of livercells. So, it is most suitable for the subsequent experiments of gene therapy. （3） The PLLmodified nHAP can induce the apoptosis and necrosis of the HepG2 while effect little tothe nomal cell L02 in some proper dosage, which can induce the p53 expession in thecancer cells. Moreover, the antitumoral effect of nHAP itself can add to that effect ofanti-oncogene though the effect is later than the liposme. PLL modified nHAP is suitablefor the gene therapy of hepatoma cell in vitro.PartⅣThe in vivo study of lipiodol-hydroxyapatite nanoparticle as thetumor specific gene transfer vectorObjective To evaluate the application of hydroxyapatite nanoparticle as thetumor specific gene transfer vector and the possible mechanism.Methods （1） The safe dosage of the nHAP-PLL nanoparticle was evaluated by the effect ofdifferent concentration to the liver function of the rabbits. （2） To investigate the effect ofdifferent dosage form, different administrations and liposome on the transfection efficiency of nHAP, the group are set up as follows with each one including 3 animals: A:NS+PEGFPC2 group （transarterial）, B: nHAP+PEGFPC2 group （transarterial）, C:nHAP+PEGFPC2 group （local injection）, D: lipodiol-nHAP+PEGFPC2 group（transarterial）, E: lipodiol-nHAP+PEGFPC2 group （local injection）, F: liposome+PEGFPC2 group （transarterial）, G: liposome+ PEGFPC2 group （local injection）, H:liposome+lipodoil-nHAP+PEGFPC2 group（transarterial）。The tumor cells were harvestedby the trypsinization method three days after the infusion or injection of the gene transfersystems. The tansfection efficiency of the cells was investigated by the flow cytometry. Thehighest system was applied in the subsequent test. （3） Recombinant expressing plasmidsharboring wild type p53 or Rb were cotransferred or transferred separately to the rabbithepatic VX2 tumor by the emulsion of Pll-nHAP nanoplex and lipodiol through the hepaticartery in a tumor target manner. Subsequent expressions of p53 and Rb protein within thetreated tumors were analyzed by western blotting. The therapeutic effect was evaluated bythe tumor growth velocity and the survival time of animals. Eventually, the realtimeRT-PCR and ECL western bolt were applied to investigate the expressive changes ofimportant genes related to the above. The group were set up as follows including 120animals: Lipoidol （A, 26）, lipoidol/nanoplex （B, 35）, lipoidol/nanoplex-p53 （C, 40）,lipoidol/nanoplex-Rb（D, 35） and lipoidol/nanoplex-（p53+Rb, 35）（E）.Results （1） The nanoplex was safe to the liver function of the animal when the dosage is5-50mg/kg, which can not only inhibit the growth of the tumor and prolong the thesurvival time of the animal, but also has reversible effect to the liver function. So, for thesafety of animal, the subsequent dosage we used is 5mg/kg and 0.33mg/kg for the nHAPand DNA respectively. （2） When the polyplex is infused through hepatic artery, the nHAPcan’t induce the gene to the tumor in the liver, which can be achieved by thecombination of lipodiol and nHAP. When the polyplex is injected in the local tumor, thepolplex can induce the gene delivery alone, with the enhancement of transfectionefficiency by combing the lipodiol and polyplex. However, the injection groups all has very low transfecton activity （almost no green fluorescence under the fluorescencemicroscope）. The transfection of the transarterial group is more than the local injectiongroup. There is synergism of gene delivery for the administration of transarterial infusionand the application of lipodoil. （3） The lipodiol-nHAP and the gene can affect the liverfunction in a shor time, which can recover within a week. The lipodiol-nHAP can diliverthe antioncogene to the tumor cells and inhibit the tumor growth in a shor time. However,the long-term effect has no significant difference to the control group. The lipodiol-nHAPand it induced antioncogene can prolong the survival of the anmimal, with the two genestherapy better result.Conclusion: Rb work synergistically with p53 in combined therapy mediated bytransaterial infusion of Pll-nHAP nanoplex and lipodoil to augment the antitumoral effectthrough the down regulated expression of important genes related to apoptosis andnecrosis, growth and differentiation, multidrug resistance of tumor cells.更多还原