【作者】 李锴男；

【导师】 刘彦仿；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2004， 硕士

【摘要】 肝细胞性肝癌(hepatocellular carcinoma,HCC)简称肝癌，是恶性程度极高的肿瘤，在我国有着较高的发病率和死亡率，目前，临床对肝癌的诊治仍然十分困难。随着现代分子生物学，基因工程技术和蛋白质工程技术的发展，基因工程抗体的诞生为肿瘤的诊断和治疗带来了希望，尤其是单链抗体的应用成为免疫基因治疗的热点。我室成功的构建了一二硫键稳定的人源化抗肝癌的单链抗体(hdsFv)，提高了抗体的稳定性，降低了亲本鼠源性抗肝癌单链抗体的免疫原性，而其亲和性和特异性无明显下降。CD3是主要分布于成熟T淋巴细胞表面的分子，与TCR非共价结合形成TCR／CD3复合物，构成T淋巴细胞活化信号，而CD3ζ链在TCR／CD3信号转导中起重要作用。本研究将抗肝癌hdsFv的基因与人T细胞活化信号CD3ζ基因融合，构建了肝癌靶向性T细胞的嵌合受体，并在人PBMC和Jurkat T淋巴瘤细胞中表达，为制备肝癌靶向性T细胞奠定了实验基础。 1．构建真核表达载体pcDNA3-hdsFv-CD3ζ。采用RT-PCR从正常人外周血T淋巴细胞总RNA中克隆CD3ζ的胞内区及跨膜区基因，经测序，与GenBank公布的序列一致。用PCR从含有抗肝癌单链抗体的原核表达载体第四军医大学硕士学位论文 pET一22b(+)一hdsFv中克隆其基因序列，经测序，与已知序列相同。将二者序列重组导入真核表达载体pcDNA3，构建重组表达载体peDNA3一hdsFv一CD3乙，经酶切鉴定序列分析与GenBank公布的CD3乙和已知的hdsFv序列相同。 2.肝癌靶向性T淋巴细胞活化融合基因hdsFv一CD3心的表达与定位检验。将构建好的真核表达载体pcDNA3一hdsFv一CD3心用脂质体法转染JurkatT淋巴瘤细胞，经G418筛选获得正常生长的阳性克隆，用Westernblotting检测表明转染后Jurkat细胞中有hdsFv一CD3心融合蛋白的表达。再经细胞化学染色鉴定和灰度分析，hdsFv--CD3心表达于Jurkat细胞的细胞膜上，经修饰的Jurkat细胞可表达较高量的CD3心分子。用RT一PCR从经hdsFv一CD3心修饰的Jurkat细胞中检验出了hdsFv的表达。 3.hdsFv一CD3心修饰的Jurkat和PBMC的功能检验。将hdsFv一CD3心修饰的Jurkat细胞与HepGZ细胞共培养，发现Jurkat细胞粘附在HepGZ上，说明了经hdsFv一CD3乙修饰的Jurkat细胞具有良好的靶向性。再将pcDNA3一hdsFv一CD3心用脂质体法转染pBMC，经细胞化学染色鉴定，CD3心的表达量增加，证实hdsFv一CD3心导入PBMC中。由于活化的T细胞表达CD25，用hdsFv一CD3心修饰活化的的PBMC，经直接免疫荧光染色和FCM分析CD25的表达增加。用hdsFv一CD3心修饰活化的PBMC的培养液上清进行IL一2的生物活性检测，结果证实修饰后的PB毗的IL一2分泌增加。hdsFv一CD3心修饰的PBMC，与肝癌H即G2细胞共培养，以未转染的PBMC与HepGZ细胞共培养做对照，培养6h后，AnnexinV荧光染色检测HepGZ细胞的凋亡，结果表明经hdsFv一CD3心修饰活化的PBMC可以较好的诱导HepGZ细胞的凋亡。将hdsFv一CD3心、hdsFv修饰的PBMC与未转染的PBMC与肝癌HePGZ细胞和宫颈癌H。1a细胞按50:1的效靶比共培养，结果表 4第四军医大学硕士学位论文明，hdsFv一CD3乙、hdsFv修饰的PBMC均可显著杀伤HepGZ细胞，但以hdsFv一CD3心修饰的PBMC杀伤作用最强，共培养6h后，HepGZ细胞即有死亡，12h后死亡细胞进一步增加，18h时，转染hdsFv和hdsFv一CD3心的PBMC对H即G2细胞的杀伤率分别可达到62.39%和75.85%。转染hdsFv一CD3心的PBMC也可显著杀伤Hela细胞，18h时可达63.13%。转染hdsFv一CD3心的PB毗对Hela细胞杀伤率较高除与CD3乙有活化T细胞的功能外，还与PBMC培养时加入了IL一2引起的非特异性活化有关，应属于非特异性活化杀伤。 本研究成功的克隆了CD3心的胞内区及跨膜区基因，构建了抗肝癌hdsFv与T淋巴细胞活化信号CD3心嵌合受体的真核表达载体penNA3一hdsFv一Cn3心，转染入PB讹和JurkatT淋巴瘤细胞，并以此为基础进行了T淋巴细胞活化与功能检验。修饰后的PBMC和肝癌H即G2细胞共培养，与未修饰的PBMC相比，能较好的诱导H即G2细胞的凋亡。 综上所述，我们首次构建了抗肝癌单链抗体融合CD3心的基因，制备了具有肝癌靶向性的PBMC和JurkatT淋巴细胞。该实验结合了杀伤性T细胞的抗肿瘤作用与协同刺激分子两大研究热点，使体液免疫与细胞免疫相结合，利用了TCR/CD3心中抗原识别和信号转导的独立性，将抗体分子的导向作用和T细胞活化信号相结合，构建的肿瘤特异性T细胞的杀伤功能由于不受栩C限制具有一定的临床应用价值，可以提高抗肿瘤综合免疫治疗的水平。更多还原

【Abstract】 Hepatocellular carcinoma (HCC)> one of the most common malignant tumors, often happens with high incidence and mortality in China.Clinical diagnosis and treatment are very difficult.With the development of molecular biology, genetic engineering and protein engineering, genetically engineered antibody is a new way for diagnosis and treatment to tumors. Above all, the application of single chain Fv is a hot point in immunogenetic therapy. Our group has successfully constructed a humanized disulfide-stabilized scFv (hdsFv), which can reduce its immunogenicity of mouse-derived scFv and keep the high stability, specificity and affinity of its parent antibody to some extent. The CD3 is present on mature human T cells. It is associated with the T cell receptor (TCR) and is responsible for the signal transduction of the TCR. The 4 chain of CD3 plays a crucial role in the signal transduction of T cell activation. We made a basic study on HCC-specific T cell by constructing a chimeric receptor which was comprised of the CD3ζ gene fused to the anti-HCC hdsFv, putting it into a eukaryotic expression vector and transfecting it into Jurkat cells and PBMC.1.The construction of a eukaryotic expression vector pcDNA3-hdsFv-CD3ζ. The transmembrane and intracellular domains of CD3ζ cDNA was amplified from human T lymphocytes by RT-PCR. The hdsFv gene was cloned from pET-22b(+) - hdsFv by PCR.The CD3ζ fragments were ligated to thedownstream of the anti-HCC hdsFv cDNA and sequence were verified. The sequence of hdsFv was in accordance with the known sequence. The sequence of cDNA of the transmembrane and intracellular domains of CD3ζ was conformed to the sequence of Genebank.2. The expression and location of hdsFv-CD3ζ in transfected Jurkat cells. The pcDNA3-hdsFv-CD3ζ was transfected into human CD4+ Jurkat cells through lipofectamine. The positive cells was filtrated by G418. The protein of the hdsFv-CD3ζ was confirmed by Western blotting. Immunocytochemical staining was done with anti-CD3ζ mAb before and after transfection. We observed the expression of fused protein on cell membrane and the increment of CD3ζ expression after the transfection and detected hdsFv in transfected Jurkat cells by RT-PCR.3. The detection of function of transfected Jurkat cells and PBMC. When transfected Jurkat cells were incubated with HepG2 cells, the Jurkat cells adhered to the tumor cells, which indicated the cell-specific targeting of Jurkat T cells. The pcDNA3-hdsFv-CD3ζ was transfected into PBMC through lipofectamine. Immunocytochemical staining was done with anti-CD3ζ mAb before and after transfection. We observed increment of CD3ζ expression after the transfection, which confirmed hdsFv-CD3ζ in PBMC. Activated T cell revealed by CD25 up-regulation, which was proved in transfected PBMC by FCM. Robust IL-2 secretion were observed when the hdsFv-CD3ζ PBMC were stimulated by incubated with HepG2 cells. We cocultured transfected and untransfected cells with HepG2 cells and detected the apoptosis of HepG2 cells by Annexin V immunofluorscence.The results showed that transfected PBMC could induce the apoptosis of HepG2 cells better.Transfect pcDNA3-hdsFv-CD3ζ and pcDNA3-hdsFv into PBMC, and the PBMC were cocultured with HepG2 cells and Hela cells.Untransfected PBMC were cocultured with HepG2 cells and Hela cells as a control group. As a result,hdsFv-CD3ζ and hdsFv grafted PBMC could lyse HepG2 cells distinctly. The mortality of HepG2 reached 75.85 % and 62.39 % separately. hdsFv-CD3ζ grafted PBMC could lyse Hela cells, too.The mortality reached 63.13%. The high mortality of Hela cells by cocultured with hdsFv-CD3ζ PBMC is probably related with function of CD3ζ and presence of IL-2 which was put into PBMC. But such killing is unspecific.In our study, we have successfully cloned the transmembrane and intracellular domains of CD3ζ from T cell, constructed a eukaryotic expression vector pcDNA3-hdsFv-CD3ζ which was successfully expressed in Jurkat cells更多还原