【作者】 阴继凯；

【导师】 马庆久； 梁英民；

【作者基本信息】 第四军医大学 ， 外科学， 2004， 硕士

【摘要】 原发性肝脏恶性肿瘤主要有肝细胞癌(hepatocellular carcinoma,HCC)和胆管细胞癌，而前者的发病率约为后者的十倍。全世界每年大约有一百万新增病例，并且有明显的地理分布性。我国是肝癌高发地区，全世界每年新发病例中大约45％在中国。每年由肝硬化发展为肝细胞癌的病例呈现出逐年上升的趋势(大约1％-6％)。目前主要的治疗方法包括有肝切除术、肝移植术、冷冻治疗、射频治疗、微波治疗等等。由于单一治疗模式效果往往都不是非常理想，现多采用多种治疗方式联合应用的综合疗法。 诱导肿瘤细胞凋亡是现在肿瘤治疗研究中的新领域，相关的生物学药物既达到了杀伤肿瘤细胞的目的，又避免了由于本身的毒副作用和肿瘤细胞死亡后的炎性反应的影响，为恶性肿瘤的治疗开辟了新的前景。 BCR-ABL是与慢性粒细胞白血病发病密切相关的恶性蛋白，梁英民等利用基因重组技术将bcr-abl基因中编码SH3结构域的基因与HIV病毒的编码蛋白转导结构域(PTD)的基因进行融合表达，获得了一种相对分子量为23kD的PTD-BCR／ABL SH3融合蛋白。表达的PTD-BCR／ABL SH3融合蛋白由于PTD的存在，融合蛋白可以有效的转入细胞内，并且直接 第四军医大学硕士学位论文定位于细胞核，该蛋白可以诱导白血病细胞K562发生调亡。 SH3结构域是指蛋白酪氨酸激酶在与下游蛋白相作用的时候都享有的共同结构Src同源区3结构域。同时含有SHZ和SH3结构域的蛋白酪氨酸激酶复合作用于生长调节的信号网络，包括那些含有小G蛋白和磷酸肌醉第二信使的信号转导过程。因为SH3结构域参与生长调节信号转导中许多特殊的蛋白间的相互作用，所以作为新的抗增殖药物的潜在的靶位点吸引了研究人员的关注。 人类免疫缺陷病毒(HIV)·1所编码的反式激活蛋白工叮的基序中含有蛋白转导结构域(R旧teinT]甩几妇讹tion Domain，PTD)，可以穿越细胞膜性结构，直接进入细胞浆，其特点是转导速度快，效率高.将PTD于其它外源性效应分子融合后，可介导提高效应分子向细胞内的转运，从而提高细胞杀伤效应。 基于以上实验基础及相关理论，本文将该蛋白与肝癌细胞进行共培养，试图观察这种来源于血液病的相关蛋白对实体瘤细胞是否具有同样促凋亡活性，同时对其促凋亡机理进行讨论。通过免疫细胞化学染色、光镜及电镜观察等方法证实， PTD一BCR/ABL SH3融合蛋白与肝癌细胞共培养后，首先，融合蛋白进入肝癌细胞内主要分布在细胞核，胞浆亦有少量分布。细胞与蛋白共培养而加蛋白组在加入蛋白后4h，细胞形态上开始发生变化，部分细胞皱缩，失去原来伸展生长的成纤维细胞样的形态，细胞透光度下降。24小时后实验组内约80%细胞死亡。细胞计数、MTT检测发现，对照相比，加入PTD一BC侧ABL SH3融合蛋白的细胞生长出现下降，蛋白浓度七0.14mg/ml时，随着浓度的增加和作用时间的延长，细胞生长受抑制的程度增加，呈现剂量依赖性和时间依赖性。当蛋白浓度低至0.08mg/m】时，36h后细胞又逐渐开始生长，说明这个浓度不足以抑制所有细胞，经过对数生长期，存活的细胞再次恢复生长，因此，低于此浓度将不会第四军医大学硕士学位论文有效抑制肿瘤细胞生长。进一步电镜观察显示肝癌细胞在PTD一BC侧ABLSH3融合蛋白的作用下细胞死亡，但胞膜完整，有凋亡小体形成，呈现出凋亡细胞的明显特征。 将融合蛋白肿瘤局部注射荷有肝癌细胞HHCC肿瘤的裸鼠，可观察到实验组瘤体的生长速度明显慢于对照组，治疗治疗组裸鼠存活时间延长。HE染色表明，对照组肿瘤细胞生长活跃并可见异常核分裂向，而治疗组可见嗜酸性小体样结构，而其它组织如心、肝、脾、肾中未检测到异常形态。 综上所述，PTD一BCRjABL SH3融合蛋白可以顺利导入肝癌细胞内，通过诱导肝癌细胞凋亡从而杀伤肝癌细胞，具有高效性;在裸鼠体内可有效抑制肿瘤的生长，延长动物生存时间.因此，本实验为将PTD一BCR/ABL SH3融合蛋白运用于肝癌的介入治疗提供理论依据。更多还原

【Abstract】 There are two kinds of primary malignant liver tumours, hepatocellular carcinoma (HCC) and cholangiocarcinoma. Hepatocellular carcinoma is 10 times more frequent than cholangiocarcinoma. The overall incidence is estimated to be 1 million cases per year with a wide geographic variability. China is a countary with high risk of HCC, 45 percent of the new cases of the world are occurred in China approximately. There also appears to be a clear association with cirrhosis with an annual risk of developing HCC in such cases of 1% to 6%. Until now, the main treatments include surgical resection, liver transplantation, cryoablation, radiofrequency (RF), microwave therapy, etc. But the effect of single therapy mode is usually not good enough, onetreatment always combine with others.The thought of inducing tumor cell apoptosis will lead to the emergence of novel antitumor therapy with high efficacy and low toxicity and inflammatory reaction.SH3 domain belongs to Src homology domain which exists in protein-tyrosine kinase and its downstream proteins. Protein-tyrosine kinases include both SH2 and SH3 domains participate in signal net, which alsocontains signal transduction of small G protein and phosphocreatine second messenger. Because SH3 domain is involved with growth-adapting signal transduction, it attracts many attention of being applied in targeted anti-proliferation drugs.PTD derives from tat of HIV-1 can translocate across cell membrane and get into cytoplasma directly effiently and promptly. When fused with some effectors it can lead these factors into cytoplasma, which can improve the cytotoxity of them.In this paper, hepatocellular carcinoma cells were co-cultured with PTD-BCR/ABL SH3 fusion protein. We wanted to know whether this fusion protein could induce stereo-tumor apoptosis and discuss the mechanism of apoptosis.After hepatocellular carcinoma cells were co-cultured with PTD-BCR/ABL SH3 fusion protein, most fusion protein was localized in nuclei, and little in cytoplasma. After co-ccultured 4h some cells shrinked, and after 24h 80% died by immunocytochemistry staining and light microscope observation. The results of cell counting and MTT assay in expriment group showed that the cell growth was inhibited, and when the protein’s concentration >0.14mg/ml, the higher protein concentration was given, the more inhibition cells suffered. It was dosage and time dependent. But when protein concentration declined to 0.08mg/ml, cell groethed again after 36h, which indicated this dosage couldn’t inhibit cell growth effectively. Electron microscopy analyses displayed shrinkage nuclei, chroatin condensation, plasma membrane bledding and chromatin breakage fragments and apoptotic bodies.We next tested antitumor activity of PTD-BCR/ABL SH3 fusion protein in nude mouse models. It was observed that tumor local administration of PTD-BCR/ABL SH3 fusion protein suppressed HHCC tumor. PTD-BCR/ABL SH3 fusion protein was administrated into the nude micewith HHCC xenograft. The growth rate of tumor of treament group was distinctly slower than that of control group, and survival time of treament group mice was longer than that of control group. Tumor cells of control group growth actively and showed pathologic karyokinestic image, and treament group showed many acidophilia bodys. While in other tissues, such as heart, liver, spleen, lung, kidney displayed normal morphology.In summary, PTD-BCR/ABL SH3 fusion protein can enter into hepatocellular carcinoma cells and induce these cells apoptosis, showes high cytotoxity, and in vivo it can suppress tumor growth and prolong nude mice’ life span. Therefore the present study probably provides laboratory data for the application of PTD-BCR/ABL SH3 fusion protein into interventional therapy of hepatocellular carcinoma.更多还原