CN118027175A - 冻存prp与干细胞外泌体治疗无菌性炎症疾病 - Google Patents
冻存prp与干细胞外泌体治疗无菌性炎症疾病 Download PDFInfo
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Abstract
本发明涉及冻存prp与干细胞外泌体治疗无菌性炎症疾病。具体的,采用冻存prp与干细胞外泌体治疗关节炎。本发明的prp和外泌体能够较好的促进骨关节炎软骨细胞增殖。将prp和外泌体以及本发明筛选的活性肽一起使用能够有效的治疗骨关节炎模型小鼠的关节炎症状,能够显著的降低关节炎相关基因的表达量,具有较好的治疗效果。
Description
技术领域
本申请涉及生物领域,具体的涉及冻存prp与干细胞外泌体治疗无菌性炎症疾病。
背景技术
“无菌性炎症”是人体发生机体障碍疾病和顽固疼痛的部位没有细菌感染,病理检查和组织切片找不到任何微生物侵害的迹象,从病理变化上来看是无菌性的,没有病源菌的炎症,因而抗生素治疗无效。有的人对某些物质过敏时也可引起炎症反应,如过敏性鼻炎、接触性皮炎、药物性皮炎、湿疹等。这些疾病虽然也有一个“炎”字,但不是因细菌感染引起的,如果使用抗菌消炎药物治疗,不但无效,有时反而因引起新的过敏反应而加重病情。治疗过敏性炎症应给以抗过敏药物,如息斯敏、扑尔敏或激素类抗过敏药物等。还有一些运动系统疾病,如关节炎、纤维组织炎、腱鞘炎、强直性脊柱炎等,这些炎症也不是病原微生物引起,不要用抗菌消炎药。
骨关节炎是一种常见的致残疾病,2006-2016年间全球伤残损失健康生命年百分比增长了31.5%,导致巨大的社会经济成本,对个体健康和卫生保健系统有显著影响。膝骨关节炎是骨关节炎最常见的亚型,主要影响老龄人群及肥胖人群,临床症状为膝关节疼痛、肿胀、僵硬和活动能力下降。膝骨关节炎的病理改变包含关节软骨退变、软骨下骨改变、骨赘形成和非细菌性滑膜炎,其发病机制复杂,起初认为与“磨损”这种机械应力有关,后发现微环境和遗传因素在疾病恶化过程中相互作用,最终导致关节软骨变性、关节内炎症伴滑膜炎和软骨下骨变化。软骨细胞产生和分泌的细胞外基质和成纤维细胞样滑膜细胞分泌的滑液是维持微环境的2种重要成分,但是软骨细胞仅占软骨体积的2%,所以膝骨关节炎发生时可能首先被来自滑膜或软骨下骨的炎症信号激活,从而改变细胞外基质合成和降解之间的平衡,导致关节软骨自我再生潜力有限。
目前关节炎治疗方法已经有多种形式。富血小板血浆(PRP)是新鲜全血离心后的自体产物,含有许多促进组织修复的生长因子(如血小板衍生生长因子AB、转化生长因子β1和血管内皮生长因子)。已有大量研究报道富血小板血浆对于年轻及影像学分级较低(Kellgren-lawrence分级2-3级)的膝骨关节炎患者能发挥疼痛缓解、功能提升及改善生活质量的作用。通过对80只胫骨近端骨折骨松质形成的观察研究表明,使用最高剂量PRP组显微CT骨体积/总体积显著增高,组织形态学上新骨面积和骨向内生长的深度也最高。
当然,除了PRP之外,还有多肽也能够用于关节炎的治疗。比如ZL200510040378.5高效抑制血管生成多肽及其制备方法和应用,还介绍了一种多肽,该多肽序列可以有效地结合于整合素亚型,并且该序列中含有新生血管抑制序列,能够新生血管形成。先前专利中只是对黑色素瘤做了研究,本发明针对该序列做了进一步研究,发现其对类风湿性关节炎有治疗作用,增加了其适用症,拓展了其社会价值和经济价值。
此外,MSCs已被许多研究报道具有免疫调节及抗炎作用,且仍然主要通过务分泌机制所分泌的EVs发挥作用。据报道脂肪来源的MSCs所分泌的EX(AD-MSCS-EX)对T细胞的增殖、分化和活化具有抑制作用。最近研究发现在IL-1β刺激的OA软骨细胞中,AD-MSCS-EVs减少了炎症介质(IL-6、TNF、前列腺素E2、NO等)的产生。IL-1B对抗炎细胞因子IL-10有抑制作用,而AD-MSCs-EVs能够抵消其抑制作用,并显示出对IL-10的表达有一定的增强作用。还有研究发现,MSCS-EVs可诱导IL-10和TGF-B1高表达,同时抑制IL-1B、IL-6、TNF-α、IL12/P40的表达。由此看来,MSCs-EVs具有一定的抗炎及免疫调节潜能。目前抗炎作用已在OA模型中得到证实,而免疫调节作用尚未在OA模型中得到证实。最近有研究表明,来自小鼠的MSCs-EVs可抑制CD8T淋巴细胞的增殖以及B淋巴细胞的增殖激活化,并且还增加了调节性T淋巴细胞群体,但对CD4 IFN-T淋巴细胞没有影响。因此,MSCs-EVs可能通过其抗炎及免疫调节潜能在OA治疗中发挥重要作用。
目前,有效治疗骨关节炎的组合物还不够多,特别是疗效好的具有自主知识产权的组合物类型也有待于进一步的丰富和完善。
发明内容
本发明一方面,提供了一种冻存PRP在制备治疗无菌性炎症疾病关节炎的药物中的用途。
更进一步的,所述的药物中还含有干细胞外泌体。
所述的干细胞外泌体是本领域常用的脂肪间充质干细胞外泌体。
所述的外泌体为采用本领域常用的外泌体制备方法制备获得的。
更进一步的,所述的药物中还含有具有鹿茸多肽活性肽,其氨基酸序列如SEQ IDNO:1所示。
更进一步的,所述的活性肽还可以被修饰或者保守性取代。
本发明的术语“保守取代”是指用具有相似结构和/或化学性质的不同氨基酸取代一个氨基酸。这种氨基酸取代通常可以基于残基的极性、电荷、溶解性、疏水性、亲水性和/或两亲性质的相似性而发生。在整个说明书中,使用了用于天然发生的氨基酸的常规单字母和三字母代码。此外,本文提及的氨基酸根据IUPAC-IUB的命名规则缩写如下。丙氨酸Ala、A精氨酸Arg、R;天冬酰胺Asn、N天冬氨酸Asp、D;半胱氨酸Cys、C谷氨酸Glu、E;谷氨酰胺Gln、Q甘氨酸Gly、G;组氨酸His、H异亮氨酸Ile、I;亮氨酸Leu、L赖氨酸Lys、K;甲硫氨酸Met、M苯丙氨酸Phe、F;脯氨酸Pro、P丝氨酸Ser、S;苏氨酸Thr、T色氨酸Trp、W;酪氨酸Tyr、Y缬氨酸Val、V;同时,任何氨基酸都可以描述为Xaa、X。进一步的,在具有侧链的带电荷氨基酸中,带正电荷(碱性)氨基酸包括精氨酸、赖氨酸和组氨酸,带负电荷(酸性)氨基酸包括谷氨酸和天冬氨酸;在具有侧链的不带电荷氨基酸中,非极性氨基酸包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸和脯氨酸,极性或亲水氨基酸包括丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺和谷氨酰胺。在非极性氨基酸中,芳香族氨基酸包括苯丙氨酸、色氨酸和酪氨酸。
进一步的,本发明的还提供一种治疗骨关节炎的药物组合物,所述药物组合物含有本发明的鹿茸多肽活性肽,其氨基酸序列如SEQ ID NO:1所示。
进一步的,所述的药物组合物还含有药学上可接受的载体。
本发明的药物组合物可以进一步含有崩解剂、粘合剂、增溶剂、流化剂、甜味剂、发泡剂、表面活性剂、防腐剂、pH调节剂、着色剂、香料。
进一步的,本发明的载体包括赋形剂。其中所述赋形剂选自:甘露醇、乳糖、山梨醇、木糖醇、葡萄糖、麦芽糖、甘氨酸、可溶性糊精、黄原胶、明胶、水解明胶、阿拉伯胶、果胶、瓜尔胶、桃胶、黄芪胶、金合欢胶、羧甲基纤维素钠、聚乙烯吡咯烷酮、卡波姆、羟丙基纤维素、羟丙基甲基纤维素、海藻酸盐、或其组合。上述制剂所用赋形剂可以为乳糖、蔗糖、甘露醇、葡萄糖、山梨醇、葡聚糖等糖的衍生物,淀粉、糊精等淀粉衍生物;微晶纤维素等纤维素衍生物;硅酸钙、硅铝酸镁等硅酸盐衍生物;磷酸氢钙等磷酸盐;海藻酸盐;碳酸钙等碳酸盐;硫酸钙等硫酸盐等无机类赋形剂。润滑剂可以为硬脂酸、硬脂酸钙、硬脂酸镁等硬脂酸金属盐;滑石粉;蜂蜡等蜡类;硼酸;己二酸;硫酸钠等硫酸盐;乙二醇;富马酸;月桂基硫酸钠等月桂基硫酸盐;硅酸水合物等硅酸类;淀粉衍生物。粘合剂可以为羟丙基纤维素、羟丙甲基纤维素、聚乙烯吡咯烷酮、聚乙二醇以及上述兼具粘合作用的物质。崩解剂可以为低取代羟丙基纤维素、羧甲基纤维素钙、羧甲基纤维素、交联羧甲基纤维素钠等纤维素衍生物;羧甲基淀粉,羧甲基淀粉钠,交联聚乙烯吡咯烷酮,纤维素类,上述淀粉衍生物。矫臭矫味剂可以为甜味剂、酸味剂、香料等。基质可以为可可豆脂、脂肪酸甘油酯等硬脂基料、甘油明胶基料、聚乙二醇类及它们的混合物。气雾剂载体可以为氢氟烷HFA类。粉雾剂载体可以为乳糖、甘露醇、葡萄糖、果糖、木糖醇、山梨醇,苏氨酸、天冬氨酸、谷氨酸、异亮氨酸、精氨酸、亮氨酸、甘氨酸等氨基酸。在实际操作中优选水溶性辅料。
其中所述药物组合物中还包括酸碱调节剂。在一个实施方案中,所述的酸碱调节剂选自氢氧化钠、氢氧化钾、磷酸二氢钠、磷酸氢二钠、磷酸二氢钾、磷酸氢二钾、盐酸、磷酸、硝酸、硫酸、或其组合。在一个实施方案中,所述的酸碱调节剂是盐酸溶液或者氢氧化钠溶液,例如1M盐酸溶液或者1M氢氧化钠溶液。
本发明的药物组合物进一步的还含有增塑剂。“增塑剂”的例子包括药物制剂通常使用的增塑剂。具体地说,例如,酯,例如柠檬酸三乙酯,中链甘油三酯,邻苯二甲酸二乙酯,邻苯二甲酸二丁酯,三醋酸甘油酯(三乙酰甘油),丁基邻苯二甲酰基丁基乙醇酸酯,辛酸甘油基酯等等;醇,例如甘油,丙二醇,聚乙二醇,等等。对于增塑剂,优选化学式[HOCH2(CH2OCH2)nCH2OH(n=2-870的整数)]的化合物,尤其优选聚乙二醇(PEG)。实际上用作增塑剂的PEG的例子包括聚乙二醇(Sanyo chemical industries,Ltd.生产)。尽管对PEG的平均分子量没有特别限制,但优选不小于200,更优选200-20000,这是由于小平均分子量可以提高吸湿性。当将PEG加入到用于膜包衣片剂等等的包衣试剂中时,优选,PEG的含量是包衣试剂的大约5-大约30wt%,尤其是大约10-25wt%,更优选大约10-大约20wt%。
进一步的,本发明的药物组合物可以是冻干形式。在制备本发明冻干粉针剂时,所配制的药液中,固形物含量是为1~20%(w/v),优选1~15%(w/v),再更优选1~10%。由于冻干粉针剂通常是在管状西林瓶中进行冷冻干燥得到,本领域技术人员理解这种产品在获得成品甚至在供医生使用之前,通常均呈现一个圆饼状,尽管该圆饼的体积理论上讲会比原有水溶液的体积少(稍有缩小),然而通常这种缩小通常不会缩小到原水溶液体积50%,通常会在原水溶液体积的80-120%之间,更通常在原水溶液体积的90-100%之间,而从终产品西林瓶内可观察到原水溶液液面痕迹(主体饼状物因冻干缩小后残留在瓶壁上的液面痕迹,即便西林瓶中的冻干品因各种原因例如碰撞等原因而呈粉末状,通常仍然可以保留原有的液面痕迹),据此痕迹亦可估计出该冷冻干燥组合物在冷冻干燥之前的水溶液体积。因此,虽然本发明提供的是一种基本无水的冷冻干燥粉针剂,然而根据该粉针剂仍然可以大致估计出其在配制时,至少在冷冻干燥开始之前的药液体积,根据该估计出的体积以及西林瓶中的干燥终产物的重量,亦可计算到在制备本发明冻干粉针剂时,所配制的药液中的固形物的含量。因此,根据本发明第一方面的冻干粉针剂,其在配制时的药液的固形物含量是为1~20%(w/v),优选1~15%(w/v),再更优选1~10%。
有益效果
本发明提供了一种采用冻存prp与干细胞外泌体治疗无菌性炎症疾病的用途,具体的,采用冻存prp与干细胞外泌体治疗关节炎。本发明的prp和外泌体能够较好的促进骨关节炎软骨细胞增殖。将prp和外泌体以及本发明筛选的活性肽一起使用能够有效的治疗骨关节炎模型小鼠的关节炎症状,能够显著的降低关节炎相关基因的表达量,具有较好的治疗效果。
附图说明
图1 prp和外泌体促进软骨细胞增殖效果图
图2活性肽促进软骨细胞增殖效果图
具体实施方式
通过下面的实施例可以对本发明进行进一步的描述,然而,本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。以下实施例进一步说明本发明,而不是限制本发明。任何依据本发明构思所作出的仅仅为形式上的而非实质性的等效变换都应视为本发明的技术方案范畴。
实施例1PRP的制备及激活及冻干制备
收集小鼠全血,并将其以8:1(V/V)的比例添加到BAC001-ACD抗凝剂中,将样品轻轻搅拌以混合.样品进行两步离心:将20ml混合物在50ml离心管中以250g离心15min,将包含血小板的最上面两层转移到新的离心管中,并以1000g的速度离心15min。丢弃大部分上清液血浆和贫血小板血浆(PPP)层,并将沉淀的血小板重新悬浮在残留血浆中共获得1.8ml的PRP。
随后用10%CaCl2与前述PRP 1:9的比例迅速混匀,37℃放置1h后置于4℃冰箱过夜。次日见大量淡黄色澄清液体析出,即为激活态PRP。将试管置于离心机内,4℃,5000r/min离心30min。上清液即为激活态的PRP。
采用鼠TGF-β、IGF、PDGF、bFGF和VEGF检测试剂盒,检测激活态PRP与未激活的PRP相比,表达量均大幅得到提高,表明激活态PRP制备成功。
将激活态PRP进行冷冻干燥存储。具体的方法为:赋形剂是海藻糖和乙酸钠组成,二者浓度分别为2%和0.03%;其在与激活态PRP以体积比1:1混合后,所得混合液中海藻糖浓度为1%、乙酸钠浓度为0.015%;将混合液放入预降温至-46℃的冻干机进行真空干燥,保持真空度为<15Pa,干燥12h后取出,即为冻干激活态PRP,室温密封干燥保存4周后,使用前加入该样本冻干前等体积灭菌注射用水充分混匀复水。
实施例2脂肪间充质干细胞外泌体的制备
取小鼠BMSCs(编号CSI031Mu01,武汉云克隆动物有限公司)在脂肪间充质干细胞无血清培养基(货号NC0103+NC0104.S,诺扬生物)培养60h,收集细胞培养上清液,将收集的上清液通过以下差速离心步骤收集BMSCs-Exo:300×g,10min;2000×g,10min;10000×g,30min;120000×g,90min。超速离心后,弃去上清液,沉淀即主要为BMSCs-Exo,用1mLPBS重悬,0.22μm滤膜过滤,-80℃保存。将20μLBMSCs-Exo悬液滴加在铜网上,室温1min后滴加20μL磷钨酸溶液,1min后吸去磷钨酸溶液,红外灯下烘烤10min,吸去多余液体,透射电镜下观察Exo的粒径几种在95-110nm,圆形囊泡双层膜结构。使用BCA法调整外泌体浓度为1mg/mL备用。
实施例3激活态PRP和外泌体活性验证
小鼠软骨细胞(购买自联迈生物,货号LM-003)培养在含10%FBS和1%青霉素/链霉素的DMEM/F12培养基中培养软骨细胞,培养条件为含5%CO2的37℃湿润的无菌细胞培养箱,1d换液1次,后续实验均采用对数生长期细胞。
按照CCK-8试剂盒说明检测软骨细胞的增殖。软骨细胞分为对照组(正常软骨细胞)、IL-1β组(模型组)、IL-1β+激活态PRP(50mg/L)组、IL-1β+激活态PRP(100mg/L)组、IL-1β+BMSCs-Exo(50mg/L)组、IL-1β+激活态PRP(50mg/L)+BMSCs-Exo(50mg/L)组进行相应处理。在120h时,向每个孔中加入10μl CCK-8溶液和100μl新鲜培养基,并在37℃下孵育4h.用酶标仪在450nm的波长下检测光密度值。软骨细胞的存活/增殖表示为测试孔的光密度值减去空白孔的光密度值。结果如图1所示。
从图1结果可以看出,IL-1β抑制了细胞增殖(P<0.05,与对照组相比),而激活态PRP和BMSCs-Exo都能够有效促进骨关节炎软骨细胞增殖,而且激活态PRP和BMSCs-Exo联合使用,能够更有效的促进软骨细胞增殖(P<0.05,相对于模型组)。
实施例4鹿茸多肽高活性肽筛选和鉴定
梅花鹿鹿茸2kg,剁成小块(1cm2左右),用4℃蒸馏水冲洗至无血色,粉碎,胶体磨匀浆,不断添加预冷的匀浆液(pH3.5 HAc溶液),9000r/min,离心20min,取上清,加入95%乙醇使其终浓度达到65%,4℃放置并搅拌30min,随后9000r/min离心20min,取上清,55℃真空旋转蒸发,回收乙醇,冻干得到鹿茸粗提物,-20℃保存。
将鹿茸提取物采用胰蛋白酶(4×104U/g)按照底物浓度5%,酶底比为10%,温度为40摄氏度,pH为9的条件下酶解4h。煮沸10min灭酶,冷却后调至pH7。在8000rpm下离心15min去沉淀,通过超滤,收集分子量小玉5Kda的组分,然后采用SephadexG-25(1.6cm×60cm)对肽液进行分离,加样量为1mL,以水为洗脱剂,流速为1.5mL/min。在280nm处检测吸光度,收集各洗脱峰,进行真空浓缩干燥,检测各峰对软骨细胞增殖效果较好的2号峰,采用Superdex75凝胶过滤层析进行再分离,色谱条件如下:玻璃柱:柱长*直径(1.6cm×80cm)流动相:纯水(微孔滤膜过滤)流速:3mL/min温度:4℃检测波长:280nm上样体积3.0mL,收集活性较好的2-II号峰进行HPLC高效液相色谱层析,色谱柱:Agilent公司300SB-C18;规格:4.6mmX250mm;流速:0.5mL/min;温度:20℃;检测波长:214nm,230nm280nm;流动相A:0.1%三氟乙酸的水溶液;流动相B:0.1%三氟乙酸的乙腈溶液,经C18反相层析柱分离纯化后共得到4个组分,体外软骨细胞增殖活性鉴定活性组分主要集中在峰2部分。将峰2部分委托百泰派克生物科技进行测序,鉴定氨基酸序列如SEQ ID NO:1所示,人工合成所述多肽,HPLC鉴定纯度为99%以上。
小鼠软骨细胞(购买自联迈生物,货号LM-003)培养在含10%FBS和1%青霉素/链霉素的DMEM/F12培养基中培养软骨细胞,培养条件为含5%CO2的37℃湿润的无菌细胞培养箱,1d换液1次,后续实验均采用对数生长期细胞。
按照CCK-8试剂盒说明检测软骨细胞的增殖。软骨细胞分为对照组、IL-1β组(模型组)、IL-1β+活性肽(10mg/L)组、IL-1β+活性肽(50mg/L)组、IL-1β+活性肽(100mg/L)组、IL-1β+活性肽(200mg/L)组进行相应处理。在120h时,向每个孔中加入10μl CCK-8溶液和100μl新鲜培养基,并在37℃下孵育4h,用酶标仪在450nm的波长下检测光密度值。软骨细胞的存活/增殖表示为测试孔的光密度值减去空白孔的光密度值。结果如图2所示。
从图2可以看出,本发明分离得到的活性肽具有剂量依赖性的促进软骨细胞增殖的效果(与模型组相比,P<0.05)。
实施例5大鼠骨关节炎模型的构建及治疗
实验前适应性喂养7d,随后从SPF级SD雄性大鼠中随机抽取10只作为空白组,其余大鼠建造膝骨关节炎模型,造模过程无大鼠死亡。造模方法:麻醉药物选择10%的水合氯醛溶液,抓取大鼠以3mL/kg剂量对其腹腔注射。待大鼠充分麻醉后,将大鼠膝关节待注射处毛发剃净,棉签蘸取体积分数75%的乙醇消毒,然后屈曲大鼠膝关节至45°左右,以髌韧带胫骨附着点外上方约1mm处为进针点,然后抽取4%木瓜蛋白酶0.2mL+0.03mol/L的L-半胱氨酸混合溶液,经皮肤刺入有落空感即表明针头到达关节腔内,随即进行注射,注射完毕退出针头后,使用棉签压迫针口以防出血,另一膝关节采用同样方法完成木瓜蛋白酶注射,所有注射完成后给予20万U青霉素钠肌注以防大鼠感染。每隔3d(即第1,4,7天)进行1次上述造模操作,共3次。
把所有膝骨关节炎模型大鼠随机分为模型组、低剂量多肽组、高剂量多肽组、高剂量多肽+激活态PRP+BMSCs-Exo组、阳性对照组,每组10只大鼠。造模后所有大鼠跑步1周,然后每组随机抽取1只大鼠处死,取膝关节做组织切片,显微镜下观察关节软骨表面粗糙,个别可见裂隙,组织结构紊乱,软骨细胞弥漫增多,参照本领域常见的改良版Mankin评分标准(参见Acoustic stifness and changein plug cartilage over time after autologousosteochondral grafting:correlation between ultrasound signal intensity andhistological score in a rabbit model.),确定造模成功。
其余大鼠确定造模成功当天即开始膝关节显微注射。空白组不做任何处理;模型组每天予以体质量相关剂量生理盐水注射;低剂量多肽组予10mg/kg多肽微量注射;高剂量多肽组予50mg/kg多肽微量注射;高剂量多肽+激活态PRP+BMSCs-Exo组予50mg/kg多肽微量注射,间隔2h后微量注射激活态PRP 10mg/kg,再加个2h后微量注射BMSCs-Exo 10mg/kg;阳性对照组予18mg/kg的塞来昔布胶囊水溶液灌胃。所有干预每日1次,共给药4周。
末次干预结束后禁食不禁水24h,使用10%水合氯醛溶液麻醉所有大鼠,对其进行腹主动脉采血。采血完毕后,使用颈椎脱臼法处死大鼠,屈曲右膝关节,使用剪刀剪开膝关节处皮肤,手术刀沿髌骨内侧从上至下打开关节囊并去除髌骨,剪刀等小心剥离胫骨平台周围韧带等软组织,使软骨暴露,使用骨剪轻轻取下右膝胫骨平台表面软骨置于2mL冻存管中,-80℃保存备用;分离左膝关节软组织并暴露胫骨平台软骨后,咬骨钳于胫骨约1/2处咬断胫骨,附带关节软骨的胫骨整个置于装有40g/L多聚甲醛的离心管中固定,以便随后石蜡标本的制作。针对大鼠软骨番红固绿染色及Mankin评分;结果如表1所示。
表1各组大鼠Mankin评分
注:a与空白组相比,P<0.05;b与模型组相比;P<0.05。
从表1可以看出,干预4周后与空白组相比,其余各组软骨Mankin评分均较高(P<0.05);与模型组相比,各治疗组评分均较低(P<0.05),而多肽联合外泌体以及激活态PRP具有相对于阳性对照组更好的治疗效果(P<0.05),也比单独治疗的效果要好。
此外,荧光定量PCR检测软骨中Col2α1mRNA表达分别取50mg左右各组大鼠的软骨组织,使用Trizol法提取总RNA,洗涤后无RNase水溶解RNA,根据反转录试剂盒说明书进行反转录操作得到cDNA,将上述cDNA模板与缓冲液、dNTP、引物、TaqDNA聚合酶等离心混匀于EP管构建反应体系,于PCR仪器上进行扩增。采用2-ΔΔCt法计算Col2α1mRNA的相对表达量。所述的引物序列如下:Col2α1Forward:5’-CATCGAGTACCGATCACAGAAG-3’;Reverse:5’-GCCCTATGTCCACACCAAAT-3’;GAPDHForward:5’-GCAAGGATACTGAGAGCAAGAG-3’;Reverse:5’-GGATGGAATTGTGAGGGAGATG-3’。
表2各组大鼠关节软骨Col2α1mRNA表达结果
#与空白组相比,P<0.05;!与模型组相比,P<0.05
从表2可以看出,模型组的Col2α1mRNA表达相较空白组显著上调(P<0.05),而各治疗组与模型组相比,表达量则显著降低,有显著性意义(P<0.05)。由于Col2aα1是膝关节软骨中间接反映软骨修复作用,模型组的Col2α1mRNA表达水平高是由于软骨的损伤刺激了机体的代偿作用导致Col2α1mRNA的表达增加,而各治疗组能够降低Col2α1mRNA表达水平,进而对早期膝骨关节炎大鼠的软骨具有一定的保护作用。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (6)
1.一种能够促进软骨细胞增殖的活性肽,所述的活性肽的氨基酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的活性肽在制备促进软骨细胞增殖的药物中的用途。
3.一种用于治疗无菌性关节炎的药物组合物,其特征在于由冻存的富血小板血浆prp、脂肪间充质干细胞外泌体以及活性肽组成,所述的活性肽的氨基酸序列如SEQ ID NO:1所示;所述的prp是激活态的prp冻干粉。
4.如权利要求3所述的药物组合物,其特征在于还含有药学上可接受的载体。
5.如权利要求3所述的药物组合物,其特征在于所述的无菌性关节炎是大鼠或者小鼠的关节炎。
6.冻存的富血小板血浆prp、脂肪间充质干细胞外泌体以及活性肽在制备治疗无菌性关节炎的药物组合物中的用途,所述的活性肽的氨基酸序列如SEQ ID NO:1所示;所述的prp是激活态的prp冻干粉。
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