本文選題：NCAM　切入點：PSA　出處：《江南大學》2015年碩士論文

【摘要】：神經細胞粘附分子(Neural cell adhesion molecule,NCAM)是一種定位在細胞膜上的糖蛋白,屬于免疫球蛋白超基因家族,它不僅能介導細胞與細胞之間和細胞與細胞外基質間的粘附作用,還影響神經細胞增殖、遷移以及神經突觸可塑性等。多聚唾液酸(Polysialic acid,PSA)是唾液酸單體通過α-2,8鍵連接的長鏈線性聚合物,PSA主要粘附在NCAM,多聚唾液酸轉移酶II(STX)和多聚唾液酸轉移酶IV(PST)通過在NCAM免疫球蛋白域的第五個和第六個N-糖基化位點催化合成PSA。在非小細胞癌、神經母細胞瘤和腎母細胞瘤等惡性腫瘤均發(fā)現PSA的過量表達。PSA-NCAM在細胞的識別以及腫瘤的侵潤、生長起著一定的作用。上皮間質轉化(Epithelial-mesenchymal transition,EMT)過程與腫瘤的轉移密切相關。EMT不僅存在于許多細胞生物的胚胎發(fā)育過程中,還在原發(fā)腫瘤細胞的轉移和侵襲過程中扮演著重要角色,而轉化生長因子(Transforming growth factor,TGF-β)可以促進誘導正常細胞發(fā)生EMT過程。本研究以正常乳腺上皮細胞和惡性乳腺腫瘤細胞為研究對象,通過建立EMT模型,研究NCAM對細胞形態(tài)、運動、增殖等細胞行為,以及NCAM和PSA-NCAM介導EGFR、STAT3信號通路的影響,闡述NCAM和PSA-NCAM在乳腺癌發(fā)生發(fā)展中的作用。首先,對比研究NCAM在正常乳腺細胞和惡性乳腺腫瘤細胞中的表達情況,發(fā)現人源惡性腫瘤細胞NCAM表達水平明顯高于正常乳腺細胞。當用TGF-β誘導正常乳腺細胞MCF10A發(fā)生EMT后,NCAM表達水平上調。通過在正常乳腺上皮細胞MCF10A穩(wěn)定轉染鼠源NCAM-140,MCF10A細胞過表達NCAM-140后發(fā)現E-cadherin表達水平下調,N-cadherin表達水平上調,細胞運動能力明顯增強,細胞增殖能力不變,激活EGFR、STAT3信號通路,這些變化在乳腺腫瘤細胞MCF7瞬時轉染過表達NCAM-140得到驗證,因此說明NCAM促進細胞發(fā)生EMT過程。利用高效液相色譜檢測發(fā)現惡性乳腺腫瘤細胞的游離唾液酸含量比正常乳腺細胞高。通過在MCF7細胞瞬時轉染NCAM-140和唾液酸轉移酶PST、STX,發(fā)現分別過表達NCAM和PSA-NCAM均能激活EGFR、STAT3信號通路。將構建NCAM-140的第五個和第六個N-糖基化位點突變質粒瞬時轉染到MCF10A細胞,發(fā)現去除多聚唾液酸化的NCAM仍能激活EGFR、STAT3信號通路。本論文說明了NCAM是非依賴于PSA,通過激活EGFR、STAT3信號通路,促進細胞發(fā)生EMT過程,揭示NCAM在乳腺腫瘤發(fā)生發(fā)展中起到的重要作用。
[Abstract]:Neural cell adhesion molecule (NCAM) is a glycoprotein located on the cell membrane and belongs to the immunoglobulin supergene family. It not only mediates the adhesion between cells and cells and between cells and extracellular matrix. It also affects the proliferation of nerve cells, Migration and synaptic plasticity. Polysialic acidase (PSAs) is a long chain linear polymer linked to sialic acid monomers via 偽 -2O8 bond. Poly sialic acid transferase IIHSTX and polysialic acid transferase IVPSTs are mainly adhered to NCAM via NCAM. The fifth and sixth N-glycosylation sites of the immunoglobulin domain catalyze the synthesis of PSAs. Malignant tumors such as neuroblastoma and nephroblastoma all found overexpression of PSA. PSA-NCAM was recognized in cells and infiltrated by tumor. Epithelial-mesenchymal transition (EMTT) is closely related to tumor metastasis. EMT not only exists in the embryonic development of many cellular organisms, but also plays an important role in the metastasis and invasion of primary tumor cells. Transforming growth factor- 尾 (TGF- 尾) can promote the induction of EMT in normal cells. In this study, normal mammary epithelial cells and malignant breast tumor cells were studied by establishing EMT model to study the morphology and motility of NCAM cells. Proliferation and other cell behaviors, and the effects of NCAM and PSA-NCAM on the EGFR STAT3 signaling pathway were discussed. The role of NCAM and PSA-NCAM in the development of breast cancer was discussed. Firstly, the expression of NCAM in normal breast cells and malignant breast tumor cells was compared. It was found that the expression level of NCAM in human malignant tumor cells was significantly higher than that in normal mammary gland cells. The expression level of EMT was up-regulated in normal breast MCF10A cells induced by TGF- 尾. The expression level of NCAM-140 MCF10A was stably transfected into normal mammary epithelial cells by MCF10A transfection. After overexpression of NCAM-140, the expression of E-cadherin was down-regulated and the expression of N-cadherin was up-regulated. The activation of EGFR STAT3 signaling pathway was demonstrated by the transient transfection of NCAM-140 into breast tumor cells by transient transfection of NCAM-140. It is concluded that NCAM promotes the development of EMT. The free sialic acid content in malignant breast tumor cells was found to be higher than that in normal breast cells by high performance liquid chromatography. NCAM-140 and sialic acid transferase were transduced by transient transfection of NCAM-140 and sialic acid transferase in MCF7 cells. It was found that overexpression of NCAM and PSA-NCAM could activate EGFR STAT3 signaling pathway. The fifth and sixth N-glycosylation site mutant plasmids of NCAM-140 were transiently transfected into MCF10A cells. It is found that NCAM can still activate EGFR r-STAT3 signaling pathway. This paper shows that NCAM is independent of NCAM. By activating EGFR r-STAT3 signaling pathway, it can promote the development of EMT in cells and reveal the important role of NCAM in the development of breast tumors.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R737.9

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