CN1612756A - 治疗伤口的组合物和方法 - Google Patents
治疗伤口的组合物和方法 Download PDFInfo
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- CN1612756A CN1612756A CNA028268121A CN02826812A CN1612756A CN 1612756 A CN1612756 A CN 1612756A CN A028268121 A CNA028268121 A CN A028268121A CN 02826812 A CN02826812 A CN 02826812A CN 1612756 A CN1612756 A CN 1612756A
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Abstract
治疗人或非人哺乳动物伤口促进其愈合的组合物，其包括活性量的toll受体(或toll样受体)配体或其前体，和合适的载体。配体可以是来源于昆虫，例如黑腹果蝇(Drosophilamelanogaste r )或丝光绿蝇(Luciliasericat a )的链条或链条样蛋白。发明也涉及包含将根据发明的组合物应用到伤口上来治疗伤口的方法，和包含带有根据本发明的组合物的支架的用于伤口的敷料。
Description
导言
本发明涉及伤口的治疗。更特别地，本发明涉及促进伤口愈合的物质、组合物、掺入了这些物质的敷料以及使用这些物质治疗伤口的方法。
有效的伤口愈合是涉及许多机制的复杂的生理过程，这些机制包括细胞迁移、生长因子的分泌、血管发生、组织重建以及以协调方式和以明显地阶段性的方式有助于加速受控组织再生的伤口的内在蛋白酶/抗蛋白酶的平衡。
在现代医疗实践中，特别是对于有慢性创伤或烧伤疾病患者的治疗，伤口护理产品是必不可少的。以前已经建议了许多不同的具有有助于伤口愈合活性的物质。这些以前建议的物质包括链激酶、胶原酶和链道酶(所有都是从细菌来源中获得)、菠萝蛋白酶(从菠萝中获得)、纤溶酶和胰蛋白酶(从牛中获得)和杀死酶(从甲壳动物中获得)。临床试验数据表明这样的物质在促进伤口愈合中仅仅是部分有效的。
已知绿蝇，丝光绿蝇(
Lucilia sericata)的幼虫(蛆)作为活的生物体具有显著的伤口愈合特性。使用丝光绿蝇(
Lucilia sericata)幼虫的清创处理，已经成为被广泛接受的临床实践。然而，在文献中还几乎没有关于这些幼虫进行伤口清创，使常规不能治疗的伤口达到愈合程度的方式的报道。
尽管是有效的，但是许多患者讨厌活幼虫，并且许多患者和许多开业医生不能接受在伤口上使用活幼虫以及当使用幼虫时不可避免地会发生将幼虫的天然分泌物引入到伤口中。使用活的生物体也增加了患者感染或发生变态反应的风险。
发明陈述
大体上说，本发明涉及治疗人或非人哺乳动物伤口以促进其愈合的组合物，其包括活性量的toll受体配体，或其前体，和合适的载体。在本说明书中，术语“toll受体”应当被理解为包括果蝇属(
Drosophila)toll受体的人和非人的同源物，该同源物在现有技术中经常被称为toll样受体(TLR’s)，代表先天免疫识别受体的保守家族，其与在哺乳动物、昆虫和植物中保守的信号传导通路偶联，介导天然免疫防御基因活化。因此，为了避免疑问，这里使用的术语“toll受体”意思是toll受体和toll样受体，这里使用的术语“toll受体配体”也作相应地解释。在本说明书中术语“配体”应当被理解为包括天然产生的配体本身和与天然配体具有相同功能的任何其合成类似物。根据本发明的特别实施方式，配体可以选自人toll受体的组成型或诱导型配体，配体可能被或不被蛋白酶，例如丝氨酸蛋白酶加工。
典型地，toll受体配体或配体前体是蛋白的半胱氨酸结超家族成员，或其活性类似物。这个家族的每个成员都包括在活性C-端结构域群集的七个半胱氨酸残基。尽管在每个实例中二聚化的方式不同，但是家族的每个成员能够形成二聚体并且结合到特定的受体上。蛋白都采纳独特的三维折叠-半胱氨酸结，半胱氨酸结以延长的β-链和三个显示独特连接的二硫桥为特征。可以在发明的组合物中使用并且属于蛋白半胱氨酸结超家族的特别适合配体的实例是来自果蝇属(
Drosophila)的链条蛋白，或其活性部分，例如在Cell 76，677-688中描述的C-端106个氨基酸的肽。在TIBS 1998，July 23(7)(239-242)中描述了来自这个家族的其它配体。在本发明特别优选的实施方式中，发明的toll受体配体是在具有幼虫生命周期的昆虫的幼虫阶段所表达的链条样蛋白，或其合成类似物。这样的昆虫的实例是黑腹果蝇(
Drosophila melanogaster)和丝光绿蝇(
Lucilia sericata)。
根据本发明的包含toll受体配体前体的组合物可以进一步包括适合加工toll受体配体前体以形成活性toll受体配体的蛋白酶。典型地，蛋白酶将是丝氨酸蛋白酶，例如胰蛋白酶样或胰凝乳蛋白酶样酶。合适的胰蛋白酶样蛋白酶的特征是：
(i)它是生物体丝光绿蝇(
Lucilia sericta)分泌的；
(ii)它在pH8.0-8.5下显示对FITC酪蛋白的最适蛋白水解活性；
(iii)它具有对Tosyl-Gly-Pro-Arg-AMC的水解能力但对Suc-Ala-Ala-Phe-AMC无蛋白水解能力；
(iv)它对FITC-酪蛋白和Tosyl-Gly-Pro-Arg-AMC的蛋白水解活性被丝氨酸蛋白酶抑制剂PMSF和APMSF所抑制；和
(v)它被固定化的氨基苄脒结合。
在本发明组合物中有用的蛋白酶天然存在于丝光绿蝇(
Lucilia sericata)幼虫所排泄/分泌(ES)的分泌物中。
当水解荧光蛋白底物异硫氰酸荧光素-酪蛋白(FITC-酪蛋白)时，幼虫ES分泌物表现出经典的8.0-8.5的最适pH。在监测FITC-酪蛋白的水解之前，用不可逆的低分子量抑制剂4-(脒基苯基)甲烷磺酰氟化物(APMSF；所有胰蛋白酶样丝氨酸蛋白酶的抑制剂，但不是胰凝乳蛋白酶样丝氨酸蛋白酶的抑制剂)或者用苯基甲烷磺酰氟化物(PMSF；所有丝氨酸蛋白酶的抑制剂)预先培养幼虫的ES分泌物，显示幼虫的ES分泌物具有两类丝氨酸蛋白酶活性；胰蛋白酶样活性和胰凝乳蛋白酶样活性。通过监测荧光多肽底物Tosyl-Gly-Pro-Arg-AMC(对胰蛋白酶样蛋白酶有选择性)和Suc-Ala-Ala-Pro-Phe-AMC(对胰凝乳蛋白酶样蛋白酶有选择性)证实了双重活性，其中“AMC”代表7-氨基-4-甲基香豆素，“Suc”代表琥珀酰基。
在丝光绿蝇(
Lucilia sericata)的ES分泌物中除了检测到占优势的丝氨酸蛋白酶活性外，也检测到了其它不占优势的活性。尽管没有显示半胱氨酰活性，但是已经检测到存在天冬氨酰和金属蛋白酶的活性。通过监测FITC-酪蛋白的水解显示在pH5.0时具有天冬氨酰的活性，并且被这类特异的抑制剂胃蛋白酶抑制剂A成功地抑制。通过ES分泌物水解亮氨酸氨基肽的能力证实存在金属蛋白酶的活性，揭示存在外肽酶。外肽酶识别肽中游离NH2氨基酸。亮氨酸氨基肽被丝光绿蝇(
Lucilia sericata)ES的水解仅仅被经典的金属蛋白酶抑制剂，Zn2+鳌合物邻菲罗啉抑制。这种抑制反映存在具有金属蛋白酶酶性质的外肽酶。
ES分泌物具有经计算大约是0.88单位/升的α-淀粉酶活性。另外，尽管当与蛋白酶相比这种活性大约低50倍，但是在幼虫的ES分泌物中存在磷酸酶活性(正磷酸单酯键的水解)。也鉴定了脂酶的活性(脂肪酸酯中酯键的水解)。当用抑制剂PMSF预培养ES分泌物时没有检测到这种脂酶的活性，提示这种水解应归因于分泌物中的丝氨酸蛋白酶。
从我们的研究中能够推断在幼虫的ES分泌物中占优势的活性是丝氨酸蛋白酶活性，并且存在两类丝氨酸蛋白酶活性；一种来源于胰凝乳蛋白酶，另一种来源于胰蛋白酶。
可以通过层析方法从天然ES分泌物中获得基本上纯化形态的加工蛋白酶。从丝光绿蝇(
Lucilia sericata)幼虫中收集ES分泌物并且使用固定化的氨基苄脒进行亲和层析。氨基苄脒是胰蛋白酶样丝氨酸蛋白酶的可逆的抑制剂。从层析方法中收集“流通”的物质，即没有被固定剂结合的物质之后，通过添加游离的氨基苄脒可以洗脱被固定剂结合的酶并且分别收集。
如上面描述的，本发明的配体能够根据现有技术中已知的肽合成和纯化的常规途径进行合成制备和纯化。可以保护配体抗氨基肽酶活性以提高活性和/或延长配体在伤口区保持有活性的时间。例如，可以通过使用非编码的异常氨基酸在配体中的COOH的取代位置发生酰胺化和/或通过电子等排物取代CO-NH酰胺键，获得抗氨基肽酶活性的保护。
可以将发明的配体应用到伤口中以诱导有助于愈合的生长因子的轮廓。例如，以纯的形式存在或在无菌载体中的一种或更多配体，能够被洒在伤口区域或者被掺入载体中应用到伤口上。例如，配体能够被掺入或者被用胶囊包到能将配体以缓慢释放或可控释放方式释放到伤口中的合适的材料中。这样合适载体的实例是可以配制用于以可控释放方式释放肽的聚(丙交酯共乙交酯)或PLGA颗粒。
可选择地，可以将一种或多种配体掺入应用到伤口的敷料中。这些敷料的实例包括掺入了含有伤口愈合材料的缓慢释放的水胶体颗粒的阶段性的或分层的敷料，或者任选地被常规敷料覆盖的含有伤口愈合材料的海绵。可以将当前正在使用的水胶体类敷料，例如那些可以在“Granuflex”商标下买到的，进行修饰用以将配体释放到伤口中的。
本发明也涉及包含将根据发明的组合物应用到伤口上的处理人或非人的伤口以促进其愈合的方法。在进一步方面中，本发明提供用于伤口的敷料，其中包含带有根据本发明组合物的支架。
发明详述
1.本发明的加工蛋白酶的分离和分析
通过丝光绿蝇(
Lucilia sericata)ES在氨基苄脒琼脂糖上的亲和层析纯化胰蛋白酶样丝氨酸蛋白酶。用20ml含有0.5M氯化钠的pH8.0的0.025M Tris-盐酸缓冲液平衡柱基质(1ml)。在上柱前用等体积的缓冲液稀释天然ES(0.5ml，70μg/ml蛋白)。收集通过层析的馏分(0.5ml)。在用6.5倍柱体积的缓冲液洗涤以除去未结合的蛋白后，使用游离的氨基苄脒配体(2ml 400μM)以得到结合物质的洗脱液。使用馏分在280nm的吸光度读数以确定未结合(流通)和结合峰的位置和接着收集结合峰用于分析。在图1中显示洗脱轮廓图。
氨基苄脒琼脂糖结合胰蛋白酶样丝氨酸蛋白酶。在将幼虫酶分泌物上柱后，未结合的物质直接通过并且作为“流通”物(峰I)被收集。向柱缓冲液中添加游离氨基苄脒得到结合的蛋白酶(峰II)的洗脱液。未结合(流通)物质含有不受APMSF(可能包括胰凝乳蛋白酶样酶)影响的蛋白酶活性，而在氨基苄脒洗脱峰中的活性基本上被APMSF消除(80％)，提示胰蛋白酶样丝氨酸蛋白酶活性的纯化。在图2中显示柱馏分的残余活性。
将柱馏分在非还原性SDS样品缓冲液(含有4％SDS pH6.8的0.5MTris-盐酸、20％甘油和0.02％溴酚蓝)中在含有0.1％人血红蛋白的12％SDS聚丙烯酰胺凝胶上通过电泳进行检查。通过在2.5％Triton X-100(1小时)和蒸馏水(15分钟)中洗涤以除去SDS。通过在37℃、pH8.0的0.1M Tris-盐酸缓冲液中孵育过夜，凝胶中血红蛋白底物的蛋白酶解在相应于蛋白酶的位置上产生了考马斯亮蓝蛋白染色所显示的清晰条带(图3)。起始馏分和流出的馏分每个都显示了几种蛋白酶的活性，然而用氨基苄脒洗脱的馏分显示了单一条带。因此先前在用氨基苄脒洗脱的馏分中鉴定的胰蛋白酶样酶(图2)显示具有～25Kda的分子量(图3)。
2.用FITC-酪蛋白研究幼虫酶(ES)的蛋白水解行为
使用下面ES的不同呈现方式(0.25μg)研究pH8时在FITC-酪蛋白水解中丝光绿蝇(
Lucilia sericata)的ES的活性。
A、 ES+水
B、 ES+乙醇
C、 用0.2mM PMSF预培养的ES
D、 用0.6mM PMSF预培养的ES
E、 用1mM PMSF预培养的ES
F、 用0.04mM APMSF预培养的ES
G、 用0.12mM APMSF预培养的ES
H、 用0.2mM APMSF预培养的ES
在用不可逆的丝氨酸蛋白酶抑制剂PMSF预培养后丝光绿蝇(
Lucilia sericata)ES的蛋白水解活性被抑制。在用1mM PMSF预培养ES的实例中蛋白水解活性被完全抑制。PMSF溶于乙醇，溶剂对ES活性的影响可以忽略。相反，在用不可逆的“胰蛋白酶样”特异性抑制剂APMSF预培养ES的实例中检测到ES的大约50％残余丝氨酸蛋白酶活性。在APMSF存在下的残余活性表明存在胰凝乳蛋白酶样酶。得到的活性值(％)如下：
A、 100％
B、 85.5％
C、 13.8％
D、 18％
E、 0％
F、 43.5％
G、 47％
H、 54％
这些结果以图示方式显示在图4中。
3.幼虫酶(ES)对特定底物的蛋白水解活性的研究
使用下面不同呈现方式的ES研究存在APMSF和PMSF时丝光绿蝇(Lucilia sericata)ES(0.25μg)对Tosyl-Gly-Pro-Arg-AMC(a)和对Suc-Ala-Ala-Phe-AMC(b)的活性。
(a)
A、 ES
B、 用0.025mM APMSF预培养的ES
C、 用0.05mM APMSF预培养的ES
D、 用1mM PMSF预培养的ES
(b)
E、 ES
F、 用0.2mM APMSF预培养的ES
G、 用1mM PMSF预培养的ES
得到的残余活性值(％)如下：
(a)
A、 100％
B、 14.3％
C、 3.6％
D、 0％
(b)
E、 100％
F、 86.8％
G、 1.3％
这些结果以图示方式显示在图5中。
(a)的结果揭示在丝光绿蝇(
Lucilia sericata)ES中存在“胰蛋白酶样”丝氨酸蛋白酶活性。Tosyl-Gly-Pro-Arg-AMC的水解(对丝氨酸蛋白酶凝血酶和纤溶酶有选择性)被1mM PMSF和0.05mMAPMSF抑制。然而，丝光绿蝇(
Lucilia sericata)ES对胰凝乳蛋白酶底物Suc-Ala-Ala-Phe-AMC的水解作用仅仅被PMSF(1mM)抑制而不被过量的APMSF(其不抑制胰凝乳蛋白酶)抑制。该结果提供了进一步的证据表明在ES中存在两种不同丝氨酸蛋白酶亚类。
4.toll和toll样受体的配体
如上面提到的，根据特别的实施方式，可以在本发明组合物中使用的配体可以选自人toll受体的组成型和诱导型配体，可以被或不被蛋白酶，如丝氨酸蛋白酶加工。特定的实例是未被加工和已加工形式的链条蛋白，一种从黑腹果蝇(
Drosophila melanogaster)获得的toll受体配体。在Cell(1994)，
76，677-688中描述和刻画了该链条蛋白的特性。
来自丝光绿蝇(
Lucilia sericata)不同发育阶段的链条样蛋白(链条蛋白同源物或类似物)，在本发明中也可以被用作toll受体配体。可以使用形成的抗果蝇属(
Drosophila)链条蛋白的抗体鉴定这些蛋白。这些来自丝光绿蝇(
Lucilia sericata)不同发育阶段的富含链条样蛋白的物质可以通过在生理盐水中提取，获得提取物，接着将提取物应用到抗体亲和层析柱中以获得提纯的方法而被纯化。可以检验这样鉴定的链条样蛋白结合人白细胞上toll受体的能力。可以用来自丝光绿蝇(
Lucilia sericata)的链条样蛋白与人外周血单核(HPBM)细胞共培养，接着用胸苷掺入的方法测定HPBM细胞的增殖。一前一后地，与已知的Toll配体(LPS-细菌脂多糖)一起监测绿蝇属(
Lucilia)的链条蛋白同源物或类似物诱导细胞因子(TNF-α)分泌的能力。
Cytokine and Growth Factor Reviews 11(2000)219-232中描述了toll样受体的配体。
实验方面
进行了在诱导的蛆血淋巴(使用丝光绿蝇(Lucilia sericata)的幼虫)中鉴定LPS样活性的研究。
使丝光绿蝇(L.sericata)幼虫在存在(诱导的)或缺乏(非诱导的)铜绿假单胞菌(Pseudomoma aeruginosa)的无菌肝/琼脂溶液中生长。
从外科材料测试实验室SMTL(Princess of Wales Hospital，Bridgend CF31 1RQ)获得丝光绿蝇(L.sericata)的无菌幼虫。幼虫在Sherman(1995)描述的，包含分解的猪肝和细菌培养用琼脂的培养基上生长，在允许容器内外之间气体和潮气交换但是防止细菌进入的密闭容器中通过高压灭菌消毒。在容器的底部为幼虫提供薄薄的一层营养培养基。
在200μl无菌磷酸缓冲盐溶液中悬浮无菌的第一龄期幼虫(200)，并且转移到容器中。允许幼虫在28℃潮湿室内无菌环境下生长约48小时，以便让幼虫定居。将铜绿假单胞菌(Pseudomoma aeruginosa)突变株PAO P47接种到10ml Luria Bertani(LB)培养基中，在37℃振荡生长过夜。将1ml培养物(～108活细胞计数)接种到容器中，让幼虫在存在细菌的条件下生长。
重复上面描述的过程，但例外的是不使用铜绿假单胞菌(P.aeruginosa)培养物接种。
培养48小时后，将从上面描述的过程中得到的新的第二龄期幼虫分别进行如下加工。
在无菌条件下将幼虫从肝-琼脂溶液中移出并且转移到无菌普通试管内。用冷的无菌PBS冲洗蛆，接着在70％乙醇中洗涤，最后在滤纸上干燥。接着使用无菌外科刀片将幼虫钩状物的底部切开。接着用带有无菌黄色Eppendorf枪头的20μl移液器收集血淋巴，并且转移到含有20μg/ml抑酶肽(蛋白酶抑制剂)和40μM苯硫脲(melinisation抑制剂)的预冷的Eppendorf试管中。在4℃，15000g离心10分钟后，将上清液收集到预冷的试管中，在-80℃保藏直到需要为止。通过在LB溶液或平板中的过夜培养物判断，当使用这种方法时肠内容物没有污染血淋巴，并且血淋巴也没有被铜绿假单胞菌(P.aeruginosa)污染。
使用“三明治”ELISA方法在人外周血单核细胞(PBMCs)上检测诱导的和非诱导的血淋巴上清液(根据上面描述的方法制备的)对TNF-α释放的影响，与LPS进行比较。
血样本是从三名征得同意的健康志愿者(供血者1，2和3)中获得的。通过浮力密度离心法用Histopaque 1077(Sigma，Poole，UK)以600g离心20分钟从三名供血者的每份肝素化的全血中分离人外周血单核细胞(PBMC)。用RPMI 1640培养基把从中间层收获的PBMC冲洗两次，重悬浮在AIM-V培养基中。
接着将105PBMCs铺到96孔平板上，用100μl浓度逐渐增加的非诱导的/诱导的血淋巴培养，来自大肠杆菌血清型055：B5的LPS作为阳性对照。培养24小时后，收集细胞上清液，加到用小鼠抗人TNF-α抗体预先覆盖的96孔平板上。包括从20ng/ml开始的标准人TNF-α的系列稀释液作为平行对照。留下预期的TNF-α过夜用以捕捉，用0.05％(V/V)PBS/Tween 20三次冲洗后，添加生物素化的小鼠抗人TNF-α抗体来检测捕捉抗体。用0.05％(V/V)PBS/Tween 20冲洗最后一次后，向孔中加入链霉抗生物素蛋白-辣根过氧化物酶，用四甲基联苯胺作为底物显色10分钟，在Dynex平板读数器中在450nm处读取显色读数。所有的分析都进行两次。
结果以图示方式显示在图6中。在图6中(A)栏显示获得的，表示检测到的TNF-α(ng/ml)与用于对来自三名供血者中每一个的PBMCs进行LPS处理的LPS(μg/ml)之间关系的图。(B)栏显示所生产的TNF-α占最大LPS反应的％(用LPS％表示)与每个血淋巴处理的PBMC样品的血淋巴浓度的变化关系。(B)栏中的图显示针对诱导的和非诱导的血淋巴的结果。研究表明诱导的血淋巴刺激从人PBMCs中分泌TNF-α。很重要地提及的是，根据在LB溶液或平板中的过夜培养物所做的判断，血淋巴不存在任何铜绿假单胞菌(P.aeruginosa)的污染。
Claims (9)
1、用于治疗人或非人哺乳动物伤口促进其愈合的组合物，其包含活性量的toll受体配体或其前体和合适的载体。
2、如权利要求1中所述的组合物，其中所述的toll受体配体或配体前体是蛋白的半胱氨酸结超家族成员或其活性类似物。
3、如权利要求1或2中所述的组合物，其中toll受体配体或配体前体是来源于昆虫的蛋白质、其活性部分或两者之一的活性类似物。
4、如权利要求3中所述的组合物，其中所述的蛋白质来源于黑腹果蝇或丝光绿蝇。
5、如权利要求3或4中所述的组合物，其中所述蛋白质的所述活性部分包括C-端的106个氨基酸的多肽。
6、如权利要求1-5中任一项的组合物，它进一步包括适合加工toll受体配体前体以形成活性toll受体配体的蛋白酶。
7、如权利要求6中所述的组合物，其中所述蛋白酶的特性在于：
(i)它是由生物体丝光绿蝇分泌的；
(ii)它在pH8.0-8.5下具有FITC酪蛋白的最适蛋白水解活性；
(iii)它对Tosyl-Gly-Pro-Arg-AMC具有水解能力，但对Suc-Ala-Ala-Phe-AMC无蛋白水解能力；
(iv)它对FITC-酪蛋白和Tosyl-Gly-Pro-Arg-AMC的蛋白水解活性被丝氨酸蛋白酶抑制剂PMSF和APMSF所抑制；和
(v)它能被固定化的氨基苄脒结合。
8、治疗人或非人哺乳动物伤口促进其愈合的方法，其包括将根据权利要求1-7中任意一项的组合物应用到伤口上。
9、用于伤口的敷料，其包括带有根据权利要求1-7中任意一项的组合物的支架。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011075958A1 (zh) * | 2009-12-22 | 2011-06-30 | 中山大学中山眼科中心 | Toll-likereceptor-3激动剂在制备促进伤口愈合的药物中的应用 |
| CN102388135A (zh) * | 2009-03-03 | 2012-03-21 | B.R.A.I.N.生物技术研究和信息网络公司 | 用于创伤调理和皮肤护理的新型蛋白酶 |
| CN102675409A (zh) * | 2012-04-25 | 2012-09-19 | 大连医科大学附属第一医院 | 可保持五谷虫分泌排泄物活性的收集液 |
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| GB0607495D0 (en) * | 2006-04-13 | 2006-05-24 | Secr Defence | Larval enzymes |
| JP4998999B2 (ja) * | 2007-09-11 | 2012-08-15 | 国立大学法人 岡山大学 | ウジムシ治療用のカバードレッシング |
| CN111285922B (zh) * | 2020-03-03 | 2022-06-10 | 南京中医药大学 | 一种促进组织修复的果蝇多肽及其制备方法与应用 |
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| US2187766A (en) * | 1936-12-10 | 1940-01-23 | Standard Chemical And Mineral | Therapeutic composition |
| EP1798288B1 (en) * | 1997-05-07 | 2009-09-16 | Schering Corporation | Human Toll-like receptor proteins, related reagents and methods |
| DE29924318U1 (de) * | 1999-01-14 | 2002-09-19 | Fleischmann Wilhelm | Verbandsmaterial |
| GB9925005D0 (en) * | 1999-10-22 | 1999-12-22 | Univ Nottingham | The treatment of wounds |
| US20030134283A1 (en) * | 2000-10-03 | 2003-07-17 | Peterson David P. | Genes regulated in dendritic cell differentiation |
| DE10138303A1 (de) * | 2001-08-10 | 2003-03-06 | Aventis Pharma Gmbh | Verwendung von Enzymisolaten aus Fliegenlarven zur Wundbehandlung |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388135A (zh) * | 2009-03-03 | 2012-03-21 | B.R.A.I.N.生物技术研究和信息网络公司 | 用于创伤调理和皮肤护理的新型蛋白酶 |
| CN102388135B (zh) * | 2009-03-03 | 2013-07-24 | B.R.A.I.N.生物技术研究和信息网络公司 | 用于创伤调理和皮肤护理的新型蛋白酶 |
| WO2011075958A1 (zh) * | 2009-12-22 | 2011-06-30 | 中山大学中山眼科中心 | Toll-likereceptor-3激动剂在制备促进伤口愈合的药物中的应用 |
| CN102675409A (zh) * | 2012-04-25 | 2012-09-19 | 大连医科大学附属第一医院 | 可保持五谷虫分泌排泄物活性的收集液 |
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| Publication number | Publication date |
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| JP2005518355A (ja) | 2005-06-23 |
| AU2002366000A1 (en) | 2003-06-10 |
| EP1450872A1 (en) | 2004-09-01 |
| AU2002366000B2 (en) | 2007-01-18 |
| GB2399500A (en) | 2004-09-22 |
| GB0412321D0 (en) | 2004-07-07 |
| WO2003043669A1 (en) | 2003-05-30 |
| CA2467246A1 (en) | 2003-05-30 |
| GB0127618D0 (en) | 2002-01-09 |
| US20050053597A1 (en) | 2005-03-10 |
| GB2399500B (en) | 2006-07-05 |
| WO2003043669A8 (en) | 2003-10-02 |
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