【作者】 周恩相；

【导师】 钟德玝；

【作者基本信息】 中南大学 ， 普通外科学， 2011， 博士

【摘要】 第一章蜈蚣提取液对三阴性乳腺癌MDA-MB-231细胞体外抗癌作用及机制的研究目的：研究蜈蚣提取液对乳腺癌细胞株的敏感性、作用的细胞周期时相及作用机制,为乳腺癌的临床治疗提供理论依据。方法：体外培养三阴性乳腺癌细胞MDA-MB-231,采用不同浓度蜈蚣提取液(Extract of Centipede, ECP)予以干预,通过MTT实验、细胞的药物浓度一时间生长曲线研究其对乳腺癌细胞株的敏感性；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Bcl-2及Bax的表达情况。结果：①MTT法揭示各浓度ECP对人乳腺癌细胞MDA-MB-231生长均有抑制作用。ECP在80mg/ml、40mg/ml.20mg/ml.10mg/ml.5mg/ml、2.5mg/ml及1.25mg/ml下对MDA-MB-231的抑制率依次为(71.26±2.64)％、(62.48±6.67)％.(56.26±3.17)％.(48.63±4.48)％.(42.13±4.73)％、(34.5±2.98)％和(26.89±2.95)％,中效浓度为12.5mg/ml.②细胞生长曲线图显示MDA-MB-231活细胞数目与ECP浓度及药物作用时间相关。治疗组浓度在12.5mg/ml与25mg/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48小时后,浓度低于10mg/ml对MDA-MB-231细胞无明显的抑制作用,浓度在10mg/ml以上时,MDA-MB-231活细胞数减少,细胞形态变圆变小,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,染色质凝集固缩,染色质边集,核固缩、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(36.7+4.2)%,浓度12.5mg/ml为(56.5±4.1)％,25mg/ml组最高,为(67.3±4.4)%。(B)细胞增殖指数(PI)：在对照组为(63.3±3.2)%,浓度12.5mg/ml时为(43.5±3.5)％,25mg/ml时为(32.7±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：培养及作用48小时后对照组的自发凋亡率(2.15±0.13)%,浓度12.5mg/ml时为(13.01±1.51)％,25mg/ml时为(21.3±1.98)%,各浓度组间差异明显(P<0.05)。⑤免疫组化法检测MDA-MB-231细胞的Bcl-2和Bax表达,对照组中Bcl-2的OD值最大,染色最深、最多,随ECP浓度加大,Bcl-2在MDA-MB-231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能抑制人三阴性乳腺癌细胞MDA-MB-231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、高浓度有直接杀伤肿瘤细胞作用。③ECP作用MDA-MB-231细胞的G1/GO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治疗三阴性乳腺癌MDA-MB-231细胞后能促进Bax基因的表达,抑制Bcl-2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乳腺癌是否有抑制作用,并探讨蜈蚣提取液抑制乳腺癌细胞生长的机制。方法：建立裸鼠MDA-MB-231人异位乳腺癌移植模型,以蜈蚣提取液予以灌服,观察肿瘤生长、乳腺癌转移情况。②运用免疫组化法对肿瘤组织标本进行Bcl-2、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MDA-MB-231人异位乳腺癌移植模型成功率100%②蜈蚣提取液对裸鼠MDA-MB-231移植瘤有明显抑制作用,在移植后第25天,对照组的肿瘤平均体积为(1011.52±212.68)mm3,治疗组为(238.83±65.45)mm3；裸鼠处死后,对照组的肿瘤平均重量为(983+97.3)mg,治疗组为(556+98.1)mg,抑制率达44.2%,两组有显著差异性。③在对照组中,Bcl-2、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：蜈蚣提取液能抑制裸鼠MDA-MB-231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。第三章蛋白质组学技术和方法分离治疗组乳腺癌细胞的差异表达蛋白目的：筛选蜈蚣提取液治疗乳腺癌细胞后的相关差异蛋白,探讨药物治疗乳腺癌的机理。方法：运用蛋白质组学技术和方法,采用一步抽提法制备乳腺癌MDA-MB-231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MDA-MB-231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12个蛋白质斑点作为差异表达的质谱分析,分析出11个差异蛋白。5个蛋白质即：S100A9、CALML5、CK-19、Gal-7和α-烯醇化酶在MDA-MB-231治疗组中表达上调；而6个蛋白质即GST01-1、PGAMA、HSP90-β、AKP-P. IMPDH和GAPDH在MDA-MB-231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11个差异蛋白,S100A9、 CALML5、CK-19、Gal-7和α-烯醇化酶在MDA-MB-231治疗组中表达上调；而6个蛋白质即GST01-1、PGAMA、HSP90-β、AKP-P、IMPDH和GAPDH在MDA-MB-231治疗组中表达下调。提示蜈蚣提取液能多途径抑制乳腺癌细胞的生长,诱导乳腺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-blot技术对S100A9蛋白、细胞角蛋白19、半乳凝素-7在MDA-MB-231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：S100A9蛋白、细胞角蛋白19、半乳凝素-7在MDA-MB-231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MDA-MB-231细胞株治疗组和对照组存在差异表达。更多还原

【Abstract】 Chapter Ⅰ Assay sensibility of brest cancer MDA-MB-231Cell line to ECP in vitroObjective:To investigate sensitivity,targeted cell cycle phase and possible mechanism of the effect of centipede extract on breast cancer cell lines and provide evidences for further clinic therapy of breast cancer.Methods:Cultured breast cancer cells MDA-MB-231are intervened with different concentration of centipede extract. MTT assay and concentration-time curve are used to study the effect sensitivity of centipede extract on breast cancer cell lines; Inverted light microscope and electron microscope are used to observe cell changes in morphology and ultrastructure; flow cytometry is applicated to investigate cell cycle and apoptosis rate in centipede extract-treated group.Results:①The results of MTT assay showed that different concentrations of centipede extract all can inhibit the growth of human breast cancer cells MDA-MB-231.The inhibition rates were (71.26±2.64)%,(62.48±6.67)%,(56.26±3.17)%,(48.63±4.48)%,(42.13± 4.73)%,(34.5±2.98)%and (26.89±2.95)%respectively,while the Corresponding concentration of centipede extract were80mg/ml,40mg/ml,20mg/ml,10mg/ml,5mg/ml,2.5mg/ml and1.25mg/ml. IC50of centipede extract to MDA-MB-231was12.5mg/ml.②Drug concentration-time curve revealed that the viable cell counts of MDA-MB-231has direct relations with the concentration and administrative time of centipede extract. When treated with12.5mg/ml and25mg/ml centipede extract, viable counts of MDA-MB-231sharply decrease after24hours treatment and the difference among treatment groups is significant (P<0.01).③After48hr intervention, there were no inhibitory effect observed by light microscopy in groups with concentration less than10mg/ml; viable counts of MDA-MB-231decreased and the cells transformed to round and small when concentration were more than10mg/ml; With treated with80mg/ml, large number of necrotic cells and irregular cell debris were seemed. Meanwhile, margination and condensation of chromatin, fragmentation and condensation of nucleus and formation of apoptotic bodies were found in cells by electron microscope which had higher density④The result of flow cytometry displayed that (A)The ratio of G0/G1phase was varied by different concentrations of centipede extract:(36.7±4.2)%in control group,(56.5±4.1)%in12.5mg/ml group, and (67.3±4.4)%which was the highest in25mg/ml group.(B) Cell proliferation index (PI) of MDA-MB-231cells was significantly different as well (P<0.05):the values in control group,12.5mg/ml group and25mg/ml group were (63.3±3.2)%,(43.5±3.5)%, and(32.7±2.0)%,respectively. With the increased concentration of centipede extract, the "sub-G1peak" corresponding to apoptotic cells was gradually rising.(C) Apoptosis ratio: After48hr intervention, spontaneous apoptosis ratio in control group was (2.15±0.13)%, while in12.5mg/ml group it was (13.01±1.51)%and (21.3±1.98)%in25mg/ml group. The differences were statistically significant (P<0.05);⑤Immunohistochemistry had detected that the expression of Bcl-2gene was gradually weaken but that of Bax enhanced in MDA-MB-231cells with the increased concentration of centipede extract.Conclusion:①Extract of centipede can inhibit the growing of human breast cancer cells MDA-MB-231and the effect was concentration-time dependent;②Extract of centipede can induce MDA-MB-231cell line to apoptosis in low concentration and directly kill it in middle or high concentration;③Extract of centipede to MDA-MB-231cells were held in G1/G0phase,inhibition of its growing,and can induce apoptosis in concentration-time depedent effects.④One of the possible mechanisms of Centipede extract treatment may be owned to its ability of promoting the expression of Bax gene and inhibiting that of Bcl-2gene in MDA-MB-231. Chapter II Investigation to the effect and mechanisms of breast cancer MDA-MB-231treatment with ECP in vivoObjective:To investigate the effects and mechanisms of breast cancer MDA-MB-231treatment with ECP.Method:①To set up model of heterotopic grafting carcinoma for breast cancer for MDA-MB-231in nude mouse, and observe its tumor growth and metastasis by intragastric administration of ECP.②To detect Bcl-2、Bax、VEGF and Ang-2in tumor tissue with immunohistochemical methodsResult:①The achievement ratio for hefterotopic grafting carcinoma for breast cancer MDA-MB-231in nude mouse is100%.②It has distinctive inhibitive effect for ECP to hefterotopic grafting carcinoma for breast cancer MDA-MB-231in nude mouse. At the25th day postgraft of nude mouse,the average grafting carcinoma volume of the contrast is (1011.52±212.68)mm3, but for the treatment group is (238.83±65.45) mm3;The average grafting carcinoma weight of the contrast is (983±97.3)mg, but for the treatment group is (556±98.1)mg, The inhibitive ratio reaches to44.2%. there are significant difference for them (P<0.05).③The staining cells population, staining intensity and the optical density (OD) of Bcl-2,VEGF and Ang-2for the contrast group are higher than the treatment group, but they are lower for Bax. there are significant difference for them (P＜0.01)Conclusion:ECP can inhibit hefterotopic grafting carcinoma for breast cancer MDA-MB-231in nude mouse, the mechanisms relate to inhibit tumor Angiogenesis and to promote cells to apoptosis.. Chapter Ⅲ:Application of the techniques and methods of Proteomics into separating differential expression proteins in breast cancer cells treatment with ECPObjective:To screen the relevant differential expression proteins after treatment with ECP, and to investigate the molecule mechanism of ECP for breast cancer MDA-MB-231.Method:we use proteomic Techniques and methods to analysis the mechanism of treatment with ECP for breast cancer MDA-MB-231. Firstly, comparative two-dimensional gel electrophoresis (2-DE) technology was applied to separate the total protein of MDA-MB-231 treatment group by ECP and its control group respectively. The well-resolved, reproducible2-DE patterns of treatment group of ECP and control group were established. Then, PDQuest software was used to analyze2-DE images, and the differential expression proteins between the two groups were identified by both peptide mass fingerprint (PMF) and peptide sequence tag (PST) based on MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry).Result:We select the differential expression proteins for between treatment group and control group which spots variance is over double to analyse with MALDI-TOF-MS after spots enzymolyed,and found out76protein spots at difference level of expression (p<0.05), including41spots decreasing in treatment group, and35spots increasing in control groups.12of the total pieces of PMF were be matched searching in SWISS-PROT/TREMBL database by Mascot software. Among the identified12protein spots, the expression level of proteins which up-regulates in treatment group is5in total including:S100A9、 CALML5、CK-19、Gal-7、Alpha-enolase. But6proteins in treatment group is down-regulation, which include GSTO1-1、PGAMA、HSP90-β、 AKP-P、IMPDH and GAPDH.Conclusion:①Through analysis of two-dimensional gel electrophoresis (2-DE),76protein spots at difference level of expression (p<0.05) have been found out, including41spots decreasing in treatment group, and35spots increasing in control groups.②Among the identified11of the total of protein spots, the expression level of proteins which up-regulates in treatment group is5in total including:S100A9、 CALML5、CK-19、Gal-7、Alpha-enolase and which down-regulates in treatment group is6proteins in total, including:GSTO1-1、PGAMA、 HSP90-β、AKP-P、IMPDH and GAPDH.. It hint that ECP can inhibit MDA-MB-231cell line to growth by inducing it to apoptosis or directly to death in multi-channels. Chapter Ⅳ:Verification of the differential expression levels of the partial proteins by western-blotting analysis and its function studyObjective:To verify the differential expression levels of the partial proteins S100A9、CK19and Gal-7which identified with compared proteomic techniques.Method:To verify the differential expression levels of S100A9、 CK19and Gal-7again by Western-blot methods, which express in MDA-MB-231treatment with ECP group and control group ever identified with comparative proteomic techniques.Result:The proteins of S100A9、CK19and Gal-7express up-regulation in treatment group with ECP, and the results were identical with the proteome analysis.Conclusion:The differential expression proteins of S100A9、CK19and Gal-7screened by proteome analysis are indeed differential expression in treatment group and control group of MDA-MB-231cell.更多还原