本文選題：Caspase3　切入點：Caspase8　出處：《天津醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的 本研究擬通過動物實驗觀察Caspase家族成員Capsase3、Capase8、Caspase9及其所介導(dǎo)的凋亡在腫瘤血管生成擬態(tài)形成過程中的作用。并通過明膠酶譜和免疫組織化學(xué)技術(shù)觀察藥物干預(yù)后各組腫瘤組織基質(zhì)金屬蛋白酶(Matrix Metalloproteinase, MMP)的活性水平和表達,基因芯片技術(shù)檢測Caspase3抑制劑組原代細胞差異表達的基因,分析VM形成的分子機制,為臨床治療存在VM的高度惡性腫瘤提供實驗依據(jù)。 研究方法 1)建立惡性黑色素瘤動物移植瘤模型,并隨機分為Caspase3抑制劑組、Caspase8、Caspase9抑制劑聯(lián)合組、對照組,待成瘤后每日分別注射Caspase3抑制劑和Caspase8、Caspase9抑制劑。每日測量腫瘤體積繪制生長曲線來觀察不同組間腫瘤的生長速度。 2)通過免疫組化和組織化學(xué)雙染的方法觀察各組腫瘤組織VM的數(shù)量,通過明膠酶譜和免疫組化方法觀察各組腫瘤組織MMP-2和MMP-9的活性水平和表達,進一步分析Caspase家族成員對VM形成的影響。 3)將Caspase3抑制劑干預(yù)后的惡性黑色素瘤細胞和對照組細胞進行原代培養(yǎng),待細胞達到5×106時提取總RNA。應(yīng)用18000個克隆的雜交膜進行cDNA微陣列雜交。芯片數(shù)據(jù)標準化處理后,采用倍數(shù)法篩選Caspase3抑制劑組B16原代細胞和對照組B16細胞之間的差異表達基因,閾值為1.0倍。采用DAVID(http://david.abec.nciferf.gov/)在線分析工具,對差異表達基因進行日本京都基因和基因組百科全書代謝通路(KEGG pathway)功能富集。 4)通過傷口愈合實驗觀察Caspase3抑制劑組腫瘤原代細胞與對照組相比遷移能力的變化。 研究結(jié)果 1)與對照組相比,在腫瘤生長早期,Caspase3抑制劑組和Caspase8、 Caspase9抑制劑聯(lián)合組腫瘤體積的生長速度較緩慢,差異有統(tǒng)計學(xué)意義(P0.05), Caspase3抑制劑組和Caspase8和Caspase9抑制劑聯(lián)合組腫瘤組織中VM的數(shù)量小于對照組,差異有統(tǒng)計學(xué)意義(P0.05)。 2)對照組MMP-2以及MMP-9的活性和表達強于Caspase3抑制劑組,差異有統(tǒng)計學(xué)意義(P0.05),對照組MMP-9的活性和表達強于Caspase8、Caspase9抑制劑聯(lián)合組,差異有統(tǒng)計學(xué)意義(P0.05)。 3)基因芯片數(shù)據(jù)顯示Caspase3抑制后的惡性黑色素瘤細胞有1103個基因差異表達,經(jīng)DAVID的KEGG pathway分析顯示差異表達的探針富集于近200條代謝通路,包括TGF-β信號通路,促分裂原活化蛋白激酶途徑,粘著斑通路,細胞周期,谷胱甘肽代謝信號通路,胰島素信號通路,Wnt信號通路等。 4) Caspase3抑制劑組腫瘤原代細胞與對照組相比遷移能力明顯下降。 結(jié)論 1)Caspase家族及其介導(dǎo)的凋亡可能參與惡性黑色素瘤VM的形成。 2) Caspase3抑制劑可能通過下調(diào)腫瘤細胞MMP-2和MMP-9活性及表達,并降低細胞的遷移能力,從而抑制VM的形成。 3) Caspase3抑制劑還可能通過下調(diào)Id1和Id3的表達抑制VM的形成。 4) Caspase3不僅參與了凋亡的過程,還可能參與TGF-β信號通路的信號傳遞,當抑制Caspase3的表達后,這些信號通路的多個基因出現(xiàn)差異表達。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.5

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