【作者】 李玉华；

【导师】 陈誉华；

【作者基本信息】 中国医科大学 ， 发育生物学， 2010， 硕士

【摘要】 背景及目的P190蛋白是一种神经元跨膜蛋白,由1384个氨基酸组成。最初发现于有髓神经纤维郎飞氏节的节旁侧,主要在脊椎动物脑内表达,另外在卵巢、胰脏、肠、肺、心脏及睾丸等组织内也有少量表达。P190蛋白与髓鞘蛋白NF155等结合,在神经轴突的节旁侧形成节旁连接(Paranodal junction),这种连接结构类似于间隔连接(Septate junction)。间隔连接广泛存在于果蝇等无脊椎动物的血-神经屏障处,与脊椎动物血脑屏障处的紧密连接功能类似。目前研究表明,脊椎动物内由P190、NF155等蛋白形成的节旁连接在维持神经冲动的跳跃式传导、轴突1胶质细胞间信息快速传递、轴突特化区域的形成及轴-胶间隙与细胞外基质间屏障形成中发挥重要作用。P190蛋白表达异常可产生严重的神经系统功能紊乱,P190-／-型小鼠的神经冲动传导速度明显低于正常小鼠,而且在出生后不久即因神经元变性坏死而导致死亡。另外有研究显示多发性硬化症(Multiple Sclerosis, MS)患者脑内P190表达水平明显降低。P190表达降低可进一步导致髓鞘脱离轴突,促进轴突变性坏死,进而形成神经元变性损伤的恶性循环。在糖尿病性神经病变的大鼠中,P190的表达水平也明显降低。由此可见,P190蛋白的功能是至关重要的,但其具体功能和作用机制还不清楚。血脑屏障(blood-brain barrier, BBB)是由脑微血管内皮细胞、星型胶质细胞、基膜及周细胞等共同组成的高度特化的神经血管结构单元。其中脑微血管内皮细胞间的紧密连接和粘附连接是构成血脑屏障结构的基础,也是维持血脑屏障功能的关键。目前已知的内皮细胞间连接相关蛋白已有很多,包括跨膜蛋白和细胞内的衔接蛋白；同时对内皮细胞间紧密连接和粘附连接的作用机制也有了深入认识,但仍有待发现更多的连接蛋白及更深入的了解内皮细胞屏障功能机制。我室研究人员发现P190蛋白除了在神经轴突有表达外,在人脑微血管内皮细胞(Human brain microvascular endothelial cell, HBMEC)内也有大量表达,而且表达的P190蛋白在细胞间连接处有聚集的倾向。基于此,本研究通过构建P190蛋白的一系列突变体作为基础,进一步检测P190显性缺失突变对HBMEC功能的影响；同时利用酵母双杂交的方法,筛选与P190蛋白的胞内段互作的蛋白,期望找出P190对内皮细胞功能作用的机制。方法1、根据已有的P190全长基因序列,利用重叠PCR的方法获得P190不同结构域缺失的基因序列,克隆至真核细胞表达载体pcDNA3.1/myc-His B上,并稳定转染至第26代人脑微血管内皮细胞(HBMEC)中,Western blot法验证阳性细胞株。2、体外血管形成实验检测P190不同结构域缺失突变对HBMEC形成血管的能力是否有影响。3、悬滴聚集实验(Hanging-drop aggregation assay)检测P190不同结构域缺失突变对HBMEC的细胞间黏附力是否有影响。4、用HRP通透性实验,检测P190不同结构域缺失突变对HBMEC形成的细胞单层的通透性是否有影响。5、利用蛋白质免疫印迹的方法检测P190不同结构域缺失突变对HBMEC的紧密连接相关蛋白ZO-1和黏附连接蛋白VE-cadherin的表达是否有影响。6、利用细胞免疫荧光方法检测P190不同结构域缺失突变对HBMEC胞间连接结构及对ZO-1和VE-cadherin的分布是否有影响。7、利用酵母双杂交实验筛选人胎脑文库中与P190胞内段互作的蛋白,并用GST-pull down法进行验证。结果1、成功获得P190不同结构域缺失的基因序列,经测序验证后,成功转染至人脑微血管内皮细胞,获得相应的细胞株。2、体外血管形成实验结果显示,P190不同结构域缺失突变对HBMEC的血管形成能力无影响。3、悬滴聚集实验表明,P190胞内段缺失突变的内皮细胞其细胞间黏附力明显低于对照组细胞。4、HRP通透性实验表明,P190胞内段缺失型内皮细胞对HRP的通透性明显高于对照组细胞,且有统计学意义；而SH3或band4.1结构域分别缺失的内皮细胞与对照组细胞相比,通透性无明显变化。5、Western blot结果显示,细胞连接蛋白ZO-1和VE-cadherin在各种突变型细胞和对照组细胞内,表达水平趋于一致。6、免疫荧光实验结果显示,对照组细胞和SH3、band4.1结构域缺失的内皮细胞的细胞间连接结构较完整,紧密连接蛋白ZO-1和黏附连接蛋白VE-cadherin在细胞间连接处呈连续的线状分布；而P190胞内段全部缺失的内皮细胞,细胞间连接结构破裂不完整,而且ZO-1和VE-cadherin在细胞间连接处呈点状或交错的刺状分布。7、酵母双杂交法筛选得到14个阳性克隆,根据文献资料选择ATP1B3进行验证。结论人脑微血管内皮细胞中P190蛋白胞内段(1305-1384aa)缺失后,可使内皮细胞单层的通透性明显提高,同时人脑微血管内皮细胞间的黏附能力降低,进而破坏血脑屏障的完整性。但这种作用的具体机制及P190是否直接参与人脑微血管内皮细胞间的连接还有待进一步研究。更多还原

【Abstract】 Background and PurposeP190 is a neuronal transmembrane protein, which was initially found at the paranodes of the myelinated fibers, and mainly expressed in vertebrate brain tissues. Ovary, pancreas, intestines, lung, heart and testis also have low expression for P190 protein. P190 can contact with NF155 and together form paranodal junction at the paranodes of myelinated fibers. Paranodal junction is similar with septate junction, which is widely distributed at blood-nerve barrier in invertebrate such as Drosophila. Present studies indicate that the main functions of septate like junction in vertebrate are maintaining rapid and efficient propagation of action potential, messages transmition between axon and glia, specific axon domain formation, and barrier formation between axon-glia gap and extracellular matrix. The abnormal expression of P190 can cause serious nervous system disorder. P190-/-type mouse have low nerve impulse conduction speed and ill or death with neural degeneration after delivery. Other studies indicate that the expression of P190 is obviously low in multiple sclerosis patients. Low expression of P190 protein can cause axon demyelination and nerve degeneration. The diabetic neuropathies rats also have low P190 expression. Those indicate the important function of P190 protein, but its specific functions and mechanisms still need studies.The blood-brain barrier (BBB) is a highly specialized neurovascular system which is composed of brain microvascular endothelial cells, astrocytes, basilar membrane and pericytes. The brain microvascular endothelial cells are the most important components for BBB, and they are connected together by tight junctions and adhesion junctions. Many central nervous diseases have dispersed endothelial cell-cell junctions, including Alzheimer’s disease and multiple sclerosis. Now people already have certain understanding on the function mechanism of tight junctions and adhesion junctions, but the mechanism is still not so clear. Our laboratory found that besides nerve axons, HBMECs also express P190 highly, and the P190 potentially assembled on the membrane at cell-cell junctions. Based on this phenomenon, we construct different P190 mutants and test the function of different mutants and normal HBMEC. We also exploit yeast two hybrid method to screen proteins interacting with P190 intracellular region form human fetal brain library, and wish to find out the P190 protein function mechanism.Methods1、Overlap PCR method to obtain P190 band4.1 binding domain deletion gene sequences. Then eukaryotic expression plasmid pcDNA 3.1/myc-His B-P190 was constructed by molecular cloning technology, and then was transfected into P26 HBMECs.2、In vitro tube formation assay detect the tube formation ability of different mutated endothelial cells or control cells.3、Hanging-drop aggregation assay test the cell-cell adhesion of different mutated cells or control cells.4、Test the permeability of cell monolayers formed by different mutated cells or control cells to HRP.5、Western blot investigate the expression of tight junction protein ZO-1 and adhesion junction protein VE-cadherin in different mutated cells or in control cells.6、Immunocytochemical method investigate the distribution of tight junction protein ZO-1 and adhesion junction protein VE-cadherin in different mutated cells and in control cells.7、Screening proteins interact with P190 intracellular domain by yeast two hybrid method and confirm the interaction by GST-pull down. Results1、Successfully obtained band4.1 binding domain deletion gene sequences, and stable transfected to P26 HBMECs.2、In vitro tube formation assay suggested that there is no significant difference between P190 mutated cells and the control cells3、Hanging-drop aggregation assay indicated that the intercellular adhesion was obviously weak in P190 intracellular domain deleted cells than in the control cells or other kinds of mutated cells.4、P190 intracellular domain deleted cells monolayer show a high permeability to HRP.5、The expression of tight junction protein ZO-1and adhesion junction protein VE-cadherin has no difference in P190 different mutated cells or control cells.6、Immunofluorescence show a line continuous distribution of junctional protein ZO-1 and VE-cadhetin at cell-cell junctions in control cells and SH3 or band4.1 domain deleted cells; While there is an intermittent distribution of ZO-1 and VE-cadhetin and interrupted cell-cell junction in P190 intracellular domain deleted cells.7、Obtained 14 interacted gene sequence but need further certification.ConclusionDeletion of the intracellular domain (1305-1384aa) of P190 can decrease the adhesion between human brain microvascular endothelial cells and increase the permeability of the endothelial cells monolayer. But whether the P190 protein participates in or regulates the junctions between HBMECs is still need further study.更多还原