【作者】 张斌；

【导师】 钟德玝；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 肝细胞性肝癌(hepatocellular carcinoma, HCC)是全世界最常见的恶性肿瘤之一,死亡率在癌症中占第三位,在亚洲和非洲平均每年100,000人中就有约50-150人死于HCC。近20年来,我国HCC的治疗尽管取得了长足的进步,早期诊断率、手术切除率、术后生存率及生存质量均有较大提高,但HCC的发病率和死亡率仍占癌症中的第二位。治疗方法选择主要取决于肿瘤的分期及肝功能情况。手术切除(包括部分肝切除和肝移植)可能治愈小肝癌,而对进展期和转移性HCC疗效有限。对于不能手术切除或全身情况差不能耐受手术者,可采取肿瘤的局部和全身治疗相结合的方法。尽管目前治疗方法很多,但每种方法各有其局限性,对中、晚期HCC的疗效有限。故寻找有效的治疗药物是提高中、晚期HCC存活率及改善生活质量的重要手段,具有重要意义。在我国,蜈蚣用于疾病的预防和治疗已有两千余年的历史。近年研究表明蜈蚣全虫提取液有多种作用,其抗肿瘤的效果亦有少量报道。为此,我们以肝癌细胞株Bel-7402为研究对象,采用分离纯化的蜈蚣提取液产物处理细胞,通过观察其对Bel-7402细胞的增殖及形态等的影响,进一步了解蜈蚣提取液治疗肝癌的效果并探讨其作用机制,为蜈蚣提取液临床治疗肝癌提供理论基础。第一章蜈蚣蛋白质体外抗癌作用的研究目的：提取ECP总液并将其分离为上清液和蛋白质,分别研究三者对肝癌细胞株Bel-7402增殖的抑制作用。方法：体外培养肝癌细胞株Bel-7402,分别采用不同浓度ECP总液、上清液及蛋白质处理细胞,利用倒置光学显微镜观察Bel-7402细胞的形态变化,并通过MTT法研究肝癌细胞Bel-7402对它们的敏感性。同时体外培养正常肝细胞株L02,观察不同浓度ECP总液处理后,L02细胞形态学改变及对ECP总液的敏感性。结果：①光镜下和细胞计数结果示,ECP总液、ECP蛋白质和5-Fu作用48小时后,Bel-7402细胞数量减少明显,部分未坏死细胞形态变圆变小,而ECP上清液作用Bel-7402细胞、ECP总液作用L02细胞48小时后,表现为细胞数量减少不明显。②MTT法示,ECP总液、ECP蛋白质和5-Fu对Bel-7402细胞的抑制率分别为0.788、0.453、0.944,而ECP上清液对Bel-7402细胞的抑制率为0.198,ECP总液对L02细胞的抑制率为0.095。③MTT法示ECP总液在12mg/ml、1.2mg/ml、0.12mg/ml、0.012mg/ml、0.0012mg/ml时,对肝癌细胞株Bel-7402的抑制率分别为0.788、0.608、0.308、0.207、0.099,其半数抑制浓度(IC50)为9.597mg/ml。ECP蛋白质在2.9mg/ml、0.29mg/ml、0.029mg/ml、0.0029mg/ml、0.00029mg/ml时,对肝癌细胞株Bel-7402的抑制率分别为0.453、0.208、0.145、0.077、0.063, IC50为1.372mg/ml。④细胞生长-抑制率曲线示对Bel-7402细胞抑制率,随ECP总液和ECP蛋白质的浓度的增加而依次提高,与Bel-7402阴性对照组比较差异有统计学意义(P<0.05)。⑤MTT法示ECP总液在12mg/ml、1.2mg/ml、0.12mg/ml、0.012mg/ml、0.0012mg/ml时,对肝细胞株L02的抑制率分别为0.095、0.040、0.037、0.022、0.005。结论：①ECP总液中抑制肝癌细胞株Bel-7402生长的主要是ECP中的蛋白质成分,ECP总液和ECP蛋白质能抑制Bel-7402细胞的增殖,且抑制作用呈剂量依赖效应；ECP上清液对肝癌细胞株Bel-7402的增殖没有明显影响。②ECP总液对肝细胞株L02的增殖没有明显影响。第二章蜈蚣蛋白质抗肿瘤活性成分的分离纯化目的：逐层分离纯化蜈蚣总蛋白质,观察肝癌细胞株Bel-7402对所得产物的敏感性并进行有效成分鉴定。方法：①体外培养肝癌细胞株Bel-7402,采用Bestarose Cross Link-6B (CL-6B)凝胶过滤、DEAE Sepharose FF离子交换层析、Sephadex G-75凝胶过滤对ECP蛋白质进行分离纯化；将得到的产物分别以不同浓度干预Bel-7402细胞,采用MTT法进行体外抑瘤活性检测；②利用倒置光学显微镜,观察经不同浓度ECP蛋白质的分离物处理后Bel-7402细胞形态的变化,以找出蜈蚣主要抗肿瘤活性蛋白质；③通过SDS-PAGE电泳对有效成分进行分子量测定。结果：①ECP蛋白质经过CL-6B凝胶过滤层析后,得到5种成分。光镜下、细胞计数和MTT法示,C、E液抑制Bel-7402细胞生长明显,其所含蛋白质分子量范围分别在1.6X106～2.2X106、1X104～4X105。MTT法和细胞生长-抑制率曲线图示,对Bel-7402细胞抑制率随C、E液的浓度的增加而依次提高。与Bel-7402阴性对照组比较差异有统计学意义(P<0.05)。②将C、E液合并通过DEAE Sepharose FF离子交换层析后,得到7种成分,光镜下、细胞计数和MTT法示G、I液抑制Bel-7402细胞生长明显,其所含蛋白质等电点在6.8以下。MTT法和细胞生长-抑制率曲线图示,对Bel-7402细胞抑制率随G、I液的浓度的增加而依次提高。与Bel-7402阴性对照组比较差异有统计学意义(P<0.05)。③将G、I液合并通过Sephadex G-75凝胶过滤层析后,得到4种成分,光镜下、细胞计数和MTT法示,O、P液抑制Bel-7402细胞生长明显,其所含蛋白质分子量范围分别在4×104～6×104、2×104～4×104。MTT法和细胞生长-抑制率曲线图示,对Bel-7402细胞抑制率随O、P液的浓度的增加而依次提高。与Bel-7402阴性对照组比较差异有统计学意义(P<0.05)。O液的IC50为1.132mg/ml, P液的IC50为1,129 mg/ml。④SDS-PAGE电泳结果示,O、P液含多种蛋白质,其中O液包括7条含量较高的蛋白质,其分子量分别在120kD (1kD=103),60 kD,48 kD,35 kD,33 kD,25 kD,20 kD左右。P液中存在6条含量较高的蛋白质,其分子量分别在120kD,80 kD,35 kD,33 kD,25 kD,20 kD左右。⑤光镜下、细胞计数和MTT法示不同浓度的A、B、D、H、J、K、L、M、N液对肝癌细胞株Bel-7402的生长抑制作用不明显。结论：①经CL-6B、DEAE FF、Sephadex G-75得到的抗肿瘤活性成分,两者均为酸性蛋白质混合物,其分子量范围在2×104-6×104之间；②O液和P液均能抑制Bel-7402细胞的增殖,其作用呈剂量依赖性,O、P液体外抑瘤活性较ECP总液均提高了约8.5倍；第三章ECP总液对肝癌细胞株Bel-7402的作用及机制的研究目的：研究ECP总液对肝癌细胞株Bel-7402中Galectin-7和p-JNK1表达的影响,并进一步探讨其作用机制。方法：以半数抑制浓度的ECP总液(1Omg/ml)诱导肝癌细胞株Bel-7402不同时间(0h、3h、6h、12h、24h、48h),通过Western blot、Real time-PCR、免疫荧光检测细胞内Galectin-7和p-JNK1的蛋白及mRNA表达；用Galectin-7-siRNA预处理Bel-7402细胞,再予lOmg/ml ECP总液诱导48 h,观察Galectin-7的蛋白及mRNA表达和p-JNK1的蛋白表达变化。结果：①Western blot、Real time-PCR、免疫荧光均显示,ECP总液干预呈时间依赖性上调肝癌细胞株Bel-7402中Galectin-7和p-JNK1的蛋白及mRNA,与对照组相比差异有统计学意义(P<0.01)。②Western blot、Real time-PCR、免疫荧光均显示,Galectin-7-siRNAs可显著抑制ECP总液对Galectin-7的蛋白及mRNA表达的诱导作用,与ECP总液刺激组相比差异有统计学意义(P<0.05)。③Western blot显示,Galectin-7-siRNAs可显著抑制ECP总液对p-JNK1蛋白表达的诱导作用,与ECP总液刺激组相比差异有统计学意义(P<0.05),与对照组相比差异有统计学意义(P<0.05)。结论：ECP总液呈时间依赖性上调肝癌细胞株Bel-7402中Galectin-7的蛋白及mRNA和p-JNK1的蛋白及mRNA,提示ECP总液可能通过Galectin-7-JNK途径促进肝癌细胞株Bel-7402的凋亡。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) was one of the most common malignancy tumor in worldwide. It was the third leading cause of death attributable to cancer. The incidence of 50-150 HCC cases per 100,000 population and year in parts of Africa and Asia where HCC was responsible for a large proportion of cancer deaths. In recent 20 years, despite the emergence of various therapeutic modalities such as diagnosis at an early stage, hepatic resection and radiofrequency ablation were therapy and chemotherapy. In China, HCC was the second the incidence of and mortality of cancer.The treatment for the patient mainly depended on the stage of the tumor and the liver function. As we all known, although surgery (partial hepatectomy or total hepatectomy with orthotopic liver transplantation) could be curative for localized small liver tumors, therapeutic options for patients with advanced or metastatic HCC were limited. The patients who had no chance to undergo hepatic resection or could not bear the surgery may be cured by combination of region treatment with integration treatment. There were many methods for it, But all treatments had their own respective limitations; and the therapeutic effect for the patients with advanced stage of HCC was still poor. Further elucidation of the molecular pathogenesis of HCC and exploration of an effective drug may facilitate the development of more effective therapeutic interventions for HCC.Centipede as a drug applied to prophylaxis and treatment of diseases had a history of more than two thousand years. The extract of whole centipede was proved to have many effects in disease treatment in recent years. But the reports about its effect in carcinomas’ treatment were in odds and ends, and the method for experiment of tumor therapy only limits to MTT. Up to now, little information of its detailed mechanism of action had been known to us. In order to investigate the sensibility and mechanisms of HCC cell to extract of centipede (ECP),we took HCC Bel-7402 cell line and model of heterotopic grafting carcinoma for HCC Bel-7402 to research treatment with ECP by a series of methods.What we did aim to give its theory to clinic treatment with ECP for HCC. ChapterⅠAssay sensibility of HCC Bel-7402 cell line to ECP in vitroObjective:To investigate the inhibitory effect of ECP, supernatant and protein from ECP on human HCC Bel-7402 cell line.Method: Bel-7402 cell line were cultured in vitro; ECP、the supernatant from ECP and its protein were applied to the interference of the growth of Bel-7402 with different drug concentration; MTT method and cell amounts were employed to investigate the sensibility to them. We observe cell morpheus changes through inversion light microscope. LO2 cell line was cultured in vitro. ECP were applied to the interference of the growth of LO2 with different drug concentration; MTT method and cell amounts were employed to investigate the sensibility to them.Result:①According to light microscope and cell amounts after 48 hours, we found out that these were viable counts of Bel-7402 decrease at the ECP、the protein of ECP and 5-Fu, no evident depressant effect for cells Bel-7402 at the supernatant from ECP and for cells LO2 at the ECP.②The result of MTT method showed that the inhibition ratio to Bel-7402 of ECP、the protein of ECP、the supernatant from ECP and 5-Fu was 0.788、0.453、0.198 and 0.944, respectively. The inhibition ratio to LO2of ECP was 0.095.③The result of MTT method showed that the inhibition ratio of various concentration of ECP at 12mg/ml、1.2mg/ml、0.12mg/ml、0.012mg/ml and 0.0012mg/ml was 0.788、0.608、0.308、0.207 and 0.099, respectively, and IC50 of ECP to Bel-7402 was 9.597mg/ml. The inhibition ratio of various concentration at 2.9mg/ml、0.29mg/ml、.029mg/ml、.0029mg/ml and 0.00029mg/ml was 0.453、0.208、0.145、0.077 and 0.063, respectively, and IC50 to Bel-7402 was 1.372mg/ml.④Drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with ECP and the protein of ECP concentration. There existed distinct discrepancies for different groups (P<0.05)⑤The result of MTT method showed that the inhibition ratio of various concentration of ECP at 12mg/ml、1.2mg/ml、0.12mg/ml、0.012mg/ml and 0.0012mg/ml was 0.095、0.040、0.037、0.022 and 0.005, respectively.Conclusion:①ECP and the protein of ECP could inhibit the growth of Bel-7402 cells. The inhibitory effect was concentration dependent. The supernatant from ECP had no transparent inhibiting on Bel-7402.②ECP had no transparent inhibition on LO2. ChapterⅡIsolation, Purification the anti-tumor protein extracted from ECPObjective:To investigate the sensibility of HCC Bel-7402 cell line by treatment of the protein of ECP was isolated and purified by gel filtration, ion-exchange chromatography. The molecular weights of proteins were identified by SDS-PAGE electrophoresis method.Method:①Bel-7402 cell line were cultured in vitro. The protein of ECP was isolated and purified by gel filtration on CL-6B, DEAE-Sepharose-FF anion-exchange chromatography and gel filtration on Sephadex G-75, applied to the interference of the growth of Bel-7402 with different drug concentration; MTT method and cell amounts were employed to investigate the sensibility to them.②We observe cell morpheus changes through inversion light microscope.③The molecular weights of effective components were identified by SDS-PAGE.Result:①A、B、C、D、E were separated from the protein of ECP by gel filtration on CL-6B column. According to light microscope、the cell amounts and MTT after 48 hours, we found out that the proteins compound C and E had remarkable suppressive on the proliferation of Bel-7402 in vitro. The protein molecular weights of C and E were mainly distributed between 1.6×106～2.2X 106、1×104～4×105, respectively. The result of MTT method and drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with C and E concentration. The cell proliferation was slower than that of contrast group (P＜0.05)②F、G、H、I、J、K、L were separated from the compound C and E by DEAE-Sepharose-FF anion-exchange chromatography. According to light microscope、the cell amounts and MTT after 48 hours, we found out that the proteins compound G and I had remarkable suppressive on the proliferation of Bel-7402 in vitro. The pI of G and I less than 6.8. The result of MTT method and drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with G and I concentration. The cell proliferation was slower than that of contrast group (P＜0.05)③M、N、O、P were separated from the compound G and I by gel filtration on Sephadex G-75 column. T According to light microscope、the cell amounts and MTT after 48 hours, we found out that the proteins compound O and P had remarkable suppressive on the proliferation of Bel-7402 in vitro. The protein molecular weights of O and P were mainly distributed between 4×104～6×104、2×104～4×104 respectively. he result of MTT method and drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with G and I concentration. The cell proliferation was slower than that of contrast group (P<0.05).IC50 of O and P to Bel-7402 was 1.132mg/ml and 1.129mg/ml, respectively.④In SDS-PAGE,7 major bands were showed in O (molecular weights: 120Kd(1=103),60 kD,48 kD,35 kD,33 kD,25 kD,20 kD.6 major bands were showed in P (molecular weights: 120kD,80 kD,35 kD,33 kD,25 kD,20 kD).⑤A、B、D、F、H、J、L、M、N from ECP could inhibit the growth of HCC Bel-7402 cell line at different concentration, but the growth inhibiting rate were low.Conclusion:①O、P were separated from the protein of ECP by gel filtration on CL-6B column、DEAE-Sepharose-FF anion-exchange chromatography、gel filtration on Sephadex G-75 column, which were compound of acidic protein. The protein molecular weights of O and P were mainly distributed between 2×104～6×104.②O、P could inhibit the growth of the Bel-7402. The inhibitory effect was concentration dependent. And its suppressive effect on the proliferation of Bel-7402 was nearly 8.5 times more than the crude proteins of ECP. ChapterⅢTo explore the mechanisms in human Bel-7402 cell line treated with ECPObjective: To explore the effect of antitumor molecular mechanisms, the HCC Bel-7402 cell line were treated with ECP.Method:Bel-7402 cells line were treated with ECP (10mg/ml) in different time (0、3、6、12、24、48h). The expression of Galectin-7 and p-JNK1 protein expression and mRNA were examined by Western Blot, Real Time-PCR and immunofluo-rescence staining. We treated Bel-7402 cells line with Galectin-7-siRNA, then Bel-7402 cells line were treated with ECP (10mg/ml) in 48h. The expression of Galectin-7 protein expression and mRNA, and p-JNK1 protein expression were examined by Western Blot, Real Time-PCR and immunofluorescence staining.Result:①Western Blot, Real Time-PCR and Immunofluorescence staining showed that expression of Galectin-7, p-JNK1 protein and mRNA were up regulated in a time-dependent manner in Bel-7402 stimulated with ECP. There was significantly difference among ECP groups and contrast group (P<0.01)②Western Blot, Real Time-PCR and Immunofluorescence staining showed that expression of Galectin-7, p-JNK1 protein and mRNA were decreased in Bel-7402 stimulated with ECP in the presence of Galectin-7-siRNAs compared with Bel-7402 stimulated with ECP (P<0.05)③Western blot showed that induction of p-JNK1 protein was decreased in Bel-7402 stimulated with ECP in the presence of Galectin-7-siRNAs compared with Bel-7402 stimulated with ECP (P<0.05). There was significantly difference amongs Galectin-7-siRNAs groups and contrast group (P<0.05)Conclusion:Galectin-7, p-JNK1 protein and mRNA were up regulated in a time-dependent manner in Bel-7402 stimulated with ECP. ECP maybe activate Galectin-7-JNK pathway to induce apoptosis of Bel-7402.更多还原