治疗基因向脑内递送选修课件

1、治疗基因向脑内递送选修治疗基因向脑内递送选修掌握要点1.血脑屏障的功能2.物质透过血脑屏障的途径掌握要点1.血脑屏障的功能第一节 血脑屏障及血脑肿瘤屏障一、阻止药物进入脑肿瘤内的屏障 （一）脑屏障 脑屏障是在生理情况下,存在于脑和脊髓内的毛细血管与神经组织之间的一个调节界面。它能选择性地调节进入神经组织的物质,从而使神经细胞和神经胶质细胞保持恒定的内、外环境，保证正常的生理活动。脑屏障包括：血脑屏障血脑脊液屏障脑脊液-脑屏障第一节 血脑屏障及血脑肿瘤屏障一、阻止药物进入脑肿瘤内的屏障Blood Supply of the Brain Arterial supply of the brain i

2、s derived from 2 pairs of vessels: Internal Carotid Arteries and the Vertebral Arteries.Blood Supply of the Brain A治疗基因向脑内递送选修课件1.血-脑屏障血脑屏障（blood brain barrier, BBB）由脑毛细血管内皮细胞、基膜和神经胶质细胞的终足三层隔膜共同构成。 1.血-脑屏障血脑屏障（blood brain barrie脑毛细血管： 总长：约600 km 表面积：约20 m2 每cm3脑组织：1 km长 每个神经元都有自己的供给人脑血管铸型图脑毛细血管： 总长：

3、约600 km人脑血管铸型图血脑屏障血脑屏障The Blood-Brain BarrierBody capillaries:Loose junction between endothelial cells.Permeable to many substances.Brain capillaries:Tight junction between endothelial cells.Covered by astrocyte feetImpermeable to many substances.The Blood-Brain BarrierBody ca治疗基因向脑内递送选修课件内皮细胞的紧密连接(

4、tight junction, TJ)TJ是中枢神经系统毛细血管特有的解剖学特征，也是BBB的重要结构基础。TJ的存在极大的限制了BBB的细胞旁转运途径。内皮细胞的紧密连接(tight junction, TJ)T紧密连接紧密连接Protein components:ClaudinOccludinJunction Adhesion MoleculesAccessory proteins：ZO-1,2,3 cingulin, etcProtein components:The TJ consists of three integral membrane proteins: Claudin, Oc

5、cludin and Junction adhesion molecules.Cytoplasmic accessory proteins: ZO-1 (zonula occludens), ZO-2, ZO-3, cingulin, and others.The primary cytoskeletal protein, actin, has known binding sites on all of the ZO proteins, and on claudin and occludin.The TJ consists of three integClaudins: make up the

6、 backbone of Tight Junction strands by forming dimers and binding homotypically to claudins on adjacent cells to produce the primary seal of the tight junction.Occludin: functions as a dynamic regulatory protein, whose presence in the membrane is correlated with increased electrical resistance acros

7、s the membrane and decreased paracellular permeability.Zonula occludens proteins (ZO1, ZO2 and ZO3): belong to a family of proteins known as MAGUKs (membrane associated guanylate kinase-like proteins), which serve as recognition proteins for Tight Junction placement and as a support structure for si

8、gnal transduction proteins. Claudins: make up the backboneCingulin: is a double-stranded myosin-like protein that serve as a scaffolding protein that links Tight Junction accessory proteins to the cytoskeleton.Actin: A dense band of actin and myosin filaments circumscribe the cell with actin filamen

9、ts that extend into the Tight Junctions. ZO1 binds to actin filaments and the C terminus of occludin, which couples actin to the paracellular barrier. Several studies suggest that tight junctional integrity is dependent on structural organization of actin.Cingulin: is a double-strandedRegulation of

10、Tight Junctions Factors which increase permeability (Extracellular Factors): Membrane permeability increases after exposure to:Cytokines: TNF, IL-1 and IFN-Growth factors: VEGF, FGF2Nitric Oxide, ATP, histamine, bradykinin, 5-HT, Endothelin-1, thrombin, substance P, platelet activating factor and fr

11、ee radicals Aminoacids: glutamate, AspartateHypoxic insults leading to ATP depletion, such as would occur with an ischemic strokeLeukocyte migration into brain has been shown to trigger signal transduction cascades leading to loss of Tight Junction molecules including occludin and zonula occludens a

12、nd BBB breakdown. Regulation of Tight Junctions Intracellular signaling Factors which increase permeabilityZO and occludin molecules are primary regulatory proteins of tight Junctions that modulates BBB permeability. Occludin and ZO1 are phosphorylated on Serine, Threonine and Tyrosine residues. Tyr

13、osine phosphorylation correlates with decreased occludin expression, and increased permeability.Intracellular cGMPActivation of the Rho family of small GTP binding proteins triggers reorganization of perijunctional actin, which leads to increased paracellular diffusion.ATP depletion leads to marked

14、reorganization of the cytoskeleton and increased permeability.Intracellular signaling FactorFactors which decrease permeability (extracellular):Glial Cell Derived Neurotrophic FactorTransforming Growth Factor 1HydrocortisoneAstrocyte proximity Factors which decrease permeabIntracellular factors:Prot

15、ein Kinase C is a major regulator of tight Junction formation and regulation. Raising extracellular calcium increases resistance across the membrane and decreases the permeability mediated through heterotrimeric G protein and protein kinase C (PKC) signaling pathways.cAMPIntracellular factors:Protei

16、n The Ventricular system细胞直接或间接利用代谢产生的能量将物质逆浓度梯度或电位梯度进行跨膜转运的过程，是物质通过血脑屏障的主要方式。-small fat soluble molecules (ethanol)室管膜上皮间仅有缝隙连接和中间连接，一般无紧密连接。1 = paracellular diffusion , 2 = transcellular diffusion , 3 = ion channel 4 = ion-symport channel 5 = ion-antiport channel 6 = facilitated diffusion , 7 = ac

17、tive efflux pump 8 = active-antiport transport , 9 = receptor mediated endocytosisClaudins: make up the backbone of Tight Junction strands by forming dimers and binding homotypically to claudins on adjacent cells to produce the primary seal of the tight junction.常用的治疗基因递送途径及方式 ：具有巨噬细胞功能, 能够积极参与BBB 调

18、控的神经免疫网络。The primary cytoskeletal protein, actin, has known binding sites on all of the ZO proteins, and on claudin and occludin.控制葡萄糖和乳酸盐进入脑内。Regulation of Tight Junctions Factors which increase permeability (Extracellular Factors):常用的治疗基因递送途径及方式 ：基膜（basement membrane）是围绕在毛细血管内皮周围的一层完整连续的网状膜，形成血脑屏障

19、的第二道隔膜。它主要由IV型胶原和层粘蛋白构成，起支持作用，防止由于静水压和渗透压改变引起的血管变形。 The Ventricular system基膜（basem基膜基膜神经胶质细胞：是血脑屏障的第三道隔膜。神经胶质细胞（主要是星形胶质细胞）的突起形成伪足，伸向血管壁周围，包绕基膜，起着机械的屏障作用，限制毛细血管的通透性。星形胶质细胞有诱导和维持血脑屏障完整性的作用。 神经胶质细胞：是血脑屏障的第三道隔膜。周细胞 是BBB 的重要细胞成分, 在脑血管发生、内皮细胞紧密连接形成、BBB 分化和结构稳定性中起着调节作用。持续产生转化生长因子- (TGF-) 来诱导和上调BBB 的功能。具有巨噬

20、细胞功能, 能够积极参与BBB 调控的神经免疫网络。周细胞 是BBB 的重要细胞成分, 在脑血管发体外血脑屏障模型的建立体外血脑屏障模型的建立体外血脑屏障模型通透性的评价体外血脑屏障模型通透性的评价Paracellular transport / sodium fluoresceine (Na-F, 376Da)体外血脑屏障模型通透性的评价Paracellular transport / sodiuBrain pericytes are plated at the lower side of inserts (A), astrocytes at the bottom of the wells

21、(B), and brain endothelial cells at the upper side of the same inserts (C) to reconstitute the BBB in vitroApericytesBastrocytesCendothelial cells 体外血脑屏障模型建立的过程Brain pericytes are plated at 体外血脑屏障模型特性的评价Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, Kataoka Y, Niwa M: Pericytes from br

22、ain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cellular and Molecular Neurobiology 27(6):678-694, 2007体外血脑屏障模型特性的评价Nakagawa S, Deli 体外血脑屏障模型特性的评价体外血脑屏障模型特性的评价SubstancesSubstances that enter into the brain-O2, CO2, N2 -glucose, amino acids, certa

23、in ions-small fat soluble molecules (ethanol)-water soluble molecules (aqueous channel protein)SubstancesSubstances that enteSubstancesSubstances that cannot enter into the brain-large protein-toxins-antibodies-monoamines (e.g: neurotransmitter)SubstancesSubstances that cann脑室周围小器官（ Circumventricula

24、r organs, CVO）或“脑的特殊区” 脑的某些部位：海绵窦、垂体后叶、正中隆起、松果体、视丘、穹隆下器官、脑极后区、脉络丛等区域缺少血-脑屏障。 脑室周围小器官（ Circumventricular org血脑屏障的功能1.阻止外源性毒素及全身性递质进入脑内。2.避免脑组织自身神经递质的流失。3.控制葡萄糖和乳酸盐进入脑内。4.酶屏障阻止一些代谢产物进入脑内，选择 性地将脑内一些有害物质泵出脑外。 血脑屏障的功能1.阻止外源性毒素及全身性递质进入脑内。2血脑脊液屏障血脑脊液屏障（blood cerebrospinal fluid barrier）是存在于血液与脑脊液之间的生理性屏障，主

25、要位于脑室脉络丛处。基本结构由毛细血管内皮细胞、软脑膜和室管膜上皮三层组成。 毛细血管内皮细胞 软脑膜室管膜上皮：单层立方上皮，顶端连接紧密2血脑脊液屏障血脑脊液屏障（blood cerebrospThe ventricles are a complex series of spaces and tunnels through the center of the brain.The ventricles secrete cerebrospinal fluid, which suspends the brain in the skull.CSF is produced by structure

26、called the choroid plexus in lateral, third and fourth ventriclesThe ventricles also provide a route for chemical messengers that are widely distributed through the central nervous system.The Ventricular System & CSFThe ventricles are a complex sThe Ventricular systemCSF flows from lateral ventricle

27、- 3rd ventriclethrough IVF3rd ventricle & 4th ventricle are connected by Cerebral aqueductCSF flows into Subarachnoid space through foramen of LuschkaThe Ventricular systemCSF flow血脑脊液屏障血脑脊液屏障血-脑脊液屏障的作用：1.维持脑脊液成分恒定。2.阻止血液中的有害物质进入脑脊液内。3.特殊转运的酶系统，维持细胞内环境稳定。4.伸长细胞有主动运输物质的作用。 血-脑脊液屏障的作用：1.维持脑脊液成分恒定。室管膜细

28、胞（伸长细胞）室管膜细胞（伸长细胞）持续产生转化生长因子- (TGF-) 来诱导和上调BBB 的功能。The Blood-Brain Barrier（一）被动扩散 (Simple diffusion )室管膜上皮间仅有缝隙连接和中间连接，一般无紧密连接。是围绕在毛细血管内皮周围的一层完整连续的网状膜，形成血脑屏障的第二道隔膜。Activation of the Rho family of small GTP binding proteins triggers reorganization of perijunctional actin, which leads to increased par

29、acellular diffusion.细胞直接或间接利用代谢产生的能量将物质逆浓度梯度或电位梯度进行跨膜转运的过程，是物质通过血脑屏障的主要方式。室管膜上皮间仅有缝隙连接和中间连接，一般无紧密连接。Occludin: functions as a dynamic regulatory protein, whose presence in the membrane is correlated with increased electrical resistance across the membrane and decreased paracellular permeability.脑肿瘤有时

30、会出现复杂的血管变化：Paracellular transport / sodium fluoresceine (Na-F, 376Da)脑室周围小器官(CVO)特点：-large proteinFactors which decrease permeability (extracellular):P-Glycoprotein Does make a Difference:ZO and occludin molecules are primary regulatory proteins of tight Junctions that modulates BBB permeability.也称为

31、受体介导的转运 (receptor mediated transport, RMT)，是指大分子物质或物质的团块借助于细胞膜形成吞饮泡的方式进入细胞的过程。3脑脊液-脑屏障脑脊液-脑屏障（brain cerebrospinal fluid barrier） 位于脑室和蛛网膜下隙的脑脊液与脑、脊髓的神经细胞之间，其结构基础为室管膜上皮、基膜和星型胶质细胞。 能阻止脑脊液中的有害物质进入脑脊髓实质。 室管膜上皮间仅有缝隙连接和中间连接，一般无紧密连接。 持续产生转化生长因子- (TGF-) 来诱导和上调BB治疗基因向脑内递送选修课件（二）血肿瘤屏障血肿瘤屏障（blood tumor barrier

32、, BTB）是存在于脑肿瘤细胞与脑毛细血管及神经组织之间的物质交换屏障。基本结构包括：毛细血管内皮基膜肿瘤细胞膜（二）血肿瘤屏障血肿瘤屏障（blood tumor barrBBBBTBBTBBBBBTBBTB在体血肿瘤屏障模型的建立在体血肿瘤屏障模型的建立脑肿瘤模型的评价脑肿瘤模型的评价血-瘤屏障通透性复杂多样脑肿瘤有时会出现复杂的血管变化：血管密度增加、管径增大，血管内皮细胞数量增加、吞饮小泡增加、小泡运输功能亢进、毛细血管缺乏连续性等。血-瘤屏障通透性复杂多样脑肿瘤有时会出现复杂的血管变化：血-瘤屏障存在异质性和个体差异 不同肿瘤的组织间液压 脑肿瘤内微血管结构、微血流动力学、血管内及间质

33、氧分压、间质pH 不同类型的肿瘤以及同一肿瘤的不同生长 阶段 血-瘤屏障存在异质性和个体差异 不同肿瘤的组织间液压 第二节 物质通过血-脑屏障的方式和途径 脑的毛细血管长，其表面积达20平方米，毛细血管内每分钟扩散药物的距离可达40m。物质透过血-脑屏障主要有四条途径：1.小分子直接经细胞间隙扩散2.脂溶性分子的融膜扩散3.特异受体介导的吞饮转运4.特异载体通道和酶系统第二节 物质通过血-脑屏障的方式和途径 脑的毛Transport mechanisms at the BBB. 1 = paracellular diffusion , 2 = transcellular diffusion ,

34、 3 = ion channel 4 = ion-symport channel 5 = ion-antiport channel 6 = facilitated diffusion , 7 = active efflux pump 8 = active-antiport transport , 9 = receptor mediated endocytosis Transport mechanisms at the BB（一）被动扩散 (Simple diffusion )一些物质以被动扩散方式通过血脑屏障。 通透率：物质分子量的大小分子的极性与血浆蛋白的结合程度（一）被动扩散 (Simpl

35、e diffusion )一些（二）易化扩散 (Facilitated diffusion)通过“镶嵌”在血脑屏障界面上的蛋白载体或离子通道来完成。易化扩散的特点：顺浓度差：不需消耗能量需要载体和酶系统参与竞争性专一性饱和性（二）易化扩散 (Facilitated diffusion（三）主动转运 (Active transport ) 细胞直接或间接利用代谢产生的能量将物质逆浓度梯度或电位梯度进行跨膜转运的过程，是物质通过血脑屏障的主要方式。载体种类：(1)氨基酸载体；(2)胺类载体； (3)单羧酸载体；(4)腺苷载体； (5)Na+-K+-ATP酶转运系统；(6)P-糖蛋白 (7)有机阴离

36、子转运体(OAT) 主动转运的特点：逆浓度差和逆电化学梯度：需要消耗能量竞争性专一性（三）主动转运 (Active transport ) The most important efflux transporters which so far identified at the bloodbrain barrier belong to the class of ATP binding-cassette (ABC) transportersPharmacol Rev 60:196209, 2008The most important efflux tranP-Glycoprotein IHC-M

37、idBrain CapillariesClin Cancer Res 1669 2007;13(6)March15, 2007P-Glycoprotein IHC-MidBrain CaP-Glycoprotein Does make a Difference:Paclitaxel+Valspodar (Valspodar is a potent inhibitor of P-gp)Paclitaxel AloneEffect of valspodar coadministration on the paclitaxel brain levels in nude mice after intravenous injectionEffect of valspodar/paclitaxel coadministration on the intracerebralgrowth of the human U 118 MG glioblastoma in nude mice.P-Glycoprotein Does make a Dif（四）吞饮也称为受体介导的转运 (receptor mediated transport, RMT)，是指大分子物质或物质的团块借助于细胞膜形成吞饮泡的方式进入细胞的过程。蛋白