摘要
基因治疗的载体可以分为病毒载体和非病毒载体两大类。病毒载体是目前基因治疗的主要载体，但是病毒载体具有携带外源基因片段大小受到限制(7kb左右)、易引起机体免疫反应以及制备、贮存、质量控制比较困难等固有缺点，这促进了人们对非病毒载体的研究。在过去的十多年里，人们对受体介导的基因转移进行了广泛的研究，并取得了较大的进展。受体介导的基因转移有安全、低免疫原性、相对靶向性、载体制备简单、易规模化等许多优点。常用的受体有去唾液酸糖蛋白受体、胰岛素受体、血管内皮细胞生长因子受体、转铁蛋白受体、甘露糖受体、表皮生长因子受体、叶酸受体等。
肿瘤转移是肿瘤患者死亡的重要原因，尿激酶(urokinase,uPA)以及尿激酶受体(urokinase receptor,uPAR)在肿瘤转移过程中发挥着重要的作用。尿激酶受体以及纤溶系统中某些其它成员成为抑制肿瘤转移的靶标。靶向uPA和uPAR的单克隆抗体、可溶性uPAR、能阻断uPA和uPAR相互作用的uPA衍生肽都可以抑制肿瘤细胞的侵袭和转移，uPAR反义核酸表达质粒对肿瘤细胞的侵袭和转移也有明显的抑制作用。
为了观察尿激酶受体反义核酸对肿瘤细胞的作用以及探讨尿激酶受体介导的基因转移的可能性，我们先利用重组腺病毒技术，在人肺巨细胞癌高转移株95D细胞中表达了尿激酶受体的反义基因，并观察其作用：同时表达纯化了尿激酶氨基末端的突变体，与报告基因表达质粒组装成复合物，观察该基因转移系统的基因转移效率，并介导尿激酶受体反义核酸靶向尿激酶受体的基因转移。
一．重组腺病毒介导的uPAR反义基因转移
为了观察uPAR反义核酸对肿瘤细胞体外侵袭的抑制作用，用PCR方法扩增uPAR cDNA 5’端-46bp至+454bp一段长500bp的序列，克隆到病毒载体pAdeno-X中，构建出重组腺病毒载体pAdeno-X-uPAR(-)和pAdeno-X-uPAR(+)。PacI限制性内切酶线性化重组腺病毒载体，转染HEK293细胞，7天后可以获得滴度(pfu／ml)分别为1.5×10~8和0.5×10~8的uPAR反义和正义核酸表达重组腺病毒，分别命名为Ad-uPAR(-)和Ad-uPAR(+)。以不同的病毒感染指数(MOI)感染人肺巨细胞癌高转移株95D肿瘤细胞，3天后用RNA印迹法可以检测到肿瘤细胞中uPAR正义和反义核酸表达，随着MOI的升高，在Ad-uPAR(-)感染的肿瘤细胞中uPAR蛋白质水平逐渐下降，肿瘤细胞的体外侵袭能力也明显下降，
而感染Ad一uPAR(+)的肿瘤细胞无明显变化。结果表明重组腺病毒可以表达uRAR
正义和反义核酸，uPAR反义核酸可以下调95D肿瘤细胞uPAR蛋白质水平，并
明显抑制人肺巨细胞癌高转移株95D肿瘤细胞在体外的侵袭能力。
二.尿激酶受体介导的报告基因转移
利用能够被细胞内吞的重组蛋白发展靶向性非病毒载体系统，并进行基因转
移是一个很有希望的研究方向。融合蛋白ATF一lyS10的氨基末端是尿激酶氨基末
端135个氨基酸，起结合尿激酶受体的作用;梭基末端是10个赖氨酸，起结合
质粒DNA的作用。在大肠杆菌中表达的ATF一lyslo主要以可溶形式存在，并具
有人尿激酶的抗原性。纯化后的ATF一lyslo可以阻滞质粒DNA的迁移，并具有
和细胞表面的尿激酶受体结合的能力。质粒DNA与ATF一lyslo以及多聚赖氨酸
组装的基因转移复合物可以介导报告基因的转移，氯喳可以明显提高基因转移的
效率，流感病毒血凝素融合肚的重组蛋白不能提高基因转移的效率。实验结果表
明:①以ATF一lyslo重组融合蛋白组装的基因转移系统可以介导基因的转移②具
有DNA结合能力和细胞靶向性的重组蛋白质ATF一lyslo为非病毒基因转移系统
提供了一种新的途径。
三.尿激酶受体介导的ul】AR反义基因转移
将uPAR反义核酸表达质粒与ATF一lyslo以及多聚赖氨酸组装成基因转移复
合物，并转染95D细胞。结果发现，AfF一lyslo介导的u队R反义核酸基因转移
可以下调细胞uPAR蛋白水平，并降低细胞与纤连蛋白的粘附能力。Rl’- PCR可
以检测到u队R反义核酸的表达，但uPAR mRNA水平没有明显下降。
ATF一lyslo介导的基因转移具有uPAR靶向性，而且基因转移复合物制备相
对比较简单。其主要缺点有:①基因转移效率低;②基因转移复合物不够稳定，
体内实验比较困难;③基因转移复合物存在非特异性结合细胞的可能。
Great progresses have been made in gene therapy in recent years, and virus vectors become the leading vector for gene delivery. But virus vectors have many disadvantages such as limited capacity for foreign genes, immunogenicity, difficulty in manufacture and storage, which result in the development of nonvirus vectors. In recent years, more and more researchers focus on receptor-mediated gene transfer, which have many advantages such as very low immunogenicity, relatively simple manufacturing and storage, safety and relatively targeting. Asialoglycoprotein receptor, transferrin receptor and receptors for lactoferrin, insulin, epidermal growth factor, folic acid and mannose have been used in receptor-mediated gene delivery.
Tumor metastasis is the important cause for death of most cancer patients. Urokinase receptor and some members in plasminogen activation system play important roles in tumor metastasis, and they have become the main target for tumor therapy. Several technical methods affecting tumor growth and metastasis by targeting the uPA system have been explored, which are monoclonal antibodies directed to uPA or uPAR, soluble recombinant uPAR, uPA-derived peptides to block uPA/uPAR interaction and antisense gene of uPAR.
To observe the effect of antisense gene of uPAR on tumor cells and investigate the possibility of receptor-mediated gene delivery targeting uPAR, we expressed antisense gene of uPAR in human lung giant caner cell lines 95D using recombinant adenovirus and ligand-conjugated poly-L-lysine-DNA complexes as vector to mediate uPAR antisense gene delivery for inhibiting invasive ability of 95D cells in vitro.
Section 1 Recombinant adenovirus mediated antisense uPAR gene delivery
In order to study the effect of uPAR antisense gene on inhibition of invasive ability of human lung giant caner cell lines 95D, 500bp fragment of uPAR cDNA between -46bp~+454bp was amplified, and recombined into plasmid pAdeno-X. The recombinant vectors were named pAdeno-X-uPAR(-) and pAdeno-X-uPAR(+) respectively. 7 days after transfecting HEK293 cell with linearized pAdeno-X-uPAR(-) and pAdeno-X-uPAR(+), the recombinant adenovirus can be obtained, which were named Ad-uPAR(-) and Ad-uPAR(+) respectively. The virus titre(pfu/ml) of
Ad-uPAR(-) was 1.5 x108, and the virus titre of Ad-uPAR(+) was 0.5x108. 95D cells were infected with Ad-uPAR(-) in different multiplicity of infection (MOI). Northern blot analysis could detect the expression of antisense and sense RNA for 500bp fragment of uPAR gene. With the increase of MOI, Western blot analysis indicated that the uPAR protein level decreased, and modified Boyden's Chamber assay suggested that the invasive ability of 95D cells also decreased obviously. The results indicated that adenovirus mediated uPAR antisense gene delivery could downregulate uPAR protein level and inhibit the invasive ability of 95D human lung cancer cells.
Section2 Urokinase receptor mediated reporter gene delivery
Recombinant fusion proteins that combine different functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of nonviral vectors for targeted gene delivery. Here, we described a novel protein termed ATF-lys10 which composed of amino-terminal fragment of urokinase and ten lysines at the carboxyl terminus and facilitated cell-specific gene transfer via receptor-mediated endocytosis. Bacterially expressed ATF-lys10 proteins existed in soluble form, and had antigenicity of human urokinase. Purified ATF-lys10 specifically bound to uPAR-expressing cells and formed protein-DNA complexes with plasmid pGL3-control. These complexes after compensation of excess negative charge with poly-L-lysine, served as a specific gene delivery vector for uPAR-expressing cells. Lysosomotropic compounds such as chloroquine drastically increased ATF-lys10 mediated gene delivery, but not fusion peptide of hemagglutinin. Our results suggest that fusion protein with the properties of DNA binding and cell type targeting repres
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