Research On Anti-hepatoma Efficacy And Mechanism Of Combining Two Genes Of Endostatin And Angiostatin With Lipiodol In WistarRats

Hepatoma is one of the main diseases severely endangering humanexistence, promulgated by World Health Organization. The treatment efficacy ofthe common therapeutic measures, such as operation, radiotherapy, Chemtherapy,interventional therapy and Liver graft, is insufficient. So many scientificresearchers are devoting themselves to investigating the new treatment plans.With gradually recognizing the associativity of various malignant tumorsand gene, researchers try to deploy gene therapy to treat hepatoma. At present theprinciple therapeutic schemes on hepatoma by way of gene therapy contain: 1.Immunogene therapy;2.Suiside gene therapy;3.Gene therapy of inhibitingcancer cells;4. Antisense nucleotide technique;5.multi-drug resistance genetherapy;6. Gene therapy of inhibiting tumor blood vessel;7. Allied genestherapy. In these treatment plans, since the formation of tumor blood vessel has agreat deal to do with tumorous growth and migration, gene therapy of inhibitingtumor blood vessel is one of the investigative warm spots on treating hepatomathrough gene therapy. It's mechanism of action included: from expression anddelivery of genes levelly inhibiting the angiogenin, to expression and delivery ofpromoting anti-angiogenin, to induction ofvascular endothelial apoptosis.The vascular inhibiting genes that the research adopts were endostatin geneand angiostatin gene . All of them are genes that can affect on tumorous newblood vessels, and their principal effect is to inhibit the generation and growth oftumorous new vessels and then achieve a goal of constraining the tumorousgrowth and migration. The research was to adopt gene therapy to treatimplanted liver cancer of Wistar rats by way of locally direct injection andtranshapetic artery intervention. On the one hand, we could observe the effect oftwo genes therapeutic alliance on liver cancer;on the other hand, we could studythe therapeutic effect on hepatoma of combining twin genes with lipiodol by wayof trans-hapetic artery intervention.The precise ascitic passage of Wistar rats were highly important tomaintaining the physiological nature of the Walker-256 cell. Moreover theconsistency of the tumorigenic function of the Walker-256 cell was premise andfoundation in the whole experimental research, thus the research firstly analyzedthe influential factors of Wistar rats ascitic generation.1 Research on the reasons affecting ascitic generation of Wistar rats1.1 The ascitogenous difference after inoculating ascitae with differentdensity in rat.32 rats were divided randomly into 4 groups. In the first group(Highconcentration hydrogaster group), the abdominal cavity of rat was injected into0.3 mL ascitic stock solution and the cell population was about 9×107. In thesecond group(Dilution group), the abdominal cavity of rat was injected into 0.3mL diluted ascites by 5 times and the cell population was 1.8×107;In the thirdgroup(Ablution group), the abdominal cavity of rat was injected into 0.05mLcentrifugated concentrated hydrogaster and 0.25mLsaline, the cell population was3.5×107. The eight rats were injected into 0.3mL saline as control group. On the8th day after injecting ascites, flow cytometer be used to measure numbers of Tlymphocytes and their subgroup and the density of immunoglobulin IgG, IgMand IgA of the rats in the different groups .The percentages of ascitogenous in high concentration hydrogaster group,dilution group and ablution group respectively were 37.5%, 67.5% and 87.5%.The result proved that ascitic inoculation with different density would lead todifferent possibility of Wister rat's ascitic generation, and inoculation of asciticstock solution with high concentration may degrade the probability of rattishascitic generation.1.2 Analysis on cellular and humoral immune function of the rats inoculatedby the cancerous ascites with different concentration.CD3+,CD4+,CD8+and IgG,IgM,IgA content in the blood of the ratsrespectively in the control group, high concentration hydrogaster group, dilutiongroup and centrifugated ablution group were measured and analyzed. Theconsequence showed that the indexes of T lymphocytes and their subgroup, andimmunoglobulin in the high concentration hydrogaster group were all higherthan the corresponding ones in the dilution group, ablution group and controlgroup. The high concentration cancerous ascites made immune function of theinoculated rats strong.The consequence showed that the ascitogenous possibility of the rats,which were inoculated by high-concentration cancerous ascites, obviouslylowered. And this situation had a great deal to do with rats' humoral and cellimmunity being activated by high-density cancerous ascites, which wasdisadvantageous to the establishment of liver cancer models.2.Comparative study of the two ways of direct intraperitoneal injection andneoplastic pieces transplantation.30 male Wistar rats were divided into two groups: two groups of rats wereused to establish hepatic cancer models respectively by way of directintraperitioneal injection and neoplastic piece transplantation . After 7 days, thediameter of the solid tumors in the direct injection was larger than the one in thetransplantation group, P<0.001, owning the statistical significance. Thesuccessful rate of models in the two groups was respectively 86.7% and 80%, notowning the statistical significance. The direct intraperitioneal injection methodwas noted for the short time of forming tumor and convenient operation;theoperation of neoplastic piece transplantation was rather complicated, but it wasnot easy to cause the infiltration and transference on neighboring organs andformation of transferred ascites. The successful rate of tumorous formationthrough the two methods was equally high. The life span of the bearing cancerrats would meet the need of the most experimental research.According the requirement of the laboratory objective and process, theresearch adopted the direct intraperitioneal injection method to attain hepaticcancer models, convenient for evaluating the effect of treating liver cancer byway of gene therapy.3. The laboratory research on treating hepatic cancer model by way ofcombining vascular inhibiting gene with lipiodol.3.1. The expression of gene product of endostatin and angiostatin inside theextra-organismal Walker-256 cancer cell.pCMV-Endostatin plasmid and pCMA-Angiostatin plasmid were mingledand transfected with LipofectAmine-2000 in the Walker-256 tumor cells. Afterthe cellular collection and spallation, western blot was adopted to measure geneproduct of endostatin and angiostatin.Consequence: In the 20KD and 38KD zone in the electrophoretic strap ofthe western blot, the gene product idio-strap of endostatin and angiostatin, andthe limpid expression of cancer cells co-transfected by the two genes, were seen.And it showed that the two genes could equally be transfected into theWalker-256 cancer cell and could produce corresponding proteinic product. Theconsequence provided the strong support for adopting gene therapy to treathepatic cancer by way of locally direct injection and trans-hapetic arteryintervention.3.2 The corresponding mRNA expression of Endostatin and AngiostatinRT-PCR was adopted to measure the mRNA expression of Endostatin andAngiostatin of cancer cells respectively in endostatn group, angiostatin group,and endostatin plus angiostatin group. In the electrophoretic strap, the idio-strapsof 650bp Endostatin mRNA and 1400bp Angiostatin mRNA were seen. The twoidio-straps were seen expression in the two gene therapy group. It shows that theobjective genes adopted by the research could be transfected into the Walker-256tumor cells either in vivo or vitro.3.2.1 Inhibiting effect of endostatin and angiostatin genes on hepatic cancerby way of locally direct injection.The Wistar rats were divided into control group, endostatin gene therapygroup and angiostatin gene therapy group. The hepatic cancer was locallyinjected into 200 μL physiologic saline and 200μL gene plasmid owningliposome encapsulation function. After 3, 6, 9, 13, 15 and 18 days, 10 rats wererandomly selected in each group to measure the voluminal growth rate(expressedby tumor growth rate: f=V/V0).After 18 days, the tumor weight(expressed byweight variation rate : F=W/W0), cancer tissular microvessel density(MVD),vascular endothelial growth factor(VEGF), apoptotic index(AI), the life span ofrat and the numbers of pulmonary metastasis, of the rats in each group, weremeasured.The consequence shows that, after 6 days, comparing with the control group,each gene therapy group equally owned the statistic difference(P<0.001);at thesame time, comparing with the single gene therapy group, the two genes therapygroup also had the statistic significance (P<0.05). The tumor growth in thecontrol group is the fastest, and its growth in endostatin plus angiostatin genetherapy group is lowest of all the groups. After 18 days, comparing with thecontrol group, the tumor weight,cancer tissular microvessel density(MVD),vascular endothelial growth factor(VEGF), apoptotic index(AI), rattish life spanand the numbers of pulmonary metastasis, of the rats in each gene therapy group,had the obvious statistic difference(P<0.05～P<0.001). The gene therapeuticeffect of endostatin plus angiostatin was greatest, and the effect of the controlgroup was poorest. Comparing with other groups, the above index also hadstatistic significance. VEGF(100%) expression of the 8 rats in the control groupcould be seen;VEGF(50%) expressin in the endostatin and the angiostatin genetherapy groups could be seen;VEGF(25%) expression of the only two rats inthe twin gene therapy group could be seen.The consequence proved that the two genes therapeutic alliance had moreobviously cooperative effect on hepatic cancer than the single gene therapy has.3.2.2 Analysis of the therapeutic efficacy and its mechanism of genemedicine combining lipiodol administered via hepatic artery.The rattish hepatic cancer models were randomly divided into 8 groups, andthen were respectively injected via hepatic artery into 200μL hyper-liquidlipiodol,endostatin gene,angiostatin gene,endostatin gene plus angiostatingene,endostatin plus lipiodol mixture,angiostatin plus lipiodol,endostatin plusangiostatin plus lipiodol and physiological saline .After administrating gene medicine via hepatic artery, the volume growthrate of rattish hepatic cancer in the different groups is more different than before.After 6 days, the tumor volume growth rates in the different therapy groups weresmaller than one in the control group(P<0.001). The therapeutic efficacy in twingene plus lipiodol therapy group was the most obvious and had more differencecomparing with other therapy group(P<0.01).After 18 days, the tumor weight growth rate, the numbers of pulmonarymetastasis, MVD and VEGF in the therapeutic groups are smaller than thecorresponding indexes in the control group. Apoptotic index(AI) and Life spanof rat are obviously higher than ones in the control group, P<0.05～0.001. Theconsequence showed that inhibiting cancer effect in twin genes plus lipiodoltherapeutic group was the greatest, and had statistic significance. It also showedthat twin genes plus lipiodol via hepatic artery treating liver cancer mightproduce the definite therapeutic efficacy and prolong the growth life of rats .During the therapeutic progress of gene plus lipiodol, there existed an effectof lipiodol on direct tumorous blood-supply vessel embolish and then on tumorcellular apomorphosis, thanatosis and apoptosis;also anti-hepatoma effect ontumorous new vessels of vascular inhibiting gene . Moreover combining twingenes with lipiodol has obvious anti-hepatoma therapy efficacy because thevascular inhibiting gene can strongly check the vascular proliferation induced bylipiodol.In one word, the anti-hepatoma therapy efficacy of two genes of endostatinand angiostatin either through tumorous local injection or via hepatic arteryintervention was better than one gene therapy. Moreover the therapy efficacy ofcombining two vascular inhibiting genes with lipiodol on rattish inoculatedhepatoma was better than the therapy merely by genes.