CN110585423A - Il-6在制备治疗腰椎间盘突出药物中的应用 - Google Patents
Il-6在制备治疗腰椎间盘突出药物中的应用 Download PDFInfo
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Abstract
本发明公开了IL‑6在制备治疗腰椎间盘突出药物中的应用。IL‑6通过JAK/STAT信号通路调控TSLP的表达,TSLP可以通过诱导单核细胞趋化因子‑1(monoeyte chemotactic protein‑1,MCP‑1)的表达升高来促进巨噬细胞向椎间盘组织浸润,进而促进突出腰椎间盘组织的重吸收过程。
Description
技术领域
本发明涉及医学研究领域,具体的涉及IL-6在制备治疗腰椎间盘突出药物中的应用。
背景技术
腰椎间盘突出是指腰椎间盘退行性改变或外伤所致纤维破裂造成髓核从椎间盘破裂脱出,压迫腰椎神经,从而出现要退放射性疼痛,此称为腰椎间盘突出症。目前,只有少数脱出型椎间盘突出症及经保守治疗症状未缓解的腰椎间盘突出症患者接受手术治疗。俞振翰等研究表明,突出的椎间盘组织可通过重吸收过程达到自愈,但其潜在机制仍未明确。王志伟和元虎提出,巨噬细胞浸润参与重吸收的过程,可以使突出的椎间盘组织明显缩小。这可能与巨噬细胞直接或间接介导的一些因子释放有关,包括基质金属蛋白酶(mmatrixmetalloproteinases,MMPs)和血管内皮生长因子(vascular endothelial growthfactor,VEGF)。
胸腺基质淋巴细胞生成素(thymic stromal lym-phopoietin,TSLP)是1994年由Friend等在小鼠胸腺基质细胞的上清液中发现的IL-7样细胞因子,主要由非造血细胞分泌,如纤维母细胞、上皮细胞和基质细胞等。TSLP mRNA主要在胸腺、肺、皮肤、小肠等组织的上皮细胞及基质细胞和肥大细胞中表达,并与哮喘、气道变态炎性反应密切相关。Moret等发现,TSLP在风湿关节炎中扮演重要角色;Koyama等在对类风湿关节炎和骨关节炎患者滑膜成纤维细胞的体外实验研究中证实了TSLP的表达增加,说明在骨组织中也有TSLP的表达,并且参与炎症反应。Ohba等研究证实,在小鼠及人类的椎间盘组织中同样有TSLP的表达,并且在体外实验中证实肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)可通过核因子-κB(nuclear factor-κB,NF-κB)上调TSLP的表达,而TSLP可通过促使单核细胞趋化因子-1(monocyte chemotactic protein-1,MCP-1)的表达增加而引起巨噬细胞向椎间盘组织浸润,同时作者在椎间盘突出患者的椎间盘组织中也发现了TSLP表达增加,认为TSLP为介导重吸收的重要因子。然而,椎间盘突出后,机体是否上调了TSLP的表达、如何调控TSLP,目前我们仍不知道。
IL-6是炎症发生过程中另一个重要的炎症因子,当出现因感染或损伤而引起的组织损伤和炎症时,机体会诱导IL-6生成并发挥细胞增殖,分化和凋亡,免疫应答和急性期反应,同时表现出促炎和抗炎的双重作用。Weber等人发现腰椎间盘突出症患者血清IL-6明显升高。祝勇教授所在的团队发现汉族人群中IL-6的基因多态性(rs1800796,rs1524107,rs2069840)所致的IL-6启动子区域中的遗传变异可能与腰椎间盘突出症的发生风险升高有关,并导致异常的细胞转录和表达,从而影响个体对各种疾病的易感性。现在多将IL-6的较高表达水平作为腰椎间盘突出的临床诊断依据,IL-6在制备治疗腰椎间盘突出药物中的应用还未见研究报道。
发明内容
本发明的目的是提供IL-6在制备治疗腰椎间盘突出药物中的应用。
申请人在前期研究时发现,使用炎症因子TNF-α刺激鼠椎间盘组织模拟腰椎间盘突出症的炎性环境时,通过ELISA法观测到与对照组相比,实验组培养上清液中TSLP、MCP-1以及IL-6升高。申请人由此假设:IL-6参与到了腰椎间盘突出症的重吸收过程中,并通过上调TSLP的表达来促进重吸收过程。
本发明研究了IL-6介导的JAK/STAT信号通路通过对TSLP的表达调控,从而参与突出椎间盘的重吸收过程,提供了IL-6在制备治疗腰椎间盘突出药物中的应用。
进一步的,IL-6通过JAK/STAT信号通路调控TSLP的高表达从而促进突出椎间盘组织的重吸收。
与现有技术相比,本发明的有益效果为:本发明提供了IL-6在制备治疗腰椎间盘突出药物中的应用,揭示了IL-6介导的JAK/STAT信号通路通过调控TSLP的表达参与突出椎间盘的重吸收过程的机理,为今后研究腰椎间盘突出的病理生理过程提供新的分子理论基础,并为寻求最佳抗炎时机及今后利用机体自生修复机制保守治疗腰椎间盘突出提供新的靶点。
附图说明
图1是大鼠髓核细胞培养图,2x为2倍放大,4x为4倍放大。
图2是免疫荧光检测鉴定髓核细胞图,其中A为CollagenII染色(200倍),B为细胞核染色(200倍),C为CollagenII染色(400倍),D为细胞核染色(400倍)。
图3是免疫组化法检测组织中NF-κB磷酸化水平图,细胞核染色为棕黄色或棕褐色为阳性,ABC法x40,A为对照组,B为TNF-α组,C为TGF-β组,D为TGF-β抑制剂组(HTS组)。
图4是使用10ng/ml TNF-α、10ng/ml TGF-β、1uM HTS(TGF-β通路抑制剂)培养鼠椎间盘组织0、3、6、12、24h后,免疫印迹法(Western-blot)检测p-NFκB蛋白表达情况。
图5是IL-6、JAK/STAT抑制剂(BP-1-102)作用下使用qRT-PCR技术检测各基因表达情况图。A为IL-6(10ng/ml)刺激下,JAK 1、STAT 3、TSLP mRNA的表达情况;B为JAK/STAT抑制剂(BP-1-102,500nM)作用下STAT 3、TSLP mRNA的表达情况。(*表示P<0.05,**表示P<0.01,***表示P<0.001)
图6是IL-6、JAK/STAT抑制剂(BP-1-102)作用下使用Western-blot技术检测TSLP蛋白的表达情况图。
图7是IL-6、TGF-β/SMAD通路抑制剂、JAK/STAT通路抑制剂作用72h后细胞培养上清液中TGF-β的表达情况图。(*表示P<0.05)。
具体实施方式
下面结合附图和具体实施方式对本发明技术方案做进一步详细描述:
一、主要材料及试剂
DMEM/F12、胰蛋白酶、胎牛血清(Gibico,USA);Trizol(Ambion,USA);反转录试剂盒(Termo,USA)qPCR试剂盒(KAPA,瑞士);II型胶原酶(Biofoxx,瑞典);鼠抗-Smad2、兔抗-Smad7、鼠抗-p-STAT3、兔抗-TSLP、HRP标记的二抗(上海生工生物工程有限公司,上海,中国);培养瓶、培养板(NEST,Thermo,USA)。
二、大鼠髓核细胞的分离及培养
4周龄雌性SD大鼠(由西安交通大学动物实验中心提供)腹腔过量注射麻醉剂处死,75%乙醇浸泡10min。无菌实验台上延背部分离脊柱,剥离脊柱周围附着组织,无菌PBS冲洗3次,显微镜下分离椎间盘(保留上下终板),切开纤维环,小心收集胶冻状髓核组织至1.5ml离心管,将收集好的的髓核组织先后使用有抗生素和无抗菌素的PBS液清洗髓核组织3遍,1500rpm,离心5min,弃上清;加入0.25%胰酶37℃消化30min,每15min摇晃一次,使用含血清的DMEM终止消化后1000rpm离心5min,弃上清;使用含0.2%II型胶原酶的DMEM/F12(20%FPS、双抗)培养基重悬,37℃静置消化4h,每隔1h摇晃一次,待絮状组织消失后用70um细胞滤网过滤,滤过液900rpm,离心5min,弃上清;DMEM/F12(20%FPS、双抗)重悬,接种于25cm2透气培养瓶中,置于37℃、饱和湿度、体积分数5%的CO2培养箱中。5天后第一换液,以后每2天换液一次。于显微镜下观察细胞贴壁及生长情况,如图1所示。大鼠髓核细胞附壁生长融合率达到80%时将细胞进行传代培养。PBS液清洗两次,弃上清液,0.25%胰蛋白酶消化细胞。显微镜下观察,消化1-2min,可看到细胞相互分离变圆,即消化完成。快速弃去胰酶,加入完全培养基,吹打细胞,制成单细胞悬液,按1:4的比例传代,37℃、5%CO2饱和湿度条件下扩大培养,取第二、三代大鼠髓核细胞进行后续试验。
三、免疫荧光标记鉴定髓核细胞
将培养好的对数生长期的大鼠髓核细胞用0.25%胰蛋白酶消化处理,收集于1.5ml EP管中,无菌玻片酸化处理后,将细胞滴加在玻片上。放入37℃,5%CO2培养箱培养。待细胞附壁融合率达到80%时,取出玻片,弃上清。PBS浸洗3次,每次3min。用4%多聚甲醛滴加固定玻片,15分钟后PBS再次浸洗玻片3次,每次3min。洗掉多余的多聚甲醛。0.5%Triton X-100(PBS配制)室温通透20min。PBS浸洗玻片3次,每次3min。使用吸水纸吸干多余的PBS,将提前准备好的正常山羊血清滴加在玻片上,完成后将玻片放置于室温下封闭。30min后再次使用吸水纸吸掉封闭液,不经过润洗,再次在每张玻片上滴加足够量的兔抗鼠Ⅱ型胶原抗体和聚合蛋白抗体(1∶100)并放入湿盒,4℃孵育过夜。次日取出,PBS浸洗玻片3次,每次3min。吸水纸吸干爬片上多余PBS,将稀释好的荧光(Cy3)标记羊抗兔IgG(1∶100)滴加在玻片上,从加荧光二抗起,后面所有后续操作步骤都转移到暗室进行。将加好二抗的玻片置于湿盒中20-37℃条件下进行孵育1h。取出后使用PBS浸洗玻片3次,每次3min,滴加DAPI对标本进行染核。避光孵育5min后,PBS润洗玻片6次,每次5min,洗去多余的DAPI;完成后将玻片上的多余液体用吸水纸吸干。用含抗荧光淬灭剂的封片液封片,然后在荧光显微镜下观察制备好的玻片并采集图像,如图2所示。
四、冰冻研磨提取大鼠椎间盘组织蛋白
4周龄雌性SD大鼠(由西安交通大学动物实验中心提供)腹腔过量注射麻醉剂处死,75%乙醇浸泡10min。无菌实验台上延背部分离脊柱,剥离脊柱周围附着组织,无菌PBS冲洗3次,显微镜下分离椎间盘,全程注意无菌操作。分离椎间盘使用含双抗的DMEM冲洗3次,分别接种于24孔板中,每孔1个椎间盘组织,分别加入含IL-6(10ng/ml)、TGF-β通路抑制剂(SB431542,150ng/ml)、JAK/STAT通路抑制剂(BP-1-102,500ng/ml)的DMEM,37℃、5%CO2培养72h,用眼科剪将组织剪成1mm3大小,称取0.1g组织置于灭菌研钵中,加入液氮10ml迅速旋转研磨至粉状,加入含1%蛋白酶抑制剂(PMSF)的细胞裂解液150ul充分裂解细胞(全程冰上操作),4℃,12000rpm离心5min,BCA法测定蛋白浓度,所提蛋白用于Western-Blot试验。
五、实时荧光定量PCR(qRT-PCR)
调整细胞数量至2.5x 105细胞/ml,接种于6孔板中,37℃、5%CO2培养12h生长至70%,分别加入IL-6(10ng/ml)、TGF-β通路抑制剂(SB431542,150ng/ml)、JAK/STAT通路抑制剂(BP-1-102,500ng/ml),对照组使用α-MEM,37℃、5%CO2培养72h,使用Trizol法提取mRNA,使用qRT-PCR技术检测JAK1、JAK2、STAT3、SMAD2/3/7、TSLP mRNA的表达情况。引物由北京擎科新业生物技术有限公司合成(引物序列见表1)。具体条件如下:95℃、3min,1个循环;95℃、3s和60℃、20s,40个循环,使用非特异性扩增法的解离曲线测定。
六、免疫印记试验(Western-Blot)
调整细胞数量至2.5x 105细胞/ml,接种于6孔板中,37℃、5%CO2培养12h生长至70%,分别加入IL-6(10ng/ml)、TGF-β通路抑制剂(SB431542,150ng/ml)、JAK/STAT通路抑制剂(BP-1-102,500ng/ml)37℃、5%CO2培养72h,使用PBS清洗3次,加入含1%蛋白酶抑制剂(PMSF)的细胞裂解液充分裂解细胞(全程冰上操作),4℃,12000rpm离心5min,10%SDS-聚丙烯酰胺凝胶(SDS-PAGE)上分离1h,湿转印法1.5h转印至聚偏二氟乙烯膜(PVDF膜),5%脱脂奶粉室温封闭1h,加入一抗,4℃孵育过夜,使用Tris-HCl缓冲盐溶液(TBST)中洗涤3次,加入HRP标记的二抗室温孵育1小时,TBST洗涤3次,每次5min,曝光拍照。
七、酶联免疫吸附实验(ELISA)
收集上述72h细胞培养上清液,每个聚苯乙烯板的反应孔中分别加入IL-6(10ng/ml)、TGF-β通路抑制剂(SB 431542,150ng/ml)、JAK/STAT通路抑制剂(BP-1-102,500ng/ml)细胞培养上清液100ul,4℃避光过夜。次日,37℃避光孵育1h,弃上清,1%TBST清洗3次,每次3min,加入一抗,37℃避光孵育1h,TBST清洗3次,加入二抗37℃避光孵育1h后,避光加入显色剂,37℃显色30min,加入终止剂并在分光光度计上检测OD 450nm值。
统计学分析
采用Excel统计软件(Microsoft,USA)建立数据库并进行统计分析。两组均数比较用独立样本t检验,P<0.05为差异有统计学意义。
结果分析
一、IL-6介导的JAK/STAT信号通路通过调节TSLP的表达促进突出椎间盘组织的重吸收过程。
分别使用IL-6、JAK/STAT通路抑制剂(BP-1-102)处理大鼠MSC后,JAK/STAT通路相关因子JAK1、STAT3及TSLP mRNA的表达(见图5)。IL-6刺激可上调大鼠细胞中JAK1、STAT3、TSLP mRNA的表达。而在JAK/STAT通路的抑制(BP-1-102)存在时STAT 3、TSLP的表达明显下降。在蛋白表达方面,使用免疫印记试验(Western-blot)同样观察到IL-6刺激下,TSLP蛋白的表达明显增加,而在JAK/STAT抑制剂存在的情况下,TSLP蛋白的表达则明显减弱(见图6)。这提示IL-6介导的JAK/STAT信号通路通过上调TSLP的表达参与了突出椎间盘组织的重吸收进程。
二、内源性TGF-β对NF-κB通路具有抑制作用
申请人前期实验发现,将鼠椎间盘组织与10ng/ml TNF-α、10ng/ml TGF-β、1uMHTS(TGF-β通路抑制剂)共培养6h后,使用免疫组化法检测组织中NF-κB磷酸化水平,细胞核染色为棕黄色或棕褐色为阳性,结果显示:TNF-α组阳性细胞数量最多,其次为TGF-β抑制剂组(HTS组),而TGF-β组、对照组仅见少量阳性细胞,如图3所示。对照组仅见少量磷酸化NF-κB阳性细胞,为9.4%±3.3%;BTNF-α组磷酸化NF-κB阳性细胞最多,为92.4%±1.4%;TGF-β组表达磷酸化NK--κB阳性细胞数量较少,为33.7%±8.8%;TGF-β抑制剂组(HTS组)阳性细胞数量仅次于TNF-α组为74.1%±6.7%。
各组比较差异有统计学意义(F=167.669,P=0.000);任意两组比较差异均有统计学意义(均P=0.000)。
鼠椎间盘组织与10ng/ml TNF-α、10ng/ml TGF-β、1uM HTS(TGF-β通路抑制剂)共培养0、3、6、12、24h后,冰冻研磨法提取组织蛋白,使用免疫印迹试验(Western-blot)检测磷酸化NF-κB蛋白表达,如图4所示:培养3、6h后,TNF-α组、TGF-β抑制剂组(HTS组)与对照组相比,磷酸化NF-κB蛋白表达明显增加,这提示内源性TGF-β会抑制NF-κB的磷酸化。
结果显示在培养3、6h后,TGF-β抑制剂组(HTS组)和TNF-α组与对照组相比,磷酸化NF-κB表达明显增高,表明抑制了TGF-β的活性(HTS组)与TNF-α组均具有诱导磷酸化NF-κB蛋白表达的作用。
三、IL-6对大鼠髓核细胞中TGF-β表达的影响。
分别使用IL-6(10ng/ml)、TGF-β通路抑制剂(SB431542,150ng/ml)、JAK/STAT通路抑制剂(BP-1-102,500ng/ml)培养大鼠髓核细胞,收集72h时细胞培养上清液,应用酶联免疫吸附实验(ELISA)检测上清液中TGF-β浓度,结果显示:在IL-6刺激下,细胞培养上清液中TGF-β浓度升高,TGF-β通路抑制剂组、JAK/STAT通路抑制剂组较对照组降低(见图7)。
与对照组相比,IL-6刺激下细胞培养上清液中TGF-β浓度较对照组升高;抑制TGF-β、JAK/STAT通路后,观察到上清液中TGF-β浓度下降。
实验结论
一、IL-6通过JAK/STAT信号通路调控TSLP的表达参与突出椎间盘组织的重吸收。
研究表明,TSLP可以通过诱导单核细胞趋化因子-1(monoeyte chemotacticprotein-1,MCP-1)的表达升高来促进巨噬细胞向椎间盘组织浸润,进而促进突出椎间盘组织的重吸收过程。本实验中,申请人观察到在加入外源性IL-6刺激72h后,JAK 1、STAT 3以及TSLP mRNA呈现高表达状态,而在加入JAK/STAT通路抑制剂(BP-1-102)后,JAK/STAT通路关键因子STAT 3、TSLP mRNA的表达显著降低。免疫印记试验也证实,在加入IL-6刺激后,TSLP蛋白表达显著增加。这提示IL-6通过JAK/STAT信号通路诱导TSLP的表达上升,进而参与椎间盘突出的重吸收过程。
二、IL-6通过JAK/STAT信号通路影响TGF-β信号通路的活性,从而影响TSLP的表达,进而参与椎间盘突出的重吸收过程。
申请人的研究证实,生理条件下椎间盘组织中TGF-β信号通路呈现一定的活化状态,并抑制TSLP的表达。本次实验中,申请人观察到在加入IL-6刺激后,TGF-β通路标志物——Smad 2的mRNA表达显著升高,而在加入JAK/STAT通路抑制剂后(BP-1-102)后,Smad2的表达则明显下降,免疫印记试验(Western-blot)也显示在加入IL-6后Smad 2蛋白表达明显增加。同时在酶联免疫吸附实验(ELISA)中我们观察到加入IL-6后细胞培养上清液中TGF-β浓度升高(P<0.05),抑制JAK/STAT通路后上清液中TGF-β浓度下降(P<0.05)。由此可得出在IL-6刺激下,JAK/STAT信号通路被激活,进而引起TGF-β通路的激活。
正常生理条件下,TSLP的表达持续受到抑制,而这种抑制作用是由椎间盘组织持续、微量表达TGF-β造成的。当腰椎间盘突出时,这种抑制作用被打破,TSLP表达升高,进而使MCP-1表达升高,而后者可以诱导巨噬细胞向突出椎间盘组织浸润,引起并促进重吸收过程。本次实验中,申请人发现炎症因子IL-6可以激活JAK/STAT通路上调TSLP的表达,这为研究腰椎间盘突出症的炎症发生及重吸收机制提供新的思路,为保守治疗腰椎间盘突出症提供了新的靶点。另外,IL-6/JAK/STAT家族可影响TGF-β/Smads家族,这意味着IL-6在维持椎间盘局部微环境稳态中存在着潜在的重要作用。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何不经过创造性劳动想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应该以权利要求书所限定的保护范围为准。
序列表
<110> 内蒙古医科大学第二附属医院
<120> IL-6在制备治疗腰椎间盘突出药物中的应用
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Claims (2)
1.IL-6在制备治疗腰椎间盘突出药物中的应用。
2.根据权利要求1所述的IL-6在制备治疗腰椎间盘突出药物中的应用,其特征在于,IL-6通过JAK/STAT信号通路调控TSLP的高表达参与突出腰椎间盘组织的重吸收。
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