臺灣博碩士論文加值系統

前言
由於盛行的病毒性肝炎及肝硬化，肝細胞癌(hepatocellular carcinoma)是東亞地區最常見的癌症。目前公認主要而有效的治療方法是手術切除，然而由於早期症狀不明顯以及病人常合併肝硬化，往往發現時可切除率僅達18%左右，而手術後每年仍有25%的病人會復發。除了手術切除之外，肝臟移植也是另一個可達長期存活率的選擇，可惜由於捐贈器官之缺乏及往往病人在診斷時已有遠端轉移，能受惠的病人有限。絕大部分的肝癌病患最後接受經肝動脈血管栓塞術( Transcatheter arterial embolization )及酒精注射治療( Alcohol injection)，然而目前這些療法之成效均不能令人滿意。而其中最廣為採用的經肝動脈血管栓塞術，往往無法完全根除肝癌病灶，腫瘤仍然不斷復發，而無法達到滿意的療效。如何尋求更好的治療策略，改善肝癌病人之存活率，是當前首要之急。
假說
本研究提出一個假說：當肝癌腫瘤在缺血狀態下，會誘發殘餘癌細胞進行特異的新血管生成，這狀態反而加強了癌細胞增值的能力，結果影響了預後。於是本實驗設計的目的在於創造出肝癌缺血的環境，再施以抗血管新生之基因治療。在不同控制組的對照之下，探討腫瘤缺血時的新生血管生成，抗血管新生基因療法對肝癌之效果及合併抗血管新生療法及腫瘤缺血模式對抑制肝癌是否有加成作用，以期找出新的肝癌治療策略。
材料及方法
本實驗選用二十四隻200-250克重之雄性 F344大鼠，以肝被膜下植入法植入3 x 106 MP7TB大鼠肝癌細胞株。七天後施以基因治療, 所使用之載體為攜帶人類endostatin( h-endostatin)基因加上綠色螢光蛋白(Green fluorescent protein)之腺病毒(pAdEasy-GFP-endo)，以及用以做對照組之僅攜帶綠色螢光蛋白之腺病毒(pAdEasy-GFP)。將所有大鼠以肌肉注射Ketamin麻醉( 30 mg/kg)，於操作型顯微鏡下由胃十二指腸動脈(gastroduodenal artery)處置入PE10 polythelene導管固定，並結紮遠端胃十二指腸動脈。接著隨機取12隻大鼠以0.5 ml/min之速度由肝動脈導管打入帶有人類endostatin 基因之 pAdEasy-GFP-endo (5 x 109 IU / 0.5 ml)，其中6隻結紮總肝動脈(common hepatic artery)作為 ”缺血及治療組”，其他6隻為 ”治療組”，另外12隻大鼠打入pAdEasy-GFP (5 x 109 IU / 0.5 ml)，其中6隻結紮總肝動脈作為 ”缺血組”，剩下之6隻為”對照組”。
於治療後第二，三，五，七及十日內每日由眼窩後取血(retro-orbital method)，各抽血樣0.5 ml 以ELISA 法測human endostatin之濃度。術後第28天，各組犧牲一半大鼠，切除肝臟，肝臟放血後稱重並紀錄，切片並計數腫瘤數目及大小，用螢光顯微鏡檢視切片後以hematoxylin & eosin法染色，並作CD31免疫組織化學染色( immunohistochemistry staining)以及TUNEL assay。
結果
術後對照組及缺血組之每日血清中endostatin濃度均在10ng/ml以下,治療組及缺血治療組則在第二天達到400 ±40 ng/ml之高峰然後逐日遞減至第十天仍有22 ±3 ng/ml。比較28日後之鼠肝腫瘤大小各為對照組 3.5 ±0.3 cm3, 缺血組 4.3 ±0.5 cm3，治療組 2.4 ±0.4 cm3，缺血及治療組 0.3 ±0.1 cm3。依one-way ANOVA之檢定對照組及治療組，對照組及缺血組，對照組跟缺血治療組,缺血組及缺血治療組，治療組及缺血組之間有均顯著差異。
經CD31染色後之腫瘤樣本可計算出微血管濃度(microvessel density (MVD) )，所得結果分別為： 對照組 52 ±3.0，缺血組 63 ±4.3，治療組 34 ±2.0，缺血及治療組 36 ±1.0，依one-way ANOVA之檢定治療組及對照組，缺血組及缺血治療組，對照組及缺血組之間有顯著差異。
TUNEL assay分別為 對照組 1 ±0.3,缺血組 2.3 ±0.4, 治療組 8 ±1.0, 缺血及治療組 14 ±4.3依one-way ANOVA之檢定對照組及治療組, 對照組跟缺血治療組,缺血組及缺血治療組,治療組及缺血組之間有均顯著差異。
結論
由本實驗目前成果得知，腫瘤面臨缺血時，的確會促進新生血管生成，並且加速腫瘤生長，最後可能使預後變差。目前對於治療肝癌所使用的局部治療法例如經肝動脈血管栓塞術若作用不完全很有可能反而造成殘餘腫瘤旺盛的血管新生。
在本大鼠模型中，endostatin之基因治療可有效抑制腫瘤之血管新生並得到有統計意義的療效，而在結紮肝動脈的帶腫瘤鼠身上施用, 更可有效廓清腫瘤細胞而達到一加成作用(synergistic effect)。未來可利用此帶肝內導管大鼠肝細胞癌動物模型繼續朝運用不同抗血管生成因子或是不同轉染 (transfection) 途徑嘗試不同抗癌方法，並研究腫瘤缺氧造成血管生成之機制，以期造福病患。

Introduction
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, especial in the Asia. The only curative treatment is surgery. In fact, only 15-30% of patients with HCC is suitable for surgical management, so the outcome of HCC is always dismal. Most patients received the treatment of transcatheter arterial embolization(TAE), to disrupt the blood supply of tumor and make tumor necrosis. Unfortunately, TAE seemed to be a palliative method for treatment of HCC, most tumors received TAE were recurrent. It is necessary to find out an more effective strategy for management of inoperable HCC.
HCC is generally considered as a hypervascular solid tumor. In recent 30 years, there were some new concepts about tumorogenesis established. Numerous studies have shown that tumor vascularization is necessary to the growth of solid tumors . Inhibition of this process therefore may indirectly inhibit tumor cell growth and lead to tumor dormancy. The progression of tumors to larger, more invasive lesions requires the formation of new blood vessels-so called angiogenesis. This process is regulated by stimulators and inhibitors of angiogenesis. There were also many research devoted to characterize the therapeutic potential of angiogenesis inhibitors as anticancer agents. Endostatin, a specific angiogenesis inhibitor produced through enzymatic cleavage of a protein precursor, collagen XVIII, is a potent anticancer agent in animal models. The MW 20,000, 184 amino acid, endostatin protein was shown to specifically inhibit the proliferation of endothelial cells and to stop the growth of Lewis lung carcinoma experimental metastases in mice without signs of drug toxicity. Furthermore, endostatin suppressed, in a dose-dependent fashion, the growth of a panel of animal primary tumors. Tumors could be subjected to repeated treatment cycles, without acquired resistance to the therapy.
However, the treatment requires prolonged administration and high doses of recombinant protein because of the short half-lives of these compounds, and the regressed tumors may reawaken when therapy is suspended. However, many of these biological agents are unstable in vitro and difficult to produce in large quantities , and producing large quantities of biologically active proteins has proven difficult. Consequently, antiangiogenic gene therapy has been proposed as an alternative to the delivery of recombinant protein. The goal of this therapy would be to create a situation where the body becomes an endogenous "factory," producing high circulating levels of the gene product. Because high levels of transgene products have been reported using adenoviral vectors, we investigated the ability of an adenoviral vector to elevate circulating endostatin levels in rats as an approach to cancer treatment.
This study established an orthotopic liver tumor model using a transformed administration of adenoviral vector that expresses a human endostatin(h-ED) gene, but only partial therapeutic response was obtained, and the large tumors were rather resistant to this type of anti-angiogenic therapy . As a result, more powerful therapeutic strategies for treating animals with orthotopic liver tumors remain to be developed.
Materials and methods
Cell lines
GP7TB is a cell line derived from chemically transformed hepatic epithelial cells in Fischer 344 rats. The 293 cell line is a kind of adenoviral E1 transformed human embryonic kidney cells). ECV304 is an immortalized human umbilical vein endothelial cell (HUVEC) line.
Plasmid and adenoviral vector construction
Human endostatin (hED) cDNA was obtained by RT-PCR of the RNA prepared from human liver. The DNA fragment coding for the 24 amino acids (MAAGPRTSVLLAFALLCLPWTQEV) of the signal peptide (SP) of porcine growth hormone was obtained by PCR from a plasmid. The two PCR products, SP and hED, were digested with Bam HI + Nar I and Nar I + Xba I, respectively, and cloned together into pCA4 vector digested with Bam HI and Xba I, under the control of the CMV promoter. Plasmid DNA was amplified in DH5 cells and the SP-hED sequences were confirmed to be correct and in the right reading frame by the dideoxynucleotide termination method. The resulting SP-hED/pCA4 plasmid was used for the following construction of adenoviral vector.
Recombinant adenoviruses were constructed using the AdEasy system. The structures of the resultant recombinant vectors were confirmed by restriction enzyme digestion and PCR analysis.
In vitro and in vivo Western blot and ELISA analysis of the secreted proteins from adenovirus infection
GP7TB cells were infected with recombinant adenoviruses at a multiplicity of infection (MOI) of 30. Eight hours later, the supernatant was harvested. On the one hand, it was concentrated 80-fold by cellulose columns and Western blot analysis with a mouse polyclonal anti-hED antibody. Meanwhile, the levels of hED in the conditioned supernatant were directly measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. For in vivo detection of transgene expression, tumor cells (3 x 106 cells) were first inoculated in the liver. Seven days after tumor implantation, adenoviruses of 3 x109 pfu in a final volume of 100 ul were trans-arterially injected (n = 3 for each group). Blood samples were collected from the retro-orbital sinus on days 1, 2, 3, 5, 7 and 10 after adenoviruses injection, and serum levels of hED were determined by ELISA, too.
In vitro endothelial cell proliferation assays
To determine the biological activity of hED, ECV304 cells or GP7TB cells were plated on collagen-coated 96-well plates at 3000 cells/well. After overnight incubation, the medium was aspirated and replaced with 10ul, 30ul, or 50ul of the 80-fold-concentrated conditioned medium from the adenovirus-infected GP7TB cells. Finally, proliferation of ECV304 or GP7TB cells was analyzed using a WST-1 kit.
In vivo anti-angiogenesis assays
Each rat (N=9) was injected with 0.5 ml of unpolymerized Matrigel containing heparin and basic fibroblast growth factor subcutaneously. Rats were given one intravenous injection of 3 x109 pfu of Ad/ED (N=3), Ad/GFP (N=3), or PBS (N=3) via the tail vein 3 days before Matrigel injection. The Matrigel plugs were harvested 7 days later. Half of the Matrigel plugs were stained with hematoxylin and eosin (H&E). The number of infiltrating endothelial cells from the five most vascular areas was obtained at 200 magnification, and the mean number was calculated. The remaining half of the Matrigel plugs was assayed for hemoglobin content
Generation of orthotopic liver tumors and hepatic arterial catheterization
The experiment used 24 male Fischer 344 rats aged 7-8 weeks. The rats were anesthetized with 100 mg/kg ketamine, their abdominal cavities were opened, and 3 x106 GP7TB cells injected into the left liver lobe on day 0. A single tumor nodule ( 75-100 mm3) was generally observable 7 days after tumor inoculation. Another surgery was performed for insertion of a PE10 catheter into hepatic artery under dissecting microscopy on the same day.
Anti-angiogenic gene therapy
A single injection of adenoviruses with endostatin gene (3 x109 pfu in a volume of 100 ul ) was administered via hepatic arterial catheter on days 7 after tumor implantation(N=12). The other 12 rats received injection of adenovirus with GFP gene for control. Hepatic artery was ligation on the half of the both groups(N=6).The rats received Ad/ED treatment only is Ad/ED group, received Ad/PBS treatment is control group, received Ad/PBS +hepatic artery ligation treatment is ischemia group. Tumors were removed and measured using calipers on day 28 on the half of the 4 subgroup (N=3).The residual 3 rats in per group left for observation of life span.
Immunohistochemistry and TUNEL assays in tumor sections
The sections of tumors were probed with mouse anti-rat CD31 (TLD-3A12), followed by a secondary antibody, rabbit anti-mouse immunoglobulin. The slides were examined and the five most vascularized areas of the tumors in each section were then identified. Microvessel counts were obtained at 200 magnification and the mean number of these five fields for each tumor was calculated. The data obtained is microvessel density(MVD), and MVD mean angiogenic index.
Terminal dUTP nick-end labeling (TUNEL) assay was used to detect cell apoptosis in the tumor area. After two washings with PBS, the sections were incubated with proteinase K to digest virally expressed GFP protein and to prevent interference with the green fluorescence of the TUNEL reagent. Finally, apoptotic cells from five random fields per slide were counted under a fluorescence microscope at 400 magnification
Statistical Analysis
All data are presented as the mean ± SE. Comparisons between groups were
performed using one-way ANOVA. Student's t test was used to evaluate the statistical
significance of the difference in tumor volumes between two groups, and two-tailed P
< 0.05 indicates statistical significance.
Results
The effect of hepatic artery ligation
No rat was dead due to hepatic artery ligation, and the HCC on rats those hepatic artery was ligated is larger than control group(4370 ± 235 mm3 vs 3050 ±740 mm3 , p=0.021).and the mean life span of ischemia group is longer than that of control group(45 ± 2.9 day vs 39 ±1.6 day , p=0.027).
In vitro Western blot and ELISA analysis of the secreted proteins from adenovirus infection
GP7TB cell line was infected in vitro with the adenoviruses at an MOI of 30. Forty-eight hours later, cultured supernatant was harvested, concentrated 80-fold, and analyzed by Western blot to determine hED expression, the MW 20 kda product was noted, then analyzed by ELISA to determine hED secretion. The supernatant level of hED was 160 ±10 ng after infection with Ad/ED 48 hours later.
In vivo ELISA analysis of human endostatin.
In vivo expression of hED was also evaluated following a single trans-arterial injection of 3 x109 pfu adenoviruses into the 7-day-old tumors implanted in the liver. Serum levels of hED were measured by ELISA on days 1, 2, 3, 5, 7 and 10 after injection. Peak expression after Ad/ED infection was observed 2 days after injection, at which time plasma concentrations were 400 ± 40 ng/ml. However, the expression was transient, lasting only about 1 week. On day 7, plasma levels remained significantly elevated (22 ± 3 ng/ml vs 8 ± 1.5 ng/ml in Ad/GFP-treated animals; p = 0.04)
In vitro endothelial cell proliferation assays
We tested the conditioned supernatant of GP7TB cells transduced with various adenoviruses (Ad/ED and Ad/GFP) in a proliferation inhibition assay using ECV304 cells. Significant inhibition of ECV304 cell proliferation could be demonstrated using the conditioned supernatant derived from the cells infected with Ad/ED, but not from those infected with Ad/GFP （proliferation inhibition assay of 10ul，30ul，50ul medium is 10 ± 0.8，15 ± 1.0，38 ± 2.0）. However, the conditioned supernatant did not influence the growth of GP7TB cells.
In vivo anti-angiogenesis assays
In vivo anti-angiogenic activity of the adenovirally expressed hED was tested by a Matrigel assay, the hemoglobin content of Matrigel implants from rats infected with Ad/ED(51 ± 17) (unit : mg Hb/ g Matrigel) was significantly lower than in the animals treated with PBS(173 ± 12) or Ad/GFP(199 ± 9) (P<0.05). The number of infiltrating endothelial cells in the Matrigel samples was also examined to provide another measure of anti-angiogenic effect. The mean cell number in the Matrigel samples from animals infected with Ad/ED(30 ± 4) was significantly lower than that in the Matrigel samples from the animals treated with PBS(90 ± 11) or Ad/GFP(92 ± 8)(P<0.05). This assay confirmed the anti-angiogenic activity of hED in vivo.
Anti-angiogenic gene therapy
When the animals were sacrificed on the day 28, it reveals significant reduction in tumor volume in animals treated with Ad/ED(2.4 ± 0.4 cm3 ) compared with the control group(3.5 ± 0.3 cm3) or the ischemia group(4.3 ± 0.5 cm3) (P < 0.05). Furthermore, the Ad/ ED + hepatic artery ligation treatment almost completely regressed the tumors(0.3 ± 0.1 cm3).All tumor specimens were observed under fluorescent microscopy, and all slides showed abundant fluorescent staining.
Immunohistochemistry and TUNEL assays in tumor sections
The effects of endostatin expression on tumor vascularization were determined by immunohistochemical staining. Expression of endostatin mediated by Ad/ED infection caused significant reduction in MVD compared with the control group(34 ±2.0 vs 52 ± 3.0 , p<0.05 ,one-way ANOVA). Interestingly, the increase in MVD between control group(34 ± 2.0) and ischemia group(63 ± 4.3) was statistically significant, and the MVD of Ad/ED + hepatic artery ligation group is 36 ± 1.0. These MVD data thus indicate that adenovirally expressed endostatin significantly reduces tumor vasculature in liver tumors, and ischemia induced vigorous angiogenesis.
We then measured the numbers of apoptotic cells in the tumor areas by TUNEL assays. The number of apoptotic cells in the Ad/ED-treated group(8 ± 1.0) was significantly higher than in the control groups (1 ± 0.3) (P < 0.05). The number in the ischemia group is 2.3 ± 0.4. More importantly, the combination therapy with hepatic artery ligation and endostatin further increased the number of apoptotic cells compared with the Ad/ED monotherapy (8 ± 1.0 vs 14 ± 4.3, P < 0.001, one-way ANOVA).
The improvement of life span
All the residual rats were dead of tumor rupture, the mean life span in control group is 47 ± 3.0 day, ischemia group is 37 ± 1.5 day, Ad/ED group is 51 ± 2.9 day and Ad/ED +hepatic artery ligation group is 80 ± 6.0 day. The ischemia status of HCC shortened the life span of rats, and Ad/ED treatment could improve the outcome of the rats with hepatic artery ligation(p<0.05).
Discussion
We selected an adenoviral vector to deliver the human endostatin gene because of previously reported high circulating levels of transgene product, as well as known biodistribution mostly to the liver. This model allowed us to investigate the hypothesis that the host, in particular the host liver, can be used as a "factory" for generating angiogenesis inhibitors. The ability of Ad/ED to generate endostatin in vivo was assessed. Rats receiving 109 pfu Ad/ED demonstrated 100% survival and appeared healthy. Autopsy revealed no significant liver damage.
Cancer gene therapy using anti-angiogenic molecules has been shown to be a potential strategy for regressing tumors to dormant, microscopic nodules, but the eradication seemed to be impossible. Several studies have reported the inhibition of tumor growth and metastasis in mice by adenoviral vector-mediated delivery of endostatin, no strong activity against preexisting tumors was reported via this method. The results of this study support this conception, demonstrating that a synergistic effect on treating an orthotopic liver tumor model could be achieved by combining anti-angiogenic therapy with hepatic artery ligation.. The combination of GM-CSF with endostatin gene therapy has been reported previously, and also revealed synergistic antitumor activity.
The angiogenesis of implanted HCC was triggered by tumor ischemia, and the unusual condition results in rapid growth of the tumors. It hints that incomplete treatment of TAE may make the prognosis worse. In the experiment, the anti-angiogenic therapy using endostatin did inhibit the angiogenesis of ischemic tumor, and dramatically shrink the tumors. The apoptosis of the treated tumor was proved to be increased. Life span of these treated rats was prolonged significant. In summary, this is a demonstration of anticancer efficacy of endostatin in implanted rat HCC model, it is also the demonstration that the hypoxia induced angiogenesis can be suppressed─although not completely eliminated─by short-term endostatin therapy.
We demonstrated inhibition of tumor growth associated with high circulating endostatin levels in a tumor model. The more specific anti-angiogenic factor for HCC may be more effective in anticancer treatment. We are currently investigating new gene delivery systems to prolong systemic expression of anti-angiogenic agents, making use of the host as the "factory" for production.

目錄
一、中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧1
二、緒論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧5
背景‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧5
血管新生對腫瘤之重要性‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧8
抗血管生成因子的角色 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧11
血管新生之機制‧‧‧‧‧‧ ‧‧‧‧‧‧‧‧‧‧‧‧‧‧12
肝細胞癌和血管新生之關係 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧14
缺氧及腫瘤血管新生之關係 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧15
實驗目的及假說 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16
三、研究方法與材料 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧17
材料 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧17
細胞株 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧17
質體設計 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧18
病毒載體 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧18
實驗動物 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧19
研究方法 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧20
生體外抗血管生成因子檢定 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧20
生體內抗血管生成因子檢定 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧21
植入式肝腫瘤模 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧22
帶腫瘤鼠之缺血反應實驗‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 22
經肝動脈基因治療 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧24
免疫組織染色 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧26
細胞凋亡分析 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧27
統計方法 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧28
四、結果 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧29
第一部份 缺血性肝癌模型 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧29
第二部份 帶Endostatin蛋白腺病毒定性定量檢定 ‧‧‧‧ 31
第三部份 大鼠肝腫瘤抗血管生成基因治療 ‧‧‧‧‧‧‧34
五、討論 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧37
肝細胞癌之治療及衍生之問題 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧37
結紮肝動脈對腫瘤之影響 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧38
抗血管新生療法之發展 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧39
Collagen XVIII在肝臟組織之角色 ‧‧‧‧‧‧‧‧‧‧‧‧41
Endostain在腫瘤治療之角色 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 42
基因治療的優點 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧43
使用腺病毒載體之考量 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧44
本實驗之Endostatin作用為何不明顯‧‧‧‧‧‧‧‧‧‧‧ 45
為何肝動脈結紮和 endostatin 之治療有加成作用 ‧‧‧‧‧46
抗血管新生療法的前景在於合併其他療法 ‧‧‧‧‧‧‧‧‧47
六、展望 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧48
七、英文簡述‧‧‧‧ ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧56
八、參考文獻 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧71
九、附圖 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧87
圖一：重組腺病毒之示意圖 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧88
圖二：肝動脈結紮與否之腫瘤大小 ‧‧‧‧‧‧‧‧‧‧‧‧89
圖三：西方墨點法確認產物為endostatin‧‧‧‧‧‧‧‧‧ 90
圖四：所表現之endostatin蛋白定量分析‧‧‧‧‧‧‧ ‧‧91
圖五：生體外內皮細胞增殖分析 ‧‧‧‧‧‧‧‧‧‧‧‧‧92
圖六：生體內合成endostatin定量分析‧‧‧‧‧‧‧‧‧‧ 93
圖七：生體內抗血管生成因子生物活性檢定 ‧‧‧‧‧‧‧‧94
圖八：基因治療後各組大鼠肝癌之大小比較 ‧‧‧‧‧‧‧‧95
圖九：CD31免疫組織染色‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 96
圖十：細胞凋亡分析 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧97

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