治療基因向腦內(nèi)遞送選修課件

1、治療基因向腦內(nèi)遞送選修治療基因向腦內(nèi)遞送選修掌握要點1.血腦屏障的功能2.物質(zhì)透過血腦屏障的途徑掌握要點1.血腦屏障的功能第一節(jié) 血腦屏障及血腦腫瘤屏障一、阻止藥物進入腦腫瘤內(nèi)的屏障 （一）腦屏障 腦屏障是在生理情況下,存在于腦和脊髓內(nèi)的毛細血管與神經(jīng)組織之間的一個調(diào)節(jié)界面。它能選擇性地調(diào)節(jié)進入神經(jīng)組織的物質(zhì),從而使神經(jīng)細胞和神經(jīng)膠質(zhì)細胞保持恒定的內(nèi)、外環(huán)境，保證正常的生理活動。腦屏障包括：血腦屏障血腦脊液屏障腦脊液-腦屏障第一節(jié) 血腦屏障及血腦腫瘤屏障一、阻止藥物進入腦腫瘤內(nèi)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治療基因向腦內(nèi)遞送選修課件1.血-腦屏障血腦屏障（blood brain barrier, BBB）由腦毛細血管內(nèi)皮細胞、基膜和神經(jīng)膠質(zhì)細胞的終足三層隔膜共同構(gòu)成。 1.血-腦屏障血腦屏障（blood brain barrie腦毛細血管： 總長：約600 km 表面積：約20 m2 每cm3腦組織：1 km長 每個神經(jīng)元都有自己的供給人腦血管鑄型圖腦毛細血管： 總長：

3、約600 km人腦血管鑄型圖血腦屏障血腦屏障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治療基因向腦內(nèi)遞送選修課件內(nèi)皮細胞的緊密連接(

4、tight junction, TJ)TJ是中樞神經(jīng)系統(tǒng)毛細血管特有的解剖學(xué)特征，也是BBB的重要結(jié)構(gòu)基礎(chǔ)。TJ的存在極大的限制了BBB的細胞旁轉(zhuǎn)運途徑。內(nèi)皮細胞的緊密連接(tight junction, TJ)T緊密連接緊密連接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細胞直接或間接利用代謝產(chǎn)生的能量將物質(zhì)逆濃度梯度或電位梯度進行跨膜轉(zhuǎn)運的過程，是物質(zhì)通過血腦屏障的主要方式。-small fat soluble molecules (ethanol)室管膜上皮間僅有縫隙連接和中間連接，一般無緊密連接。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.常用的治療基因遞送途徑及方式 ：具有巨噬細胞功能, 能夠積極參與BBB 調(diào)

18、控的神經(jīng)免疫網(wǎng)絡(luò)。The primary cytoskeletal protein, actin, has known binding sites on all of the ZO proteins, and on claudin and occludin.控制葡萄糖和乳酸鹽進入腦內(nèi)。Regulation of Tight Junctions Factors which increase permeability (Extracellular Factors):常用的治療基因遞送途徑及方式 ：基膜（basement membrane）是圍繞在毛細血管內(nèi)皮周圍的一層完整連續(xù)的網(wǎng)狀膜，形成血腦屏障

19、的第二道隔膜。它主要由IV型膠原和層粘蛋白構(gòu)成，起支持作用，防止由于靜水壓和滲透壓改變引起的血管變形。 The Ventricular system基膜（basem基膜基膜神經(jīng)膠質(zhì)細胞：是血腦屏障的第三道隔膜。神經(jīng)膠質(zhì)細胞（主要是星形膠質(zhì)細胞）的突起形成偽足，伸向血管壁周圍，包繞基膜，起著機械的屏障作用，限制毛細血管的通透性。星形膠質(zhì)細胞有誘導(dǎo)和維持血腦屏障完整性的作用。 神經(jīng)膠質(zhì)細胞：是血腦屏障的第三道隔膜。周細胞 是BBB 的重要細胞成分, 在腦血管發(fā)生、內(nèi)皮細胞緊密連接形成、BBB 分化和結(jié)構(gòu)穩(wěn)定性中起著調(diào)節(jié)作用。持續(xù)產(chǎn)生轉(zhuǎn)化生長因子- (TGF-) 來誘導(dǎo)和上調(diào)BBB 的功能。具有巨噬

20、細胞功能, 能夠積極參與BBB 調(diào)控的神經(jīng)免疫網(wǎng)絡(luò)。周細胞 是BBB 的重要細胞成分, 在腦血管發(fā)體外血腦屏障模型的建立體外血腦屏障模型的建立體外血腦屏障模型通透性的評價體外血腦屏障模型通透性的評價Paracellular transport / sodium fluoresceine (Na-F, 376Da)體外血腦屏障模型通透性的評價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 體外血腦屏障模型建立的過程Brain pericytes are plated at 體外血腦屏障模型特性的評價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體外血腦屏障模型特性的評價Nakagawa S, Deli 體外血腦屏障模型特性的評價體外血腦屏障模型特性的評價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腦室周圍小器官（ Circumventricula

24、r organs, CVO）或“腦的特殊區(qū)” 腦的某些部位：海綿竇、垂體后葉、正中隆起、松果體、視丘、穹隆下器官、腦極后區(qū)、脈絡(luò)叢等區(qū)域缺少血-腦屏障。 腦室周圍小器官（ Circumventricular org血腦屏障的功能1.阻止外源性毒素及全身性遞質(zhì)進入腦內(nèi)。2.避免腦組織自身神經(jīng)遞質(zhì)的流失。3.控制葡萄糖和乳酸鹽進入腦內(nèi)。4.酶屏障阻止一些代謝產(chǎn)物進入腦內(nèi)，選擇 性地將腦內(nèi)一些有害物質(zhì)泵出腦外。 血腦屏障的功能1.阻止外源性毒素及全身性遞質(zhì)進入腦內(nèi)。2血腦脊液屏障血腦脊液屏障（blood cerebrospinal fluid barrier）是存在于血液與腦脊液之間的生理性屏障，主

25、要位于腦室脈絡(luò)叢處?；窘Y(jié)構(gòu)由毛細血管內(nèi)皮細胞、軟腦膜和室管膜上皮三層組成。 毛細血管內(nèi)皮細胞 軟腦膜室管膜上皮：單層立方上皮，頂端連接緊密2血腦脊液屏障血腦脊液屏障（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血腦脊液屏障血腦脊液屏障血-腦脊液屏障的作用：1.維持腦脊液成分恒定。2.阻止血液中的有害物質(zhì)進入腦脊液內(nèi)。3.特殊轉(zhuǎn)運的酶系統(tǒng)，維持細胞內(nèi)環(huán)境穩(wěn)定。4.伸長細胞有主動運輸物質(zhì)的作用。 血-腦脊液屏障的作用：1.維持腦脊液成分恒定。室管膜細

28、胞（伸長細胞）室管膜細胞（伸長細胞）持續(xù)產(chǎn)生轉(zhuǎn)化生長因子- (TGF-) 來誘導(dǎo)和上調(diào)BBB 的功能。The Blood-Brain Barrier（一）被動擴散 (Simple diffusion )室管膜上皮間僅有縫隙連接和中間連接，一般無緊密連接。是圍繞在毛細血管內(nèi)皮周圍的一層完整連續(xù)的網(wǎng)狀膜，形成血腦屏障的第二道隔膜。Activation of the Rho family of small GTP binding proteins triggers reorganization of perijunctional actin, which leads to increased par

29、acellular diffusion.細胞直接或間接利用代謝產(chǎn)生的能量將物質(zhì)逆濃度梯度或電位梯度進行跨膜轉(zhuǎn)運的過程，是物質(zhì)通過血腦屏障的主要方式。室管膜上皮間僅有縫隙連接和中間連接，一般無緊密連接。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.腦腫瘤有時

30、會出現(xiàn)復(fù)雜的血管變化：Paracellular transport / sodium fluoresceine (Na-F, 376Da)腦室周圍小器官(CVO)特點：-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.也稱為

31、受體介導(dǎo)的轉(zhuǎn)運 (receptor mediated transport, RMT)，是指大分子物質(zhì)或物質(zhì)的團塊借助于細胞膜形成吞飲泡的方式進入細胞的過程。3腦脊液-腦屏障腦脊液-腦屏障（brain cerebrospinal fluid barrier） 位于腦室和蛛網(wǎng)膜下隙的腦脊液與腦、脊髓的神經(jīng)細胞之間，其結(jié)構(gòu)基礎(chǔ)為室管膜上皮、基膜和星型膠質(zhì)細胞。 能阻止腦脊液中的有害物質(zhì)進入腦脊髓實質(zhì)。 室管膜上皮間僅有縫隙連接和中間連接，一般無緊密連接。 持續(xù)產(chǎn)生轉(zhuǎn)化生長因子- (TGF-) 來誘導(dǎo)和上調(diào)BB治療基因向腦內(nèi)遞送選修課件（二）血腫瘤屏障血腫瘤屏障（blood tumor barrier

32、, BTB）是存在于腦腫瘤細胞與腦毛細血管及神經(jīng)組織之間的物質(zhì)交換屏障?；窘Y(jié)構(gòu)包括：毛細血管內(nèi)皮基膜腫瘤細胞膜（二）血腫瘤屏障血腫瘤屏障（blood tumor barrBBBBTBBTBBBBBTBBTB在體血腫瘤屏障模型的建立在體血腫瘤屏障模型的建立腦腫瘤模型的評價腦腫瘤模型的評價血-瘤屏障通透性復(fù)雜多樣腦腫瘤有時會出現(xiàn)復(fù)雜的血管變化：血管密度增加、管徑增大，血管內(nèi)皮細胞數(shù)量增加、吞飲小泡增加、小泡運輸功能亢進、毛細血管缺乏連續(xù)性等。血-瘤屏障通透性復(fù)雜多樣腦腫瘤有時會出現(xiàn)復(fù)雜的血管變化：血-瘤屏障存在異質(zhì)性和個體差異 不同腫瘤的組織間液壓 腦腫瘤內(nèi)微血管結(jié)構(gòu)、微血流動力學(xué)、血管內(nèi)及間質(zhì)

33、氧分壓、間質(zhì)pH 不同類型的腫瘤以及同一腫瘤的不同生長 階段 血-瘤屏障存在異質(zhì)性和個體差異 不同腫瘤的組織間液壓 第二節(jié) 物質(zhì)通過血-腦屏障的方式和途徑 腦的毛細血管長，其表面積達20平方米，毛細血管內(nèi)每分鐘擴散藥物的距離可達40m。物質(zhì)透過血-腦屏障主要有四條途徑：1.小分子直接經(jīng)細胞間隙擴散2.脂溶性分子的融膜擴散3.特異受體介導(dǎo)的吞飲轉(zhuǎn)運4.特異載體通道和酶系統(tǒng)第二節(jié) 物質(zhì)通過血-腦屏障的方式和途徑 腦的毛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）被動擴散 (Simple diffusion )一些物質(zhì)以被動擴散方式通過血腦屏障。 通透率：物質(zhì)分子量的大小分子的極性與血漿蛋白的結(jié)合程度（一）被動擴散 (Simpl

35、e diffusion )一些（二）易化擴散 (Facilitated diffusion)通過“鑲嵌”在血腦屏障界面上的蛋白載體或離子通道來完成。易化擴散的特點：順濃度差：不需消耗能量需要載體和酶系統(tǒng)參與競爭性專一性飽和性（二）易化擴散 (Facilitated diffusion（三）主動轉(zhuǎn)運 (Active transport ) 細胞直接或間接利用代謝產(chǎn)生的能量將物質(zhì)逆濃度梯度或電位梯度進行跨膜轉(zhuǎn)運的過程，是物質(zhì)通過血腦屏障的主要方式。載體種類：(1)氨基酸載體；(2)胺類載體； (3)單羧酸載體；(4)腺苷載體； (5)Na+-K+-ATP酶轉(zhuǎn)運系統(tǒng)；(6)P-糖蛋白 (7)有機陰離

36、子轉(zhuǎn)運體(OAT) 主動轉(zhuǎn)運的特點：逆濃度差和逆電化學(xué)梯度：需要消耗能量競爭性專一性（三）主動轉(zhuǎn)運 (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）吞飲也稱為受體介導(dǎo)的轉(zhuǎn)運 (receptor mediated transport, RMT)，是指大分子物質(zhì)或物質(zhì)的團塊借助于細胞膜形成吞飲泡的方式進入細胞的過程。蛋白