【作者】 樊强；

【导师】 李建民；

【作者基本信息】 山东大学 ， 骨外科学（专业学位）， 2014， 博士

【摘要】 实验背景及目的骨肉瘤(osteosarcoma)是临床上最常见的恶性成骨性肿瘤之一。在我国,骨肉瘤发病率在原发恶性骨肿瘤中居首位。化疗的出现为骨肉瘤的治疗开辟了崭新的天地。现代骨肉瘤的治疗主要采取以手术为主的综合治疗,包括手术前化疗、手术和术后定期化疗,同时配合免疫和生物疗法等综合治疗方法。化疗在治疗过程中的作用不可替代,但联合辅助化疗长期用药对机体的毒副作用以及肿瘤细胞耐药性的产生又成为近年来困扰临床的一大难题。这一现实驱使人们不断探索新的化疗药物用于骨肉瘤的治疗。蜂毒素(MEL, melittin)是蜂毒的主要活性成分,具有高度的生物学活性。近年来,有关蜂毒素的抗肿瘤作用日益引起人们的重视。研究表明,蜂毒素具有广泛的抗瘤活性,其抗瘤机制包括直接杀伤、免疫调节,诱导细胞凋亡、抑制转移和血管生成等。虽然很多实验已经证明蜂毒素具有抑制多种肿瘤细胞增殖和促进凋亡的作用,但其对于骨肉瘤细胞的作用效果,国内外鲜有文献报道。本实验选取人骨肉瘤细胞株MG63作为研究对象,观察蜂毒素对MG63细胞活力及凋亡的影响,并进一步研究其可能作用机制、为其应用于临床提供理论和实验依据。实验材料1.细胞及蜂毒素人骨肉瘤MG63细胞株,人胎儿成骨细胞Hfob1.19细胞株由中国科学院细胞库提供。蜂毒素由第二军医大学长海医院中医科实验室提供。2.主要试剂及仪器MTT-华美生物工程公司,RTPCR试剂盒-美国invitrogen公司,Annexin V-FITC/PI凋亡试剂盒-南京凯基公司,Western blot实验装置-美国GE公司,流式细胞仪-美国BD公司。实验方法1.细胞培养：人骨肉瘤细胞MG63及人胎儿成骨细胞Hfob1.19贴壁生长于含10%热灭活胎牛血清、100U/ml青霉素、100μ g/m1链霉素的DMEM高糖型培养液中,培养瓶置于37℃,含5%C02的饱和湿度孵育箱中培养。每隔2-3天进行传代,取对数生长期的细胞进行实验。2.构建含蜂毒素基因质粒并转染实验细胞：提取蜂毒素肽总RNA,反转录反应合成cDNA,根据genebank设计引物,反克隆MEL基因。MEL基因与载体连接,构建真核表达载体,并转染实验细胞。3.实验分组：蜂毒素基因转染的MG63、Hfobl.19细胞为实验A组(MEL-express组),蜂毒素肽孵育的MG63、Hfobl.19细胞为实验B组(MEL-pep组),未处理细胞为对照组(con组)4.蜂毒素孵育实验细胞：使用生理盐水配制蜂毒素溶液,调整溶液浓度,加入容纳实验细胞的培养瓶中,摇匀,使蜂毒素终浓度约为32μg/ml,37℃、5%C02条件下培养箱中孵育24小时。5.采用四甲基偶氮唑盐(MTT)比色法检测蜂毒素对人骨肉瘤细胞MG63和人胎儿成骨细胞Hfobl.19的活力抑制作用：取蜂毒素转染、孵育及未处理的处于对数生长期细胞并制成单细胞悬液,加入MTT及二甲亚砜,全自动酶联免疫检测仪上测定各孔光密度值。计算活细胞率。实验重复3次。6.采用流式细胞仪检测细胞凋亡(Annexin V-FITC/PI标记法)：实验细胞用0.25%的胰酶消化,离心,置于流式管中,加入FITC标记的AnnexinV5μl,10μl PI (Propidium Iodide)染液(50mg/L),1小时内上流式细胞仪完成检测。使用CELLQuest软件检测细胞凋亡率。7.采用Western-blotting法检测各组细胞内IRE-a、p-Perk、AFT-6, Caspase-12和CHOP蛋白表达：用数字成像仪对硝酸纤维素膜进行图像扫描分析,比较不同组之间蛋白表达峰面积的差异。8.RT-PCR分析XBP1, EIF-2a, TRAF2mRNA水平的变化：采用kodak2.0软件对PCR琼脂糖凝胶电泳产物进行扫描分析,数据处理。结果人骨肉瘤细胞MG63和人胎儿成骨细胞Hfobl.19经蜂毒素转染或蜂毒素孵育处理后细胞内均有较高水平的蜂毒素存在。MTT分析显示,人胎儿成骨细胞Hfobl.19经蜂毒素转染或蜂毒素孵育处理后,活细胞率与对照组比较无显著性差异,而人骨肉瘤细胞MG63经蜂毒素转染或蜂毒素孵育处理后,活细胞率显著低于对照组,差异具有统计学意义。流式细胞仪检测细胞凋亡结果显示,人胎儿成骨细胞Hfob1.19经蜂毒素转染或蜂毒素孵育处理后,凋亡细胞比例与对照组比较没有显著性差别,人骨肉瘤细胞MG63经蜂毒素处理后,凋亡细胞比例显著高于对照组,差异具有统计学意义。蜂毒素转染和蜂毒素孵育两组间差异无显著性。Western-blot显示,入骨肉瘤细胞MG63和人胎儿成骨细胞Hfobl.19经蜂毒素转染或蜂毒素孵育处理后,细胞表达未折叠蛋白反应因子IRE-a, p-Perk, AFT-6较对照组均有增强,但只有人骨肉瘤细胞MG63中IRE-a的表达明显增高,与对照组相比,差异具有显著性。人胎儿成骨细胞Hfob1.19经蜂毒素转染或蜂毒素孵育处理后,细胞中Caspase12和CHOP蛋白的表达与对照组比较无显著性差异,但人骨肉瘤MG63细胞表达CHOP蛋白明显增强,差异具有统计学意义。RT-PCR检测内质网应激下游蛋白XBPl,EIF2-a, TRAF2mRNA结果显示,人骨肉瘤细胞MG63经蜂毒素处理后,细胞表达XBP1mRNA水平较对照组明显增高,差异具有统计学意义。结论1.MEL能特异性抑制MG63细胞活力并诱导细胞凋亡。2.MEL抑制MG63细胞活力并诱导细胞凋亡这一过程可能是通过活化内质网应激未折叠蛋白反应的IRE-a信号通路实现的。3. CHOP蛋白参与其中,并发挥重要作用。意义1.证实了蜂毒素可特异性的诱导人骨肉瘤MG63细胞凋亡2.初步探讨了蜂毒素诱导人骨肉瘤细胞MG63凋亡的可能机制,为进一步明确其分子机制提供了一种可能的研究方向。更多还原

【Abstract】 Background and objectiveOsteosarcoma is one of the most common malignant bone tumor in clinic. The incidence of osteosarcoma ranks first in the primary malignant bone tumor in our country. Chemotherapy plays a very crucial role in the treatment of osteosarcoma. Modern treatment of osteosarcoma mainly adopts the combined therapy based on operation, including operation, chemotherapy before operation and postoperative regular chemotherapy. Effect of chemotherapy in the treatment process should not be replaced, but the side effects of chemotherapy has become a puzzling problem in recent years.This reality constantly drives people to explore new chemotherapeutic agents for the treatment of osteosarcoma. Melittin (MEL, melittin) is the main active component in bee venom and has high biological activity. In recent years, the antitumor effect of melittin has been paid more and more attention. Research shows that, melittin has broad antitumor activity, its antitumor mechanisms include direct killing, immune regulation, cell apoptosis, inhibition of metastasis and angiogenesis. Although many experiments had demonstrated that melittin can induce apoptosis of tumor cells, but the mechanism of its effect on osteosarcoma cells is not clear. This experiment selects the human osteosarcoma cell line MG63as the research object, to observe the effects of melittin on MG63cell and take a further research on its possible mechanism, and provide theoretical and experimental basis for its application in clinic.Experimental materials1.Cells and melittin Human osteosarcoma cell line MG63, human fetal osteoblast cell line Hfobl.19provided by cell bank Chinese Academy of sciences. Melittin provided by the laboratory of traditional Chinese medicine, Changhai Hospital of Second Military Medical University.2.The main reagents and instruments MTT-Huamei biotech company, RTPCR Kit-USA Invitrogen company, Annexin V-FITC/PI apoptosis Kit-KGI Nanjing company, Western blot experimental device.-GE America, Flow cytometry-America BD companyExperimental method1.Cell culture:human osteosarcoma cells MG63and human fetal osteoblasts Hfobl.19grow in DMEM containing10%heat inactivated bovine serum, penicillin(100U/ml), streptomycin(100μg/ml) in the culture bottles in37℃,5%CO2saturated humidity incubator, logarithmic growth phase cells for experiments2.Constructs containing melittin gene Plasmid and transfects experiment cells: Extracted total RNA from melittin peptide, reverse transcription reaction synthesis of cDNA, the primers were designed according to genebank, MEL gene cloning. Connect the MEL gene and the vector. Construct the eukaryotic expression vector, transfect the experimental cell.3.The cells were divided into three groups. Control group:no intervention. Melittin transfection group:to construct the plasmid containing melittin gene which were transfected into human osteosarcoma cell line MG63and the human fetal osteoblastic Hfob1.19cells. Melittin incubation group;use the final concentration (32μg/ml) of melittin to incubate human osteosarcoma MG63cells and human fetal osteoblastic Hfobl.19cells.4.The four methyl thiazolyl tetrazolium (MTT) assay detects the inhibitory effect of melittin on human osteosarcoma MG63cells and human fetal osteoblastic Hfobl.19cells.The logarithmic growth phase cells were made into single cell suspension, adding MTT and DMSO, use the optical density determination of full automatic enzyme immunoassay instrument to calculate the living cell rate. The experiment was repeated3times5.The cell apoptosis was detected by flow cytometry (Annexin V-FITC/PI notation).6.Using Western-blot method to detect the intracellular IRE-a, p-perk, XBP1, EIF2-a, AFT-6, Caspase-12and CHOP protein expression.7.Using RT-PCR to detect XBP1, EIF2-a, TRAF2mRNA level to explore the possible molecular mechanism.ResultsThe human osteosarcoma cell line MG63and human fetal osteoblastic Hfob1.19, after melittin transfection or incubations,a high level of melittin was in cells.MTT showed that human fetal osteoblastic Hfobl.19cells transfected by melittin or incubation, The cell survival rate compared with the control group had no significant difference. While the MG63human osteosarcoma cells transfected or incubated by melittin, the cell activity was significantly lower than that of the control group, the difference was statistically significant. Flow cytometry results showed that human fetal osteoblastic Hfob1.19cells transfected or incubated by melittin, the proportion of apoptotic cells compared with the control group had no significant difference, but MG63human osteosarcoma cells treated by melittin, the proportion of apoptotic cells was significantly higher than that in the control group, the difference was statistically significant. Western-blot display, IRE-a, CHOP proteins in human osteosarcoma MG63cells were significantly increased, compared with the control group, the difference was significant. RT-PCR detect the endoplasmic reticulum stress unfolded protein response downstream proteins XBP1, EIF2-a, TRAF2mRNA,results showed, MG63human osteosarcoma cells treated by melittin,the expression of XBP1mRNA was significantly higher than the control group, the difference was statistically significant. Conclusion1.Melttin can specifically inhibit the activity of MG63cells and induce apoptosis.2.This process may be realized through activating IRE-a signal transduction protein in the endoplasmic reticulum stress reaction.3.CHOP protein is involved, and play an important roleSignificance1. Confirmed that melittin can specifically induce human osteosarcoma MG63cells apoptosis2. Investigated the possible mechanism of apoptosis of human osteosarcoma cell line MG63induced by melittin and provide a possible research direction for the further elucidation of the molecular mechanism.更多还原