【作者】 陶冀；

【导师】 王亚贤；

【作者基本信息】 黑龙江中医药大学 ， 中西医结合基础， 2012， 博士

【摘要】 目的：探讨乳腺癌组织中nm23-H1、c-erbB2和MMP-9蛋白表达及其与相关指标和转移的关系；研究红花多糖(SPS)、表阿霉素(EPI)对人乳腺癌细胞MCF-7增殖、凋亡的影响,观察凋亡和转移相关基因BAX、Bcl-2、MMP-9、 nm23-H1mRNA和蛋白表达变化,通过Transwell小室法证实红花多糖、表阿霉素对乳腺癌细胞转移能力的影响,并进行对比及统计学分析,明确中药红花多糖与化疗药物表阿霉素对乳腺癌细胞作用的区别,探讨红花多糖抗MCF-7细胞作用及规律。方法：1.随机抽取乳腺癌组织标本120例,正常乳腺组织标本77例。采用免疫组化SP法检测乳腺癌组织及正常乳腺组织中nm23-H1、C-erbB2和MMP-9的表达情况,结合临床病理资料,分析三者表达与年龄、临床分期、淋巴结转移等临床病理特征的关系。2. MTT比色法观察不同浓度的红花多糖、表阿霉素对人乳腺癌MCF-7细胞体外增殖的抑制作用,并通过流式细胞仪(FCM)分析细胞凋亡。3. Real-time PCR检测不同浓度红花多糖、表阿霉素作用于乳腺癌MCF-7细胞后BAX、Bcl-2、MMP-9、nm23-H1mRNA表达水平；western blot检测不同浓度红花多糖、表阿霉素作用于乳腺癌MCF-7细胞后BAX、Bcl-2、MMP-9、 nm23-H1蛋白表达水平。4. Transwell小室法研究不同浓度红花多糖、表阿霉素对乳腺癌细胞MCF-7转移能力的影响。结果：1. nm23-H1、c-erbB2和MMP-9在乳腺癌组织中均显著表达；三者中仅C-erbB2蛋白表达与患者年龄有关；nm23-H1蛋白在早期、无淋巴结转移及远处转移的乳腺癌中呈高表达,而c-erbB2和MMP-9则在晚期、有淋巴结转移及远处转移的乳腺癌中呈现高表达。2.红花多糖、表阿霉素均能抑制乳腺癌MCF-7细胞增殖。表阿霉素对MCF-7细胞的抑制作用随浓度的升高抑制作用增强,随着时间进行作用减弱；红花多糖对MCF-7细胞的抑制作用随着时间的进行作用增强,并在一定浓度内,随着浓度的升高作用增强,超过一定浓度后作用反而降低。3.流式细胞仪检测,MCF-7细胞经SPS、EPI作用后24h后,其早期凋亡率、细胞坏死或晚期凋亡率显著增加,呈现明显的浓度依赖性。4.Real-time PCR和western blot检测发现,SPS、EPI作用后的MCF-7细胞的Bcl-2、MMP-9mRNA及蛋白表达量明显下降,BAX、nm23-H1mRNA表达量明显升高。5.SPS、EPI对乳腺癌MCF-7细胞的转移能力均有一定的抑制作用。结论：1.c-erbB2、MMP-9高表达及nm23-H1低表达与乳腺癌发生浸润转移密切相关。联合检测这些指标可作为乳腺癌患者转移复发,早期诊断及预后判断的参考指标,同时也为抗肿瘤药物的研究提供重要靶点及理论依据。2.中药SPS对人乳腺癌MCF-7细胞体外的抑制作用与表阿霉素有区别,SPS对MCF-7细胞的抑制随着浓度的升高、时间的延长作用增强。3.SPS、EPI诱导人乳腺癌MCF-7细胞凋亡和坏死,作用具有剂量依赖性；同一浓度的SPS诱导乳腺癌细胞凋亡和坏死的作用较EPI弱。4. SPS、EPI均可以诱导Bcl-2、MMP-9基因表达下降,BAX、nm23-Hl上升。5.SPS、EPI均可以抑制乳腺癌MCF-7细胞的转移能力,二者的抑制作用无统计学差异。更多还原

【Abstract】 Objective:To investigate nm23-H1, c-erbB-2and MMP-9protein expression in breast cancer tissue and its relationship with the relevant indicators and transfer; to study the influence of safflower polysaccharides (SPS), epirubicin (EPI) on the proliferation and apoptosis of the human breast cancer cells MCF-7, and observe the changes of the apoptosis and metastasis related genes of BAX, Bcl-2and MMP-9, nm23-H1mRNA and protein expression, to confirm using Transwell chamber assay that safflower polysaccharide, epirubicin can suppression the metastatic ability of breast cancer cells. And make sure that Chinese medicine safflower polysaccharide has the ability of anti-MCF-7cells by certain mechanism.Methods:1. Random collecting120cases of breast cancer tissue samples and77cases of normal breast tissue samples. We try to use SP immunohistochemistry method to detect the expression of nm23-H1, c-erbB-2and MMP-9in breast cancer tissue and normal breast tissue. To combine with clinical and pathological data, and analysis of the three exppression with age, clinical stage, lymph node metastasis the relationship of the clinical and pathological features.2. To observe the proliferation inhibition and apoptosis of human breast cancer MCF-7cell effected by safflower polysaccharide or epirubicin by using MTT assay and flow cytometry (FCM) analysis.3.To detect the expression levels of BAX and Bcl-2and MMP-9, nm23-Hl mRNA in MCF-7cell by using real-time PCR; to detect the expression levels of BAX and Bcl-2and MMP-9, nm23-H1mRNA in MCF-7cell by using western blot.4. Transwell chamber experiment was used to investigate effects on the metastatic ability of breast cancer cell line MCF-7by giving different concentrations of safflower polysaccharide or epirubicin.Results:1. Nm23-H1, c-erbB-2and MMP-9were highly expressed in breast cancer; only the c-erbB-2level related with age; expression of nm23-H1protein was high in the early stage of breast cancer and breast cancer with no lymph node metastasis or distant metastasis; however, expression of c-erbB-2and MMP-9were high in the late stage, lymph node metastasis and distant metastasis of breast cancer.2. Safflower polysaccharide and epirubicin can both inhibit the proliferation of breast cancer MCF-7cell. The depressant effect of Epirubicin on MCF-7cells increased with the concentration of Epirubicin, and decreased with time; the inhibition of safflower polysaccharide on MCF-7cells increased with time progresses, and in a certain concentration, as the concentration increased, the enhanced role increased, till the concentration achieved or larger than the certain concentration, and then the inhibition effect decreased.3. Be treated wiht SPS or EPI for24hours, flow cytometry was applied. Results showed that the early apoptosis rate, cell necrosis and late apoptosis rate of MCF-7cells were significantly increased, showing a significant concentration-dependent.4. Real-time PCR and western blot results showed that, treated with SPS and EPI, the expression of Bcl-2and MMP-9(both in RNA and protein) were decreased, and the level of BAX and nm23-H1mRNA were increased.5. Both SPS and EPI had the ability to inhibit metabasis of breast cancer MCF-7cells.Conclusion:1. High expression of c-erbB-2and MMP-9and low expression of nm23-H1were closely related to invasion and metastasis in breast cancer. Testing these index can be helpful in early diagnosis of metastasis and recurrence of breast cancer, providing evidence for estimating the outcome, and providing theoretical basis on anticancer targets of reference2. Inhibition effect of Traditional Chinese medicine SPS and EPI on breast cancer cell MCF-7cells was significant different. The inhibition of safflower polysaccharide on MCF-7cells increased with time progresses only in a certain concentration.3. Both SPS and EPI induced human breast MCF-7apoptosis and necrosis in a dose dependent; the effect of apoptosis and necrosis by SPS is weaker than that by EPI. 4. Both SPS and EPI can induce Bcl-2and MMP-9gene lowly express, and induce BAX and nm23-H1gene highly express.5. SPS and EPI both can inhibit the metastatic potentia of breast cancer MCF-7cells, with no significant difference.更多还原