CN102256608B - 含有血单核细胞培养物的上清液的药物制剂 - Google Patents
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Abstract
本发明涉及一种用于治疗炎症性皮肤疾病,优选与局部缺血相关的疾病的局部药物制剂,包括通过在无增殖PBMC和活化PBMC的物质的生理溶液中培养外周血单核细胞(PBMC)或其亚群至少1h可获得的生理溶液的上清液。
Description
技术领域
本发明涉及一种用于治疗体内炎症性皮肤疾病,优选与局部缺血相关的体内皮肤疾病的药物制剂。
背景技术
当肺被损害或者血流降低时可能会出现氧不足,一种低氧状况。局部缺血,血流减少,可能是由血块(血栓)或任何外来循环事件(栓塞物)或血管紊乱如动脉粥样硬化阻塞动脉或静脉而引起的。血流减少可能会突然发病而且持续时间短(急性局部缺血),或者可能会缓慢发病并持续时间长或频繁复发(慢性局部缺血)。急性局部缺血常常伴随局部的,不可逆的组织坏死(梗塞),而慢性局部缺血通常伴随瞬时的含氧量低的组织创伤。但是,如果灌注减少延长或者严重,慢性局部缺血可发生梗塞。梗塞通常出现在脾,肾,肺,脑和心脏中,产生紊乱如肠梗塞形成,肺梗塞形成,缺血性中风和心肌梗死。
局部缺血性紊乱中的病变取决于局部缺血的持续时间和严重程度,以及患者存活的时间。在最初的24h可以在梗塞内看到坏死,而且在与梗塞邻近的活组织中发生急性炎症性应答,白细胞迁移到坏死组织的区域内。在接下来的几天,通过吞噬作用使梗塞内的细胞逐渐裂解和去除,并被胶原样的或神经胶质疤痕取代。
一个器官内的灌注不足或梗塞通常影响其他的器官。例如,如由肺栓塞引起的肺局部缺血,不仅影响肺,还使心脏和其他器官如脑处于含氧量低的应激中。心肌梗塞,其常常涉及归因于心脏的血栓形成,动脉壁血管痉挛或病毒感染心脏的冠状动脉阻塞,可导致充血性心力衰竭和全身性血压过低。如果心搏停止延长,及持续的灌注不足,会发生二级并发症如总体的局部缺血性脑病。脑局部缺血,大多数通常由归因于动脉粥样硬化的血管闭塞所引起,严重程度从暂时性缺血性发作(TIA)到脑梗塞或中风。虽然TIA的症状是暂时的和可逆的,但是TIA易于复发而且常常接着是中风。
闭塞的动脉疾病包括可导致心肌梗塞的冠状动脉病,和外周的动脉疾病,其可影响腹主动脉,它的重要分支,以及腿动脉。外周的动脉疾病包括血栓闭塞性血管炎,雷诺氏病和手足发绀。尽管外周的动脉疾病通常由动脉粥样硬化引起,其他的重要起因包括,例如,糖尿病等等。与外周的动脉疾病相关的并发症包括严重的腿痛性痉挛,绞痛,心率失常,心力衰竭,心脏病,中风和肾衰竭。
局部缺血和含氧量低的紊乱是发病和致死的主要原因。心血管疾病引起全世 界30%的死亡。各种心血管疾病当中,局部缺血性心脏病和脑血管疾病引起大约17%的死亡。
目前,局部缺血和含氧量低的紊乱的治疗集中在减轻症状和治疗引起的紊乱。例如,心肌梗塞的治疗包括硝化甘油和镇痛药,以控制疼痛和减轻心脏的工作负荷。其他的药物,包括地高辛,利尿剂,氨利酮,β-阻断剂,降低脂质的试剂和转变血管紧张素的酶抑制剂,用于稳定疾病,但是这些治疗都没有直接解决由局部缺血和氧不足产生的组织损伤。
由于目前治疗中的缺陷,仍然需要能有效治疗涉及氧不足的疾病的方法。还需要能有效预防由局部缺血引起的组织损伤的方法,局部缺血的发生是由于例如,动脉粥样硬化,糖尿病和肺疾病。
与局部缺血和氧不足相关的疾病通常伴有炎症。因此也需要减轻炎症的手段和方法。
发明内容
本发明的一个目标是提供能有效治疗炎症性疾病,优选与局部缺血相关的炎性疾病的方法。
本发明涉及一种用于治疗炎症性皮肽疾病,优选与局部缺血相关的皮肤疾病的局部药物制剂,其包括通过在无增殖PBMC和活化PBMC的物质的生理溶液中培养外周血单核细胞(PBMC)或其亚群至少1h获得的生理溶液的上清液。
结果表明把如上限定的药物制剂施用给遭受炎症性皮肤疾病,优选与局部缺血相关的皮肤疾病的患者,导致各自症状的减轻和痊愈过程。
本发明的药物制剂包括培养的PBMC或其亚群的上清液。培养PBMC的过程中,这些细胞表达并分泌如细胞因子类物质,其不同于活化的PBMC中表达和分泌的那些物质。这意味着本发明的PBMC的分泌蛋白质组与活化的PBMC的分泌蛋白质组不同。本发明的细胞进行非细胞表面部分触发的分泌蛋白质组产生。因此令人惊讶的是,PBMC的上清液,其没有与活化PBMC的物质如PHA或LPS接触,可以用于治疗炎症性皮肤疾病,其表明这些细胞的分泌蛋白质组包括支持治疗所述疾病的物质。该上清液尤其适于治疗局部缺血的皮肤疾病。
本发明的PBMC上清液通过在不包含增殖PBMC和活化PBMC的物质的生理溶液中培养PMBC来获得的。但是,PBMC在生理溶液中孵育至少1h。这种最低时间的培养是PBMC分泌细胞因子及其他有益的物质所需的。
本发明制剂的PBMC部分,可使用本领域已知的方法如ficoll密度梯度,低渗裂解等等获自全血。这些方法是本领域熟知的。
药物制剂的PBMC可获自供体库或被施用该制剂的相同个体。
获得上清液的生理溶液包括每毫升溶液或每剂量单位至少500,优选至少1000,更优选至少105,更加优选至少106个细胞。
这里使用的“生理溶液”,指在PBMC用于本发明的药物制剂之前培养于其中的一种液体溶液。
“生理溶液”还指一种不会导致PBMC在1小时,优选30分钟内死亡的溶液。如果活的PBMC数在一种溶液中1小时,优选30分钟内减少75%,更优选90%,该溶液不被认为是这里限定的“生理溶液”。当PMBC与所述溶液接触时,“生理溶液”不会导致PBMC的自发裂解。
在本文中,“培养(cultivating)”或“培养(culturing)”步骤包括“孵育”的步骤或由“孵育”的步骤组成,该步骤中,在通常用于培养PBMC的条件下,该细胞与一种溶液接触规定的时间(至少1h,优选至少4h,更优选至少8h,更加优选至少12h)。
在本发明的上下文中,术语“与局部缺血相关的皮肤疾病”可与术语“局部缺血的皮肤疾病”互换使用,而且表示其中人或动物体的区域丧失足够的供氧,而导致组织损伤或功能缺失的任何状况,疾病或紊乱。病理状况可以是特征在于皮肤或其部分内供血减少或消失,其可能是由血管收缩或阻塞引起的。这里术语“局部缺血”或“局部缺血相关的皮肤疾病”或“涉及局部缺血的皮肤疾病”都一起指这种状况。心脏疾病中,例如,局部缺血常常用于描述由于狭窄的或阻断的冠状动脉,心脏肌肉没有获得适当量的富氧血液。局部缺血的症状取决于“局部缺血”的器官。对于心脏,局部缺血常常导致心绞痛。脑中,局部缺血可导致中风。局部缺血疾病伴随有炎症。
涉及炎症,特别是局部缺血的病理性皮肤疾病的非限制性例子包括,伤口,慢性伤口,糖尿病伤口,皮肤溃疡,牛皮癣等等。
尽管以上所述,本发明的上下文中病理性疾病的特征在于内皮细胞损伤或功能紊乱,即伤口。可以通过使用本发明的制剂进行治疗的伤口的非限制性例子是慢性伤口,糖尿病伤口,溃疡,灼伤,炎症性皮肤病和肠疾病。
这里使用的“生理溶液”,优选是这样的溶液,其显示不会导致破坏PBMC或其亚群的渗透压,而且可以直接施用给个体。
术语“无增殖PBMC和活化PBMC的物质的生理溶液”指不含有活化PBMC并诱导PBMC或其亚群增殖的物质的生理溶液。这些物质包括PHA,LPS等等。
根据本发明的一个优选的实施方案,炎症性皮肤病选自炎症,氧不足诱导的炎症和自身免疫性疾病,优选牛皮癣,粉刺,红斑痤疮,坏疽性脓皮病,皮炎,特应性皮肤病,接触性皮炎,皮下脂溢性皮炎,结节性红斑,由细菌,病毒,真菌,寄生虫感染引起的感染皮肤病,刺伤,咬伤和荨麻疹,以及与局部缺血相关的皮肤疾病。尤其优选的皮肤疾病是与局部缺血相关的皮肤疾病。
根据本发明的一个特别优选的实施方案,与局部缺血相关的皮肤疾病选自伤口,组织缺血,慢性伤口,糖尿病伤口,皮肤溃疡,皮肤灼伤,整形手术和牙齿移植以后的组织再生中的皮瓣。
外周血单核细胞(PBMC)的亚群优选T细胞,B细胞或NK细胞。当然使用这些细胞的组合也是可能的:T细胞和B细胞;T细胞和NK细胞;B细胞和NK细胞;T细胞,B细胞核NK细胞。提供和分离所述细胞的方法是已知的。
令人惊讶地结果是本发明的PBMC可以培养于任何种类的溶液中,只要所述溶液不含有不是药学上可接受的,导致PBMC立即死亡(如上面限定的),活化PBMC和刺激PBMC增殖的物质。因此,待使用的溶液至少显示不导致PBMC裂解的渗透特性。生理溶液优选是生理盐溶液,优选生理学NaCl溶液,全血,血液级分,优选血清或细胞培养基。
细胞培养基优选选自RPMI,DMEM,X-vivo和Ultraculture。
根据本发明的一个特别优选的实施方案,在应激诱导条件下培养本发明的细胞。
这里使用的术语“应激诱导条件下”,指导致被应激的细胞的培养条件。导致对细胞应激的条件其中包括加热,化学制剂,放射,氧不足,渗透压等等。
对本发明的细胞另外的应激导致进一步增加了有益于治疗炎症性皮肤疾病,尤其是与局部缺血相关的皮肤疾病的物质的表达和分泌。
根据本发明的一个优选的实施方案,应激诱导条件包括氧不足,臭氧,加热(例如比PBMC的最佳培养温度也即37℃超过2℃,优选超过5℃,更优选超过10℃),放射(例如紫外辐射,γ辐射),化学制剂,渗透压(也即渗透条件比常规在体液,尤其是血液中存在的渗透条件提高至少10%)或其组合。
如果放射用于应激本发明的PBMC,该细胞优选用至少10Gy,优选至少20Gy,更优选至少40Gy进行辐射,其中Cs-137铯优选用作辐照源。
根据本发明的一个优选的实施方案,在培养基中培养未活化的PBMC或其亚群至少4h,优选至少6h,更优选至少12h。
本发明的药物制剂进行局部施用。因此所述制剂优选以凝胶,优选水凝胶,以软膏,以皮肤贴片,以药学上可接受的基质,优选位于胶原蛋白/弹性蛋白基质上(参见,例如,HaslikW等J Plast Reconst Aesth Surg(2008):“Management of full-thickness skin defects in the hand and wrist region:first long-term experiences with the dermal matrix Matriderm”),以糊状物,以乳剂,以粉末,以涂抹剂或以洗液提供。
本发明的药物制剂可包含药学上可接受的赋形剂如稀释剂,稳定剂,载体等等。根据剂型,本发明的制剂包含各自的组分。制备制剂的方法是本领域技术人员熟知的。
为了增加本发明制剂的贮存期限,溶液a)或上清液b)被冻干。冻干这种制品的方法是本领域技术人员熟知的。
在使用之前,冻干制剂可以与水或水溶液接触,水溶液包括缓冲液,稳定剂,盐等等。
本发明的另一个方面涉及如上限定的制剂用于制备治疗炎症性皮肤疾病,尤其是与局部缺血相关的皮肤疾病的药物的应用。
仍然是本发明的另一个方面,涉及一种用于制备如本文公开的局部药物制剂的方法,包括以下步骤:
a)提供外周血单核细胞(PBMC)或其亚群,
b)在无增殖PBMC和活化PBMC的物质的生理溶液中培养步骤a)的细胞至少1h,
c)分离步骤b)的上清液,和
d)使用步骤c)的上清液制备药物制剂。
可通过在生理溶液中孵育或培养PBMC至少1h,优选至少4h,更优选至少8h,更加优选至少12h获得本发明的制剂。在该步骤期间,PMBC开始合成和分泌可用于治疗炎症性疾病的物质。培养步骤之前,之后和期间,不通过添加活化PBMC的物质如PHA或LPS活化PBMC细胞。培养步骤之后,分离细胞和/或培养物上清液以进一步用于制备最终的药物制剂。如上所述,药物制剂可包含培养的PBMC,其中所述细胞已经被孵育的培养物的上清液,或者培养的PBMC以及培养基。
根据本发明的一个优选的实施方案,在步骤b)之前或期间,细胞经受应激诱导条件,其中所述应激诱导条件包括氧不足,臭氧,热量,放射,化学制剂,渗透压(例如通过添加盐,尤其是NaCl进行诱导,以便提供高于血液中的渗透压),pH改变(也即通过添加酸或氢氧化物进行pH改变,以提供6.5到7.2或7.5到8.0的pH值)或其组合。
根据本发明的一个优选的实施方案,步骤b)之前或期间,细胞被至少10Gy,优选至少20Gy,更优选至少40Gy辐射,用臭氧,用高温或用紫外辐射。
本发明的另一个方面涉及一种通过如上所述的方法可获得的制剂。
本发明的另一个方面涉及一种通过给需要其的个体施用合适量的本发明药物制剂治疗炎症性皮肤疾病,尤其是与局部缺血相关的皮肤疾病的方法。
本发明更进一步地通过下面的附图和实施例进行描述,但是它们不是对本发明的限制。
附图说明
图1显示了外周血单核细胞(PBMC)来源的培养物上清液(SN)对人初级角化细胞(KC)和皮肤成纤维细胞(FB)中细胞迁移的作用。
KC和FB分别生长于KC生长培养基和DMEM(补充有10%FBS)中。达到汇合以后,细胞单层用移液管尖刮涂,并用PBMC来源的SN进一步培养16h。我们只检测到来自活PBMC(LL)的SN对KC很少的作用,而来自凋亡PBMC(APO)的SN强烈诱导KC迁移。相反,SN(LL和APO)都强烈诱导皮肤FB中的细胞 迁移。
图2显示了PBMC来源的SN对KC和FB的细胞周期进展的作用。
增殖的KC和FB培养于PMBC来源的SN中。24h以后,细胞用BrdU孵育2h,并进一步如用户手册中所述的进行处理(BrdU-FACS流式细胞周期试剂盒,BD Biosciences)。如图2所示,用PMBC来源的SN刺激FB,导致增殖细胞群体的降低,同时伴有G2/M期细胞的增加。相反,发现用LL和APO-SN都显著增加了增殖的KC。但是,来源于凋亡细胞的SN的作用在KC中更明显。
图3显示了PBMC来源的SN强烈增强体内的伤口愈合。
包含LL(图3a,b)或APO(图3b)的乳剂,PBMC-SN在创伤后立即用于B6/129小鼠背部的6mm打孔活检伤口。创伤后8天,处死小鼠,并通过H&E-染色分析伤口。如图3a和3b所示,两种SN强烈增强皮肤的真皮和表皮部分的伤口愈合。
图4显示了PBMC来源的SN强烈增强体内的伤口痊愈。
测量创伤后第一个5天到形成痂期间的伤口尺寸。如图4中所示,在全部5天期间,发现用含有PBMC来源的上清液的乳剂处理以后,伤口闭合比单独用乳剂处理要快得多。用乳剂单独处理的伤口尺寸在第一个2天稍微增加,而用PBMC来源的上清液处理的伤口在创伤和应用乳剂以后的第一个24h开始闭合。
图5显示用PBMC来源的SN处理以后,小鼠体内伤口中增加的血管形成。
通过VIII因子(图5a)和CD31(图5b)的免疫组织化学,两者都是用于血管的标记,发现相对于对照,SN处理的伤口中CD31阳性细胞大量增加,其指示增加的血管形成,这可能有助于增强的伤口愈合。相反,如通过Ki67染色所分析的,在该时间点没有检测到增加数量的增殖细胞(图5a)。
图6a显示未刺激的活PBMC或IA-PBMC都不分泌主要单核细胞来源的前炎症性细胞因子TNF-α。(显著性如下表示:*p=0.05,**p=0.001;n=8)。
图6b证明相比较于未刺激的PBMC,活化以后强烈诱导前炎症性干扰素-γ分泌。(显著性如下表示:*p=0.05,**p=0.001;n=8)。
图7a显示流式细胞术分析的合并结果。对PBMC根据T细胞进行分门,并评估了活化标记CD69和CD25的表达。(显著性如下表示:*p=0.05,**p=0.001;n=4)。
图7b显示了活化的(PHA,CD3mAb)PBMC的代表性FACS分析。分门(Gating)表示阳性细胞的%。
图8显示了相比较于培养于RPMI中的没有刺激的活PBMC,通过3[H]-胸腺嘧啶核苷掺入刺激的PBMC所测定的高增殖速率。
图9显示了T细胞增殖分析中,PBMC分泌蛋白质组的T细胞应答抑制。
图10显示了用PBMC进行的抗CD3和PHA刺激实验。
图11显示了用抗CD3,PHA和混合的淋巴细胞刺激的PBMC的增殖。
图12显示了用PBMC上清液孵育的CD4+细胞上清液的膜联蛋白V和PI阳性水平。
图13显示了通过PBMC上清液对CD4+细胞中CD25和CD69上调的抑制。
图14显示了停止使用IL-10和TGF-β没有增加CD4+细胞的增殖速率。
具体实施方式
实施例:
实施例1:外周血单核细胞的培养物上清液强烈增强伤口愈合。
未愈合的皮肤溃疡常常对大多数常见治疗是有抗性的。以前的研究表明外周血单核细胞(PBMC)与碱性成纤维细胞生长因子一起应用是一种对糖尿病性坏疽有用的治疗。本实施例中研究了PBMC的培养物上清液(未辐射或辐射的)是否足以在小鼠模型中诱导增强的伤口愈合。而且,分析了这些上清液对人原代成纤维细胞(FB),角化细胞(KC)和内皮细胞(EC)的作用。
通过PBMC来源的上清液孵育FB和KC,发现未被辐射和被辐射的细胞上清液都强烈诱导FB的迁移,但是它们对FB增殖没有作用。相反,两种上清液都显示对KC的迁移和增殖能力是有效的。但是,来源于被辐射细胞的上清液的作用更显著。由于PBMC来源的上清液诱导体外的迁移和增殖机制,进一步研究了这些上清液是否也能诱导体内的伤口愈合。因此制备了含有PBMC上清液的乳剂,并在创伤以后立即用于B6/129小鼠背部的6mm穿孔活检伤口。接着的4-5天直到形成痂期间,测量伤口尺寸。令人惊讶地发现,用含有PBMC来源的上清液的乳剂处理以后,在整个5天期间伤口闭合比单独用乳剂快得多。有趣地是,单独用乳剂的第一个2天内伤口尺寸稍微增加,而用PBMC来源的上清液处理的伤口在创伤和应用乳剂以后的第一个24h开始闭合。创伤以后8-10天,处死小鼠,并通过H&E-染色和对CD31的免疫组织化学对伤口进行分析,CD31是一种用于血管的标记。H&E染色显示在存在来自PBMC来源的上清液的乳剂时,皮肤的真皮和表皮部分中的伤口愈合都更提高。而且,这种伤口中CD31阳性细胞大量增加,这指示增加的血管形成,其可有助于增强的伤口愈合。
总之,PBMC来源的上清液显示导致小鼠体内增强的伤口愈合,而且这些上清液还诱导体外人细胞的增殖和迁移。含有PBMC上清液的乳剂的配方,显示对未痊愈的皮肤溃疡的治疗的较大的优势(参见图1到5)。
实施例2:静止外周血单核细胞(PBMC)证明低的活化标记和降低的炎症性细胞因子产生
活化的外周血单核细胞(PBMC)和它们的上清液(SN)认为在伤口再生中是有益的(Holzinger C等Eur J Vasc Surg.1994 May;8(3):351-6)。实施例1和2中,显示未活化的PBMC及其来源的SN在试验性的急性心肌梗塞(AMI)和创伤模型中具有有益作用。由于未活化的PBMC必须用实验方法进行证实,因此研究了PBMC 的培养是否导致增强的T细胞活化标记(CD69,CD25)或增强的炎症性细胞因子分泌(单核细胞活化=TNFα,T细胞活化=INFγ)。对照实验中,通过CD3mAb刺激或植物血细胞凝集素(PHA)触发培养的T细胞。
方法与结果
从健康的志愿者收集静脉血到EDTA-试管中。Ficoll-泛影葡胺密度梯度分离后,收集PBMC并分成活的和被辐射的凋亡细胞(IA-PBMC)。为了获得凋亡细胞,用60Gy(铯-137)辐射PBMC。为了进行流式细胞术分析,500,000PBMC培养于200μl无血清培养基中。用PHA(7μg/mL)或CD3-mAb(10μg/mL)刺激细胞,或者不进行刺激。孵育24h以后洗涤细胞,进行CD3,CD69和CD25染色(R&D System),并在FC500(Coulter)上评估表面活化标记。为了进行ELISA分析,PBMC以2.5x106细胞/ml的密度培养过夜,用或不用PHA或CD3刺激。24h以后,收集上清液并于-20℃冰冻。购买市售的用于TNF-α(R&D)和INF-γ(Bender)的ELISA试剂盒。简而言之,MaxiSorp板用抗TNF-α和INF-γ的抗体包被并储存过夜。24h以后,洗涤该板,并添加样品一式两份到每个孔中。孵育和添加检测抗体与链霉亲和素-HRP以后,添加TMB-底物到每个孔中。显色以后,通过添加硫酸停止酶促反应。在Wallac Victor3平板读数器上读取光密度值。
结果:
FACS分析:CD3和PHA刺激的T细胞显示孵育24h以后,活化标记CD69和CD25的上调。未被刺激的和凋亡的细胞只表达低量的CD69和CD25(图6a(代表性样品,图6b,直方图,n=4)。通过星号表示统计显著性(xx p<0.001,x p<0.05)。ELISA分析:虽然没有检测到未刺激的PBMC来源的上清液中的TNF-α和INF-γ,但是如通过ELISA分析表明的,来自PHA或CD3刺激的PBMC的上清液证实了较高的这些细胞因子的值(星号**p<0.001;*p<0.05,n=8)。结果清楚表明了与未被刺激的PBMC相比的炎症性细胞因子的不同分泌模式。
结论:
这些数据表明,相较于刺激的PBMC(PHA和CD3mAb),“未刺激的PBMC”表现了显著不同的表型(活化标记,细胞因子分泌)。
图6a表明,未刺激的活PBMC或IA-PBMC都不分泌主要单核细胞来源的前炎症性细胞因子TNF-α。(显著性如下表示:*p=0.05,**p=0.001;n=8)
图6b证明相较于未刺激的PBMC,活化以后强烈诱导前炎症性干扰素-γ分泌。(显著性如下表示:*p=0.05,**p=0.001;n=8)
图7a显示流式细胞术分析的合并结果。对PBMC根据T细胞进行分门,并评估了活化标记CD69和CD25的表达。(显著性如下表示:*p=0.05,**p=0.001;n=4)
图7b显示两种活化的PBMC(PHA,CD3mAb)的代表性FACS分析。分门(Gating)表示阳性细胞的%。
实施例3:培养于生理溶液中的PBMC的增殖活性
本实施例的目的是证明,相较于在2天(CD3,PHA)和5天(MLR)刺激分析中,利用特异性(CD3),非特异性(凝集素,PHA)和同种异体T细胞触发(混合淋巴细胞反应,MLR)的免疫分析,PBMC没有增殖活性。
材料和方法
通过Ficoll密度梯度离心从年轻的健康志愿者分离PBMC,并以每200μL 1*105个细胞重悬于RPMI(Gibco,USA)中,其含有0.2%硫酸庆大霉素(Sigma Chemical Co,USA),1%L-谷氨酰胺(Sigma,USA)。应答细胞通过抗CD3的MoAb(10μg/mL,BD,NJ,USA),PHA(7μL/mL,Sigma Chemical Co,USA),或用被辐射的同种异体PBMC以1∶1比例(用于MLR)刺激。平板孵育48h或5天,然后用3[H]-胸腺嘧啶核苷脉冲18h(3.7*104Bq/孔;Amersham Pharmacia Biotech,Sweden)。收获细胞,并在液体闪烁计数器中测定3[H]-胸腺嘧啶核苷掺入。
结果:
相较于培养于RPMI中的没有刺激的活的PBMC,如通过3[H]-胸腺嘧啶核苷掺入测定的,刺激的PBMC显示高的增殖速率(图8)。通过添加T细胞特异性刺激物(PHA,CD3),以及在其中通过抗原呈递细胞(MLR)触发增殖的分析中,观察到这种作用。
结论:
这组实验暗示,保持培养高达5天的活的PBMC没有增殖,但是通过不同的方式刺激的PBMC显示显著的增殖反应。推断没有刺激的PBMC的培养不导致增殖反应。
实施例4:保持在无菌培养条件下的分离的PBMC的分泌蛋白质组具有新的血管形成能力。
由于体内新血管形成与炎症强烈相关,因此对这些PBMC的分泌蛋白质组是否也显示对T细胞的抗增殖作用并因此妨碍炎症性免疫应答进行了研究。
材料和方法
通过在RPMI(Gibco,CA,USA)中孵育Ficoll密度梯度离心分离的来自年轻健康志愿者的PBMC(2.5*106/mL)24h获得分泌蛋白质组,该RPMI含有0.2%硫酸庆大霉素(Sigma Chemical Co,USA),1%L-谷氨酰胺(Sigma,USA)。从细胞级分分离上清液并储存于-80℃。为了进行增殖分析,分离后,同种异体PBMC以每200μL RPMI 1*105个细胞重悬。通过抗CD3的MoAb(10μg/mL,BD,USA)或PHA(7μL/mL,Sigma Chemical Co,USA)刺激应答细胞。添加不同稀释度的上清液。平板孵育48h,然后用3[H]-胸腺嘧啶核苷脉冲18h(3.7*104Bq/孔;Amersham Pharmacia Biotech,Sweden)。收获细胞,并在液体闪烁计数器中测定3[H]-胸腺嘧啶核苷掺入。
结果:
同种异体PBMC的分泌蛋白质组证实,相较于阳性对照,通过3[H]-胸腺嘧啶核苷掺入测定到明显降低的增殖速率(图9)。这种作用是剂量依赖的,而且在抗CD3以及PHA刺激之后就能观察到。
暗示:
这种实验暗示,从保持培养24h的活的PBMC获得的分泌蛋白质组显示显著的体外抗增殖作用。这些数据表明,来源于PBMC的上清液或其冻干形式,可用作可能的治疗剂,用于治疗与低氧诱导的炎症或其他的过度炎症疾病(例如自动免疫疾病,炎症性皮肤病)相关的人类疾病。
实施例5外周血单核细胞分泌的旁泌因子具有免疫抑制特性
实施例1中,证实了PBMC分泌蛋白质组在急性心肌梗塞(AMI)动物模型中的抗炎作用。本实施例中,表明AMI诱导以后,应用PBMC分泌蛋白质组通过大大下调免疫应答而抑制心肌的炎症性损伤。
基于这些发现,对体外实验中分泌蛋白质组的可能免疫抑制作用进行了研究。CD4+细胞在免疫应答的组织中起关键作用,因为它们在免疫学过程中对于协助其他的白细胞(例如巨噬细胞,B细胞,细胞毒性T细胞)是关键的。
材料和方法
产生PBMC分泌蛋白质组
通过Ficoll密度离心分离来自健康志愿者的PBMC。细胞以1*106个细胞/mL(sup liv)的浓度重悬于Ultra Culture培养基(Lonza,Basel,Switzerland)。为了从凋亡的PBMC产生分泌蛋白质组,通过用60Gy(supAPA)辐射诱导细胞凋亡。细胞在湿润的空气中孵育24h(5%CO2,37℃,相对湿度95%)。移取上清液,并用抗50mM乙酸铵的3.5kDa截断(Spectrum laboratories,Breda,The Netherlands)于4℃透析过夜。然后对上清液进行无菌过滤,并冻干。冻干的分泌蛋白质组于-80℃储存,而且新鲜重悬用于每个实验。分泌蛋白质组对于它们的pH值随机取样。
分离CD4细胞
利用MACS珠子系统(Miltenyi,Bergisch Gladbach,Germany),通过排除非CD4+T细胞分离CD4+细胞。新鲜制备细胞,并立即用于每个实验。
细胞凋亡的测定
使用市售的膜联蛋白V/PI试剂盒(BD,New Jersey,USA),通过流式细胞术检测细胞凋亡。通过膜联蛋白阳性染色限定细胞凋亡,通过PI阳性定义晚期细胞凋亡。
增殖实验
在一个96孔圆底孔板中,PBMC或纯化的CD4+细胞在补有0.2%硫酸庆大霉素(Sigma,St.Louis,MO,USA),0.5%β-巯基乙醇(Sigma,StLouis,MO,USA)和1% GlutaMAX-I(Invitrogen,Carlsbad,CA,USA)的Ultra Culture中稀释到1*105/孔的浓度。用PHA(7μg/mL,Sigma,USA),CD3(10μg/mL,BD,New Jersey,USA),IL-2(10μg/mL,BD,USA)刺激细胞,或者1∶1比例的同种异体辐射的(60Gy)PBMC用于MLR。用不同浓度的PBMC分泌蛋白质组,IL-10或TGF-β孵育细胞48h或5天(MLR)。然后用3[H]-胸腺嘧啶核苷脉冲细胞18h(3.7×104Bq/孔;Amersham Pharmacia Biotech,Sweden)。收获细胞,并在液体闪烁计数器中测定 3[H]-胸腺嘧啶核苷掺入。
活化标记
用抗CD3(10μg/mL)刺激纯化的CD4+细胞,并与不同浓度的PBMC分泌蛋白质组共同孵育。对细胞进行CD69和CD25染色,接着进行标准的流式细胞术染色方案,并在流式细胞仪FC500(Beckman Coulter,Fullerton,CA,USA)上进行分析。
结果
在初步实验中,从来自活细胞(sup liv)的PBMC上清液中检测到抗增殖特性。在抗CD3和PHA刺激实验中,添加分泌蛋白质组显著降低了增殖速率(n=10)。
基于这些发现,评估了PBMC分泌蛋白质组对T-辅助细胞间隔的作用,因为这些细胞在启动和保持免疫应答中起关键作用。与图10类似,通过添加分泌蛋白质组,高度纯化的CD4+细胞丧失它们的增殖能力。活的,以及凋亡的,被辐射的PBMC的上清液观察到这种现象(图11,n=5)。
下一步是确定分泌蛋白质组对细胞生活力的可能作用。因此用上清液孵育静止CD4+细胞,并评估膜联蛋白V和PI阳性。来自活的和凋亡的PBMC的上清液都证实了显著的前细胞凋亡作用(图12,n=5)。
为了检测PBMC分泌蛋白质组是否能抑制CD4+细胞活化,CD4+细胞的抗CD3刺激之后,评估T细胞活化标记CD25和CD69。PBMC分泌蛋白质组显著并剂量依赖的抑制两种标记的上调(图13,n=5)。
在最后一组实验中,通过在这些实验中添加中和抗体,检验了免疫抑制细胞因子IL-10和TGF-β的作用。IL-10和TGF-β都没发现导致我们的PBMC分泌蛋白质组的抗增殖作用,因为停止使用这些细胞因子没有增加增殖速率(图14,n=5)。
结论
这些实验第一次证明,PBMC分泌蛋白质组具有体外免疫抑制特性。表明上清液a)在抗CD3,PHA和MLR刺激实验中降低增殖速率,b)具有诱导细胞凋亡的效力,而且在T细胞触发以后就抑制CD4+细胞的活化。
Claims (24)
1.用于治疗炎症性皮肤疾病的局部药物制剂,包括通过在无增殖PBMC和活化PBMC的物质的生理溶液中培养外周血单核细胞(PBMC)或其亚群至少1h可获得的生理溶液的上清液,其中所述PBMC在培养之前或期间遭受辐射和/或低氧。
2.根据权利要求1的制剂,其特征在于所述炎症性皮肤疾病是与局部缺血相关的疾病。
3.根据权利要求1的制剂,其特征在于所述疾病选自伤口,组织缺血,皮肤溃疡,皮肤灼伤,整形手术和牙齿移植以后的组织再生中的皮瓣。
4.根据权利要求3的制剂,其特征在于所述伤口是慢性伤口、糖尿病伤口。
5.根据权利要求1的制剂,其特征在于外周血单核细胞(PBMC)的亚群是T细胞,B细胞或NK细胞。
6.根据权利要求1到5中任一项的制剂,其特征在于生理溶液是生理盐溶液,全血,血液级分,或细胞培养基。
7.根据权利要求6的制剂,其特征在于所述生理盐溶液是生理学NaCl溶液,所述血液级分是血清。
8.根据权利要求6的制剂,其特征在于细胞培养基选自RPMI,DMEM,X-vivo和Ultraculture。
9.根据权利要求1至8中任一项的制剂,其特征在于在培养之前或期间,用至少10Gy对PBMC或其亚群进行辐射。
10.根据权利要求9的制剂,其特征在于在培养之前或期间,用至少20Gy对PBMC或其亚群进行辐射。
11.根据权利要求10的制剂,其特征在于在培养之前或期间,用至少40Gy对PBMC或其亚群进行辐射。
12.根据权利要求1至11中任一项的制剂,其特征在于所述制剂以凝胶,以软膏剂,以皮肤贴膜片,以药学上可接受的基质,以糊状物,以乳剂,以粉末,以涂沫剂或以洗液提供。
13.根据权利要求12的制剂,其特征在于所述凝胶是水凝胶,所述基质是胶原蛋白/弹性蛋白基质。
14.根据权利要求1至13中任一项的制剂,其特征在于上清液被冻干。
15.根据权利要求1至14中任一项的制剂,其特征在于PBMC或其亚群在所述溶液中培养至少4h。
16.根据权利要求15的制剂,其特征在于PBMC或其亚群在所述溶液中培养至少6h。
17.根据权利要求16的制剂,其特征在于PBMC或其亚群在所述溶液中培养至少12h。
18.根据权利要求1至17中任一项限定的制剂用于制备治疗炎症性皮肤疾病的药物的应用。
19.根据权利要求18的应用,其特征在于所述炎症性皮肤疾病是与局部缺血相关的疾病。
20.用于制备根据权利要求1到17中任一项的局部药物制剂的方法,包括以下步骤:
a)提供外周血单核细胞(PBMC)或其亚群,
b)在无增殖PBMC和活化PBMC的物质的生理溶液中培养步骤a)的细胞至少1h,
c)分离步骤b)的上清液,和
d)使用步骤c)的上清液制备药物制剂,
其中所述PBMC在步骤b)之前或期间遭受辐射和/或低氧。
21.根据权利要求20的方法,其特征在于在步骤b)之前或期间,用至少10Gy对细胞进行辐射。
22.根据权利要求21的方法,其特征在于在步骤b)之前或期间,用至少20Gy对细胞进行辐射。
23.根据权利要求22的方法,其特征在于在步骤b)之前或期间,用至少40Gy对细胞进行辐射。
24.通过根据权利要求20或21中任一项的方法可获得的制剂。
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EP2198873A1 (en) | 2010-06-23 |
ZA201104349B (en) | 2012-02-29 |
US20120045418A1 (en) | 2012-02-23 |
RU2533253C2 (ru) | 2014-11-20 |
ES2429518T3 (es) | 2013-11-15 |
AU2009336668B2 (en) | 2014-05-08 |
WO2010079086A1 (en) | 2010-07-15 |
PL2379085T3 (pl) | 2014-01-31 |
CA2746862C (en) | 2017-08-29 |
JP2012512842A (ja) | 2012-06-07 |
JP5869343B2 (ja) | 2016-02-24 |
BRPI0922174A2 (pt) | 2015-12-29 |
EP2379085B1 (en) | 2013-07-10 |
US11235003B2 (en) | 2022-02-01 |
CA2746862A1 (en) | 2010-07-15 |
EP2379085A1 (en) | 2011-10-26 |
RU2011129676A (ru) | 2013-01-27 |
CN102256608A (zh) | 2011-11-23 |
AU2009336668A1 (en) | 2011-07-07 |
NZ593364A (en) | 2012-11-30 |
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