【作者】 李璟；

【导师】 温博贵；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2002， 博士

【摘要】 研究背景：肿瘤是严重危害人类健康的疾病之一，尽管肿瘤的治疗方法取得了很大的进展，但是仍然有很多病人在治疗后复发或转移，或因反复用药产生耐药性。近年来，从天然植物中寻找抗肿瘤药物已经成为肿瘤治疗领域的一大热点。到目前为止，国内外学者已从苦瓜中分离纯化了数种核糖体失活蛋白，经研究证实这些蛋白质具有抗肿瘤活性，并且可以诱导肿瘤细胞凋亡。苦瓜中还存在其它生物活性蛋白有待进一步开发和利用。 目的：从苦瓜中分离纯化具有抗肿瘤活性的蛋白质，并对其作用机制进行初步的探讨。 方法：(1)将苦瓜种仁应用丙酮分级沉淀法制备苦瓜子蛋白粗提物，用Hiprep 16／10 SOURCE 30S阳离子交换层析和HiLoad 26／60 Superdex 75凝胶过滤分离纯化，纯化产物经SDS-PAGE和双向电泳，硝酸银染色鉴定纯度，SDS-PAGE测定苦瓜子蛋白的分子量，等电聚焦测定等电点。(2)应用人非小细胞性肺癌细胞株A549、人肝细胞性肝癌细胞株BEL-7402、人早幼粒细胞性白血病细胞株HL-60和小鼠淋巴瘤细胞株P388对从苦瓜子中分离纯化的14个组分的蛋白质进行体外抗肿瘤活性的初步筛选。悬浮培养的白血病细胞应用MTT法、贴壁生长的实体瘤细胞应用SRB法进行抗肿瘤活性的筛选。(3)对初筛显示较强抗肿瘤活性的苦瓜子蛋白HP2-751和HP3-751应用白血病、肺癌、肝癌、胃 汕头大学医学院博士学位论文 中文摘要 癌、结肠癌、乳腺癌、卵巢癌等n个肿瘤细胞株进行体外抗肿瘤谱分析。（4） 将不同浓度的苦瓜子蛋白 HPZ－75、Ill，3．75和 HPS－75作用于人肝细胞性肝癌 BEL．7402细胞，应用流式细胞仪对BEL－7402细胞的DNA含量进行动态分析， 应用透射电镜观察BEL7402细胞超微结构的变化。 『结果：门）苦瓜子蛋白丙酮粗提物经阳离子交换和凝胶过滤后，共得到14 个组分的蛋白质，暂定名为pl－751、Ill，l－753、HPZ－751、HPZ－752、HP3－751、 HP3．752、HP3－753、HP4751、HPS．751、grs－752、HPS．753、HP6．751、HP6．752 和HP6．753。它们均为碱性蛋白，等电点在9刀．9．6之间，分子量在13－29KD之 间。苦瓜子蛋白 ffeZ－75和 HP3－75在双向电泳图谱中显示为单个斑点，可见 纯化的效果良好。（2）对从苦瓜子中分离纯化的！4种蛋白组分进行了体外抗肿 瘤活性的筛选，其中HPZ－75、HP3－75、HP3．753和HPS－751对人非小细胞性0 肺癌A549细胞、人肝细胞性肝癌BEL－7402细胞、人早幼粒细胞性白血病HL七0 细胞和小鼠淋巴瘤 p388细胞均有抑制作用，其汇。在 1石X10人2．4X10”’m。e （之间。不同的苦瓜子蛋白组分对不同的肿瘤细胞的抑制作用不同。人非小细胞 性肺癌A549细胞、人肝细胞性肝癌 BEL－7402细胞对 HPZ－75最为敏感，其n。 分别为7．6X 10bOCh和4．SX 10forrt。人早幼粒细胞性白血病HL七0细胞对 Hp5J 最为敏感，IC50为4．3X106moto。小鼠淋巴瘤p388细胞对Hp3刁 最 为敏感，IC扣为 1石X 10－6m。W。门）不同的肿瘤细胞株对苦瓜子蛋白 HPZ刁 和 HP3－75！的敏感性有所不同，对苦瓜子蛋白最敏感的肿瘤细胞株是人肺腺癌 SPCM，其ns。值分别为4．5 XIO“ho W opZ－75）和5．0 XIO枷ilL p3刁5卜对 HPZJ5和 HP3J5两种苦瓜子蛋白均不敏感的细胞株为人急性 淋巴母细胞性白血病MOLT4、人胃腺癌MKN28、人胃腺癌SGC－7901、人结 肠腺癌HCTll6和人乳腺癌MCF－7。人早幼粒细胞性白血病HL60、人非小细 胞性肺癌 A549和人肝细胞性肝癌 BEL－7402对苦爪子蛋白 HPZ－75和 HP3－7n 的敏感性介于两者H间，其 IC00值在 3．4 XI obeffel．0 XIO”’iOW之间。N） 2 汕头大学医学院搏士学位论文 中文摘要 苦瓜子蛋白诱导BEL7402细胞凋亡具有时间和剂量依赖性，随着剂量的增加， HPZ－75、HP3－75诱导 BEL－7402细胞凋亡率也随之升高，但是 HPS－75却呈 现下降趋势。在 3个苦瓜子蛋白组分中，HP3刁5！诱导 BEL7402细胞凋亡的能 力最强，分别加入0．5mghlll、0．25mglllll、0．125mtw的HI，3－751、HPZ－751和4”“”“’”“—“”””——”—一”“—”—”—一“”“—”—““一“一““””——－’—“”一“”—“·”－ ．0 HPS．75，BEL．7402细胞凋t率为HP3－75：90．gi％、80．47％和77．89％，明显 高于加入同等剂量的HPZ－751和pps－751（BEL－7402细胞凋亡率分别为 50?更多还原

【Abstract】 Background: Carcinoma is one of the leading causes of death from diseases in the world, hi addition to cytotoxic therapy, radiotherapy, surgery, gene therapy and immunotherapy are effective for malignant tumor. However, many patients show limited responses to these therapies or become unresponsive after treatment. The search for effective anti-tumor agents is of global importance. Recently, many investigators purified amounts of ribosome inactivating proteins (RIPs) from native Momordica charantia L, which were identified to have anti-tumor activity and exert their effects by induction of apoptosis of carcinoma cells. There are others active proteins in Momordica charantia L. whose bioactivities remain unknown.Objective: To purify proteins from Momordica charantia L that can inhibit the proliferation of tumor cells and to investigate their anti-tumor mechanisms.Methods: (1) The proteins from Momordica charantia seeds were purified by a procedure involving extract by acetones and a series of chromatographies on Hiprep 16/10 SOURCE SOS and HiLoad 26/60 Superdex 75. The protein fractions’s purity were characterized by the SDS-PAGE and 2-D electrophoresis. The molecular weight of the proteins was measured by SDS-PAGE and the isoelectric point (pi) was measured by isoelectric focusing gel electrophoresis (IEF). (2) The anti-tumor effect of 14 protein fractions from Momordica charantia was measured by MTT assay onhuman myeloblastosis HL-60 cell line and mouse lymphadenoma P388 cell line and measured by SRB assay on human non-small cell lung carcinoma A549 cell line and human hepatocellular carcinoma BEL-7402 cell line. (3) The anti-tumor activity of HP2-751 and HP3-751, which exert higher anti-tumor activity on A549, BEL-7402, HL-60 and P388, was studied on several tumor cell lines, such as leukocythemia, lung carcinoma, hepatocarcinoma, colon cancer, breast cancer, oval carcinoma and gastrocarcinoma. (4) The BEL-7402 cells were treated with different concentrations of HP2-751, HP3-751 and HP5-751 at 12hr and were treated with the same dose (0.25mg/ml)of HP2-751, HP3-751 and HP5-751 at different times. The loss of DNA fragments, after plasma-membrane of treated cells, was revealed by propidium-iodide staining, followed by flow cytometer analysis. The percentage of apoptotic cells was calculated from the area of hypodiploid peak. The ultrastructure of BEL-7402 cells that were treated by proteins from Momordica charantia was surveyed by electron micrograph.Result: (1) 14 protein fractions were obtained after cation exchange chromatography and gel filtration chromatography, which were transiently called as HP1-751, HP1-753, HP2-751, HP2-752, HP3-751, HP3-752, HP3-753, HP4-751, HP5-751, HP5-752, HP5-753, HP6-751, HP6-7523 and HP6-753. They are all strong basic proteins, whose pi are between 9.0-9.6 and whose molecular weight are between 13-29KD. HP2-751 and HP3-751 appeared as a single point on the figure of 2-D eletrophoresis. (2)The anti-tumor effect of 14 protein fractions was evaluated in vitro on human myeloblastosis HL-60 cells, mouse lymphadenoma P388 cells, human non-small cell lung carcinoma AS49 cells and human hepatocellular carcinoma BEL-7402 cells, of which, HP2-751, HP3-751, HP3-753 and HP5-751 inhibit the proliferation of all the 4 tumor cell lines, the ICso value between 1.6 X10"6 and 2.4X10’5moI/L. Different tumor cell lines exhibit different sensitivities to the same protein fraction from Momordica charantia and so do the same tumor cell line to different protein fractions. The most sensitive tumor cell lines to HP2-751 were human non-small cell lung carcinoma A549 cells and human hepatocellular carcinoma BEL-7402 cells with IC50 value 7.6 X10"6 mol/L and 4.8 X KT6 mol/L respectively. The most sensitive tumor cell lines to HP5-751 were human myeloblastosis HL-60 cells with ICso value 4.3 X 10"6 mol/L. The most sensitive tumor cell lines to HP3-751 were mouse lymphadenoma P388 cells with IC50 value 1.6X10"6 mol/L. (3) The anti-tumor activity of HP2-751 and HP3-751 to several tumor cell l更多还原