神经细胞粘附分子对肿瘤细胞形态、生长和运动的影响

发布时间：2018-03-30 01:22

本文选题：NCAM　切入点：PSA　出处：《江南大学》2015年硕士论文

【摘要】：神经细胞粘附分子(Neural cell adhesion molecule,NCAM)是一种定位在细胞膜上的糖蛋白,属于免疫球蛋白超基因家族,它不仅能介导细胞与细胞之间和细胞与细胞外基质间的粘附作用,还影响神经细胞增殖、迁移以及神经突触可塑性等。多聚唾液酸(Polysialic acid,PSA)是唾液酸单体通过α-2,8键连接的长链线性聚合物,PSA主要粘附在NCAM,多聚唾液酸转移酶II(STX)和多聚唾液酸转移酶IV(PST)通过在NCAM免疫球蛋白域的第五个和第六个N-糖基化位点催化合成PSA。在非小细胞癌、神经母细胞瘤和肾母细胞瘤等恶性肿瘤均发现PSA的过量表达。PSA-NCAM在细胞的识别以及肿瘤的侵润、生长起着一定的作用。上皮间质转化(Epithelial-mesenchymal transition,EMT)过程与肿瘤的转移密切相关。EMT不仅存在于许多细胞生物的胚胎发育过程中,还在原发肿瘤细胞的转移和侵袭过程中扮演着重要角色,而转化生长因子(Transforming growth factor,TGF-β)可以促进诱导正常细胞发生EMT过程。本研究以正常乳腺上皮细胞和恶性乳腺肿瘤细胞为研究对象,通过建立EMT模型,研究NCAM对细胞形态、运动、增殖等细胞行为,以及NCAM和PSA-NCAM介导EGFR、STAT3信号通路的影响,阐述NCAM和PSA-NCAM在乳腺癌发生发展中的作用。首先,对比研究NCAM在正常乳腺细胞和恶性乳腺肿瘤细胞中的表达情况,发现人源恶性肿瘤细胞NCAM表达水平明显高于正常乳腺细胞。当用TGF-β诱导正常乳腺细胞MCF10A发生EMT后,NCAM表达水平上调。通过在正常乳腺上皮细胞MCF10A稳定转染鼠源NCAM-140,MCF10A细胞过表达NCAM-140后发现E-cadherin表达水平下调,N-cadherin表达水平上调,细胞运动能力明显增强,细胞增殖能力不变,激活EGFR、STAT3信号通路,这些变化在乳腺肿瘤细胞MCF7瞬时转染过表达NCAM-140得到验证,因此说明NCAM促进细胞发生EMT过程。利用高效液相色谱检测发现恶性乳腺肿瘤细胞的游离唾液酸含量比正常乳腺细胞高。通过在MCF7细胞瞬时转染NCAM-140和唾液酸转移酶PST、STX,发现分别过表达NCAM和PSA-NCAM均能激活EGFR、STAT3信号通路。将构建NCAM-140的第五个和第六个N-糖基化位点突变质粒瞬时转染到MCF10A细胞,发现去除多聚唾液酸化的NCAM仍能激活EGFR、STAT3信号通路。本论文说明了NCAM是非依赖于PSA,通过激活EGFR、STAT3信号通路,促进细胞发生EMT过程,揭示NCAM在乳腺肿瘤发生发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 伪 -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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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