CN1109749C - 压力介导的分子或微颗粒的细胞内输送 - Google Patents
压力介导的分子或微颗粒的细胞内输送 Download PDFInfo
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Abstract
本发明提供一种增强细胞吸收原子、分子或尺寸小于10μm的颗粒的方法。该方法包括:(a)将细胞和包含有所述原子、分子或颗粒的液体介质相接触;(b)维持细胞和液体介质在一封闭空间内;以及(c)使所封闭的细胞和液体介质承受足够的培养压力,以便增强细胞对原子、分子或颗粒的吸收。
Description
发明背景
本发明涉及促进活细胞细胞外物质的吸收。
细胞和分子生物学的研究已阐明在活细胞的膜内存在一个复杂的物理和生化环境。这些膜提供了一个复杂的半渗透屏障,使之在不同细胞类型中执行专门功能;所有活细胞的膜由脂类双分子层组成,它把生命发生的显微水空间分开。通过被动排除不能渗透细胞边界脂类双分子层的大和/或疏脂的(lipophobic)物质,或者利用专门蛋白通过细胞膜的分子或颗粒的主动控制的双向运动,所有的细胞调节它们细胞内空间的分子组成。一些这样的蛋白可以联合形成孔或通道,以提供进入细胞的普通通路或只允许进入特定元素。
原子、分子和显微颗粒导入细胞已变成生物科学家和医学临床医师的必要装置,并为他们仔细分析生命的细胞过程以及设计药理学策略以提高防止、诊断或治疗疾病的能力提供基础。细胞膜精细的功能本质经常对带电或大的分子的输送提出严峻的挑战,并因此限制这些物质在细胞内使用的潜在应用。此外只有通过细胞膜凹陷的过程例如内吞作用或胞饮作用,许多复杂物质才可以由细胞吸收。在大部分情况下,形成含有以前细胞外物质的内吞小泡,然后与含有严酷酶环境的溶酶体融合,产生内吞物质的消化分解。
发明简述
本发明的方法包括将物质输送至活细胞或细胞的细胞外环境,在细胞周围创造一封闭空间并在封闭空间内建立一提高的培养压力以足够加强细胞的物质吸收。物质大小范围为从小分子至微颗粒,对吸收它的细胞可以施加一系列的影响或行使一系列的功能。例如,物质可以是小分子(如药物)、糖、脂肪酸或脂肪酸衍生物,或蛋白(如抗体、酶,或激素)。细胞能在体外、在组织或器官中存在,或形成活生物体如哺乳类例如人的一部分。
相应地,本发明的目的是在靶细胞或细胞群内诱导比预期的或已实现的更大程度的显微或分子种类的细胞内吸收。因而此方法可用以促进大量物质的细胞内输送(而此物质正常情况下只小量地由细胞吸收)或允许物质由细胞吸收,否则此物质在细胞外环境中被细胞膜排除在外。本发明使靶群体被吸收的细胞比例更大,因而吸收更有效,并允许靶向特定器官或组织。使用本发明的方法进行的细胞内输送在不引起组织损伤(如扩张)或创伤并且不对细胞有潜在毒性上也有好处。此外,在许多情况下,本发明的细胞内输送方法在无需溶酶体隔室化下提供一种向细胞质输送的方法,因而提供一种保护,使输送的物质免受溶酶体酶的破坏。此方法也可以加强物质通过细胞内屏障的运动,进入空间如细胞核。
本发明其它特征和优点从具体描述、附图和权利要求上将体现出来。
附图简述
图1-A描述本发明的输送系统。图1-B表示含有要传递物质的溶液向血管输送之前,缚在血管自由末端的图1-A系统。图1-C表示含有要传递物质的溶液向血管内皮输送过程中图1-B的系统和血管。图1-C′表示含有要输送物质的溶液向血管内皮和外表面输送过程中图1-B的系统和血管。
图2表示向定义为加压封闭空间边界部分的部分血管的输送。
图3-A描述向血管的细胞内输送物质的另一种方法。图3-B表示图3-A的血管,其中血管被机械加压。
图4-A描述适于将含有要输送物质的溶液输进血管的两气囊导管。图4-B表示具有充气状态气囊的图4-A的导管。图4-C表示具有气囊以及将含有要输送物质的溶液输送到血管壁的内部小管的导管系统。
图5A表示向器官中血管的输送。图5-B表示对器官加压的气囊导管的应用。
图6表示含有加压室的输送系统。
图7-A表示根据本发明对转染的人隐静脉转染效率所施压力的函数。图7-B表示防扩张护套对FITC-ODN转染的人隐静脉转染效率的影响。图7-C表示IL-6反义ODN转染进人隐静脉后IL-6蛋白合成的抑制。
图8-A表示IL-6反义ODN转染进第一个实验对象的人隐静脉后IL-6 mRNA合成的抑制。图8-B是与图8-A相似的对第二个实验对象的图形。图8-C是与图8-A相似的对第三个实验对象的图形。
图9-A表示通过荧光显微镜测定的对兔颈动脉体内转染的转染效率。图9-B表示用编码荧光虫虫荧光素酶的DNA体内转染兔颈动脉后对对照、健康的、及动脉粥样硬化的细胞的虫荧光素酶活性。
图10表示对根据本发明的方法体外转染的大鼠血管平滑肌细胞的转染效率。
图11表示对用含有编码荧光虫虫荧光素酶基因的质粒DNA所灌注大鼠肾的虫荧光素酶活性。
图12-A表示大鼠主动脉细胞压力对转染效率的影响。图12-B表示在有和无压力介导的反义PCNA ODN转染的移植大鼠主动脉中局部缺血诱导的PCNA表达。图12-C表示在有以及无压力介导的反义cdc 2激酶ODN转染的移植大鼠主动脉中局部缺血诱导的cdc 2激酶的表达。图12-D表示产生于压力介导的PCNA和cdc 2激酶的反义ODN进行的转染,同系移植、局部缺血损伤的大鼠主动脉腔收缩的下降。
图13-A表示有和无压力下用FITC-ODN exvivo转染的大鼠心脏的转染效率。图13-B表示在有和无压力介导的反义ICAM-1ODN转染的移植大鼠心脏中ICAM-1的表达。图13-C表示由压力介导的用ICAM-1的反义ODN进行的移植大鼠心脏转染所诱导的长期移植接受。
图14-A表示对未处理(对照)移植物以及用对PCNA和cdc 2激酶的反向反义(对照)ODN或反义ODN转染的移植物移植后6周和6个月时壁的厚度。图14-B表示将用E2F decoy ODN转染的静脉与未处理移植物和用量化的(serambled)ODN转染的移植物相比较的,与图14-A相似的结果。
具体描述
本发明所使用的物质类型包括(1)带电原子或离子,(2)除小核酸以外的中性或带电小分子(如分子重量≤1000的分子),(3)除核酸以外的大分子(如分子量≥1000的分子),尤其是蛋白和肽,(4)多聚体和纤维(一般最大尺寸<10μm),(5)无机原子和分子以及(6)显微颗粒(一般最大尺寸<10μm)。根据物质的类型和预计的用途,这些可输送到细胞外环境中的物质可以具有广泛范围的浓度,优选但不限制于1nM至1mM。在本发明的每一个应用中所使用的精确浓度可由使用者根据他(她)的要输送物质的化学、生物化学或功能特性的知识来确定。物质可以在细胞外环境中存在于溶液中,或者它可以包含悬浮液或胶体。
确定含有组织和细胞外环境的密封空间,并在密封空间内建立培养压力。在优选的实施方案中,基本上通过封闭的装置确定封闭空间的边界,以便靶组织(含有靶细胞的组织)受到各向同性的压力,并不扩张或受损伤。在另一个实施方案中,封闭边界的部分由组织确定。预防性装置如无弹性的护套放于组织周围,以防止组织内扩张和损伤。
尤其在血管中,封闭空间优选确定在充气气囊或绳结形成的闭合之间。含有要输送物质的溶液通过闭合之间有输送出口的导管输送至封闭空间。更一般来说,通过在器官导管(如血管)内建立闭合,在器官内确定封闭空间,因而可以对器官内的空间加压。
用于促进细胞吸收的培养压力在预先确定的培养时期保持在预先确定的水平。培养压力的大小依赖于应用,包括各种参数如培养时间、组织类型、和要输送的分子等。所用培养压力范围可以如高于环境压力50mmHg至5个大气压,例如高于环境压力300mmHg至1500mmHg,或高于环境压力0至2个大气压;如果取得有效的输送并且靶细胞或细胞未遭受反向隐藏的损伤,也可使用更高或更低的压力。
用以促进细胞吸收的培养时间根据参数如培养压力、靶组织类型、要输送的分子和所需要的剂量而变化。培养压力的持续时间范围能从如大约1秒至4小时。优选压力持续时间在20秒和30分钟之间,更优选在60秒和10分钟之间。
合适的哺乳靶组织包括血管组织(尤其用作动脉中移植物的静脉)、心脏、骨髓、结缔组织、肝、肾、生殖-泌尿系统组织、骨、肌肉、胃肠器官和内分泌以及外分泌器官。本发明的方法可应用于部分器官、整个器官(如心脏)、或整个生物体。在一个实施方案中,含有要输送物质的溶液灌注进患者的靶区域(如肾),患者在加压室中接受加压。本发明的应用也包括处理同种移植物(来源于不同实验对象而非移植患者的移植物)以及同种同基因移植物(来源移植患者的移植物)。
通过用放射活性、荧光或化学标签,然后根据物质、压力-增强的施用,通过标签确定本发明每一个应用的条件。另外可以进行生物活性的测定。相似地,通过对输送的易标记物质输送的定量,此易标记物质与所需要物质的化学和/或物理性质是相似的,可取得输送条件的最优化。
如上面所讨论的,使用本发明的方法将分子输送到细胞中的系统包括一个封闭装置以确定至少封闭空间的部分边界,以及一个加压装置,该装置在封闭空间中建立培养压力。封闭空间含有靶细胞和细胞的细胞外环境。输送装置如导管或注射器用以将分子直接或间接(如通过静脉内注射)输送至细胞外环境。
通过封闭装置并有可能通过组织确定封闭空间的边界。根据实施方案,合适的封闭装置包括加压室、不可渗透的护套或袋、以及用以闭合组织内通路的闭合装置。加压室尤其适合于处理移植物或整个生物体,而其它装置非常适于组织的手术中的处理。
在一个实施方案中,基本上通过封闭装置确定封闭空间边界。然后压力均匀从所有方向施加到靶组织上,靶组织没有受损伤的危险。在另一个实施方案中,边界的部分由组织(如靶组织)确定。形成封闭空间边界的组织只受到一面的压力,并能被扩张。保护性装置如无弹性护套置于组织周围以防止组织内扩张和损伤。下面更详细地说明本发明的方法和输送系统。
图1-A至1-C′说明将分子加压输送至血管细胞的方法。图1-A是本发明输送系统11的侧视图。系统11包括容纳含有要输送物质的溶液40的贮存器10,以及将溶液40从贮存器10中排出的输送装置。输送装置包括柱塞12和输送管14。推动柱塞12,贮存器10开放,溶液进入管14。从邻近贮存器10的顺序列出的是与管14相连的旋阀16、压力计18、移回护套20和凸起物22。护套20优选是不渗液体和非弹性的。凸起物22邻近管14的远开口端30。旋阀16开始处于关闭位置,防止溶液40从贮存器10中传送至管14中。
图1-B表示连接到系统11上的血管24。开口端30放进血管24的近端。凸起物22与组织24的近端内面贴合。护套20拉下以覆盖组织24。绳子或缚线26A在它们连接到管14的地方缚在护套20和组织24的周围,以防止组织24从开口端30滑掉。当旋阀16转到开放位置时,溶液40进入管14和护套20,通过护套20的开口端28冲掉存在的所有气体和液体。冲洗后,如图1-C和1-C′所示,绳结26B位于护套20的远开口端28以形成水密封。图1-C示出了向靶向血管24内皮的物质输送,而图1-C′示出了向血管24的内皮以及外表面的物质输送。
在图1-C中,绳结26B放置在护套20和组织24的周围。绳结26B闭合血管24。旋阀16转到开放位置时,推进柱塞12,因而溶液40在输送压力下被传送进血管24。可增加输送压力直至达到培养压力,关闭旋阀76。血管24允许培养一定的培养时间,之后解开绳结以释放压力(未说明)。
通过血管24的壁及封闭装置确定封闭空间的边界。封闭空间含有血管24的靶(内皮)细胞,以及它们的细胞外环境。如果旋阀16处于关闭位置,封闭装置包括管14、旋阀16和缚线26B。如果旋阀16处于开放位置,封闭装置包括缚线26B、管14、柱塞12和贮存器10的部分壁。封闭装置确定至少部分封闭空闭的边界。
在图1-C所示的实施方案中,血管24确定了封闭空间的部分边界。只向血管24的内面施加压力会引起血管24扩张并受损伤。护套20作为保护装置,防止血管24扩张。在一个如图1-C的装置中,护套20无弹性是很重要的。
如图1-C′中所说明的,也有可能将绳结26B只放置于护套20的周围。在这种情况下,基本上由封闭装置确定含有血管24靶细胞及它们的细胞外环境的封闭空间。如果旋阀16处于关闭位置,封闭装置包括护套20、管14、旋阀16和缚线26B′。如果旋阀处于开放位置,封闭装置包括护套20、管14、缚线26B′、柱塞12和贮存器10的部分壁。
在图1-C′所示的实施方案中,基本上通过封闭装置确定封闭空间的边界。血管周围的压力是均匀的,因而血管24并不受损伤。护套20作为封闭装置的部分,因此护套不透液体是重要的。在图1-C′的装置中,护套20无需是无弹性的,因为弹性护套的使用不会导致血管24的损伤。
图2说明体内将分子向与患者循环系统相连的血管输送的方法。管14插入仍与活体相连的血管224的腔中。扩套220包裹在血管224周围,固定件228(如热封口)连接护套220的两个边形成管。护套220作为保护装置,防止血管224的扩张。两个绳结226A和226B缚在护套220周围。绳结226A和226B作为闭合装置闭合血管224。闭合件226A和226B以及在闭合件226A和226B之间的血管壁确定了含有血管224靶细胞及它们细胞外环境的封闭空间230。溶液40注射进封闭空间,片段230允许培养一定的培养时间。一段培养时间之后去除闭合件226A和226B,允许血液流经血管224。
图3-A和图3-B说明具有独特和加压元件的输送系统,用于将分子运送至图2所示的血管中。如图3-B所示,一个刚性管形包裹物250放置于护套220周围,钳紧件252放置于包裹物250周围。包裹物250的圆周是可变的,因此它形成的管直径是可变化的,但它在轴向是刚性的,因而即使直径改变,它仍然基本上保持管形。一个加紧螺丝钉254使钳紧件252变紧,将包裹物250拉紧,在血管224内产生压力。此压力在培养期间保持,之后松开螺丝钉254。
图4-A至4-D说明将含有要输送物质的溶液通过导管314输送至血管324腔中的方法。导管314插入血管324中。导管314在末端316处封闭。导管314有两个气囊332A和332B以及气囊332A和332B之间的输送部分。开始,气囊332A和332B是放气的,如图4-A所示。导管插入血管324后,气囊332A和332B充气,如图4-B所示。气囊332A和332B闭合血管324,并在血管324内创造封闭空间334。含有要输送物质的溶液340通过出入口330被输送到封闭空间334。溶液340在压力作用下输送,因而封闭空间334被加压。一定培养时间后,气囊332A和332B放气。靶封闭空间334减压。
图4-C表示本发明另一个输送系统,其中安装在导管上的气囊具有用以将含有要输送物质的溶液输送至血管壁上的小管。气囊432有小管450,它与气囊432内导管314段中的孔452直接连接。当加压溶液440通过导管314被输送时,溶液流出孔452,经过管450抵达血管324。
图5-A说明使用系统11来将溶液输送至器官124如心脏的血液大管道(血管和/或主动脉和心室)。保护性套120包裹在器官124周围。器官124有将血液运送进来的动脉112和将血液运送出去的静脉114。管14插进动脉112的腔中,护套120包裹在动脉112和静脉114周围。绳结126A在动脉112处的护套120周围束紧,绳结126B在静脉114处的护套120周围束紧,以防止液体渗漏出器官124。绳结126A允许管14进入动脉112,然而紧紧包裹以使封闭动脉112,防止渗漏。注射含有要输送物质的溶液40,允许器官124培养。一定培养时间后,去除绳结126A和126B,允许血液再一次流经器官124。
图5-B说明用以封闭器官(如胃肠器官)的入口和出口的气囊导管的使用。具有气囊552的导管550插入与器官524相通的第一个器官导管512中,另一个具有气囊562的导管560插入导引离开器官524的第二个器官导管514。开始时气囊552和562放气(未显示)。一旦导管550、560插入它们各自的血管中,气囊552、562充气,并在导管512、514中各自建立闭合。含有要输送物质的溶液540在输送压力下输送至器官524中。
图6说明以促进物质输送至细胞的加压室的使用。容纳装置如盘子610包含含有靶细胞的组织。含有物质的溶液640放置在盘610中。盘610放在压力室650中。室650关闭并密封,加压气体通过导管660导入室650中。溶液640和组织624在培养压力下保持一个培养时间。组织624可以包含整个器官。
如图6所示的加压室尤其适于整个生物体加压的输送方法。在此方法中,含有要输送物质的溶液优选灌注进患者的血管和/或器官(如肾)内。患者位于加压室中。加压室在培养压力下保持一个培养时间。
在压力下增加分子向细胞膜的渗透有几个可能的机制。膜渗透的增加需要增加细胞和/或细胞外环境内的压力。但并不需要一个跨过细胞膜的压力梯度。有可能蛋白形成的转膜通道在高压下改变构成,因而允许分子通过通道并进入细胞质。
精确的压力、培养时间和所用浓度取决于靶组织类型。例如,如下面所描述的,在低压力(约0.5大气压)下大约5分钟的培养时间足够在人隐静脉中取得接近最大的转染效率,而在大鼠主动脉中为取得80-90%的转染效率需要在高压力(约2大气压)下经过超过1小时的培养时间.对大鼠心脏而言,为达到50%以上的转染效率,有必要在2大气压下培养30至45分钟的时间。一般情况下,在正常大气压的培养压力下,在不同组织类型中取得给定转染效率所需培养时间从几分钟至几个小时。对给定组织类型而言,熟练的技术人员很容易确定合适的培养时间和压力。
在没有外科手术过程的限制时,一般优选的加压封闭空间壁不包括活组织,因为形成部分封闭空间壁的组织受到机械压力。在一些外科手术过程中,如手术过程中与循环系统相连的血管的处理(见图3-A和3-B),需要至少部分封闭空间壁由组织确定。在这种情况下,用于防止组织扩张的保护性措施是重要的。体外处理的移植物一般在加压室中或在等压封闭空间中通过培养来处理。
本发明的方法在下面表现核酸分子的细胞内输送的实验中进一步说明。下面对各种培养压力培养时间和组织类型评估本发明方法的转染效率。在下面讨论中所用的一些缩写是:CTRL,对照;ELISA,酶联免疫吸附实验;FITC,异氰酸荧光素;FITC-ODN,FITC-标记的寡脱氧核苷酸;IL-6,白介素-6;ODN,寡脱氧核苷酸;PCR,聚合酶链式反应;VSMC,血管平滑肌细胞。字母“n”指对每个数据点所评估的受试验者的数量。给定的压力是高于环境(大气)压力的,施加到样品上的净压力。实施例1
根据与图1-C中所说明的相似的方法,用FITC-ODN转染人隐静脉。图7-A示出了对几个ODN浓度的转染效率与所用压力的函数。通过荧光显微镜测定效率为发现具有FITC-ODN核定位的内膜和肌肉层细胞的百分率。压力范围从50至760mmHg,在生理盐溶液中ODN浓度范围从5至100μm。n=6。
使用与图1-C所说明的相似方法评估防止扩张的护套对转染效率的影响。ODN浓度为20μm,压力范围从50至760mmHg。图7-B表示实验结果。n=6。通过测定在整个器官培养物中反义ODN产生的IL-6合成的抑制体外考查本发明的方法的转染效率。转染后静脉片段在生长培养基中培养24小时。根据与图1-C所说明的相似方法,在5mM、10mM和100mM转染进行10分钟。图7-C表示在生长培养基中对反义转染的培养物通过ELISA所检测的IL-6蛋白的下降。下降的水平与在未转染的对照培养物和非特异ODN-转染的静脉中发现的水平相关。n=6。实施例2
定量反向转录PCR用以测量根据本发明进行的反义-ODN转染所产生的IL-6mRNA的下降。如图1-C所说明的转染人隐静脉的3个样品,反义转染的静脉片段中mRNA的水平与未处理及反向反义(对照)ODN-转染片段中的水平相比较。3个样品的结果分别显示于图8-A、8-B和8-C。mRNA水平的降低表示反义ODN处理的序列的特异功效。实施例3
根据与图2中所说明的相似方法,用FITC-ODN体内转染兔颈动脉。转染后4小时、4天和7天测量转染效率。作为时间函数的FITC-阳性核百分率显示于图9-A中。n=2-3。用含有虫荧光素酶基因的质粒DNA构建体体内转染兔颈动脉后测量荧光虫虫荧光素酶基因的表达。如图2中所示,在压力下转染健康动脉(正常的)和动脉粥样硬化血管(CHOL/inj)。对照(CTRL)动脉在不加压下暴露于带有虫荧光素酶基因的质粒中。第5天得到动脉的结果,组织匀浆测量虫荧光素酶活性。测量结果显示于图9-B中。n=2-4。实施例4
如图6中所示,体外用FITC-ODN转染大鼠血管平滑肌细胞(VSMC)。细胞暴躁于大气压(净压力为零)或者2个大气压下45分钟。图10表示1mM和80mM FITC-ODN浓度的转染效率。n=4。实施例5
图11表示压力对如图6所示,用含荧光虫虫荧光素酶基因的质粒DNA体内灌注的大鼠肾细胞的转染效率的影响。肾灌注后大鼠暴露于大气压(0大气压)或者2个大气压中30分钟。转染后3天得到肾的结果,组织匀浆测量虫荧光素酶活性。n=7。实施例6
图12-A表示压力对大鼠主动脉细胞转染效率的影响。从供体大鼠中得到主动脉,以与图1-C′所说明的相似方式在生理溶液中于4℃培养24小时诱导局部缺血损伤。培养溶液含有40μm FITC-ODN。培养在高于大气压0大气压和2个大气压下进行。培养后,组织同系移植进大鼠主动脉,移植后24小时得到结果。通过用荧光DNA插入染料共染切片的荧光显微镜测定FITC的核定位。转染效率表示为表现FITC-ODN核定位的细胞作为总细胞的百分率。n=3,p<0.005。
图12-B表示在有和无压力介导的反义-PCNA ODN转染的移植大鼠主动脉中局部缺血诱导的PCNA的表达。转染过程与图1-C′中所说明的相似。在盐溶液(对照)或在含40μm ODN的盐溶液中4℃培养24小时诱导局部缺血损伤。在培养过程中对对照和ODN-处理样品施加高于大气压2个大气压的压力。同系移植后6天得到组织结果,通过ELISA测定组织均浆中PCAN蛋白的水平。n=7,p=0.02。
图12-C表示在有和无压力介导的反义-cdc 2激酶ODN转染的移植大鼠主动脉中局部缺血诱导的cdc2激酶的表达。转染、移植和得到结果的过程与上面图12中的相关描述相类似。通过ELISA测量cdc2激酶的蛋白水平。
图12-D表示同系移植、局部缺血损伤的鼠主动脉腔收缩的下降产生于压力介导的PCNA和cdc 2激酶反义ODN的转染。通过在盐溶液(对照)或在反义-PCNA/反义-cdc2激酶ODN溶液(每种40μM)中4℃培养24小时诱导局部缺血损伤。向所有的组织(包括对照)施加高于环境压力2个大气压的压力。如计算机图象分析所测定的,两个细胞循环调节基因表达的阻断在局部缺血损伤的同系移植物中降低了新内膜的增生和腔收缩。n=12,p=0.03。实施例7
图13-A表示在有和无压力下用FITC-ODN ex vivo转染的大鼠心脏的转染效率。主动脉交叉夹紧后FITC-ODN溶液(80μM)灌注进供体心脏的冠状动脉。如图6所示,心脏浸于FITC-ODN溶液中,于4℃暴露于高于环境压力0个大气压或2个大气压中45分钟。然后心脏异位移植进受体大鼠的腹部主动脉和腔静脉。移植后24小时通过用荧光DNA插入染料共染切片的荧光显微镜测量FITC的核定位。转染效率表示为表现FITC-ODN核定位的细胞作为总细胞的百分率。n=3,p<0.005。
图13-B表示在有和无压力介导的反义-ICAM-1 ODN转染的移植大鼠心脏中ICAM-1的表达。主动脉交叉夹紧后盐(对照)或反义ICAM-1-ODN溶液(80μM)灌注进供体PVG劳损心脏的冠状动脉。然后如图6所说明的,心脏浸在FITC-ODN溶液中,于4℃暴露于高于环境压力2个大气压中45分钟。异位移植进ACI受体后3天得到组织结果。通过ICAM-1免疫组织化学染色切片的图象分析测量ICAM-1阳性区域。n=3-6,p=0.04。
图13-C表示通过压力介导的移植大鼠心脏ICAM-1反义ODN的转染诱导长期移植物的接受。如与上面图13-B相关所描述的得到PVG劳损大鼠心脏,并用反义-ICAM-1ODN溶液(80μm)或者盐溶液(对照)体外转染。然后心脏异位移植进ACI劳损受体。移植后6天所有的动物系统施用抗-LFA-1抗体以阻断ICAM-1配体。没有施用进一步的免疫抑制。耐受报道为发现长期接受它们同种移植物的处理动物的百分率。通过在移植物中存在心脏跳动>100天确定为移植物接受。对照n=12,反义处理n=27,p=0.003。实施例8
图14-A和图14-B表示压力介导的、用设计阻断细胞循环调节基因调节的ODN的转染后,在移植进颈动脉的兔颈静脉中新生内膜增生的抑制。
图14-A表示未处理(对照)移植物、用反向反义(对照)ODN或PCNA和cdc2激酶的反义ODN转染的移植物移植后6周和6个月壁厚度。新内膜形成抑制达到6个月,而肌肉层肥大允许合适的壁增厚以降低高压动脉环境中壁压力。图14-B表示与未处理的移植物和用量化的ODN转染的对照移植物相比较,对用E2F decoy ODN转染的静脉,与图14-A中相似的结果。n=6,p<0.005。
尽管上面的描述含有许多详述,但这些不应该解释为对本发明范围的限制,而应作为其特定实施方案的说明。例如一般可以使用随时间变化的培养压力。根据应用,本领域熟练技术人员很容易设计许多封闭方法、保护方法和/或闭合方法的潜在计划。对不同的组织类型很容易确定导致所需或接近最大转染效率的各种培养压力、时间、和活性药物剂量。
Claims (17)
1.一种加强细胞吸收原子、分子或最大尺寸<10μm颗粒的方法,所述方法包括:将所述细胞和含有所述原子、分子或颗粒的液体培养基相接触,将所述细胞和所述液体培养基保持在封闭空间内,并且使所封闭的细胞和液体培养基承受培养压力,使之足以加强所述细胞对所述原子、分子或颗粒的吸收。
2.权利要求1的方法,其中所述细胞包含在哺乳类动物血管内,此血管在两个位置密封以提供所述封闭的空间。
3.权利要求1的方法,其中所述的分子是一种核酸分子。
4.权利要求1的方法,其中所述分子是分子量少于1000的治疗性的、带电有机分子。
5.权利要求1的方法,其中所述分子是分子量少于1000的蛋白或肽。
6.权利要求1的方法,其中所述培养压力在高于环境压力50mmHg和5个大气压之间。
7.权利要求6的方法,其中所述培养压力在高于环境压力200mmHg和2.5个大气压之间。
8.权利要求1的方法,其中所述细胞被包含在哺乳类动物血管或器官中,并且培养压力在血管或器官的外部和内部之间达到平衡,以便在所述外部和内部之间基本上无压力梯度。
9.一种将物质输送进细胞的系统,所述系统包括:确定至少部分封闭空间边界的封闭装置,所述的封闭空间含有所述细胞和所述细胞的细胞外环境,所述的细胞外环境含有所述的物质;在所述封闭空间内建立培养压力的加压装置。
10.权利要求9的系统,进一步包括将所述物质输送到所述细胞外环境的输送装置。
11.权利要求9的系统,其中所述封闭装置包括不可渗透的护套。
12.权利要求9的系统,其中所述封闭装置包括在组织中闭合通路的闭合装置。
13.权利要求9的系统,其中所述封闭装置包括加压室。
14.权利要求9的系统,其中所述边界基本由封闭装置来限定。
15.权利要求9的系统,其中部分所述边界由组织确定。
16.权利要求15的系统,进一步包括适合放置于所述组织周围的保护装置,以防止所述组织的损伤。
17.权利要求16的系统,其中所述的保护装置包括非弹性护套。
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EP0944715A4 (en) | 1999-12-22 |
ATE223967T1 (de) | 2002-09-15 |
DK0944715T3 (da) | 2002-12-30 |
DE69715449T2 (de) | 2003-07-31 |
CA2271244A1 (en) | 1998-05-14 |
CN1244213A (zh) | 2000-02-09 |
KR100449330B1 (ko) | 2004-09-18 |
EP0944715B1 (en) | 2002-09-11 |
EP0944715A1 (en) | 1999-09-29 |
IL129808A0 (en) | 2000-02-29 |
JP2001505419A (ja) | 2001-04-24 |
AU7002098A (en) | 1998-05-29 |
KR20000053164A (ko) | 2000-08-25 |
HK1022711A1 (en) | 2000-08-18 |
CA2271244C (en) | 2008-09-02 |
EP1279412A2 (en) | 2003-01-29 |
WO1998020109A1 (en) | 1998-05-14 |
AU736298B2 (en) | 2001-07-26 |
JP2008054681A (ja) | 2008-03-13 |
JP2008237221A (ja) | 2008-10-09 |
DE69715449D1 (de) | 2002-10-17 |
ES2183221T3 (es) | 2003-03-16 |
PT944715E (pt) | 2003-01-31 |
IL129808A (en) | 2004-06-20 |
US5922687A (en) | 1999-07-13 |
BR9713334A (pt) | 2000-05-09 |
EP1279412A3 (en) | 2003-12-17 |
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