The Experiment Studies Of Specific Target Gene Therapy Of Lipiodol-hydroxyapatite Nanoparticle On Hepatic Malignant Tumor

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.