【作者】 陈胜；

【导师】 邵增务；

【作者基本信息】 华中科技大学 ， 外科学（骨科）， 2022， 博士

【摘要】 第一部分:粘着斑相关蛋白Kindlin-2在退变椎间盘组织的表达目的:检测粘着斑相关蛋白Kindlin-2在人不同退变程度髓核(Nucleus pulposus,NP)标本和不同年龄小鼠椎间盘(Intervertebral disc,IVD)中的表达,探究Kindlin-2的表达变化与椎间盘退变(Intervertebral disc degeneration,IVDD)的相关性。方法:1.收集腰IVD突出病人的退变NP手术标本,根据病人术前MRI实行Pfirrmann分级,取II/III级为轻度退变组,IV/V级为重度退变组。对NP标本进行H/E和阿利新蓝染色进一步确定组织退变程度,进行粘着斑相关蛋白(Kindlin-1,2,3和Talin,Vinculin)免疫荧光染色检测其表达。2.对年轻小鼠(3月龄)和年老小鼠(20月龄)的腰IVD组织进行番红-固绿(Safranin O and fast green,SO&FG)染色和组织评分确定退变程度,进行上述粘着斑相关蛋白免疫荧光染色检测其表达。结果:1.在人退变NP标本中,H/E和阿利新蓝染色结果显示,重度退变组较轻度退变组NP细胞稀疏、细胞外基质(Extracellular matrix,ECM)少;荧光染色显示Kindlin-1和Kindlin-3阳性细胞几乎没有,Talin和Vinculin阳性细胞较少,Kindlin-2阳性细胞较多且随着退变严重程度增加而减少。2.在小鼠IVD标本中,SO&FG染色和组织评分显示年老小鼠较年轻小鼠IVD发生严重退变;免疫荧光染色显示Kindlin-1和Kindlin-3在小鼠IVD组织中几乎不表达,Talin和Vinculin表达较少,Kindlin-2主要在NP组织中表达且随着小鼠年龄增加而显著减少。结论:上述结果说明粘着斑相关蛋白Kindlin-2主要在IVD中的NP组织表达,并且随着退变程度和年龄增加而减少,提示Kindlin-2的表达减少和IVDD相关。第二部分:条件性敲除Kindlin-2对小鼠椎间盘组织退变的影响目的:通过构建IVD特异性Kindlin-2敲除小鼠,探究Kindlin-2在IVDD中的作用。方法:1.通过多次交配繁育出Kindlin-2fl/fl;AggrecanCre ERT2小鼠,在小鼠2月龄的时候注射玉米油(对照组)或他莫昔芬(敲除组),在小鼠2、3、5、8月龄的时候对腰椎和尾椎进行计算机化断层显像(Computerized tomography,CT)扫描,在小鼠3、5、8月龄的时候取材,进行免疫荧光染色、TUNEL染色、SO&FG染色、组织评分和Western Blot分析。2.为进一步探究异常应力的参与作用,在Kindlin-2fl/fl;AggrecanCre ERT2小鼠注射玉米油或他莫昔芬4周后对小鼠尾IVD进行针刺造成尾椎不稳,在手术前(12周龄)和手术后6周(18周龄)对尾椎进行CT扫描,在18周龄的时候取材,进行免疫荧光染色、TUNEL染色、SO&FG染色和组织评分。结果:1.在小鼠腰IVD中,与对照组相比,免疫荧光染色显示敲除组在3、5、8月龄时,Kindlin-2表达显著降低,敲除效果较好;SO&FG染色和组织评分显示,敲除组在5月龄时组织评分明显高于对照组,8月龄时更加显著;染色和Western Blot结果表明,敲除组IVD内ECM合成分解失衡,表现为ECM合成相关蛋白II型胶原(Collagen type II,Col2)和蛋白聚糖(Aggrecan,Acan)表达减少,ECM分解相关蛋白基质金属蛋白酶13(Matrix metalloproteinase 13,Mmp13)和含血小板结合蛋白基序的解聚素金属蛋白酶5(A disintegrin and metalloproteinase with thrombospondin motif 5,Adamts5)表达升高;荧光染色、TUNEL染色和Western Blot结果表明,敲除组IVD内细胞凋亡增加,表现为Caspase3和Bax水平升高,Bcl2水平降低,TUNEL阳性细胞增加;CT结果显示,IVD的高度没有显著变化。2.在小鼠尾IVD中,免疫荧光染色也显示Kindlin-2敲除效果较好;但SO&FG染色、组织评分以及CT结果显示,敲除组和对照组的尾IVD没有显著差异。3.其他结果显示针刺造成的异常应力可导致对照组小鼠尾IVD组织评分增加、IVD高度降低、ECM合成分解失衡和凋亡增加,而在敲除组中这些变化更加显著。结论:上述结果说明Kindlin-2敲除可导致小鼠腰IVD发生自发性退行性改变,在尾IVD中则没有发生显著改变;Kindlin-2敲除可以加速异常应力存在下的小鼠尾椎间盘退变。这些结果提示Kindlin-2缺失在IVDD的发生以及进展中发挥重要作用,尤其是在异常应力存在的情况下。第三部分:Kindlin-2缺失引起椎间盘退变的机制研究目的:探究Nlrp3炎症小体信号通路是否参与Kindlin-2缺失引起的IVD细胞和组织退变。方法:1.运用免疫染色分别评估人不同退变程度NP标本和Kindlin-2敲除小鼠NP组织中Nlrp3炎症小体通路激活相关蛋白水平。2.在大鼠NP细胞系中,运用加压装置模拟椎间盘内的异常压力,并运用Kindlin-2 si RNA或Kindlin-2质粒分别敲低或过表达Kindlin-2,检测NP细胞中Nlrp3炎症小体通路和ECM合成分解相关蛋白的表达以及细胞凋亡情况。3.在IL-1β或(和)异常压力处理条件下,检测NP细胞中Nlrp3炎症小体通路相关蛋白和Kindlin-2水平。4.运用Nlrp3炎症小体通路激活抑制剂MCC950分别在体外和体内阻断Nlrp3炎症小体通路激活,检测MCC950对伴或不伴异常应力条件下Kindlin-2缺失造成NP细胞和IVD组织退变的挽救情况。结果:1.与轻度退变人NP标本相比,重度退变组Nlrp3炎症小体通路激活相关蛋白表达明显升高;与对照组比,Kindlin-2敲除组小鼠NP组织中Nlrp3炎症小体通路激活相关蛋白水平显著升高。2.异常压力导致Kindlin-2和Col2a1表达减少、Mmp13表达增多、细胞凋亡增加以及Nlrp3炎症小体通路激活,Kindlin-2敲低可加重这些变化,而Kindlin-2过表达则会改善这些变化。3.IL-1β可以浓度和时间依赖性的下调Kindlin-2和上调Nlrp3炎症小体通路激活相关蛋白的表达,异常压力可加重这些变化。4.在体外实验中,MCC950抑制了Kindlin-2敲低伴或不伴异常压力刺激造成的Nlrp3炎症小体通路激活、Col2a1表达减少、Mmp13表达增多以及细胞凋亡增加。5.在体内实验中,MCC950抑制了Kindlin-2敲除伴或不伴异常应力刺激造成的Nlrp3炎症小体通路激活、组织评分增加、椎间盘高度降低、Col2a1表达减少、Mmp13表达增多以及细胞凋亡增加。结论:上述结果表明Kindlin-2缺失可激活Nlrp3炎症小体通路,引起IVD细胞和组织退变。此外,激活的Nlrp3炎症小体可释放IL-1β,使得Kindlin-2表达下降和Nlrp3炎症小体通路进一步激活,形成Kindlin-2/Nlrp3炎症小体/IL-1β恶性循环轴,造成IVDD。第四部分:过表达Kindlin-2对椎间盘退变修复作用的研究目的:探究腺相关病毒介导的Kindlin-2过表达对压力诱导的人原代NP细胞退变和大鼠尾椎加压模型造成的IVDD的保护作用,初步评估Kindlin-2在IVDD治疗中的临床转化意义。方法:1.人原代NP细胞感染Kindlin-2或对照组腺相关病毒后,进行常压或异常压力处理,检测Kindlin-2,Nlrp3,Col2a1,Mmp13和IL-1β的表达以及细胞凋亡。2.SD大鼠(3月龄)分三组,包括假手术组、大鼠尾椎加压模型+对照腺相关病毒感染组、大鼠尾椎加压模型+Kindlin-2腺相关病毒感染组。在病毒感染3周后手术,手术2周后进行MRI评估和取材,以及SO&FG染色、组织评分、免疫荧光染色和TUNEL染色。结果:1.在人原代NP细胞内,腺相关病毒介导的Kindlin-2过表达部分抑制了异常压力造成的Kindlin-2表达降低、Nlrp3炎症小体通路激活、Col2a1表达减少、Mmp13表达增多以及细胞凋亡增加。2.在大鼠尾椎加压模型中,腺相关病毒介导的Kindlin-2过表达部分逆转录了异常压力造成的椎间盘T2加权信号降低、组织评分增加、Kindlin-2表达降低、Nlrp3炎症小体通路激活、Col2a1表达减少、Mmp13表达增多以及细胞凋亡增加。结论:上述结果表明腺相关病毒介导的Kindlin-2过表达对IVDD具有部分修复作用,Kindlin-2腺相关病毒在IVDD治疗中具有一定的临床转化价值。更多还原

【Abstract】 Part I: The expression of focal adhesion protein Kindlin-2 in degenerative intervertebral disc tissue Objective: To investigate the correlation between the expression change of focal adhesion protein Kindlin-2 and intervertebral disc(IVD)degeneration(IVDD)by analyzing the Kindlin-2 expression in human nucleus pulposus(NP)samples with different degeneration degrees and IVD tissues of mice with different ages.Methods: 1.The degenerative NP specimens of patients with lumbar disc herniation were collected,and Pfirrmann grading system was used based on the patients’ preoperative magnetic resonance imaging(MRI).Grade II/III was classified as mild degeneration group,and grade IV/V was classified as severe degeneration group.The degeneration degree of the specimen was further determined by alcian blue and H/E staining,and the expressions of focal adhesion proteins(Kindlin-1,2,3 and Talin,Vinculin)were detected by immunofluorescence staining.2.The lumbar IVD tissues of young mice(3 months of age)and old mice(20 months of age)were stained with SO&FG and histological scoring was adopted to determine the degeneration degree.The expressions of above focal adhesion proteins were detected by immunofluorescence staining.Results: 1.In human degenerative NP samples,H/E and alcian blue staining showed that there were fewer NP cells and less extracellular matrix(ECM)in severe degeneration group than that in mild degeneration group.Immunofluorescence staining demonstrated that there were almost no Kindlin-1 or Kindlin-3 positive cells in human NP tissues,and Talin or Vinculin positive cells were few,but there were many Kindlin-2 positive cells and the cell number was decreased with the increase IVDD severity.2.In mice IVD samples,SO&FG staining and histological scores revealed that the IVD degeneration of the aged mice was more severe than that of the young mice.Immunofluorescence staining showed that Kindlin-1 or 3 was almost not expressed in mice IVD,and Talin and Vinculin were expressed at low levels,but Kindlin-2 was highly expressed in NP and down-regulated significantly with the increase of age in mice.Conclusions: These results indicated that Kindlin-2 was mainly expressed in the NP tissue,and its expression level was decreased with the increase of degeneration degree and age,which suggested that the decreased Kindlin-2 expression level was related to IVDD.Part II: The effect of conditional knockout of Kindlin-2 on the intervertebral disc degeneration of miceObjective: To explore the role of Kindlin-2 in IVDD by constructing IVD specific Kindlin-2 knockout mice.Methods: 1.Kindlin-2fl/fl;AggrecanCre ERT2 mice were obtained by mating and injected with corn oil(control group)or tamoxifen(c KO group)at 2 months of age.Lumbar and coccygeal vertebra was scanned by computerized tomography(CT)at age of 2,3,5 and 8 months.The mice were sampled at age of 3,5 and 8 months,and immunofluorescence staining,TUNEL staining,SO&FG staining,histological scoring as well as Western Blot detection were performed.2.In order to further explore the involvement of abnormal mechanical stress,coccygeal IVDs needle stab was performed to cause coccygeal vertebrae instability in Kindlin-2fl/fl;AggrecanCre ERT2 mice at 4 weeks after injection of corn oil or tamoxifen.CT scan of the caudal vertebra was performed before operation(12 weeks of age)and 6 weeks after operation(age of 18 weeks),and samples were collected at age of 18 weeks.Immunofluorescence staining,TUNEL staining,SO&FG staining and histological scoring were performed.Results: 1.In lumbar IVD of mice,immunofluorescence staining demonstrated that the Kindlin-2 expression in c KO group was markedly down-regulated at age of 3,5 and 8 months,compared with the control mice,indicating that the knockout effect was good;SO&FG staining and histological scoring demonstrated that the IVD histological score of the c KO mic was markedly higher than the control mice at age of 5 months,and more significantly at age of 8 months;Immunofluorescence staining and WB analysis revealed that the ECM synthesis and catabolism balance in c KO mice was disturbed,which was manifested as decreased expression of Col2a1 and Acan,and increased expression of Mmp13 and Adamts5;Immunofluorescence staining,TUNEL staining and WB indicated that the cell apoptosis was increased in c KO group,which was manifested as up-regulated expression level of active Caspase3 and Bax,decreased Bcl2 expression,and increased TUNEL-positive cells;CT images showed no significant change in IVD height.2.In coccygeal IVD of mice,immunofluorescence staining also suggested that Kindlin-2 knockout effect was good,but SO&FG staining,histological scoring and CT images demonstrated that there was no markedly difference in coccygeal IVD between c KO mice and the control mice.3.Other results showed that the abnormal mechanical stress caused by needle stab could lead to an increase in histological scores of mice coccygeal IVD,a decrease of IVD height,an imbalance of ECM synthesis and catabolism,and an increase in cell apoptosis in control mice,but the changes were more significant in c KO mice with coccygeal IVDs needle stab.Conclusions: The data suggested that Kindlin-2 knockout could result in spontaneous lumbar IVD degeneration of mice,but there was no obvious change in coccygeal IVD.Additionally,Kindlin-2 knockout could accelerate coccygeal IVD degeneration of mice suffered from abnormal mechanical stress.The results suggested that Kindlin-2 loss played an important role in IVDD initiation and progression,especially in the presence of abnormal mechanical stress.Part III: The mechanisms of intervertebral disc degeneration caused by Kindlin-2 lossObjective: To investigate whether Nlrp3 inflammasome signaling pathway is involved in cell and tissue degeneration of IVD caused by Kindlin-2 loss.Methods: 1.Immunol stainings were performed to evaluate Nlrp3 inflammasome-related protein level in NP tissues of human with different degeneration degrees and in NP tissues of Kindlin-2 c KO and control mice.2.In rat NP cell line,a pressurizing device was applied to simulate the abnormal compression loading in IVD,and Kindlin-2 si RNA or Kindlin-2 plasmid was used to knock down or overexpress Kindlin-2,respectively.The expressions of proteins related to Nlrp3 inflammasome and ECM synthesis and catabolism were detected,and cell apoptosis was evaluated.3.The expressions of Nlrp3 inflammasome-related proteins and Kindlin-2 were analyzed in IL-1β-treated NP cells with or without abnormal compression loading treatment.4.The Nlrp3 inflammasome pathway activation inhibitor MCC950 was used to inhibit Nlrp3 inflammasome activation under in vivo and in vitro conditions,respectively.The repair effect of MCC950 on the Kindlin-2 loss-induced degeneration of NP cells and IVD tissues with or without abnormal mechanical stress was detected.Results: 1.In human degenerative NP tissues,compared with mild degeneration group,the Nlrp3 inflammasome-related protein levels were markedly up-regulated in severe degeneration group.Similarly,compared with the control mice,Nlrp3 inflammasome-related protein expression was obviously up-regulated in the Kindlin-2 knockout group of mice.2.Abnormal compression loading resulted in decreased Kindlin-2 and Col2a1 expression,up-regulated Mmp13 expression,increased cell apoptosis,and activation of Nlrp3 inflammasome pathway.Kindlin-2 knockdown aggravated these changes,while Kindlin-2 overexpression ameliorated these changes.3.IL-1β down-regulated Kindlin-2 and up-regulated Nlrp3 inflammasome-related protein level in time-and concentration-dependent manner,and abnormal compression could aggravate these changes.4.In vitro,MCC950 inhibited the activation of Nlrp3 inflammasome pathway,decreased Col2a1 expression,increased Mmp13 expression,and increased cell apoptosis induced by Kindlin-2 knockdown with or without abnormal compression loading treatment.5.In vivo,MCC950 inhibited the activation of Nlrp3 inflammasome pathway,increased histological scores,decreased IVD height,down-regulated Col2a1 expression,up-regulated Mmp13 expression,and increased cell apoptosis induced by Kindlin-2 knockdown with or without abnormal mechanical stress.Conclusions: Results suggested that Kindlin-2 loss could lead to the degeneration of IVD cells and tissues via Nlrp3 inflammasome activation.Additionally,activated Nlrp3 inflammasome released IL-1β,which resulted in decreased Kindlin-2 expression and further activation of Nlrp3 inflammasome pathway,forming a Kindlin-2/Nlrp3 inflammasome /IL-1β vicious cycle,and causing IVD degeneration.Part IV: The repair effect of Kindlin-2 overexpression on the degeneration of intervertebral discObjective: To investigate protective effect of adeno-associated virus(AAV)-mediated Kindlin-2 overexpression on compression loading-induced human primary NP cell degeneration and coccygeal IVDs compression model-induced rat IVD degeneration,and to preliminarily explore the clinical transformation significance of Kindlin-2 in IVDD treatment.Methods: 1.Control AAV or Kindlin-2 AAV was used to infect human primary NP cells,then cells were treated with or without compression loading.The expressions of Kindlin-2,Nlrp3,Col2a1,Mmp13 and IL-1β were evaluated,and cell apoptosis was detected.2.SD rats(3 months old)were divided into sham group,coccygeal IVDs compression + control AAV group,and coccygeal IVDs compression + Kindlin-2 AAV group.The operation was performed 3 weeks after AAV infection,and MRI evaluation and sampling were performed 2 weeks after the operation.SO&FG staining,histological scoring,immunofluorescence staining and TUNEL staining were performed.Results: 1.In human primary NP cells,AAV-mediated Kindlin-2 overexpression partially inhibited abnormal compression loading-induced decreased Kindlin-2 expression,activation of Nlrp3 inflammasome pathway,down-regulated Col2a1 expression,up-regulated Mmp13 expression,and increased cell apoptosis.2.In rat coccygeal IVDs compression model,AAV-mediated Kindlin-2 overexpression partially reversed the abnormal compression loading-induced decreased T2 signal in MRI imaging of rat IVD,increased histological scores,decreased Kindlin-2 expression,activation of Nlrp3 inflammasome pathway,down-regulated Col2a1 expression,up-regulated Mmp13 expression,and increased cell apoptosis.Conclusions: The results demonstrated that AAV-mediated Kindlin-2 overexpression could partially repair IVD degeneration,suggesting Kindlin-2 AAV had certain clinical translational value in IVDD treatment.更多还原