Caspase家族在恶性黑色素瘤血管生成拟态形成过程中作用的初步研究

发布时间：2018-03-10 22:17

本文选题：Caspase3　切入点：Caspase8　出处：《天津医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究目的本研究拟通过动物实验观察Caspase家族成员Capsase3、Capase8、Caspase9及其所介导的凋亡在肿瘤血管生成拟态形成过程中的作用。并通过明胶酶谱和免疫组织化学技术观察药物干预后各组肿瘤组织基质金属蛋白酶(Matrix Metalloproteinase, MMP)的活性水平和表达,基因芯片技术检测Caspase3抑制剂组原代细胞差异表达的基因,分析VM形成的分子机制,为临床治疗存在VM的高度恶性肿瘤提供实验依据。研究方法 1)建立恶性黑色素瘤动物移植瘤模型,并随机分为Caspase3抑制剂组、Caspase8、Caspase9抑制剂联合组、对照组,待成瘤后每日分别注射Caspase3抑制剂和Caspase8、Caspase9抑制剂。每日测量肿瘤体积绘制生长曲线来观察不同组间肿瘤的生长速度。 2)通过免疫组化和组织化学双染的方法观察各组肿瘤组织VM的数量,通过明胶酶谱和免疫组化方法观察各组肿瘤组织MMP-2和MMP-9的活性水平和表达,进一步分析Caspase家族成员对VM形成的影响。 3)将Caspase3抑制剂干预后的恶性黑色素瘤细胞和对照组细胞进行原代培养,待细胞达到5×106时提取总RNA。应用18000个克隆的杂交膜进行cDNA微阵列杂交。芯片数据标准化处理后,采用倍数法筛选Caspase3抑制剂组B16原代细胞和对照组B16细胞之间的差异表达基因,阈值为1.0倍。采用DAVID(http://david.abec.nciferf.gov/)在线分析工具,对差异表达基因进行日本京都基因和基因组百科全书代谢通路(KEGG pathway)功能富集。 4)通过伤口愈合实验观察Caspase3抑制剂组肿瘤原代细胞与对照组相比迁移能力的变化。研究结果 1)与对照组相比,在肿瘤生长早期,Caspase3抑制剂组和Caspase8、 Caspase9抑制剂联合组肿瘤体积的生长速度较缓慢,差异有统计学意义(P0.05), Caspase3抑制剂组和Caspase8和Caspase9抑制剂联合组肿瘤组织中VM的数量小于对照组,差异有统计学意义(P0.05)。 2)对照组MMP-2以及MMP-9的活性和表达强于Caspase3抑制剂组,差异有统计学意义(P0.05),对照组MMP-9的活性和表达强于Caspase8、Caspase9抑制剂联合组,差异有统计学意义(P0.05)。 3)基因芯片数据显示Caspase3抑制后的恶性黑色素瘤细胞有1103个基因差异表达,经DAVID的KEGG pathway分析显示差异表达的探针富集于近200条代谢通路,包括TGF-β信号通路,促分裂原活化蛋白激酶途径,粘着斑通路,细胞周期,谷胱甘肽代谢信号通路,胰岛素信号通路,Wnt信号通路等。 4) Caspase3抑制剂组肿瘤原代细胞与对照组相比迁移能力明显下降。结论 1)Caspase家族及其介导的凋亡可能参与恶性黑色素瘤VM的形成。 2) Caspase3抑制剂可能通过下调肿瘤细胞MMP-2和MMP-9活性及表达,并降低细胞的迁移能力,从而抑制VM的形成。 3) Caspase3抑制剂还可能通过下调Id1和Id3的表达抑制VM的形成。 4) Caspase3不仅参与了凋亡的过程,还可能参与TGF-β信号通路的信号传递,当抑制Caspase3的表达后,这些信号通路的多个基因出现差异表达。
[Abstract]:research objective
This study based on the experimental observation of animal Caspase family members Capsase3, Capase8, Caspase9 and apoptosis mediated by the formation of function in the process of vasculogenicmimicry. And by gelatin zymography and immunohistochemistry technique to observe the drug intervention after tumor tissue matrix metalloproteinases (Matrix, Metalloproteinase, MMP) activity and expression level of expression. Gene chip technology to detect Caspase3 inhibitor group primary cell genes, analysis of the molecular mechanisms of VM formation, and provide experimental basis for clinical treatment of malignant tumors with VM.
research method
1) the establishment of malignant melanoma animal xenograft model, and were randomly divided into group Caspase8, Caspase3 inhibitor, Caspase9 inhibitor group, the control group, the tumor were daily injection of Caspase3 inhibitor and Caspase8 inhibitor, Caspase9. Tumor volume was measured daily drawing growth curves of different groups to observe the tumor growth rate.
2) the number of VM in each tumor tissue was observed by immunohistochemistry and histochemical double staining. The activity and expression of MMP-2 and MMP-9 in tumor tissues were observed by gelatinase and immunohistochemistry. Further, the influence of Caspase family members on the formation of VM was further analyzed.
3) Caspase3 inhibitor stem malignant melanoma prognosis and control group cells were cultured until the cells reached 5 * 106 cDNA microarray hybridization hybridization membrane extract total RNA. using 18000 clones. The chip data standardization processing, screening of Caspase3 inhibitor group B16 primary cells and the differences between the control group B16 cell gene expression by multiple method, the threshold is 1 times. The DAVID (http://david.abec.nciferf.gov/) online analysis tools, gene expression Kyoto Encyclopedia of genes and genomes of differences in metabolic pathways (KEGG pathway) functional enrichment.
4) through the wound healing experiment, the changes in the migration ability of the primary cells of the Caspase3 inhibitor group compared with the control group were observed.
Research results
1) compared with the control group, in the early stage of tumor, Caspase3 group and Caspase8 inhibitor, Caspase9 inhibitor combination group tumor volume growth rate is slow, the difference was statistically significant (P0.05), the number of tumor tissue inhibitor of Caspase3 group and Caspase8 group and Caspase9 inhibitor combined with VM than the control group, the difference was statistically significant (P0.05).
2) the activity and expression of MMP-2 and MMP-9 in the control group were stronger than those in the Caspase3 inhibitor group. The difference was statistically significant (P0.05). The activity and expression of MMP-9 in the control group were stronger than those in the Caspase8 group, and the difference was statistically significant in Caspase9 inhibitor combination group (P0.05).
3) microarray data showed that Caspase3 inhibited the malignant melanoma cells have differences in the expression of 1103 genes, the DAVID KEGG pathway analysis showed that differentially expressed probe enriched in nearly 200 metabolic pathways, including the TGF- beta signaling pathway, mitogen activated protein kinase pathway, focal adhesion pathway, cell cycle, glutathione metabolism signaling pathway, insulin signaling pathway, Wnt signaling pathway.
4) the metastatic ability of the tumor cells in the Caspase3 inhibitor group was significantly lower than that in the control group.
conclusion
1) the Caspase family and its mediated apoptosis may be involved in the formation of VM in malignant melanoma.
2) Caspase3 inhibitors may inhibit the formation of VM by lowering the activity and expression of MMP-2 and MMP-9 in tumor cells and reducing the migration ability of the cells.
3) Caspase3 inhibitors may also inhibit the formation of VM by downregulating the expression of Id1 and Id3.
4) Caspase3 not only participates in the process of apoptosis, but also participates in the signal transduction of TGF- beta signaling pathway, and when the expression of Caspase3 is inhibited, multiple genes of these pathways are differentially expressed.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

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