【作者】 张晨；

【导师】 凌昌全；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2004， 博士

【摘要】 蜜蜂毒是中国传统的中药,蜂毒素是蜜蜂毒中的主要成分和活性单位,其约占蜂毒干重的50%。蜂毒素的生物学作用广泛,具有抗菌、抗关节炎、抗辐射、镇痛和对心血管方面产生影响,而近年来在抗肿瘤及抗爱滋病病毒方面的研究尤其引人注目。原发性肝细胞癌是我国最常见的恶性肿瘤之一,其大部分具有原发性耐药现象,对一般化疗药物不敏感,治疗是临床上十分棘手的问题。蜂毒素具有独特和极强的细胞膜穿孔作用,对细胞的离子交换和信号传导均有影响,因而有望对肝癌的治疗产生深刻的影响。本研究是在我科实验室对蜂毒素提取纯化及抗肿瘤作用进行多年研究的基础上,建立了中压液相色谱提取纯化蜂毒素工艺,为蜂毒素的产业化奠定了基础。本研究的目的是(1)对此工艺提取纯化的蜂毒素进行鉴定和含量测定,评价该方法的可行性和可靠性;(2)在得到高纯度蜂毒素的基础上了解其是否具有抗肝癌的生物活性及对肝癌细胞生物特性的影响;(3)进一步了解蜂毒素抗肝癌细胞作用的可能机制。其意义在于为蜂毒素在肿瘤临床上的应用提供科学的实验依据。本实验所采用的方法:(1)用AKTA 蛋白纯化工艺开拓系统提取蜂毒素样品,通过质谱进行鉴定,并通过HPLC测定其含量。(2)MTT法测定蜂毒素对各种肝癌细胞系的抑制作用,了解蜂毒素抗肝癌的生物活性。(3)利用流式细胞仪方法,测定蜂毒素对细胞增殖核抗原(PCNA)和细胞周期的影响。(4)通过形态学观察、DNA电泳、流式细胞仪和TUNEL等方法,观察蜂毒素的促凋亡作用。(5)通过流式细胞仪、RT-PCR方法,研究蜂毒素促肝癌细胞凋亡的部分信号传导的机制。结果表明:(1)蜂毒素样品电喷雾质谱在全扫描一级激发的条件下,分子离子峰为m/z 2846.0,从而确定其分子量为2846 Dal,与蜂毒素的理论分子量相符;HPLC含量测定结果计算样品的平均百分含量为97.32%,RSD = 0.49%。(2)蜂毒素体外抗肝癌实验显示:蜂毒素对实验中的4种肝癌细胞系均有抑制作用,显示了良好的抗肝癌生物活性;对BEL-7402细胞生长、增殖有明显的抑制作用,其抑制率的IC50值为15.33 (g/ml,并且可降低细胞的PCNA表达,与对照组比较差异有统计学意义;蜂毒素32 μg/ml和16 μg/ml处理细胞后,细胞周期中S期细胞比例相对增多,与对照组比较有统计学差异,其干扰细胞周期的正常移行,表现为阻滞细胞于S期。(3)蜂毒<WP=7>素处理肝癌细胞后,光镜下见部分肝癌细胞出现凋亡形态学变化;DNA电泳可见典型的细胞凋亡“梯形带”;原位缺口末端标记、AnnexinV和APO2.7蛋白的流式细胞仪等定性、定量分析提示可有效诱导肝癌细胞凋亡,其凋亡率均高于空白对照组。(4)经蜂毒素体外处理BEL-7402肝癌细胞后,其Fas蛋白的表达增加,而没有FasL的表达 ;RT-PCR扩增结果显示肝癌细胞内的胞质型磷脂酶A2(cPLA2)和Fas mRNA的表达增高,而同样没有检测出FasL mRNA的表达。从本研究中初步得出以下结论:(1)采用AKTA 蛋白纯化工艺开拓系统提取蜂毒素方法可行、可靠,其纯度达到标准。(2)提取、纯化的蜂毒素体外对4种肝癌细胞均有一定的抑制作用,表明该工艺提取、纯化的蜂毒素具有抗肝癌细胞的生物活性。(3)而蜂毒素的抗肝癌细胞作用的生物活性可能与其抑制PCNA表达、干扰细胞周期的正常移行、诱导细胞凋亡、激活细胞内cPLA2活性,以及干扰细胞内信号传导通路有关。更多还原

【Abstract】 Bee venom is a natural substance that has been used medicinally as traditional Chinese medicine for more than thousands years. Melittin, a 26-residue peptide, is the major component and activity unit of bee venom, exhibits highly and extensive biological action in antibacterial, anti-arthritis, anti-radiation, analgesia, as well as effect on heart-blood vessel. In recent years, it is noticeable that melittin studies in anti-tumor and anti-virus of acquired immune deficiency syndrome (AIDS). Hepatocellular carcinoma is one of the most common malignancies in our country. It may be the important role of treatment in hepatocellular carcinoma with melittin, a unique and potent cell membrane perforation. The purpose of paper is to identify of melittin extracted with AKTA protein production explorer system, and study anti-hepatocarcinoma action and mechanisms of melittin. It is great significance that this studies provided scientific data to promote clinical application of melittin in the treatment of malignant disease. Methods: (1) The melittin extracted from bee venom with AKTA protein production explorer system were evaluated with high performance liquid chromatograph (HPLC) and mass spectrometry. (2) The efficiency of melittin in anti- hepatocarcinoma was determined MTT assay. (3) Morphologic observation, Flow cytometry, DNA electrophoresis, RT-PCR and TUNEL assay were used to study the cell cycle, proliferating cell nuclear antigen (PCNA), apoptosis and signal transduction of hepatocarcinoma cells treated by melittin. Results: (1) Mass spectrometry result of the melittin isolated and purified from bee venom showed that m/z was 2846 dalton. Content of melittin was 97.32%, and relative standard deviation was 0.49%, which was detected with HPLC. (2) MTT assay results demonstrated that melittin can inhibit 4 hepatocarcinoma cells (HepG2, Hep3B, BEL-7402 and SMMC-7721) proliferation in vitro and 50% inhibitive concentration was 36.95 (g/ml, 16.24 (g/ml, 15.33 (g/ml and 102.77 (g/ml respectively. In addition, the growth inhibition was also observed in BEL-7402 cells from 6 hour to 48 hour. (3) Melittin could block cell cycle (S arrest) and down-regulated PCNA expression. (4) Hepatocarcinoma cells presented apoptosis features: chromatin condensation, nucleic fragmentation; Agarose electrophoresis showed marked DNA ladder; Flow cytometry analysis showed Annexin V positive cells and APO2.7 expression.(5) Anther experimental results show that cytosolic <WP=9>phospholipase A2 was activated and up-regulated Fas mRNA expression, after melittin treated BEL-7402 cells.Conclusions: (1) The melittin extracted with AKTA protein production explorer system from bee venom was approved succeed and reliability. (2) 4 hepatoma cell lines, HepG2, Hep3B, SMMC-7721, and BEL-7402 were treated with melittin and the results demonstrated the suppression of cell growth and proliferation. It suggested that melittin isolated from bee venom possess significant anti-hepatocarcinoma effect. (3) Melittin has shown substantial efficacy in treating hepatocarcinoma in vitro. These actions of melittin may result in the induction of apoptosis and the inhibition of growth. Data show that melittin induce apoptosis and the inhibition of proliferation, at least in part associated with down-regulated PCNA expression, arrest cycle, and the activation of phospholipase A2, as well as Fas signal transduction.更多还原