蜈蚣提取液治疗肝细胞肝癌的实验研究
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摘要
肝细胞性肝癌(hepatocellular carcinoma，HCC)是我国最常见的恶性肿瘤之一，平均生存时间较短，临床预后很差。近20年来，我国HCC的治疗尽管取得了长足的进步，手术切除率、术后生存率及生存质量均有较大提高，但与其他肿瘤相比，HCC的疗效远未如人意。治疗方法选择主要取决于癌症的分期及肝功能情况。手术切除(包括肝移植)是公认的最有效的根治肝癌方法，应作为首选。但由于肝癌进展程度不同，以及肝硬化的存在和肝脏储备功能的差异，适合作肝切除的患者，通常切除率很低(10％～30％)。对于肿瘤不能手术切除者或全身情况差不能耐受手术者，可采取肿瘤的局部及全身治疗相结合方法，尽管目前治疗方法很多，但各种方法各有其局限性，对中、晚期肝癌疗效有限。故寻找有效的治疗药物是提高中、晚期肝癌存活率及改善生活质量的重要手段，具有重要现实意义。
在我国，蜈蚣在疾病预防和治疗中的使用已有两千余年历史。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零星报道，但研究方法仅见体外的药敏试验，且确切机理不详。为此，我们以蜈蚣提取液(Extract of centipedes，ECP)作用肝癌细胞株Bel-7404和裸鼠肝癌Bel-7404移植模型为研究对象，采用系列方法，旨在阐明ECP治疗肝癌的效果及机制，为ECP临床治疗肝癌提供理论基础。
第一章蜈蚣提取液对肝癌Bel-7404细胞体外抗癌作用及机制的研究
目的：研究蜈蚣提取液对肝癌细胞株的敏感性、作用的细胞周期时相及作用机制，为肝癌的临床治疗提供理论依据。
方法：体外培养肝癌细胞Bel-7404，采用不同浓度ECP予以干预，通过MTT实验、细胞的药物浓度—时间生长曲线研究其对肝癌细胞株的敏感性；利用倒置光学显微镜和电子显微镜观察细胞形态及其超微结构的变化；流式细胞仪检测药物治疗组的细胞周期及其凋亡率；免疫组织化学法检测细胞内凋亡相关基因X染色体连锁凋亡抑制蛋白(X-chromosome linked Inhibitor of Apoptosis Protein，XIAP)及Bax的表达情况。
结果：①MTT法揭示各浓度ECP对人肝癌细胞Bel-7404生长均有抑制作用。ECP在80mg／ml、40 mg／ml、20 mg／ml、10mg／ml、5 mg／ml、2.5 mg／ml及1.25 mg/ml下对Bel-7404的抑制率依次为(76.58±3.58)％、(71.06±3.43)％、(63.98±4.70)％、(56.74±11.30)％、(36.89±8.13)％、(31.26±3.42)％和(17.93±4.34)％，中效浓度为9.5mg／ml。②细胞生长曲线图显示Bel-7404活细胞数目与ECP浓度及药物作用时间相关。治疗组浓度在5mg／ml时，活细胞数亦先增多，在用药48小时后Bel-7404细胞数开始减少；浓度在10mg／ml与20mg／ml时，用药后24小时活细胞数就显著减少。各组间活细胞数比较差异明显(P＜0.01)。③光镜下见ECP作用48hr后，浓度低于5mg／ml对Bel-7404细胞无明显的抑制作用，浓度在10mg／ml以上时，Bel-7404活细胞数减少，细胞形态变园变小，80mg／ml时见细胞大片坏死，有较多的不规则细胞碎片，培养瓶中坏死脱落细胞增多。电镜下见治疗组细胞密度增高，染色质凝集固宿，染色质边集，核固宿、核碎裂，及凋亡小体的形成。④流式细胞仪检测发现：(A)ECP在不同浓度下，G0／G1期所占的比例是不同的。对照组为(50.8±3.5)％，浓度5mg／ml为(58.2±2.4)％，浓度10mg／ml为(65.7±4.1)％，20mg／ml组最高，为(72.1±5.3)％。(B)细胞增殖指数(PI)：在对照组为(49.2±1.9)％，浓度5mg／ml时为(41.8±4.1)％，浓度10mg／ml时为(35.3±3.7)％，20mg／ml时为(27.9±2.0)％，差异显著(P＜0.05)。并且，随着药物浓度的增高，细胞凋亡所出现的“亚G1峰”是逐步增高的。(C)细胞凋亡率：24小时后对照组的自发凋亡率(1.56±0.17)％，浓度在5mg／ml时为(4.99±0.59)％，10mg／ml时为(7.71±0.92)％，20mg／ml时为(12.1±0.63)％，各浓度组间差异明显(P＜0.05)；48小时后对照组的自发凋亡率(1.75±0.145)％，浓度5mg／ml时为(5.90±0.35)％，10mg／ml时为(9.80±0.95)％，20mg／ml时为(17.3±1.71)％，各浓度组间差异明显(P＜0.05)；在相同浓度组下，24小时的细胞凋亡率明显低于48小时的，差异非常显著(P＜0.01)。④免疫组化法检测Bel-7404细胞的XIAP和Bax表达，对照组中XIAP的OD值最大，染色最深、最多，随ECP浓度加大，XIAP在Bel-7404细胞的OD值逐渐变小，染色逐步变少、变浅。在对照组中，Bax的OD值最小，染色浅而少，随ECP浓度加大，表达Bax的细胞的OD值依次变大，染色逐步变多，颜色加深。
结论：①ECP能抑制人肝癌细胞Bel-7404的增殖，且抑制作用呈剂量和时间依赖效应；②ECP低浓度有诱导肿瘤细胞凋亡作用，中、高浓度直接杀肿瘤细胞作用。③ECP作用Bel-7404细胞的G1／G0期，抑制其增殖，并能诱导其凋亡，其作用呈剂量和时间依赖效应；④ECP治疗肝癌Bel-7404细胞后能促进Bax基因的表达，抑制XIAP基因的表达。
第二章蜈蚣提取液体内抑癌及作用机制的研究
目的：研究蜈蚣提取液在体内对肝癌是否有抑制作用，并探讨蜈蚣提取液抑制肝癌细胞生长的机制。
方法：①建立裸鼠Bel-7404人异位肝癌移植模型，以蜈蚣提取液予以灌服，观察肿瘤生长、裸鼠胸腺与脾重量及肝癌转移情况。②运用免疫组化法对肿瘤组织标本进行XIAP、Bax、血管内皮细胞生长因子(Vascular Endothelial Growth Factor，VEGF)和促血管生成素2(Angiopoietin2，Ang-2)的检测。
结果：①裸鼠Bel-7404人异位肝癌移植模型成功率100％②蜈蚣提取液对裸鼠Bel-7404移植瘤有明显抑制作用，在移植后第31天，对照组的肿瘤平均体积为(1187.5±117.4)mm~3，治疗组为(506.1±65.7)mm~3；裸鼠处死后，对照组的肿瘤平均重量为(1059±125.2)mg，治疗组为(647±122.89)mg，抑制率达38.91％；在治疗组，胸腺指数(2.15±0.43)％，脾指数为(10.63±3.7)％；在对照组，胸腺指数(0.81±0.27)％，脾指数为(7.90±2.58)％。两组有显著差异性。③在对照组中，XIAP、VEGF与Ang-2的染色细胞数、染色强度及OD值均较治疗组多而强，而Bax的染色强度、染色细胞数及OD值较治疗组要少而弱，它们比较均有显著性差异(p＜0.01)。
结论：①蜈蚣提取液能抑制裸鼠Bel-7404移植瘤的生长，其机理与抑制肿瘤血管生成、促进肿瘤细胞凋亡有关。②蜈蚣提取液能提高荷瘤裸鼠的胸腺指数及脾指数，具有增强裸鼠免疫功能作用。
第三章蛋白质组学技术和方法分离治疗组肝癌细胞的差异表达蛋白
目的：筛选蜈蚣提取液治疗肝癌细胞后的相关差异蛋白，探讨药物治疗肝癌的机理。
方法：运用蛋白质组学技术和方法，采用一步抽提法制备肝癌Bel-7404细胞株治疗组和对照组的总蛋白质，用双向凝胶电泳技术建立了重复性和分辨率均较好的Bel-7404细胞株治疗组和对照组总蛋白的双向凝胶电泳图谱。PDQuest软件对两组细胞蛋白质的双向电泳图谱进行了比较，并采用MALDI-TOF-MS对差异表达的蛋白质进行分析，获取MALDI-TOF-MS肽质量指纹图谱，数据库搜索鉴定主要差异表达的蛋白质。
结果：①共发现76个差异表达蛋白点(p＜0.05)。治疗组中41个表达下调，35个表达上调。②选择其中明显的14个蛋白质斑点作为差异表达的质谱分析，8个蛋白质即：肽基脯氨酰顺反异构酶A(PPIA)、核纤层蛋白-B1(Lamin-β1)、黄素还原酶(FRase)、转铁结合蛋白-2(Tbp-2)、唾液酸合成酶(SAS)、3-磷酸甘油醛脱氢酶(GAPDH)、周期素依赖激酶抑制蛋白1(P~(21))和半乳凝素-7(Gal-7)在Bel-7404治疗组中表达上调；而6个蛋白质即角蛋白-19(CK-19)、肌动蛋白(Actin)、过渡型内质网ATP酶(TERATPase)、ω-1谷胱甘肽转移酶(GSTO1-1)、碱性磷酸(酯)酶(ALP)和抑制蛋白-1(PFN1)在Bel-7404治疗组中表达下调。
结论：①对双向凝胶电泳图谱分析共发现76个差异表达蛋白，41个表达下调，35个表达上调。②质谱分析14个差异蛋白，PPIA、Lamin-β1、FRase、TBP-2、SAS、GAPDH、P~(21)和Gal-7在Bel-7404治疗组中表达上调；而6个蛋白质即CK-19、Actin、TERATPase、GSTO1-1、PFN1及ALP在Bel-7404治疗组中表达下调。提示蜈蚣提取液能多途径抑制肝癌细胞的生长，诱导肝癌细胞凋亡或直接死亡。
第四章部分蛋白质分子差异表达水平验证研究
目的：验证比较蛋白质组学研究结果。
方法：采用Western-blotting技术对核纤层蛋白B_1、转铁结合蛋白-2和半乳凝素-7在Bel-7404细胞株治疗组与对照组中的表达水平进行了检测验证。
结果：核纤层蛋白B_1、转铁结合蛋白-2和半乳凝素-7在Bel-7404治疗组中表达上调，与蛋白质组学研究结果一致。
结论：蛋白质组学方法筛选到的差异表达蛋白确实在Bel-7404细胞株治疗组和对照组存在差异表达。
Hepatocellular carcinoma(HCC)is one of the most commonmalignancy in China. In recent 20 years, despite the emergence of varioustherapeutic modalities such as hepatic resection, radiofrequency ablationtherapy and chemotherapy, the prognosis of HCC remains poor, and theaverage life time is very short.
The treatment for the patient mainly depends on the stage of thetumor and the liver function. As we all known, hepatic resection includeliver transplantation is the most effective treatment and the first choice tocure the HCC radically. However, due to different stages of the tumor,the exist of cirrhosis and different functions of liver, the resection rateof the patients who undergo radical hepatic resection is often low (10％～30％).
The patients who has no chance to undergo hepatic resection orcannot bear the surgery may be cured by combination of region treatmentwith integration treatment. There are many methods for it, But alltreatments have their own respective limitations; and their therapeuticeffects for the patient with advanced stage of HCC are still poor. Further elucidation of the molecular pathogenesis of HCC and exploration of aneffective drug may facilitate the development of more effectivetherapeutic interventions for HCC.
Centipede as a drug applied to prophylaxis and treatment of diseaseshas a history of more than two thousand years. The extract of wholecentipede was proved to have many effects in disease treatment in recentyears. But the reports about its effect in carcinomas' treatment are in oddsand ends, and the method for experiment of tumor therapy limits only toMTT. Up to now, little information of its detailed mechanism of actionhas been known to us. In order to investigate the sensibility andmechanisms of HCC cell to ECP, we take HCC Bel-7404 cell line andmodel of hefterotopic grafting carcinoma for HCC Bel-7404 in nudemouse as a object to research treatment with extract of centipede (ECP)by a series of methods. What we do aim to give its theory to clinictreatment with ECP for HCC.
ChapterⅠAssay sensibility of HCC Bel-7404cell line to ECP in vitro
Objective: To investigate the sensibility, cell cycle and mechanismof HCC Bel-7404 cell line by treatment of ECP, and to provideevidences of clinic therapeutic theory for HCC.
Method:①Bel-7404 cell line were cultured in vitro; ECP wasapplied to the interference of the growth of Bel-7404 with different drugconcentration; MTT method and different drug concentration-timesurvival curve are employed to investigate the sensibility of HCC cell(Bel-7404) to ECP.②We observe cell morphous and it's Ultrastructuralchanges through inversion light microscope and electron microscope.③We detect cell cycles and apoptosis ratio to ECP group by flowcytometry(FCM).④To investigate expression degrees ofrelated-apoptosis genes XIAP and Bax, we also detect HCC Bel-7404 cellline after 48 hours treated with ECP by immunohistochemical method.
Result:①ECP can inhibit the growth of HCC Bel-7404 cell lineobviously at different concentration with time-concentration dependent.The result of MTT method showed that the inhibition ratio of variousconcentration of ECP at 80mg/ml,40mg/ml,20mg/ml,10mg/ml,5mg/ml,2.5mg/ml and 1.25mg/ml is (76.58±3.58)%,(71.06±3.43)%, (63.98±4.70)%, (56.74±11.30)%, (36.89±8.13)%,(31.26±3.42)% and (17.93±4.34)% respectively, and IC_(50)of ECP to Bel-7404 is 9.5mg/ml. Inhibitionratio increases when ECP concentration adds with direct correlation tendency.②Drug concentration-time survival curve shows that the viable counts ofBel-7404 has direct relations with ECP concentration and administrativetime. The survival curve for the contrastive group show viable counts ofBel-7404 elevate firstly, and then descend gradually until to equation to cell numbers of the onset at the seventh day. As for the treatment group ofthe concentration at 5mg/ml, the curve also show viable counts ofBel-7404 elevate firstly, and then descend gradually after 48 hour. For thegroups of 10mg/ml and 20mg/ml, viable counts of Bel-7404 sharplydecrease after 24 hours treated at the concentration of 10mg/ml and20mg/ml. There exist distinct discrepancies for different groups (P＜0.01).③Observed through light microscope after 48 hours, we fred out that these isno evident depressant effect for cells Bel-7404 at the ECP concentrationof 5mg/ml,but over 10mg/ml, viable counts of Bel-7404 decrease, andcyto-morph transform to round and shrink. At the ECP concentration of80mg/ml, dead cells Bel-7404 increase, many irregular cells debris can beseen, and many dead cell cast-off from culture flask. When observedthrough electron microscope for treatment groups we see the increase of cellsdensity, chromatic agglutination, chromatin margination, nuclear pyknosis,nuclear fragmentation and apoptotic body formation.④The result ofFCM display for treatment groups that ECP mainly retard cell cycle phasein G0/G1 after 48 hours.(A)The ratio of G0/G1 is different at the differentconcentration. It's (50.8±3.5)% for the contrast After 48 hours', whentreatement with concentration of 5mg/ml, 10mg/ml and 20mg/ml,G0/G1phase account to (58.24±2.4) %,(65.7±4.1)% and (72.1±5.3)%,respectively.(B) Proliferation index(PI) for cells Bel-7404 for thecontrast is (49.2±1.9)%. For the treatment groups at the concentation of 5mg/ml,10mg/ml and 20mg/ml,PI is (41.8±4.1)%, (35.3±3.7)% and(27.9±2.0)%.(C)Apoptosis ratio:The apoptosis ratio for the contrast is(1.56±0.17) % after 24 hours for cells Bel-7404. For the treatmentgroups at the concentation of 5mg/ml, 10mg/ml and 20mg/ml,It is (4.99±0.59)%, (7.71±0.92)% and (12.1±0.63)% respectively. It is significantdifference from treatment group and control group (P＜0.05). After 48hours,the apoptosis ratio for the contrast is(1.754±0.145) %, For thetreatment groups at the concentation of 5mg/ml, 10mg/ml and 20mg/ml,Itis (5.90±0.35)%, (9.80±0.95)% and (17.3±1.71)% respectively. It issignificant difference from treatment group and control group,and also fordifferent time when in the same concentration of ECP.④It shows thatXIAP gene express gradually to weaken but Bax gene to enhance in HCCcells with the increase of ECP concentration, these are statisticalsignificance in contrast to the control (P＜0.05).
Conclusion:①ECP can inhibit the growth of the Bel-7404. Theinhibitory effect is time-concentration dependent.②ECP can induceBel-7404 cell line to apoptosis in low concentration, And directly kill it ifin middle-high concentration.③ECP mainly retard cell cycle phase ofBel-7404 in G0/G1, suppress it to proliferation and can induce it toapoptosis, the inhibitory effect is time-centration dependent.④One of themechanisms of treatment with ECP is to promote Bax gene and inhibitXIAP gene expression for HCC Bel-7404.
ChapterⅡInvestigation to the effect and mechanisms ofHCC BEL-7404 treatment with ECP in vivo
Objective: To investigate the effects and mechanisms of HCCBel-7404 treatment with ECP.
Method:①To set up model of hefterotopic grafting carcinoma forHCC Bel-7404 in nude mouse, and observe its tumor growth andmetastasis by intragastric administration of ECP, including its weight ofthymus gland and spleen.②To detect XIAP、Bax、VEGF and Ang-2 intumor tissue with immunohistochemical methods
Result:①The achievement ratio for hefterotopic graftingcarcinoma for HCC Bel-7404 in nude mouse is 100%.②It has distinctiveinhibitive effect for ECP to hefterotopic grafting carcinoma for HCCBel-7404 in nude mouse. At the 31st day postgraft of nude mouse, theaverage grafting carcinoma volume of the contrast is (1187.5±117.4)mm~3, and for the treatment group is (506.1±65.7)mm~3; The averagegrafting carcinoma weight of the contrast is (1059±125.2)mg, but forthe treatment group is (647±122.89)mg, The inhibitive ratio reaches to 38.91%. thymus gland and spleen index for the treatment group are(2.15±0.43)% and (10.63±3.7)% respectively, for the contrast group,it's(0.81±0.27)% and (7.90±2.58) % respectively, there are significantdifference for them(P＜0.05).③The staining cells population, stainingintensity and the optical density (OD) of XIAP, VEGF and Ang-2 for thecontrast group are higher than the treatment group, but they are lower forBax. there are significant difference for them (P＜0.05)
Conclusion:①ECP can inhibit hefterotopic grafting carcinoma forHCC Bel-7404 in nude mouse, the mechanisms relate to inhibit tumorAngiogenesis and to promote HCC Bel-7404 cells to apoptosis.②ECPcan't inhibit immunological function of nude mouse but can increase it.
ChapterⅢ: Application of the techniques and methods ofProteomics into separating differential expression proteinsin HCC cells treatment with ECP
Objective: To screen the relevant differential expression proteinsafter treatment with ECP, and to investigate the molecule mechanism ofECP for HCC Bel-7404.
Method: we use proteomic Techniques and methods to analysis the mechanism of treatment with ECP for HCC Bel-7404. Firstly,comparative two-dimensional gel electrophoresis(2-DE) technology wasapplied to separate the total protein of HCC Bel-7404 treatment group byECP and its control group respectively. The well-resolved, reproducible2-DE patterns of treatment group of ECP and control group wereestablished. Then, PDQuest software was used to analyze 2-DE images,and the differential expression proteins between the two groups wereidentified by both peptide mass fingerprint (PMF) and peptide sequencetag (PST) based on MALDI-TOF-MS (Matrix-assisted laserdesorption/ionization time of flight mass spectrometry).
Result: We select the differential expression proteins for betweentreatment group and control group which spots variance is over double toanalyse with MALDI-TOF-MS after spots enzymolyed, and found out76 protein spots at difference level of expression (p＜0.05), including 41spots decreasing in treatment group, and 35 spots increasing in controlgroups. 14 of the total pieces of PMF were be matched searching inSWISS-PROT/TREMBL database by Mascot software. Among theidentified 14 protein spots, the expression level of proteins whichup-regulates in treatment group is 8 in total, including: Peptidyl-prolylcis-trans isomerase A (PPIA)、Lamin-β1、Flavin reductase (FRase)、Transferrin-binding protein 2 (Tbp2)、Sialic acid synthase (SAS)、Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)、Cyclin dependent kinase proflin 1(P~(21))and Galectin-7(Gal-7). But 6 proteins intreatment group is down-regulation, which include Keratin (CK-19)、Actin、Transitional endoplasmic reticulum ATPase (TERATPase)、Glutathione transferase omega-1(GSTO1-1)、Profilin-1(PFN1) andAlkaline phosphatase (ALP).
Conclusion:①Through analysis of two-dimensional gelelectrophoresis (2-DE), 76 protein spots at difference level of expression(p＜0.05) have been found out, including 41 spots decreasing intreatment group, and 35 spots increasing in control groups.②Among theidentified 14 of the total of protein spots, the expression level of proteinswhich up-regulates in treatment group is 8 in total, including: PPIA,Lamin-β1, FRase, TBP2, SAS, GAPDH, P~(21) and Gal-7 and whichdown-regulates in treatment group is 6 proteins in total, including:Keratin (CK-19), Actin, TERATPase, GSTO1-1, PFN1 and ALP. It hintthat ECP can inhibit HCC Bel-7404 cell line to growth by inducing it toapoptosis or directly to death in multi-channels.
ChapterⅣ: Verification of the differential expression levelsof the partial proteins by western-blotting analysis and itsfunction study
Objective: To verify the differential expression levels of the partialproteins Tbp2,1aminB1 and Gal-7 which identified with comparedproteomic techniques.
Method: To verify the differential expression levels of Tbp2、1aminB1 and Gal-7 again by Western-blotting methods, which expressin HCC Bel-7404 treatment with ECP group and control group everidentified with comparative proteomic techniques.
Result: The proteins of Tbp2,1aminB1 and Gal-7 expressup-regulation in treatment group with ECP, and the results were identicalwith the proteome analysis.
Conclusion: The differential expression proteins of Tbp2,1aminB1and Gal-7 screened by proteome analysis are indeed differentialexpression in treatment group and control group of HCC Bel-7404.
在我国，蜈蚣在疾病预防和治疗中的使用已有两千余年历史。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零星报道，但研究方法仅见体外的药敏试验，且确切机理不详。为此，我们以蜈蚣提取液(Extract of centipedes，ECP)作用肝癌细胞株Bel-7404和裸鼠肝癌Bel-7404移植模型为研究对象，采用系列方法，旨在阐明ECP治疗肝癌的效果及机制，为ECP临床治疗肝癌提供理论基础。
第一章蜈蚣提取液对肝癌Bel-7404细胞体外抗癌作用及机制的研究
目的：研究蜈蚣提取液对肝癌细胞株的敏感性、作用的细胞周期时相及作用机制，为肝癌的临床治疗提供理论依据。
方法：体外培养肝癌细胞Bel-7404，采用不同浓度ECP予以干预，通过MTT实验、细胞的药物浓度—时间生长曲线研究其对肝癌细胞株的敏感性；利用倒置光学显微镜和电子显微镜观察细胞形态及其超微结构的变化；流式细胞仪检测药物治疗组的细胞周期及其凋亡率；免疫组织化学法检测细胞内凋亡相关基因X染色体连锁凋亡抑制蛋白(X-chromosome linked Inhibitor of Apoptosis Protein，XIAP)及Bax的表达情况。
结果：①MTT法揭示各浓度ECP对人肝癌细胞Bel-7404生长均有抑制作用。ECP在80mg／ml、40 mg／ml、20 mg／ml、10mg／ml、5 mg／ml、2.5 mg／ml及1.25 mg/ml下对Bel-7404的抑制率依次为(76.58±3.58)％、(71.06±3.43)％、(63.98±4.70)％、(56.74±11.30)％、(36.89±8.13)％、(31.26±3.42)％和(17.93±4.34)％，中效浓度为9.5mg／ml。②细胞生长曲线图显示Bel-7404活细胞数目与ECP浓度及药物作用时间相关。治疗组浓度在5mg／ml时，活细胞数亦先增多，在用药48小时后Bel-7404细胞数开始减少；浓度在10mg／ml与20mg／ml时，用药后24小时活细胞数就显著减少。各组间活细胞数比较差异明显(P＜0.01)。③光镜下见ECP作用48hr后，浓度低于5mg／ml对Bel-7404细胞无明显的抑制作用，浓度在10mg／ml以上时，Bel-7404活细胞数减少，细胞形态变园变小，80mg／ml时见细胞大片坏死，有较多的不规则细胞碎片，培养瓶中坏死脱落细胞增多。电镜下见治疗组细胞密度增高，染色质凝集固宿，染色质边集，核固宿、核碎裂，及凋亡小体的形成。④流式细胞仪检测发现：(A)ECP在不同浓度下，G0／G1期所占的比例是不同的。对照组为(50.8±3.5)％，浓度5mg／ml为(58.2±2.4)％，浓度10mg／ml为(65.7±4.1)％，20mg／ml组最高，为(72.1±5.3)％。(B)细胞增殖指数(PI)：在对照组为(49.2±1.9)％，浓度5mg／ml时为(41.8±4.1)％，浓度10mg／ml时为(35.3±3.7)％，20mg／ml时为(27.9±2.0)％，差异显著(P＜0.05)。并且，随着药物浓度的增高，细胞凋亡所出现的“亚G1峰”是逐步增高的。(C)细胞凋亡率：24小时后对照组的自发凋亡率(1.56±0.17)％，浓度在5mg／ml时为(4.99±0.59)％，10mg／ml时为(7.71±0.92)％，20mg／ml时为(12.1±0.63)％，各浓度组间差异明显(P＜0.05)；48小时后对照组的自发凋亡率(1.75±0.145)％，浓度5mg／ml时为(5.90±0.35)％，10mg／ml时为(9.80±0.95)％，20mg／ml时为(17.3±1.71)％，各浓度组间差异明显(P＜0.05)；在相同浓度组下，24小时的细胞凋亡率明显低于48小时的，差异非常显著(P＜0.01)。④免疫组化法检测Bel-7404细胞的XIAP和Bax表达，对照组中XIAP的OD值最大，染色最深、最多，随ECP浓度加大，XIAP在Bel-7404细胞的OD值逐渐变小，染色逐步变少、变浅。在对照组中，Bax的OD值最小，染色浅而少，随ECP浓度加大，表达Bax的细胞的OD值依次变大，染色逐步变多，颜色加深。
结论：①ECP能抑制人肝癌细胞Bel-7404的增殖，且抑制作用呈剂量和时间依赖效应；②ECP低浓度有诱导肿瘤细胞凋亡作用，中、高浓度直接杀肿瘤细胞作用。③ECP作用Bel-7404细胞的G1／G0期，抑制其增殖，并能诱导其凋亡，其作用呈剂量和时间依赖效应；④ECP治疗肝癌Bel-7404细胞后能促进Bax基因的表达，抑制XIAP基因的表达。
第二章蜈蚣提取液体内抑癌及作用机制的研究
目的：研究蜈蚣提取液在体内对肝癌是否有抑制作用，并探讨蜈蚣提取液抑制肝癌细胞生长的机制。
方法：①建立裸鼠Bel-7404人异位肝癌移植模型，以蜈蚣提取液予以灌服，观察肿瘤生长、裸鼠胸腺与脾重量及肝癌转移情况。②运用免疫组化法对肿瘤组织标本进行XIAP、Bax、血管内皮细胞生长因子(Vascular Endothelial Growth Factor，VEGF)和促血管生成素2(Angiopoietin2，Ang-2)的检测。
结果：①裸鼠Bel-7404人异位肝癌移植模型成功率100％②蜈蚣提取液对裸鼠Bel-7404移植瘤有明显抑制作用，在移植后第31天，对照组的肿瘤平均体积为(1187.5±117.4)mm~3，治疗组为(506.1±65.7)mm~3；裸鼠处死后，对照组的肿瘤平均重量为(1059±125.2)mg，治疗组为(647±122.89)mg，抑制率达38.91％；在治疗组，胸腺指数(2.15±0.43)％，脾指数为(10.63±3.7)％；在对照组，胸腺指数(0.81±0.27)％，脾指数为(7.90±2.58)％。两组有显著差异性。③在对照组中，XIAP、VEGF与Ang-2的染色细胞数、染色强度及OD值均较治疗组多而强，而Bax的染色强度、染色细胞数及OD值较治疗组要少而弱，它们比较均有显著性差异(p＜0.01)。
结论：①蜈蚣提取液能抑制裸鼠Bel-7404移植瘤的生长，其机理与抑制肿瘤血管生成、促进肿瘤细胞凋亡有关。②蜈蚣提取液能提高荷瘤裸鼠的胸腺指数及脾指数，具有增强裸鼠免疫功能作用。
第三章蛋白质组学技术和方法分离治疗组肝癌细胞的差异表达蛋白
目的：筛选蜈蚣提取液治疗肝癌细胞后的相关差异蛋白，探讨药物治疗肝癌的机理。
方法：运用蛋白质组学技术和方法，采用一步抽提法制备肝癌Bel-7404细胞株治疗组和对照组的总蛋白质，用双向凝胶电泳技术建立了重复性和分辨率均较好的Bel-7404细胞株治疗组和对照组总蛋白的双向凝胶电泳图谱。PDQuest软件对两组细胞蛋白质的双向电泳图谱进行了比较，并采用MALDI-TOF-MS对差异表达的蛋白质进行分析，获取MALDI-TOF-MS肽质量指纹图谱，数据库搜索鉴定主要差异表达的蛋白质。
结果：①共发现76个差异表达蛋白点(p＜0.05)。治疗组中41个表达下调，35个表达上调。②选择其中明显的14个蛋白质斑点作为差异表达的质谱分析，8个蛋白质即：肽基脯氨酰顺反异构酶A(PPIA)、核纤层蛋白-B1(Lamin-β1)、黄素还原酶(FRase)、转铁结合蛋白-2(Tbp-2)、唾液酸合成酶(SAS)、3-磷酸甘油醛脱氢酶(GAPDH)、周期素依赖激酶抑制蛋白1(P~(21))和半乳凝素-7(Gal-7)在Bel-7404治疗组中表达上调；而6个蛋白质即角蛋白-19(CK-19)、肌动蛋白(Actin)、过渡型内质网ATP酶(TERATPase)、ω-1谷胱甘肽转移酶(GSTO1-1)、碱性磷酸(酯)酶(ALP)和抑制蛋白-1(PFN1)在Bel-7404治疗组中表达下调。
结论：①对双向凝胶电泳图谱分析共发现76个差异表达蛋白，41个表达下调，35个表达上调。②质谱分析14个差异蛋白，PPIA、Lamin-β1、FRase、TBP-2、SAS、GAPDH、P~(21)和Gal-7在Bel-7404治疗组中表达上调；而6个蛋白质即CK-19、Actin、TERATPase、GSTO1-1、PFN1及ALP在Bel-7404治疗组中表达下调。提示蜈蚣提取液能多途径抑制肝癌细胞的生长，诱导肝癌细胞凋亡或直接死亡。
第四章部分蛋白质分子差异表达水平验证研究
目的：验证比较蛋白质组学研究结果。
方法：采用Western-blotting技术对核纤层蛋白B_1、转铁结合蛋白-2和半乳凝素-7在Bel-7404细胞株治疗组与对照组中的表达水平进行了检测验证。
结果：核纤层蛋白B_1、转铁结合蛋白-2和半乳凝素-7在Bel-7404治疗组中表达上调，与蛋白质组学研究结果一致。
结论：蛋白质组学方法筛选到的差异表达蛋白确实在Bel-7404细胞株治疗组和对照组存在差异表达。
Hepatocellular carcinoma(HCC)is one of the most commonmalignancy in China. In recent 20 years, despite the emergence of varioustherapeutic modalities such as hepatic resection, radiofrequency ablationtherapy and chemotherapy, the prognosis of HCC remains poor, and theaverage life time is very short.
The treatment for the patient mainly depends on the stage of thetumor and the liver function. As we all known, hepatic resection includeliver transplantation is the most effective treatment and the first choice tocure the HCC radically. However, due to different stages of the tumor,the exist of cirrhosis and different functions of liver, the resection rateof the patients who undergo radical hepatic resection is often low (10％～30％).
The patients who has no chance to undergo hepatic resection orcannot bear the surgery may be cured by combination of region treatmentwith integration treatment. There are many methods for it, But alltreatments have their own respective limitations; and their therapeuticeffects for the patient with advanced stage of HCC are still poor. Further elucidation of the molecular pathogenesis of HCC and exploration of aneffective drug may facilitate the development of more effectivetherapeutic interventions for HCC.
Centipede as a drug applied to prophylaxis and treatment of diseaseshas a history of more than two thousand years. The extract of wholecentipede was proved to have many effects in disease treatment in recentyears. But the reports about its effect in carcinomas' treatment are in oddsand ends, and the method for experiment of tumor therapy limits only toMTT. Up to now, little information of its detailed mechanism of actionhas been known to us. In order to investigate the sensibility andmechanisms of HCC cell to ECP, we take HCC Bel-7404 cell line andmodel of hefterotopic grafting carcinoma for HCC Bel-7404 in nudemouse as a object to research treatment with extract of centipede (ECP)by a series of methods. What we do aim to give its theory to clinictreatment with ECP for HCC.
ChapterⅠAssay sensibility of HCC Bel-7404cell line to ECP in vitro
Objective: To investigate the sensibility, cell cycle and mechanismof HCC Bel-7404 cell line by treatment of ECP, and to provideevidences of clinic therapeutic theory for HCC.
Method:①Bel-7404 cell line were cultured in vitro; ECP wasapplied to the interference of the growth of Bel-7404 with different drugconcentration; MTT method and different drug concentration-timesurvival curve are employed to investigate the sensibility of HCC cell(Bel-7404) to ECP.②We observe cell morphous and it's Ultrastructuralchanges through inversion light microscope and electron microscope.③We detect cell cycles and apoptosis ratio to ECP group by flowcytometry(FCM).④To investigate expression degrees ofrelated-apoptosis genes XIAP and Bax, we also detect HCC Bel-7404 cellline after 48 hours treated with ECP by immunohistochemical method.
Result:①ECP can inhibit the growth of HCC Bel-7404 cell lineobviously at different concentration with time-concentration dependent.The result of MTT method showed that the inhibition ratio of variousconcentration of ECP at 80mg/ml,40mg/ml,20mg/ml,10mg/ml,5mg/ml,2.5mg/ml and 1.25mg/ml is (76.58±3.58)%,(71.06±3.43)%, (63.98±4.70)%, (56.74±11.30)%, (36.89±8.13)%,(31.26±3.42)% and (17.93±4.34)% respectively, and IC_(50)of ECP to Bel-7404 is 9.5mg/ml. Inhibitionratio increases when ECP concentration adds with direct correlation tendency.②Drug concentration-time survival curve shows that the viable counts ofBel-7404 has direct relations with ECP concentration and administrativetime. The survival curve for the contrastive group show viable counts ofBel-7404 elevate firstly, and then descend gradually until to equation to cell numbers of the onset at the seventh day. As for the treatment group ofthe concentration at 5mg/ml, the curve also show viable counts ofBel-7404 elevate firstly, and then descend gradually after 48 hour. For thegroups of 10mg/ml and 20mg/ml, viable counts of Bel-7404 sharplydecrease after 24 hours treated at the concentration of 10mg/ml and20mg/ml. There exist distinct discrepancies for different groups (P＜0.01).③Observed through light microscope after 48 hours, we fred out that these isno evident depressant effect for cells Bel-7404 at the ECP concentrationof 5mg/ml,but over 10mg/ml, viable counts of Bel-7404 decrease, andcyto-morph transform to round and shrink. At the ECP concentration of80mg/ml, dead cells Bel-7404 increase, many irregular cells debris can beseen, and many dead cell cast-off from culture flask. When observedthrough electron microscope for treatment groups we see the increase of cellsdensity, chromatic agglutination, chromatin margination, nuclear pyknosis,nuclear fragmentation and apoptotic body formation.④The result ofFCM display for treatment groups that ECP mainly retard cell cycle phasein G0/G1 after 48 hours.(A)The ratio of G0/G1 is different at the differentconcentration. It's (50.8±3.5)% for the contrast After 48 hours', whentreatement with concentration of 5mg/ml, 10mg/ml and 20mg/ml,G0/G1phase account to (58.24±2.4) %,(65.7±4.1)% and (72.1±5.3)%,respectively.(B) Proliferation index(PI) for cells Bel-7404 for thecontrast is (49.2±1.9)%. For the treatment groups at the concentation of 5mg/ml,10mg/ml and 20mg/ml,PI is (41.8±4.1)%, (35.3±3.7)% and(27.9±2.0)%.(C)Apoptosis ratio:The apoptosis ratio for the contrast is(1.56±0.17) % after 24 hours for cells Bel-7404. For the treatmentgroups at the concentation of 5mg/ml, 10mg/ml and 20mg/ml,It is (4.99±0.59)%, (7.71±0.92)% and (12.1±0.63)% respectively. It is significantdifference from treatment group and control group (P＜0.05). After 48hours,the apoptosis ratio for the contrast is(1.754±0.145) %, For thetreatment groups at the concentation of 5mg/ml, 10mg/ml and 20mg/ml,Itis (5.90±0.35)%, (9.80±0.95)% and (17.3±1.71)% respectively. It issignificant difference from treatment group and control group,and also fordifferent time when in the same concentration of ECP.④It shows thatXIAP gene express gradually to weaken but Bax gene to enhance in HCCcells with the increase of ECP concentration, these are statisticalsignificance in contrast to the control (P＜0.05).
Conclusion:①ECP can inhibit the growth of the Bel-7404. Theinhibitory effect is time-concentration dependent.②ECP can induceBel-7404 cell line to apoptosis in low concentration, And directly kill it ifin middle-high concentration.③ECP mainly retard cell cycle phase ofBel-7404 in G0/G1, suppress it to proliferation and can induce it toapoptosis, the inhibitory effect is time-centration dependent.④One of themechanisms of treatment with ECP is to promote Bax gene and inhibitXIAP gene expression for HCC Bel-7404.
ChapterⅡInvestigation to the effect and mechanisms ofHCC BEL-7404 treatment with ECP in vivo
Objective: To investigate the effects and mechanisms of HCCBel-7404 treatment with ECP.
Method:①To set up model of hefterotopic grafting carcinoma forHCC Bel-7404 in nude mouse, and observe its tumor growth andmetastasis by intragastric administration of ECP, including its weight ofthymus gland and spleen.②To detect XIAP、Bax、VEGF and Ang-2 intumor tissue with immunohistochemical methods
Result:①The achievement ratio for hefterotopic graftingcarcinoma for HCC Bel-7404 in nude mouse is 100%.②It has distinctiveinhibitive effect for ECP to hefterotopic grafting carcinoma for HCCBel-7404 in nude mouse. At the 31st day postgraft of nude mouse, theaverage grafting carcinoma volume of the contrast is (1187.5±117.4)mm~3, and for the treatment group is (506.1±65.7)mm~3; The averagegrafting carcinoma weight of the contrast is (1059±125.2)mg, but forthe treatment group is (647±122.89)mg, The inhibitive ratio reaches to 38.91%. thymus gland and spleen index for the treatment group are(2.15±0.43)% and (10.63±3.7)% respectively, for the contrast group,it's(0.81±0.27)% and (7.90±2.58) % respectively, there are significantdifference for them(P＜0.05).③The staining cells population, stainingintensity and the optical density (OD) of XIAP, VEGF and Ang-2 for thecontrast group are higher than the treatment group, but they are lower forBax. there are significant difference for them (P＜0.05)
Conclusion:①ECP can inhibit hefterotopic grafting carcinoma forHCC Bel-7404 in nude mouse, the mechanisms relate to inhibit tumorAngiogenesis and to promote HCC Bel-7404 cells to apoptosis.②ECPcan't inhibit immunological function of nude mouse but can increase it.
ChapterⅢ: Application of the techniques and methods ofProteomics into separating differential expression proteinsin HCC cells treatment with ECP
Objective: To screen the relevant differential expression proteinsafter treatment with ECP, and to investigate the molecule mechanism ofECP for HCC Bel-7404.
Method: we use proteomic Techniques and methods to analysis the mechanism of treatment with ECP for HCC Bel-7404. Firstly,comparative two-dimensional gel electrophoresis(2-DE) technology wasapplied to separate the total protein of HCC Bel-7404 treatment group byECP and its control group respectively. The well-resolved, reproducible2-DE patterns of treatment group of ECP and control group wereestablished. Then, PDQuest software was used to analyze 2-DE images,and the differential expression proteins between the two groups wereidentified by both peptide mass fingerprint (PMF) and peptide sequencetag (PST) based on MALDI-TOF-MS (Matrix-assisted laserdesorption/ionization time of flight mass spectrometry).
Result: We select the differential expression proteins for betweentreatment group and control group which spots variance is over double toanalyse with MALDI-TOF-MS after spots enzymolyed, and found out76 protein spots at difference level of expression (p＜0.05), including 41spots decreasing in treatment group, and 35 spots increasing in controlgroups. 14 of the total pieces of PMF were be matched searching inSWISS-PROT/TREMBL database by Mascot software. Among theidentified 14 protein spots, the expression level of proteins whichup-regulates in treatment group is 8 in total, including: Peptidyl-prolylcis-trans isomerase A (PPIA)、Lamin-β1、Flavin reductase (FRase)、Transferrin-binding protein 2 (Tbp2)、Sialic acid synthase (SAS)、Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)、Cyclin dependent kinase proflin 1(P~(21))and Galectin-7(Gal-7). But 6 proteins intreatment group is down-regulation, which include Keratin (CK-19)、Actin、Transitional endoplasmic reticulum ATPase (TERATPase)、Glutathione transferase omega-1(GSTO1-1)、Profilin-1(PFN1) andAlkaline phosphatase (ALP).
Conclusion:①Through analysis of two-dimensional gelelectrophoresis (2-DE), 76 protein spots at difference level of expression(p＜0.05) have been found out, including 41 spots decreasing intreatment group, and 35 spots increasing in control groups.②Among theidentified 14 of the total of protein spots, the expression level of proteinswhich up-regulates in treatment group is 8 in total, including: PPIA,Lamin-β1, FRase, TBP2, SAS, GAPDH, P~(21) and Gal-7 and whichdown-regulates in treatment group is 6 proteins in total, including:Keratin (CK-19), Actin, TERATPase, GSTO1-1, PFN1 and ALP. It hintthat ECP can inhibit HCC Bel-7404 cell line to growth by inducing it toapoptosis or directly to death in multi-channels.
ChapterⅣ: Verification of the differential expression levelsof the partial proteins by western-blotting analysis and itsfunction study
Objective: To verify the differential expression levels of the partialproteins Tbp2,1aminB1 and Gal-7 which identified with comparedproteomic techniques.
Method: To verify the differential expression levels of Tbp2、1aminB1 and Gal-7 again by Western-blotting methods, which expressin HCC Bel-7404 treatment with ECP group and control group everidentified with comparative proteomic techniques.
Result: The proteins of Tbp2,1aminB1 and Gal-7 expressup-regulation in treatment group with ECP, and the results were identicalwith the proteome analysis.
Conclusion: The differential expression proteins of Tbp2,1aminB1and Gal-7 screened by proteome analysis are indeed differentialexpression in treatment group and control group of HCC Bel-7404.
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