CN116870030A - 一种猪血小板裂解液在制备防治膝骨关节炎产品中的应用 - Google Patents
一种猪血小板裂解液在制备防治膝骨关节炎产品中的应用 Download PDFInfo
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Abstract
本发明提供了一种猪血小板裂解液在制备防治膝骨关节炎产品中的应用,属于生物医药技术领域。本发明首次提出猪血小板裂解液具有治疗机体膝骨关节炎作用,且能够抗软骨纤维化、抗软骨基质降解以及具有软骨修复作用。本发明体外实验结果也证实了猪血小板裂解液在治疗膝骨关节炎时不产生免疫排斥反应,进一步的,体内外试验结果表明猪血小板裂解液能够抑制软骨纤维化和软骨基质降解,且通过SMAD2信号通路发挥作用。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种猪血小板裂解液在制备防治膝骨关节炎产品中的应用。
背景技术
膝骨关节炎(knee osteoarthritis,kOA)是一种常见的关节退行性疾病,其特征是软骨、软骨下骨、滑膜、韧带、肌肉和关节周围脂肪的逐渐损伤,从而导致关节的功能障碍、疼痛和僵硬,最终导致身体残疾。根据国际骨关节炎协会的治疗指南,目前kOA患者的治疗方法主要为非甾体抗炎药、关节镜下微骨折手术、自体软骨细胞移植和膝关节置换术。非甾体抗炎药具有抗炎、止痛的作用,但会对心血管系统、胃、肾脏产生不良影响;关节镜下微骨折手术具有创伤小、修复效果好的优点,但修复后产生的纤维软骨耐磨性远不如正常软骨;自体软骨细胞移植适用于初始治疗失败后大面积软骨缺损的患者,但该方法成本高,且临床疗效不稳定。因此,目前没有较为理想的治疗策略,kOA患者最终不得不接受膝关节置换术,但膝关节置换术容易引起感染和慢性疼痛,并且人工关节使用寿命有限。因此,仍迫切需要新的保守治疗策略。
血小板是由巨核细胞裂解产生的,含有多种生长因子,可再生组织结构,促进组织愈合。血小板通过反复冻融裂解释放大量的血小板颗粒,产生富含生长因子的血小板裂解物(Platelet lysates,PL)。近年来,PL因其在组织修复和再生中的关键作用而受到研究人员越来越多的关注。临床研究表明,人血小板裂解物(hPL)可以促进kOA患者的软骨修复,但新形成的软骨通常会转化为纤维软骨,并且随着时间的推移,纤维软骨很容易降解。此外,新生软骨组织与周围缺损处的融合往往不够紧密,从而导致软骨表面的不平整。因此,修复软骨纤维化成为影响kOA患者治疗效果的关键问题。
发明内容
有鉴于此,本发明的目的在于提供猪血小板裂解液在制备防治膝骨关节炎产品中的应用,首次提出猪血小板裂解液具有治疗机体膝骨关节炎作用,且能够抗软骨纤维化、抗软骨基质降解以及具有软骨修复作用。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了猪血小板裂解液在制备防治膝骨关节炎产品中的应用。
本发明还提供了猪血小板裂解液在制备防治软骨纤维化产品中的应用。
本发明还提供了猪血小板裂解液在制备防治软骨基质降解产品中的应用。
本发明还提供了猪血小板裂解液在制备软骨修复产品中的应用。
优选的,所述猪血小板裂解液中血小板衍生生长因子AA、BB和AB的浓度分别为1108.83pg/ml、1289.33pg/ml和159.85pg/ml。
优选的,所述猪血小板裂解液通过抑制SMAD2信号的表达来抑制软骨纤维化和软骨基质降解。
优选的,所述猪血小板裂解液的制备方法包括如下步骤:将猪外周血置于抗凝管中,4℃静置2h,离心后取上清,反复冻融后灭活,无菌处理得猪血小板裂解液。
优选的,所述猪包括巴马香猪。
优选的,所述反复冻融为-80℃冻存过夜,37℃水浴融化,反复进行三次。
本发明还提供了一种治疗膝骨关节炎、软骨纤维化、软骨基质降解或软骨修复的药物,所述药物以猪血小板裂解液为唯一活性成分。
本发明的有益效果:
本发明首次提出猪血小板裂解液具有治疗机体膝骨关节炎作用,且能够抗软骨纤维化、抗软骨基质降解以及具有软骨修复作用。本发明体外实验结果也证实了猪血小板裂解液在治疗膝骨关节炎时不产生免疫排斥反应,进一步的,体内外试验结果表明猪血小板裂解液能够抑制软骨纤维化和软骨基质降解,且通过SMAD2信号通路发挥作用。
附图说明
图1为不同组别疼痛行为评价和组织病理学结果,其中A为大鼠软骨表面的HE、SO染色结果,B为大鼠软骨表面的HE、SO染色的OARSI、Mankin′s评分及给药前后大鼠的疼痛测量数据统计图;数值以平均值±标准差表示。*P<0.05和**P<0.01vs.Model,##P<0.01vs.Sham;
图2为不同组别免疫组织化学分析结果,其中A为大鼠软骨组织中COL1和COL3的免疫组织化学分析,B为COL1阳性面积百分比和COL3阳性细胞百分比的数据统计图;数值以平均值±标准差表示,**P<0.01vs.Model,##P<0.01vs.Sham;
图3为大鼠软骨中纤维化相关蛋白的表达,左图为WB结果,由图中由上至下由左至右分别为纤维化相关蛋白COL1、COL3、α-SMA、CTGF、p-SMAD2、SMAD2的统计分析结果;数值以平均值±标准差表示,**P<0.01vs.Model组,#P<0.05和##P<0.01vs.Sham组;
图4为不同组别对大鼠软骨细胞的增殖作用以及大鼠软骨细胞中纤维化相关基因的表达结果,其中A为pPL和rPL对大鼠软骨细胞的增殖作用,B为大鼠软骨细胞中纤维化相关基因的表达结果;数值以平均值±标准差表示,**P<0.01vs.IL-1β组,##P<0.01vs.NC组;
图5为α-SMA在大鼠软骨细胞中的表达,数值以平均值±标准差表示,**P<0.01vs.IL-1β组,##P<0.01vs.NC组;
图6为大鼠软骨细胞中纤维化相关蛋白的表达,其中左图为WB结果,右图中由上至下由左至右分别为软骨细胞中COL1、COL3、α-SMA、CTGF、MMP13、p-SMAD2、SMAD2、VIMENTIN的表达结果,以平均值±标准差表示,*P<0.05和**P<0.01vs.IL-1β组,#P<0.05和##P<0.01vs.NC组。
具体实施方式
本发明提供了猪血小板裂解液在制备防治膝骨关节炎、防治软骨纤维化、防治软骨基质降解或软骨修复产品中的应用。
在本发明中,所述产品优选的包括药物。在本发明中,所述猪血小板裂解液中血小板衍生生长因子AA、BB和AB的浓度分别优选的为1108.83pg/ml、1289.33pg/ml和159.85pg/ml。本发明所述猪血小板裂解液的制备方法优选的包括如下步骤:将猪外周血置于抗凝管中,4℃静置2h,离心后取上清,反复冻融后灭活,无菌处理得猪血小板裂解液。
在本发明中,所述猪优选的为巴马香猪,抗凝管优选的为含有4%柠檬酸钠的抗凝管。在本发明中,所述离心的条件优选为185g离心5min,所述反复冻融的过程优选为-80℃冻存过夜,37℃水浴融化,如此反复进行三次,然后离心取上清,所述离心的条件优选为2000g、5min,将上清进行灭活,所述灭活的条件优选为56℃灭活30min,更优选的,每隔5min内摇一摇使其受热均匀,灭活完后优选的2000g、5min离心取上清进行无菌处理,所述无菌处理优选的为滤过无菌处理。
在本发明中,所述猪血小板裂解液优选的通过抑制SMAD2信号的表达来抑制软骨纤维化和软骨基质降解。
本发明还提供了一种治疗膝骨关节炎、软骨纤维化、软骨基质降解或软骨修复的药物,所述药物以猪血小板裂解液为唯一活性成分。
本发明对于药物中的其余辅料没有特殊限定,采用本领域常规药用辅料均可。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
下述实施例中,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中所有数据均以均值±标准差(SD)表示。采用最新版本的SPSS(R27.0.0.0)软件对数据进行统计分析。采用独立样本t检验评价组间差异。P<0.05或P<0.01认为差异有统计学意义。
实施例1
取巴马猪(6周龄,雌性)外周血,将血样置于含有4%柠檬酸钠的50ml离心管中(加入比例:全血:4%柠檬酸钠=9:1),4℃下静置2h,随后185g离心5min,离心后收集富血小板上清,转移至15ml离心管中进行冻融步骤(-80℃冻存过夜,37℃水浴融化,反复三次后2000g,5min离心取上清),随后进行灭活步骤(56℃水浴锅,30min,要隔5min内摇一摇使其受热均匀,灭活完2000g,5min离心取上清),随后进行滤过无菌处理,得猪血小板裂解液(pPL),-80℃保存备用。
对比例1
与实施例1的区别在于外周血取自SD大鼠(3周龄,雄性),其余步骤均同实施例1。得鼠血小板裂解液(rPL),-80℃保存备用。
实施例2
分别采用猪和大鼠的ELISA试剂盒(Dogesce,Beijing,China)检测实施例1所得pPL和对比例1所得rPL中血小板衍生生长因子-AA(PDGF-AA)、血小板衍生生长因子-BB(PDGF-BB)和血小板衍生生长因子-AB(PDGF-AB)的浓度。采用酶标仪(Bio-RadLaboratories,Inc.,Hercules,CA,USA)在450nm处测量光密度(OD)。通过绘制标准曲线计算样品中生长因子的浓度。结果如表1所示,pPL和rPL中PDGF-AA的浓度分别为1108.83pg/ml和603.34pg/ml,PDGF-BB的浓度分别为1289.33pg/ml和1120.84pg/ml,PDGF-AB的浓度分别为159.85pg/ml和2169.25pg/ml。
表1ELISA检测pPL和rPL中PDGF-AA、PDGF-BB、PDGF-AB的浓度
实施例3
采用大鼠软骨部分缺损(PTCD)模型诱导膝骨关节炎(kOA)模型。将大鼠左、右后肢进行消毒,沿髌骨内侧缘切开股四头肌肌腱,在膝关节中间切开1cm,露出关节囊。使用直径1.6mm的圆形钻头在股骨内侧髁上造成软骨部分缺损(深度0.1mm)。逐层缝合切口。待创面恢复后(造模后3天)进行给药治疗。
24只雄性SD大鼠随机分为4组(n=6):Sham组为正常对照组,模型组为软骨部分缺损模型组,pPL组为pPL(实施例1)治疗的模型组,rPL组为rPL(对比例1)治疗的模型组。正常对照组和模型组关节腔内注射生理盐水,给药组关节腔内注射pPL和rPL。只注射一次,注射量为50μL,注射两周后,所有大鼠麻醉处死,进行关节取样。
疼痛行为评价
采用von Frey法评估各组大鼠治疗前后的疼痛阈值。疼痛反应采用von Frey细丝(0.6-26g,UGO Basile,Italy)的“上下”模式进行测量。老鼠被放置在一个隔离板上,隔离板上有铁丝网架。待老鼠适应后,用各个型号von Frey丝刺各组大鼠足底表面,诱导其产生逃逸反应。统计数据,计算50%疼痛阈值(PWT)。
组织病理学和免疫组织化学分析
处死大鼠后,取出膝关节,放入4%多聚甲醛中浸泡48h,再用10%乙二胺四乙酸(EDTA)脱钙3个月。脱钙完成后,将样品切成4~5μm的病理切片,随后分别进行HE(苏木精-伊红)和SO(番红-固绿)染色,显微镜(Carl Zeiss,Gottingen,Germany)下观察。根据OARSI和Mankin′s评分标准,采用盲法统计来确定每张病理切片的组织病理学特征。通过免疫组织化学分析检测I型胶原酶(COL1)和III型胶原酶(COL3)的表达。将每张病理切片进行抗原修复,用不同一抗(COL1和COL3)在4℃孵育过夜,然后用相应的二抗室温孵育2h,最后用3,3′-二氨基联苯胺(DAB)进行显色,显微镜下观察。观察pPL组和rPL组与模型组相比的软骨表面恢复情况。通过统计阳性面积定量COL1和COL3的表达,所选区域阳性面积占总面积的比例用于计算最终结果。
结果如图1和图2所示。从模型组的HE和SO染色中可以看出软骨缺损和胶原破裂明显(图1A),相对于Sham组,模型组的OARSI和Mankin′s评分(图1B)也显著升高(p<0.01vs.Sham组)。而pPL组和rPL组的HE和SO染色结果显示软骨缺损和胶原破裂均得到了较为明显的修复,相对于模型组,OARSI和Mankin′s评分也均显著降低(p<0.01vs.Model组)(图1B)。免疫组织化学分析结果显示,模型组软骨组织中COL1和COL3的表达明显增多(p<0.01vs.Sham组)(图2B),而pPL组和rPL组软骨组织中COL1和COL3的异常表达明显恢复了(p<0.01vs.Model组)(图2B)。疼痛测定的结果显示,模型组大鼠感受到的痛阈值明显低于Sham组(p<0.01vs.Sham组),而pPL和rPL组可以降低模型大鼠对疼痛的灵敏度(p<0.05vs.Model组)(图1B)。从组织病理学和疼痛测定结果可以看出pPL和rPL均对PTCD大鼠模型有软骨修复和镇痛作用,免疫组织化学分析结果也表明pPL和rPL对PTCD大鼠模型的作用可能与抗软骨纤维化有关。
动物样品的Westernblot(WB)分析
为探索pPL对kOA大鼠的体内抗纤维化作用机制,采用WB实验检测大鼠关节软骨中纤维化相关蛋白COL1、COL3、α-SMA、CTGF、p-SMAD2、SMAD2的表达。处死大鼠后,提取软骨总蛋白,对其定量,变性。用SDS-PAGE分离靶蛋白,随后将靶蛋白转移到硝酸纤维素膜上,用5%脱脂奶粉在室温下封闭1h,随后用一抗β-ACTIN、COL1、COL3、α-SMA、CTGF、p-SMAD2、SMAD2在4℃冰箱孵育过夜,再与相应的二抗室温孵育2h。最后膜用ECL试剂盒孵育,用蛋白质扫描仪(BIO-RAD,California,USA)扫描出相应的蛋白条带。最后,使用ImageQuant TL7.0分析软件(GE,United States)对蛋白条带进行统计分析。
结果如图3所示,模型组COL1、COL3、α-SMA、SMAD、P-SMAD、CTGF蛋白表达均显著上调(P<0.01或0.05vs.Sham组),而pPL明显恢复了这些异常表达(P<0.01或0.05vs.Model组)。以上数据表明pPL是通过抑制SMAD2信号的表达来抑制软骨纤维化。
实施例4
原代软骨细胞的制备
采用过量麻醉(3%戊巴比妥)处死3周龄SD大鼠。将软骨切成1mm3大小,用胰蛋白酶(0.25%)和II型胶原酶(COL2:1%~2%)消化,方法为:通过过量麻醉(3%戊巴比妥)处死3周龄SD大鼠,随后浸泡在75%的乙醇里,在超净台里进行软骨细胞提取工作。暴露大鼠的膝关节股骨髁,用灭菌手术刀片刮下股骨表层的透明软骨层,放入加2%双抗的PBS中进行清洗6-10次,用手术刀片将软骨处理成1mm3大小,加入6ml胰蛋白酶(0.25%)进行消化处理30min,弃上清后加入6ml的二型胶原酶(COL2:1%-2%)消化4h,每隔2h用70μm细胞筛进行细胞过滤,最后加入10%IMDM培养基即可进行软骨细胞的贴壁培养,当细胞生长至足够的细胞量,则可进行细胞实验。消化后的软骨细胞用IMDM培养基(10%胎牛血清)培养。
体外实验
采用CCK-8实验检测PL对软骨细胞活力的影响。制备软骨细胞细胞悬液,每孔接种3.5×103个细胞/200μl培养24h,随后将pPL(实施例1)和rPL(对比例1)梯度稀释成(×10、×50、×100、×150、×200、×250、×300)。加药24h和48h后,每孔加入100μl CCK-8(10%)试剂,在37℃孵育2h后,采用酶标仪标检测450nm处的光密度值(OD)。细胞活力指数=处理OD/未处理OD。
采用qPCR实验检测相关基因表达,将软骨细胞分为NC组、IL-1β组和pPL组。将软骨细胞以5×105个/皿的密度培养24h。IL-1β组和pPL组通过IL-1β(10ng/ml)预处理7h,随后pPL组给予10%稀释浓度的pPL培养24h,提取软骨细胞总RNA,并使用NanoDrop ND-2000(NanoDrop Technologies,DE,USA)对RNA进行定量,qPCR检测程序为:提取软骨细胞总RNA,并使用NanoDrop ND-2000(NanoDrop Technologies,DE,USA)对RNA进行定量,随后将totalRNA用CDNA逆转录试剂盒合成CDNA,最后用2×SYBR Green qPCR Master Mix(low ROX)试剂盒和β-ACTIN、COL1、COL3、MMP13、VIMENTIN、α-SMA和CTGF的基因引物来配制qPCR反应液,采用QuantStudio 7Flex qPCR(Thermo Fisher Scientific,MA,USA)软件进行相关DNA的合成和数据分析,所用引物序列见表2。以β-ACTIN作为内参基因。β-ACTIN、COL1、COL3、MMP13、VIMENTIN、α-SMA和CTGF的分析采用QuantStudio 7Flex qPCR系统(Thermo FisherScientific,MA,USA)。
表2目的基因引物序列
在细胞免疫荧光实验中将软骨细胞以2×104个/孔的密度接种在24孔板中培养24h。软骨细胞按上述方法进行分组、造模和处理。给药处理24h后,对各组软骨细胞进行固定、穿透和封闭。封闭结束后,用α-SMA的一抗(1:400,v/v)在4℃冰箱中孵育过夜,次日用FITC偶联的抗兔IgG(H+L)(1:100,v/v)避光孵育2h。最后将细胞培养板带到暗室进行DAPI染色,在488nm激发波长的荧光显微镜下观察细胞爬片,每组5个重复。
在WB实验中将软骨细胞以5×105个/皿的密度接种在10cm皿中培养24h。软骨细胞按上述方法进行分组、造模和处理。后续实验处理如上,蛋白膜在经5%脱脂奶粉封闭后,与相对应一抗进行4℃孵育过夜:β-ACTIN、COL1、COL3、MMP13、VIMENTIN、α-SMA、CTGF、p-SMAD2、SMAD2,次日与相应的二抗室温孵育2h。随后,蛋白膜用ECL试剂盒孵育,用蛋白质扫描仪(BIO-RAD,California,USA)扫描蛋白条带。最后,使用ImageQuant TL 7.0分析软件(GE,United States)对蛋白条带进行统计分析。
结果如图4-图6所示。CCK-8实验结果表明在稀释浓度范围为(×10,×50,×100,×150,×200,×250和×300)内的pPL,在24h和48h时可以以剂量依赖的方式提高软骨细胞的细胞活力(P<0.01或0.05vs.control组)(图4A),并且在48h时,pPL的效果优于rPL。PCR实验结果表面光IL-1β组显著上调了COL1、COL3、CTGF、MMP13、VIMENTIN的表达(P<0.01vs.NC组),pPL组恢复了这些异常表达的基因(P<0.01vs.IL-1β组)(图4B)。细胞免疫荧光结果显示IL-1β组中α-SMA的绿色荧光表达明显增加P<0.01vs.NC组),pPL组恢复了α-SMA的绿色荧光表达量(P<0.01vs.IL-1β组)(图5)。WB实验结果表明IL-1β处理的软骨细胞中COL1、COL3、MMP13、VIMENTIN、α-SMA、CTGF、p-SMAD2、SMAD2的表达显著升高(P<0.01或0.05vs.NC组),而pPL恢复了这些异常表达的纤维化蛋白(P<0.01或0.05vs.IL-1β组)(图6)。以上结果均表明pPL可以抑制软骨纤维化和软骨基质降解,其机制可能与SMAD2信号通路有关。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.猪血小板裂解液在制备防治膝骨关节炎产品中的应用。
2.猪血小板裂解液在制备防治软骨纤维化产品中的应用。
3.猪血小板裂解液在制备防治软骨基质降解产品中的应用。
4.猪血小板裂解液在制备软骨修复产品中的应用。
5.根据权利要求1-4任意一项所述的应用,其特征在于,所述猪血小板裂解液中血小板衍生生长因子AA、BB和AB的浓度分别为1108.83pg/ml、1289.33pg/ml和159.85pg/ml。
6.根据权利要求2或3所述的应用,其特征在于,所述猪血小板裂解液通过抑制SMAD2信号的表达来抑制软骨纤维化和软骨基质降解。
7.根据权利要求1-4任意一项所述的应用,其特征在于,所述猪血小板裂解液的制备方法包括如下步骤:将猪外周血置于抗凝管中,4℃静置2h,离心后取上清,反复冻融后灭活,无菌处理得猪血小板裂解液。
8.根据权利要求7所述的应用,其特征在于,所述猪包括巴马香猪。
9.根据权利要求7所述的应用,其特征在于,所述反复冻融为-80℃冻存过夜,37℃水浴融化,反复进行三次。
10.一种治疗膝骨关节炎、软骨纤维化、软骨基质降解或软骨修复的药物,其特征在于,所述药物以猪血小板裂解液为唯一活性成分。
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