【作者】 陈琼；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 【LRRC4基因和SDF-1α／CXCR4生物学轴的研究背景】LRRC4(leucine-rich repeats containing 4)是我室从染色体7q31-32区域克隆的一个新基因，LRRC4在脑组织中相对特异性表达，是一个与脑胶质瘤级别进展呈负相关的胶质瘤抑制性基因。本实验室前期通过与细胞因子及其受体基因的cDNA微阵列杂交，发现LRRC4在脑胶质瘤U251细胞的重表达，可以明显地下调趋化因子受体基因CXCR4的表达。CXCR4为趋化因子SDF-1α(stromal cell-derived factor—1基质细胞来源因子)的受体。研究表明SDF-1α及其受体CXCR4的过表达与脑胶质瘤的进展呈正相关。SDF-1α通过ERK1／2，AKT信号通路促进脑胶质瘤细胞的增生。CXCR4的阻断剂能抑制脑胶质瘤细胞的增生和侵袭。【LRRC4通过下调SDF-1α／CXCR4生物学轴抑制脑胶质瘤细胞的运动和侵袭】LRRC4在恶性胶质瘤中的表达缺失和CXCR4的过表达均参与了胶质瘤的恶性级别进展。在SDF-1α诱导下，LRRC4转染的U251细胞中CXCR4在mRNA和蛋白表达水平较对照组细胞均明显下调，LRRC4能抑制CXCR4的表达。本研究通过MTT，黏附实验，趋化实验，基质胶侵袭实验，划痕实验和细胞间通讯能力实验，检测到SDF-1α以浓度依赖方式诱导脑胶质瘤U251细胞增殖、趋化、运动和侵袭，最佳浓度为12.5nM。而LRRC4能抑制SDF-1α诱导的脑胶质瘤细胞的增殖、趋化、运动和侵袭，但对SDF-1α(12.5nM)诱导的脑胶质瘤细胞与基质粘附无影响，却抑制瘤细胞与内皮细胞ECV304的粘附。因此，LRRC4通过下调SDF-1α／CXCR4生物学轴抑制脑胶质瘤的运动和侵袭。【LRRC4负性调控SDF-1α／CXCR4生物学轴介导的ERK1／2和AKT信号通路，并通过这两条通路调节MMP-2活性】用外源性SDF-1α(12.5nM)处理U251细胞，分别作用0min，5min，10min，15min，30min，发现SDF-1α可以使U251细胞中ERK1／2以及AKT磷酸化水平明显增高，且呈时间依赖性，15min表达达高峰。LRRC4可以有效抑制ERK1／2以及AKT的磷酸化。ERK通路抑制剂PD98059以及AKT通路抑制剂LY294002能明显抑制ERK1／2以及AKT的活化(阳性对照)。因此，LRRC4能负性调控SDF-1α／CXCR4生物学轴介导的ERK1／2，AKT信号通路。基质金属蛋白酶(MMPs)在肿瘤侵袭转移和血管形成过程中起了很重要的作用。研究表明，一些信号通路如，p38MAPK，NF-κB，PKC，PI-3K激酶及Raf／ERK，JAK／STATs通过调节MMP—2、MMP—9的分泌来影响肿瘤细胞侵袭和转移特性。本研究发现SDF-1α可以增强U251细胞MMP2酶活性，LRRC4明显的抑制了SDF-1α诱导的MMP2的酶活性增加。ERK通路抑制剂PD98059和AKT通路抑制剂LY294002处理均可抑制SDF-1α诱导的MMP2的酶活性增加(阳性对照)。因此，LRRC4能够通过SDF-1α／CXCR4生物学轴介导的ERK1／2和AKT通路抑制MMP-2的酶活性。但对MMP-9的酶活性无影响。【LRRC4诱导脑胶质瘤细胞向星形胶质样细胞分化】与对照组细胞相比(细胞呈梭形，基本无突起)，LRRC4转染后的U251细胞形态发生明显变化，细胞胞体缩小，呈圆形，突起明显延长，呈星形胶质样细胞特点。GFAP一直被视为星形细胞特异性标记物，与脑胶质瘤分化程度呈正相关，本研究发现LRRC4转染0251细胞后，GFAP表达明显上调。表明，LRRC4可以诱导脑胶质瘤U251细胞向星形胶质样细胞分化。综上所述，胶质瘤抑瘤基因LRRC4可以通过抑制CXCR4的表达，负性调控SDF-1α／CXCR4生物学轴介导的ERK1／2和AKT通路，抑制胶质瘤U251细胞趋化、运动和侵袭；LRRC4能够诱导U251细胞向星形胶质样细胞分化。更多还原

【Abstract】 【Background of LRRC4 and SDF-1α/CXCR4 axis】Glioblastomas are the most common and lethal human primary brain tumors, and are characterized by high invasiveness. Leucine-rich repeats containing 4 (LRRC4) is a potential glioma suppressive gene. The conspicuous absence of LRRC4 in high-grade gliomas directly contributes to increasing tumor grade. The reexpression of LRRC4 can significantly suppress glioblastomas U251 cells tumorigenesis in vivo and cell proliferation and invasion in vitro. LRRC4 controls glioblastoma cells proliferation by ERK/AKT/NF-κB signaling pathway. Stromal cell-derived factor SDF-1 (recently renamed CXCL12) and its CXC chemokine receptor 4 (CXCR4) are involved in normal and malignant glial cell proliferation and migration. SDF-1αexerts its activity by interacting with the CXCR4 receptor, a member of the G protein-coupled receptor superfamily. Recent data showed that CXCR4 and SDF-1 mRNAs are co-localized in glioblastomas and that their expression increase with the tumor grade and is associated with regions of necrosis and angiogenesis.【LRRC4 inhibits invasion of U251 cells through SDF-1α/CXCR4 axis】The absence of LRRC4 in high-grade gliomas and the over-expression of CXCR4 contribute to increasing tumor grade. In the present study, we demonstrate that CXCR4 is expressed in human glioblastoma U251 cell line, and that SDF-1αinduces proliferation, adhesion ability chemotaxis, mobility and invasion in CXCR4~+ glioblastoma U251 cells. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF-1α/CXCR4 axis-induced proliferative, adhesive, chemotactic and invasive effects. LRRC4 can greatly enhance GJIC of U251 cells.【LRRC4 down-regulates the SDF- 1α/CXCR4-mediated ERK1/2 and Akt signaling pathways and inhibit MMP-2 activity】LRRC4 exerts its inhibitory role in U251 cells by down-regulating the SDF- 1α/CXCR4-mediated phosphorylation activation of ERK1/2 and Akt. We also provide evidence for proMMP-2 activation involvement in the SDF-1α/ CXCR4 axis-mediated signaling pathway. LRRC4 significantly inhibits proMMP-2 activation by SDF-1α/CXCR4 axis-mediated ERK1/2 and Akt signaling pathway. We did not find impact of LRRC4 on MMP-9 activity.【LRRC4 induces U251 cells to differentiate into astrocyte-like cells】U251 cells transfected with LRRC4 become smaller and round in shape and their axon grow longer as compared with U251 cells. LRRC4 significantly increases the expression of GFAP, which is regarded as a specific mark for gliomas and is related to the differentiation of gliomas. Hence, LRRC4 can induce U251 cells to differentiate into astrocyte-like cells.Collectively, these results suggest a possible important "cross-talk" between LRRC4 and SDF-1α/CXCR4 axis-mediated intracellular pathways that can link signals of cell chemotaxis and invasion in glioblastoma, and LRRC4 induce the differentiation of gliomas, which may represent a new target for development of new therapeutic strategies in glioma.更多还原