CN106729676A - 水蛭素用于制备治疗术后肌腱粘连的药物中的应用 - Google Patents
水蛭素用于制备治疗术后肌腱粘连的药物中的应用 Download PDFInfo
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Abstract
本发明涉及水蛭素制药的应用。本发明的目的是提供水蛭素用于制备治疗术后肌腱粘连的药物中的应用。本发明过不同浓度的天然水蛭素对TGFβ1、bFGF、MMP‑2的表达作用的影响，确定了其预防肌腱粘连的作用及其机制。
Description
技术领域
本发明涉及水蛭素制药的应用。
背景技术
随着社会的进步和工业的发展，外伤所致肌腱损伤极为常见。据统计，美国每年因创伤和过量运动导致的肌腱损伤超过32万例。肌腱损伤及手术修复后发生的粘连，尤其是手外伤引起的肌腱粘连，会严重影响关节功能的恢复，成为临床亟待解决的难题。如何降低肌腱损伤及术后粘连的发生率，尽快恢复其滑动功能，同时又不影响肌腱本身的愈合，成为近年研究的焦点。
肌腱粘连与肌腱愈合有着重要联系，肌腱愈合过程中形成粘连的原因包括：①外源性愈合中，因成纤维细胞由周围组织向肌腱断端生长，形成肌腱与周围组织的粘连，这是粘连形成的主要因素。②由于炎性反应导致局部组织渗出增多，机化后加重肌腱的粘连。③肌腱细胞自身增殖过程中与周围组织形成粘连。外源性愈合所产生的瘢痕则会引起粘连形成，并影响肌腱滑动。所以通过抑制腱鞘中的成纤维细胞的增殖，来抑制损伤肌腱的外源性愈合，从而防治外伤后肌腱粘连，成为治疗的重要突破点。成纤维细胞是肌腱组织的基本功能单位，主要合成和分泌Ⅰ型胶原，维持肌腱组织的新陈代谢。
一般认为，肌腱在创伤修复过程中伴随着各类生长因子的释放与参与。细胞生长因子是由细胞分泌的具有生物活性的蛋白质或多肽类物质，是细胞因子的一类，具有调节炎性细胞趋向性移动、创伤细胞分裂激活、新生血管形成和细胞间质合成的作用。与肌腱损伤修复相关的细胞生长因子包括：TGFβ1、bFGF、MMP-2、MMP-9、BMP-12等。TGFβ1已被证实能诱导体外培养的腱鞘成纤维细胞I型胶原、纤维结合素及a-SMA的表达,揭示了TGFβ1通过干扰TGFβ/Smads信号通路以及抑制成纤维细胞转分化促进肌腱粘连产生的机制,明确了其在肌腱愈合及粘连产生中发挥了重要的作用。庞久玲等通过应用流式细胞术检测48例瘢痕疙瘩、40例增生性瘢痕及40例正常皮肤组织中TGFβ1的表达，证实TGFβ1在瘢痕疙瘩和增生性瘢痕中的表达明显高于正常皮肤组织。而另一种细胞生长因子bFGF的作用却与之相反，大量的研究显示，bFGF能显著促进鞘内肌腱的愈合，抑制肌腱粘连，调节炎性反应。Oryan等对兔屈肌腱损伤后修补模型局部注射bFGF，发现其能调节炎症过程，抑制肌腱粘连和增强肌腱细胞的增殖和成熟，增加胶原蛋白的分泌，改进胶原纤维的聚合和排列方式，改善损伤后肌腱的结构和生物力学性能。盛加根等研究发现，局部单次使用bFGF及5-氟尿嘧啶在有效促进鸡屈肌腱愈合的同时能减轻肌腱粘连。基质金属蛋白酶（MMPs）是一族含锌或其他金属离子的内源性多肽酶，又称为明胶酶。在已经了解的MMPs中MMP-2是胶原及基质降解的关键酶，此外还可以降解V、VII型胶原、弹性蛋白、纤维粘连蛋白等。MMP-2主要分布在成纤维细胞的周围，与瘢痕组织愈合早期细胞外基质的分解代谢关系密切。瘢痕粘连增生的早期，MMP-2分泌增加能促进基质分解，利于成纤维细胞、肌成纤维细胞迁移，促进创面愈合。为此，作为监测肌腱粘连的重要指标，为揭示肌腱粘连的机制，细胞生长因子TGFβ1、bFGF和MMP-2被广泛关注。
在药物防治肌腱粘连得攻坚战中，中药及中药提取物因为其显著的效果而受到大量研究。黄仲玉等临床应用海桐皮为君药的中药组方熏洗联合行气活血、舒筋利节的手法治疗手部肌腱修复术后粘连，临床结果显示疗效确切，能够迅速消除肿痛，促进指体功能的恢复。赵治伟等研究川芎嗪注射液及丹红注射液与可吸收生物膜联合应用预防肌腱及组织粘连的有效性，证实预防肌腱粘连的效果明显优于单独应用吸收生物膜。
川芎嗪、当归及丹参注射液等常规的活血中药制剂被大量用于研究，疗效明确，但仍不足以十分有效的解决术后肌腱粘连。为此，我们将研究目标锁定在另一味传统的活血化瘀类中药，水蛭，及其被广泛用于其他医学疾病的药物天然水蛭素。水蛭是一味传统的活血化瘀类中药，始载于《神农本草经》，历代本草均有记载，《本草纲目》曰其:“入肝经血分”。《中国药典》记载水蛭具有破血通经，逐瘀消癓等功效。其活性成分天然水蛭素(Hirudin)是从医用水蛭唾液中提取的凝血酶抑制剂，是一种抗凝血蛋白质，由65个或66个氨基酸残基组成的单链多肽，相对分子质量约7000，被认为是目前已知的最强效的凝血酶抑制剂之一。但是其作用远远不至于抗凝，国内外对水蛭素突出的药理、临床等方面的研究得到深入的开展,认为其还具有抗血栓形成、抗纤维化等多方面的用途。水蛭素抑制细胞增生的作用被广泛关注。李开通等通过实验探讨水蛭素对体外培养的增生性瘢痕成纤维细胞基质金属蛋白酶（MMPs）表达水平的影响，发现水蛭素能够抑制增生性瘢痕成纤维细胞的增殖，从而为烧伤愈合的患者减少瘢痕挛缩提供新途径。郭睿等通过检测水蛭素对人皮肤瘢痕成纤维细胞中bFGF、TGFβ1表达的影响，发现水蛭素能抑制bFGF、TGFβ1分泌，由此抑制成纤维细胞的生长、增殖并进一步抑制瘢痕粘连的形成。
中药及中药制剂治疗肌腱粘连，不仅能开拓临床诊疗思路，更能有效的防治肌腱粘连，同时能合理的降低医疗费用。天然水蛭素作为一种活血化瘀的传统中药，近来被广泛应用于研究抑制各种成纤维细胞的生长，特别应用于烧伤后的瘢痕愈合有十分显著的疗效。而天然水蛭素抑制肌腱长纤维细胞的生长从而防治肌腱粘连的机制及疗效有待我们进一步发掘。
发明内容
为了解决上述的技术问题，本发明的目的是提供水蛭素用于制备治疗术后肌腱粘连的药物中的应用。
本发明采用兔趾深屈肌腱细胞体外培养，确定不同浓度的水蛭素对动物肌腱成纤维细胞增殖的抑制作用，以及不同浓度的天然水蛭素对肌腱成纤维细胞TGFβ1、bFGF、MMP-2表达水平的影响。TGFβ1是纤维化促进因子，在肌腱愈合早期的外源性愈合中，能诱导肌腱成纤维细胞I型胶原（ColⅠ）、纤维结合素及a-SMA的过分表达，是目前已知的与病理性瘢痕关系最密切的细胞因子，促进肌腱细胞胶原合成的同时增加了术后瘢痕粘连组织的形成。bFGF是成纤维细胞生长因子家族原型，可通过与成纤维细胞膜上的受体结合，下调病理性瘢痕组织中I型前胶原mRNA表达，提高胶原酶mRNA水平，刺激胶原酶合成，减少硫酸软骨素粘多糖产生，抑制纤维素合成，防止过量的细胞外基质沉积，进而防止肌腱粘连。MMP-2是基质金属蛋白酶（MMPs）中的一种，是外基质中胶原及基质降解的关键酶，此外还可以降解V、VII型胶原、弹性蛋白、纤维粘连蛋白等。MMP-2分布在瘢痕粘连组织的各层，以成纤维细胞周围的染色最明显，与粘连组织早期的细胞外基质的分解代谢关系密切，是促进创面过度修复形成肌腱瘢痕粘连的关键因素。瘢痕组织增生早期，MMP-2分泌增加促进基质分解，利于成纤维细胞、肌成纤维细胞迁移，能促进肌腱的愈合。本发明过不同浓度的天然水蛭素对TGFβ1、bFGF、MMP-2的表达作用的影响，确定了其预防肌腱粘连的作用及其机制。
附图说明
图1为各浓度水蛭素（1，2，3，4，5，6，7，8，9和10U/ml）对兔肌腱成纤维细胞增殖活性抑制率的MTT实验结果曲线。
图2为各浓度水蛭素（0.75，1.5，3和6U/ml）对兔肌腱成纤维细胞TGFβ1表达结果图。
图3为各浓度水蛭素（0.75，1.5，3和6U/ml）对兔肌腱成纤维细胞bFGF表达结果图。
图4为各浓度水蛭素（0.75，1.5，3和6U/ml）对兔肌腱成纤维细胞bFGF表达结果图。
具体实施方式
1.材料
1.1、实验动物
普通级成年雌性新西兰兔5只,体重3～4kg,由浙江大学实验动物中心提供,实验室屏障环境饲养。
1.2、主要试剂天然水蛭素（广西科康生物科技有限责任公司）；胰酶、I型胶原酶(Worthington公司)；DMEM培养液、胎牛血清、胰蛋白酶(Gibco公司)；四甲基偶氮唑盐(MTT，Amresco公司)；二甲基亚砜(DMSO，Amresco公司)；TGFβ1、bFGF和MMP-2的ELISA检测试剂盒（武汉博士德生物工程有限公司）。
1.3、主要仪器恒温C02培养箱(CBl50型，Binder分司)；超净工作台(苏州净化设备公司)；电子天平(ABl04-S型，MettlerToledo公司)；电热恒温水浴槽(DK一600型，上海医疗器械五厂)；台式离心机(800型，上海手术器械厂)；全自动酶标仪(ELX800型，Bio-Tek公司)。
2.方法
2.1、兔肌腱成纤维细胞的培养
普通级成年雌性新西兰兔5只(体重3～4kg,由浙江中医药大学实验动物中心提供),用氯胺酮肌注麻醉,无菌切取前肢中趾趾深屈肌腱,共10条,采用组织块法进行原代培养。用0．05％洗必泰溶液浸泡5min，生理盐水漂洗3次，置于0．25％胰酶4℃冷消化18h，转移至培养皿中，DMEM清洗，充分剪碎，加入0.2％I型胶原酶，37℃孵育1h，用吸管反复吹打组织块，分散细胞，100目筛过滤消化液，收集滤过的细胞悬液，1000rpm离心5min，弃上清液；用含15％胎牛血清的DMEM培养液将沉淀的细胞团制成细胞悬液；进行细胞计数，调整细胞密度。以5x105／ml的密度将细胞接种于培养瓶中，加入含15％胎牛血清的DMEM培养液，于37℃、5％C02培养箱内培养。每2～3d换液一次；细胞融合至80％～90％时按常规方法接种。实验选用第4-5代细胞。
2.2、天然水蛭素对兔肌腱成纤维细胞增殖活性的抑制作用
这里需要说明的是水蛭素的单位U。本实验室是用凝血酶直接滴定法来测定。水蛭素的一个抗凝血酶活力单位U是指在37℃下，使一个单位的凝血酶失活所需水蛭素的量。实验按照水蛭素浓度分为四组：对照组（0U/ml）；1U/ml组；10U/ml组；50U/ml组。每组6孔。先每孔加入lxl04个细胞，37℃、5％C02条件下孵育12h，待细胞贴壁后，吸弃培养液，对照组每孔加入无血清DMEM培养液600μl，其余各组每孔加入含对应浓度水蛭素的DMEM培养液600μl。继续培养48h后，每孔各加5mg／ml的MTT溶液40ul，继续培养4h，吸去上清液，加入300ulDMSO，振荡15min，使紫色结晶物充分溶解，用酶标仪测490nm波长处各孔的吸光度(OD值)。
各浓度水蛭素（1，2，3，4，5，6，7，8，9和10U/ml）对兔肌腱成纤维细胞增殖活性抑制率的MTT实验结果曲线如图1所示。如图，随着水蛭素剂量增加，其对对兔肌腱成纤维细胞增殖活性抑制率逐渐增强。
2.3、不同浓度的天然水蛭素对兔肌腱成纤维细胞TGFβ1表达作用的研究
采用双抗体夹心ELISA法测定各组标本的TGFβ1表达含量，具体操作步骤严格按照试剂盒说明书进行。用酶标仪在450nm处测定样品OD值，根据样品吸光度值计算出样品浓度。实验按照水蛭素浓度分为四组：对照组（0U/ml）；1U/ml组；10U/ml组；50U/ml组。每组6孔。先每孔加入lxl04个细胞，37℃、5％C02条件下孵育12h，待细胞贴壁后，吸弃培养液，对照组每孔加入无血清DMEM培养液100μl，其余各组每孔加入含对应浓度水蛭素的DMEM培养液100ul。继续培养48h后，每孔各加生物素化的TGFβ1抗体稀释液（Biotin-antiTGFβ1）100μl，用封板胶纸封住，继续培养1h。洗板5次，且最后1次置厚吸水纸上拍干。每孔加入辣氧根过氧化物酶（HRP)100ul,混匀后避光继续培养20min。再次洗板拍干。每孔加入底物显色溶液（TMB）100μl，混匀后避光继续培养20min。每孔加入终止液50μl，混匀后即刻用酶标仪测450nm波长处各孔的吸光度（OD值）。
图2是各浓度水蛭素（0.75，1.5，3和6U/ml）对兔肌腱成纤维细胞TGFβ1表达结果图，各剂量水蛭素均可促进兔肌腱成纤维细胞分泌TGFβ1。
2.4、不同浓度的天然水蛭素对兔肌腱成纤维细胞bFGF表达作用的研究
采用双抗体夹心ELISA法测定各组标本的bFGF表达含量，具体操作步骤同2.3步骤，严格按照试剂盒说明书进行。实验按照水蛭素浓度分为四组：对照组（0U/ml）；1U/ml组；10U/ml组；50U/ml组。每组6孔。先每孔加入lxl04个细胞，37℃、5％C02条件下孵育12h，待细胞贴壁后，吸弃培养液，对照组每孔加入无血清DMEM培养液100μl，其余各组每孔加入含对应浓度水蛭素的DMEM培养液100μl。继续培养48h后，每孔各加生物素化的bFGF抗体稀释液（Biotin-antibFGF）100μl，用封板胶纸封住，继续培养1h。洗板5次，且最后1次置厚吸水纸上拍干。每孔加入辣氧根过氧化物酶（HRP)100μl，混匀后避光继续培养20min。再次洗板拍干。每孔加入底物显色溶液（TMB）100μl，混匀后避光继续培养20min。每孔加入终止液50μl，混匀后即刻用酶标仪测450nm波长处各孔的吸光度（OD值）。
图3是各浓度水蛭素（0.75，1.5，3和6U/ml）对兔肌腱成纤维细胞bFGF表达结果图.
2.5、不同浓度的天然水蛭素对兔肌腱成纤维细胞MMP-2表达作用的研究
采用双抗体夹心ELISA法测定各组标本的MMP-2的表达含量，具体操作步骤同2.3步骤，严格按照试剂盒说明书进行。实验按照水蛭素浓度分为四组：对照组（0U/ml）；1U/ml组；10U/ml组；50U/ml组。每组6孔。先每孔加入lxl04个细胞，37℃、5％C02条件下孵育12h，待细胞贴壁后，吸弃培养液，对照组每孔加入无血清DMEM培养液100μl，其余各组每孔加入含对应浓度水蛭素的DMEM培养液100μl。继续培养48h后，每孔各加生物素化的MMP-2抗体稀释液（Biotin-antibMMP-2）100μl，用封板胶纸封住，继续培养1h。洗板5次，且最后1次置厚吸水纸上拍干。每孔加入辣氧根过氧化物酶（HRP)100μl，混匀后避光继续培养20min。再次洗板拍干。每孔加入底物显色溶液（TMB）100μl，混匀后避光继续培养20min。每孔加入终止液50μl，混匀后即刻用酶标仪测450nm波长处各孔的吸光度（OD值）。
图4为各浓度水蛭素（0.75，1.5，3和6U/ml）对兔肌腱成纤维细胞bFGF表达结果图。
3.统计学方法
采用SPSS13.0软件包进行分析，数据以均数标准差（x±s）表示，组间差异比较采用t检验，P<0.05为差异有统计学意义。
本发明通过实验观察不同浓度天然水蛭素对兔趾深屈肌腱体外培养的成纤维细胞的作用，实验结果进行测定和分析，达到如下成果：
1.明确天然水蛭素对体外培养的兔肌腱成纤维细胞增殖活性具有抑制作用；
2.明确天然水蛭素对兔肌腱成纤维细胞TGFβ1的表达具有抑制作用；
3.明确天然水蛭素对兔肌腱成纤维细胞bFGF的表达具有促进作用；
4.明确天然水蛭素对兔肌腱成纤维细胞MMP-2的表达具有促进作用；
5.初步揭示不同浓度的天然水蛭素对兔肌腱成纤维细胞促进作用的强弱以及其作用机制。
Claims (1)
1.水蛭素用于制备治疗术后肌腱粘连的药物中的应用。
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Citations (3)
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|---|---|---|---|---|
| CN1213309A (zh) * | 1996-01-18 | 1999-04-07 | 汉斯－阿恩汉森 | 愈合方法的控制 |
| CN1827179A (zh) * | 2005-03-04 | 2006-09-06 | 蒋婧 | 功能性术后隔离膜及其制备方法 |
| CN103405811A (zh) * | 2013-08-16 | 2013-11-27 | 西安组织工程与再生医学研究所 | 一种防粘连生物膜及其制备方法 |
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|---|---|---|---|---|
| CN1213309A (zh) * | 1996-01-18 | 1999-04-07 | 汉斯－阿恩汉森 | 愈合方法的控制 |
| CN1827179A (zh) * | 2005-03-04 | 2006-09-06 | 蒋婧 | 功能性术后隔离膜及其制备方法 |
| CN103405811A (zh) * | 2013-08-16 | 2013-11-27 | 西安组织工程与再生医学研究所 | 一种防粘连生物膜及其制备方法 |
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| Title |
|---|
| 陈思凯: "天然水蛭素对肌腱成纤维细胞抑制作用及其机制的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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