CN110613837A - TGF-β在制备预防或治疗腰椎间盘突出药物中的应用 - Google Patents
TGF-β在制备预防或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TGF‑β在制备预防或治疗腰椎间盘突出药物中的应用。TGF‑β可通过抑制TSLP表达，维持腰椎间盘稳态，从而预防腰椎间盘突出。TGF‑β抑制剂通过抑制TGF‑β的表达，从而提高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TGF-β在制备预防或治疗腰椎间盘突出药物中的应用。
背景技术
腰椎退行性疾病已成为影响人类生活质量的主要疾病之一，每年因腰椎退变疾病带来的损失巨大。因此，研究腰椎退变的病理生理机制对治疗腰椎退变疾病尤为重要。正常情况下，机体通过一套复杂精密的调节系统维持自身稳态平衡，保证各系统之间协调运作，最近的研究表明，机体在维持椎间盘稳态、修复椎间盘退变过程中同样存在着应对调节机制。
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(tumor necrosis 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。
发明内容
本发明的目的是提供TGF-β在制备预防或治疗腰椎间盘突出药物中的应用。
申请人在研究时发现，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，抑制了TGF-β的活性有诱导磷酸化NF-κB蛋白表达的作用,而NF-κB蛋白可上调TSLP的表达。申请人由此假设：生理状况下TGF-β通路持续活化，抑制NF-κB通路活性，从而特异性抑制TSLP表达，维持椎间盘稳态、延缓椎间盘退变；抑制TGF-β表达可影响腰椎间盘突出症的重吸收，并通过上调TSLP的表达来促进重吸收过程。
本发明研究了TGF-β通过对TSLP的表达进行调控，进而参与预防腰椎间盘退变和突出椎间盘的重吸收过程，提供了TGF-β在制备预防或治疗腰椎间盘突出药物中的应用。
进一步的，TGF-β可通过抑制TSLP表达，维持腰椎间盘稳态，从而预防腰椎间盘突出。
进一步的，TGF-β抑制剂通过抑制TGF-β的表达，从而提高TSLP的表达，进而参与腰椎间盘突出的重吸收过程。
与现有技术相比，本发明的有益效果为：本发明提供了TGF-β在制备预防或治疗腰椎间盘突出药物中的应用，揭示了TGF-β通过调控TSLP的表达参与预防腰椎间盘退变疾病和突出椎间盘重吸收过程的机理，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2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3是TNF-α组、TGF-β组、HTS组实验流程图。
图4是HTS组、HTS+IMD组、HTS+MG组实验流程图
图5是临床组实验流程图。
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：
1、实验材料准备
鼠椎间盘组织的分离及培养：
选取6周龄遗传工程小鼠(C57BL/6J)，脱颈处死后切取小鼠尾部，去除表皮及皮下组织，解剖显微镜下切取10个尾椎椎间盘置于12孔培养板中，将提取的椎间盘组织放入含有0.1％小牛血清的DMEM 1mL的12孔板内，37℃，5％CO2恒温箱内培养，得到鼠椎间盘组织培养板。
2、鼠椎间盘组织中TGF-β/Smad2/3通路状态
(1)TNF-α、TGF-β、TGF-β抑制剂(HTS-466284)培养鼠椎间盘组织
将鼠椎间盘组织培养板分为对照组、TNF-α组、TGF-β组、TGF-β抑制剂组(HTS组)。TNF-α组加入10ng/ml TNF-α，TGF-β组加入10ng/ml TGF-β、TGF-β抑制剂组(HTS组)加入1umol/L TGF-β抑制剂(HTS-466284)，37℃，5％CO2恒温箱内培养0、3、6、12、24小时。
(2)鼠椎间盘组织中p-Smad2/3表达的测定
①将培养后的小鼠椎间盘组织用PBS冲洗，加入组织裂解液，物理研磨后提取蛋白质，BCA法测定蛋白质浓度，10％～15％SDS-PAGE凝胶电泳1h，湿转印法1.5h转印至聚偏二氟乙烯(PVDF)膜。质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清室温封闭PVDF膜1h，加入抗p-Smad2/3抗体(质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清1∶1000稀释)，4℃孵育过夜，加入辣根过氧化酶标记的抗兔或鼠抗体(质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清1:2000稀释)，室温孵育1h，0.5％TBS/T洗膜，加入ECL曝光液，曝光显影。
②免疫组织化学染色法检测磷酸化Smad2/3的水平：将培养6h后的小鼠椎间盘组织用质量分数为4％的多聚甲醛固定，脱水，石蜡包被，组织切片，厚度为5umol/L。用质量分数为3％的过氧化氢(含质量分数为80％的甲醛处理切片，去除组织过氧过氢酶活性后，用抗p-Smad 2/3抗体或正常山羊血清4℃孵育过夜。常规ABC法染色：磷酸缓冲盐溶液(PBS)冲洗切片后，加入二抗室温下孵育20min，PBS冲洗3次各5min，DAB显色5～10min，PBS冲洗10min，苏木精复染2min，盐酸酒精分化，自来水冲洗10～15min后，脱水，透明，封片，镜检。
3、抑制内源性TGF-β活性对TSLP表达的影响
(1)TGF-β抑制剂和NF-κB抑制剂(IMD-0354，MG132)培养鼠椎间盘组织
将鼠椎间盘组织培养板分为对照组、HTS组、HTS组+IMD组(NF-κB抑制剂IMD-0354，IMD组)、HTS组+MG组(NF-κB抑制剂组MG132，MG组)，37℃，5％CO2恒温箱内培养72小时。
(2)鼠椎间盘组织中TSLP表达的测定
收集培养上清液，利用酶联免疫吸附法(ELISA)测定TSLP表达：按照试剂盒说明书步骤操作，在ELISA检测仪上，于450nm处，测定各孔吸光度值。
4、抑制内源性TGF-β活性对磷酸化NF-κB的影响
(1)TNF-α、TGF-β、TGF-β抑制剂(HTS-466284)培养鼠椎间盘组织
将鼠椎间盘组织培养板分为对照组、TNF-α组、TGF-β组、TGF-β抑制剂组(HTS组)。TNF-α组加入10ng/ml TNF-α，TGF-β组加入10ng/ml TGF-β、TGF-β抑制剂组(HTS组)加入1umol/L TGF-β抑制剂(HTS-466284)，37℃，5％CO2恒温箱内培养0、3、6、12、24小时。
(2)鼠椎间盘组织中磷酸化NF-κB表达的测定
①将培养6h后的小鼠椎间盘组织用质量分数为4％的多聚甲醛固定，脱水，石蜡包被，组织切片，厚度为5umol/L。用质量分数为3％的过氧化氢(含质量分数为80％的甲醛处理切片，去除组织过氧过氢酶活性后，用抗p-NF-κB或正常山羊血清4℃孵育过夜。常规ABC法染色：磷酸缓冲盐溶液(PBS)冲洗切片后，加入二抗室温下孵育20min，PBS冲洗3次各5min，DAB显色5～10min，PBS冲洗10min，苏木精复染2min，盐酸酒精分化，自来水冲洗10～15min后，脱水，透明，封片，镜检。
②将培养0、3、6、12、24小时后的小鼠椎间盘组织用PBS冲洗，加入组织裂解液，物理研磨后提取蛋白质，BCA法测定蛋白质浓度，10％～15％SDS-PAGE凝胶电泳1h，湿转印法1.5h转印至聚偏二氟乙烯(PVDF)膜。质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清室温封闭PVDF膜1h，抗p-NF-κB抗体(质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清1∶1000稀释)，4℃孵育过夜，加入辣根过氧化酶标记的抗兔或鼠抗体(质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清1:2000稀释)，室温孵育1h，0.5％TBS/T洗膜，加入ECL曝光液，曝光显影。
5、内源性TGF-β对TSLP、MCP-1、IL-6、MMP-3的影响
(1)TNF-α、TGF-β、TGF-β抑制剂(HTS-466284)培养鼠椎间盘组织
将鼠椎间盘组织培养板分为对照组、TNF-α组、TGF-β组、TGF-β抑制剂组(HTS组)。TNF-α组加入10ng/ml TNF-α，TGF-β组加入10ng/ml TGF-β、TGF-β抑制剂组(HTS组)加入1umol/L TGF-β抑制剂(HTS-466284)，37℃，5％CO2恒温箱内培养72小时。
(2)ELISA法测定鼠椎间盘组织中TSLP、MCP-1、IL-6、MMP-3的表达
收集培养上清液，利用酶联免疫吸附法(ELISA)测定TSLP表达：按照试剂盒说明书步骤操作，在ELISA检测仪上，于450nm处，测定各孔吸光度值。
6、腰椎爆裂性骨折患者和腰椎退变患者椎间盘组织中TSLP蛋白的表达情况
(1)腰椎爆裂性骨折患者和腰椎退变患者组织样本的采集
选取腰椎爆裂性骨折患者10例和腰椎退变患者患者50例，两组样本术前通过影像学检查、查体、体征初步确定，术中取样最终确定：腰椎爆裂性骨折患者接受手术获取椎间盘样本，椎间盘退变患者接受PLIF融合内固定术。
TUNEL法检测椎间盘组织细胞凋亡情况
取髓核组织标本，使用4％多聚甲醛常温固定组织20min，PBS清洗三次，每次10min，随后置于透性处理液中冰上孵育2min，透性处理液包含0.1％Triton-100和1％的柠檬酸钠。配制TUNEL反应液：5ul酶缓冲液(Enzyme Solution)加45ul标记液充分混合均匀。往组织切片上滴加TUNEL反应液，充分混合均匀，覆盖整个切片，湿盒内避光孵育60min。PBS清洗三次，加抗荧光猝灭封片液。显微镜观察拍照，激发波长570-620nm。
(3)腰椎爆裂性骨折患者和腰椎退变患者椎间盘组织中TSLP及p-Smad2/3的表达测定
利用免疫组织化学法及Western blot法检测TSLP及p-Smad2/3的表达水平。
①将培养后的小鼠椎间盘组织用PBS冲洗，加入组织裂解液，物理研磨后提取蛋白质，BCA法测定蛋白质浓度，10％～15％SDS-PAGE凝胶电泳1h，湿转印法1.5h转印至聚偏二氟乙烯(PVDF)膜。质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清室温封闭PVDF膜1h，加入抗p-Smad2/3抗体(质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清1∶1000稀释)，4℃孵育过夜，加入辣根过氧化酶标记的抗兔或鼠抗体(质量浓度为5％的脱脂牛奶或质量分数为3％的小牛血清1:2000稀释)，室温孵育1h，0.5％TBS/T洗膜，加入ECL曝光液，曝光显影。
②免疫组织化学染色法检测磷酸化Smad2/3的水平：将培养6h后的小鼠椎间盘组织用质量分数为4％的多聚甲醛固定，脱水，石蜡包被，组织切片，厚度为5umol/L。用质量分数为3％的过氧化氢(含质量分数为80％的甲醛处理切片，去除组织过氧过氢酶活性后，用抗p-Smad 2/3抗体或正常山羊血清4℃孵育过夜。常规ABC法染色：磷酸缓冲盐溶液(PBS)冲洗切片后，加入二抗室温下孵育20min，PBS冲洗3次各5min，DAB显色5～10min，PBS冲洗10min，苏木精复染2min，盐酸酒精分化，自来水冲洗10～15min后，脱水，透明，封片，镜检。
组织切片用抗TSLP抗体、抗p-Smad2/3抗体或正常山羊血清孵育。
(4)ELISA法、RT-PCR检测组织样本中TSLP及Smad2/3的表达
收集培养上清液，利用酶联免疫吸附法(ELISA)测定TSLP表达：按照试剂盒说明书步骤操作，在ELISA检测仪上，于450nm处，测定各孔吸光度值。
RT-PCR：组织剪碎、研磨后Trizol法裂解细胞，然后加入200ul氯仿，混匀，室温静置15min后12000rpm高速低温离心15min；小心将上层水相移至1.5ml离心管中，加入500ul异丙醇，振荡混匀。室温放置15min沉淀RNA，12000rpm高速低温离心10min；小心弃去上清液，再在离心管中加入1ml 75％乙醇，振荡片刻，8000rpm低速离心5min；小心弃去上清液，室温静置5～10min使RNA沉淀、干燥，用DEPC水溶解。将Trizol抽提的细胞总RNA反转录进行cDNA第一链的合成：1～5ug总RNA中加1ul Oligo(dT)，70℃10min，于冰上加入5×缓冲液4ul、dNTP2ul、反转录酶0.5ul，加水至总体积20ul，42℃延伸60min后，70℃10min。RT-PCR法进行扩增：cDNA第一链1ul，Ex Taq(5u/1μl)0.5ul，10×Ex Taq buffer 5ul，dNTP mixture(2mM)4ul，Primer F(10mM)2ul，Primer R(10mM)2ul(引物设计及合成由公司完成)，dd H2O35.5ul。RT-PCR反应条件：95℃热启动10min；95℃变性15s，65℃退火20s，72℃延伸10s，35-40个循环。72℃5min，4℃保存样本。
结果分析
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1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2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。
实验结论
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，申请人发现生理状况下TGF-β通路持续活化，抑制NF-κB通路活性，从而特异性抑制TSLP表达，维持椎间盘稳态、延缓椎间盘退变；抑制TGF-β表达可影响腰椎间盘突出症的重吸收，并通过上调TSLP的表达来促进重吸收过程，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。
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。
Claims (3)
1.TGF-β在制备预防或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TGF-β在制备预防或治疗腰椎间盘突出药物中的应用，其特征在于，TGF-β可通过抑制TSLP表达，维持腰椎间盘稳态，从而预防腰椎间盘突出。
3.根据权利要求1所述的TGF-β在制备预防或治疗腰椎间盘突出药物中的应用，其特征在于，TGF-β抑制剂通过抑制TGF-β的表达，从而提高TSLP的表达，进而参与腰椎间盘突出的重吸收过程。
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