【作者】 赵晶；

【导师】 杨安钢； 王成济；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2002， 博士

【摘要】 恶性肿瘤的基因治疗是新兴的研究领域，具有广阔的发展前景。以诱导细胞死亡作为肿瘤基因治疗的手段，必须解决诱导方法的特异、高效、持续、低毒副作用等问题。 granzyme B(粒酶B，GrB)是丝氨酸蛋白酶家族的成员，是CTL(细胞毒性T淋巴细胞)和NK(自然杀伤)细胞产生的主要杀伤性分子。GrB介导的死亡途径是多层次、多水平的，GrB基因可以作为杀伤肿瘤细胞的有力工具。 本文首先证实了活性型GrBa基因异位表达具有细胞致死效应。胞浆中过表达的GrBa蛋白通过自身的丝氨酸蛋白酶活性，切割细胞内重要的结构和功能蛋白，介导转染细胞死亡。形态异常的多核巨细胞是GrBa介导的死亡途径中特殊的现象，由细胞骨架和有丝分裂异常所致。 将针对肿瘤抗原HER-2的单链抗体基因与活性型GrBa基因用绿脓杆菌外毒素(PE)的转位肽序列相连接，形成Ab-PE Ⅱ-GrBa(immunoGrB)基因。表达的immunoGrB蛋白进入肿瘤靶细胞的内吞小体后，发生Arg279和Gly280之间肽键的定点裂解，裂解后的PE Ⅱ-GrBa被转位到细胞质中。本文比较了不同长度转位肽(PE253-364aa和253-358aa)的作用效果。一方面， 男 曰 旱 匡 大 学 忆 士 学 位 论 丈 从瞬时表达、可诱导表达和组成性表达三方面研究证明，N端部分PE 11序 列①E 280习64。和280上58 aa）的存在基本不影响GrBa诱导转染细胞死 亡的作用。其中N端融合有较短肽段（PE 280－358 aa）的重组GrBa蛋白具 有较强的蛋白酶活性和细胞生长抑制率，与单独活性型GrBa的作用程度接 近。另一方面，从瞬时转染和稳定转染角度研究证实，immunoGrB分子不具 有杀伤活性，它只有当特异性内化入HER－2阳性肿瘤细胞后才转变为活性形 式。初步的动物实验结果表明，immunoGrB分子具有体内抗HER－2阳性肿 瘤活性，能特异性杀伤HER－2阳性肿瘤细胞。 将immunoGrB基因稳定转染Jurkat细胞，观察到修饰的淋巴细胞能产 生和分泌inuntmoGrB分子，并且淋巴细胞能正常生长和建株。共培养实验 表明，分泌immunoGrB蛋白的淋巴细胞特异性杀伤HER－2阳性肿瘤细胞， 而对HER－2阴性的肿瘤细胞和正常细胞没有杀伤作用。含有较短转位序列 OE 11 253习58 aa）的 nununoGrB修饰的淋巴细胞杀伤作用最强，杀伤率可 达到70％以上。上述结果为基因修饰的自体淋巴细胞回输策略提供了体外实 验依据。 综上所述，用l’－－－－－－tinoGrB基因修饰的淋巴细胞杀伤HER－2阳性肿瘤， 可以特异性地在肿瘤细胞内部引发死亡。irnmunoGrB分子对肿瘤细胞的杀伤 作用具有特异性和高效性，并且分子本身对人体为低免疫原性，有利于长期 使用，这对于肿瘤的治疗具有重要的理论意义和实用价值。更多还原

【Abstract】 Gene therapy has been widely used to cure cancers in the past decades and provides a promising way to cancer therapy. It could be reasonable to suppress tumor growth by inducing tumor cells to die. This active antitumor strategy, however, demands specificity, efficiency, persistence and minimal side-effect.Granzyme B(GrB) is member of serine proteinase family and plays an essential role in CTL/NK-mediated killing. Redundancy of GrB-mediated death pathways enables lymphocytes to eliminate unwanted cells efficiently. Therefore, GrB could be a new good candidate to kill tumor cells.Our results showed ectopic expression of active GrB(GrBa) led cells to death. This was probably because GrBa protein was overexpressed in cytoplasm and its serine proteinase activity caused proteolysis of endogenous substrates which might be important to cellular structure and function. There was a special phenomenon that cells, when subjected to GrBa-mediated death, appeared as volume extension and often in the wake of multiple nuclei. These giant cells with multilobed or multiple nuclei exhibited mitotic and cytoskeletal abnormality.In present study, the gene of single chain antibody against HER-2, a kind of tumor marker on membrane, was fused to 5’-end of GrBa gene. Sequence encoding Pseudomonas exotoxin A(PE) translocating peptide was localized between antibody gene and GrBa gene. The resulting fusion protein gene was designated as irnmunoGrB gene. In theory, on entry into endosome of tumor cells irnmunoGrB protein releases C-terminal PE II-GrBa into cytosol as a result of auto-cleavage of peptide bond between Arg279 and Gly280. Two kinds of PE translocating peptides covering 253-364 aa and 253-358 aa were compared for their endosome-disruptive function. On one hand, it was demonstrated that presence of part of PE II sequence(280-364 aa and 280-358 aa) hardly abrogated GrBa activity to induce cell death, which was proven by the data from transient expression, inducible expression and constitutive expression as well, and that the shorter N-terminal peptide PE II-GrBa fusion protein had, the closer extent of enzyme activity and cell growth inhibition to that of GrBa alone. On the other hand, immunoGrB was found inactive in transient and stable transfection research until when translocating from endosome in HER-2 positive tumor cells, it turned into an active form. We next tested antitumor activity of immunoGrB in nude mouse models. It was observed that intramuscle administration of immunoGrB gene suppressed HER-2 positive tumor.Genetically modified lymphocytes strategy was further tested hi cultured Jurkat cells. ImmunoGrB transduced Jurkat cells produced and secreted GrBa fusion protein while exhibiting a normal growth curve. By co-cultivation, these modified Jurkat cells were shown to have selective cytotoxicity to HER-2 positive tumor cells. Over 70% killing was achieved by lymphocytes transduced with immunoGrB containing PE II sequence(280-358 aa). The above data suggest that it is feasible to administrate immunoGrB-secreting autologous lymphocytes to suppress HER-2 positive tumor in vivo.hi summary, this lymphocyte-based antitumour strategy is characteristic of initiation of cell death specially in HER-2 positive tumor. Such is of value, in both theory and clinic, in that immunoGrB potently and selectively kills HER-2 positive tumor cells, and that little immunogenicity makes it possible forlong-term application.更多还原