| Background:At present the clinical conventional treatment for liver cancer is not good, the gene treatment opened a new way. To supprese individually the cancer gene or antioncogenes can only be able to alleviate the liver cancer to have in process some step, certainly cannot permanently cure the liver cancer. Treatment with antioncogene is a way of filling the lost or mutated antioncogenes into malignant hepatocytes to suppresse tumor growth. P16 and p53 are the most popular tumor suppressor gene.By using biologic technologies, p16 and p53 can be transferred into cancer cell, and prevent liver cell to proliferation and mutation, then prevent cancer growth. The appropriate vectors were needed to transfer DNA into cells, and the key to gene transferration and treatment was the celection of nanoparticles (NP). Polylactic acid-glycolic acid (PLGA) was one of the useful vectors to control drug releasing, and innocuity, bio-degenerated, bio-compatibility, which was the most appropriate carrier as nanoparticle.1. Construction of p16 and p53 gene fusion expression vectors. Objective: To creat p16-53 gene fusion expression vectors. Methods: Splice p16 A and pcDNA were excide with endonuclease EcoRI and XhoI, and splice PMAMneo53 was excide with NheI and XhoI in two splicing sites. Fetch p16 and p53 gene fragments and confuse them with spliced pcDNA, and mixed them with ligase T4, ligse T4 buffer. Put them in 16℃temperature for 20 hours, then we could get p16-53 gene fusion expression vectors. Results: Only one fragment which was 8000bp after pcDNA16-53 was excide by endonuclease HindⅢ, it is showed that 8000bp was pcDNA16-53 length. Three fragments which were 4.4Kb, 1.7Kb, 0.2Kb after pcDNA16-53 were excide by endonuclease SmaI, it is showed that it was the right direction which p16 and p53 fragments inserted in pcDNA.2. The creatment of polylactic acid-glycolic acid nanoparticles (PLGA-NP).Objective: To creat polylactic acid-glycolic acid nanoparticles (PLGA-NP).Methods: Study on preparation technology of PLGA-NP and test its size, turbid point, encapsulation and bio-compatibility.Results: 1.When the molecular weight of polymer was less than 15000, we can get high yield PLGA-NP. But the production of NP was reduced with the increase of molecular weight and increased with the augmentation of volume fraction of ethanol.2.The mean size of NP were less than 182nm, and which was influenced by ethano1.3. Encapsulation of NP was increased with the concentration of DNA, not the kind of PLGA.4.PLGA-NP was innocuity to HepG2 cell when less then 100ng/μl, and to 3T3 cell when less then 200ng/μl. If the concentration of NP was increased, the toxicity to cells was strong rapidly.3. The connection of PLGA and pcDNA16-53Objective: To connect pcDNA16-53 and PLGA nanoparticles to prepare PLOA-pcDNA16-53 nanoparticles. Its integrated efficiency and protective effect to plasmid DNA were analyzed to estimated the efficiency of gene transfer in vitro.Methods: The pcDNA16-53 and PLGA nanopartictes were prepared. Effection of pH value on binding capacity, gel block exam amd deposit experiment were determined. The anti-DNA enzyme degradation capacity of nanoparticles was detemined with enzyme-protection and serum-protection test. The pEGFP-N1 plasimid, which contained Green fluorescent protein gene as a report gene, is evaluated cellular transfection rate of the nanoparticles in vitro. Results: 1. PLGA-nanoparticles had optimal DNA binding capacity when PH value equalled to 7. The integrated efficiency is 92%, when the proportion of nanoparticles and plasmid DNA was set on 10:1. 2.PLGA-nanoparticles protect DNA from degradation of DNA enzyme and blood serum. Plasmid DNA was degradated in the same condition. 3.The EGFP expression plasmid could be transfected effectively into HepG2 and 3T3 cells by PLGA-nanoparticles in vitro. The transfection efficiency was about 47%, which was higher than that of liposome.4. The experiment research of PLGA-pcDNA16-53 treatment on liver cancerObjective: To study the effection of PLGA-pcDNA16-53 on liver cancer in vitro and vivo.Methods: 1. In vitro: HepG2 cell were divided randomly into three group, A: RPMI-1640, B: PLGA-NP, C: PLGA-pcDNA16-53. Plasmid preparated, purified and transferred into cells, western blotting were used to test the expression of p16 and p53, MTT to test cell growth and proliferation. The cell apoptosis was observed by microscope, electron microscope and DNA gel electrophoresis.2. In vivo: HepG2 cell was inoculated subcutaneously on naked mouse to treat the liver cancer model. 16 naked mouse were divided randomly into two group: treatment group(every mouse was injected slowly 100μg PLGA-pcDNA16-53 subcutaneously, every other day for 5 weeks), control group(100μg NS). The volumn and formation of tumor were observed. All mice were killed after 5 weeks, western blotting were used to test the expressions of p16 and p53, and flow cytometer to test cell apoptosis.Results: 1.The expression of p16 and p53 was tested in cells of C group by western, blot, but not in A and B group. 2. The suppression rate of A and B group-was 0.34±0.06 and 0.41±0.04, respectively, which were significantly lower than C group(p＜0.05). 3. Obviously apoptosis was in C group under microscope and electron microscope, and especially ladder was detected in DNA gel electrophoresis.4. The tumor model was created about 2 weeks.The tumor weight of treatment group was 0.84±0.19, which was significantly lower than that of control group(1.08±0.15), and the suppression rate of tumor was 53.8% in treatment group.The time for tumor formation was more than 2 weeks. 5. The expression of p16 and p53 was tested treatment group by western blot, not in control group.6.The apoptosis rate of treatment group was (11.33±3.48) %, significantly higher than that of control group (3.07±0.29) %.Conclusion:1. When pH=7 and the rate of NP/DNA was 10: 1, the capability of PLGA-NP to bind DNA was strongest, and innocuity to cells;2. PLGA-pcDNA16-53 nanoparticles can resist the digestion of nuclear acid enzyme and serum;3. PLGA-NP can transfer p16 and p53 gene into HepG2, and express in cells;4. The cells transferred with pcDNA16-53 was obviously suppressed, and apoptosis were observed under microscope and electron microscope, and especially ladder was detected in DNA gel electrophoresis;5. The expression of p16 and p53 was tested in tumor injected by PLGA-pcDNA16-53 subcutaneously, and the growth of tumor was suppressed, apoptosis cells appeared. |
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