FasL维持椎间盘免疫豁免及诱导髓核细胞凋亡的实验研究
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摘要
第一部分纤维环穿刺诱导兔椎间盘退变的动物模型研究
目的:观察采用18G皮肤穿刺针穿刺纤维环,诱导兔椎间盘退变建立动物模型。方法:新西兰大白兔24只,用持针器夹持18G皮肤穿刺针从左前外侧刺入L3/4、L4/5、L5/6椎间盘的纤维环,深度控制在5mm。术前及术后3、6、10周对造模后的椎间盘及正常的椎间盘(L2/3)行MRI扫描,并在术后3、6、10周对其行免疫组化及组织学观察。结果:MRI结果提示,从术后第3周到第10周,造模后的椎间盘T2WI信号呈现持续减弱趋势,免疫组化及组织学观察发现髓核细胞的数量及Ⅱ型胶原含量进行性减少(p<0.01)。结论:纤维环穿刺法可以诱导兔椎间盘的缓慢退变,这一模型为研究椎间盘退变提供了有效的载体。
第二部分纤维环穿刺对兔髓核细胞中FasL表达影响的实验研究
目的了解生理学屏障与FasL的分子生物学效应之间的联系,探讨椎间盘退变的发病机制。方法采用18规格的皮肤穿刺针穿刺家兔纤维环,在术后的3、6、10周收集正常及穿刺后的椎间盘组织,免疫组织化学染色观察Fas与FasL的表达,流式细胞技术分析髓核细胞凋亡指数。结果正常髓核组织中可见少许髓核细胞胞浆中FasL呈弱阳性染色,实验组髓核细胞胞浆则呈强阳性染色。FasL阳性细胞百分比在正常组与各实验组之间的差异具有非常显著性差异(P<0.001,两两比较)。三个实验组的髓核细胞平均凋亡指数明显高于正常组(P<0.001,0.002 and 0.006,两两比较)。实验组髓核细胞FasL阳性表达百分比与Fas阳性表达百分比之间具有显著相关性(r = 0.571, P = 0.0036)。结论FasL与生理学屏障的共同作用,可能是使髓核组织产生免疫豁免效应的一个重要因素。当生理学屏障受到损伤后(如穿刺纤维环),Fas/FasL系统的作用会引起髓核细胞凋亡,这也可能是椎间盘退变的发生机制之一。
第三部分FasL mRNA在人椎间盘中表达的实验研究
目的了解FasL目的基因在正常人椎间盘及椎间盘突出症患者椎间盘组织中的表达情况。方法收集16例腰椎间盘突出症患者的椎间盘组织(破裂型与非破裂型各8例)及4例正常人椎间盘组织,通过RT-PCR技术观察FasL,Fas,TNF and IL目的基因的表达情况,并比较其表达程度。结果正常髓核组织中仅有FasL的表达,突出的椎间盘组织中存在FasL,Fas,TNF and IL的表达,而且表达指数在破裂型组与非破裂型组之间具有显著性差异(P<0.05)。结论正常髓核组织中存有FasL的表达,提示FasL是使髓核组织产生免疫豁免效应的一个重要因素。当椎间盘突出时(纤维环破裂),则会在局部激发炎症反应,而髓核细胞通过自分泌或旁分泌途径导致FasL表达增高是可能的机制之一。
Part 1. A Rabbit Model of Disc Degeneration by an Anulus Needle Puncture
Objective : To establish a slowly progressive, reproducible rabbit model of intervertebral disc degeneration by puncturing the anulus with needles of defined gauges. Method: The L3/4, L4/5 and L5/6 lumbar intervertebral discs of 24 New Zealand White rabbits were stabbed by 18-gauge hypodermic needle to a depth of 5mm in the left anterolateral anulus fibrosus. Magnetic resonance imaging scans, Immunohistochemical and histologic analyses of the stabbed discs and intact L2/3 control discs were performed preoperation or at 3, 6, 10weeks post surgery. Result: Form the magnetic resonance imaging,we detected the stabbed discs exhibited a progressive decrease in signal intensity of T2-weighted images starting at 3 weeks post stab and continuing through 10 weeks. Immunohistochemical and histologic analyses revealed progressive loss of chondrocyte-like cells and type II collagen(p<0.01).Conclusion: The Stabbing approach, resulted in a slowly progressive intervertebral disc degeneration of rabbit. This model would appear suitable for studying and testing safety and efficacy of novel treatments of intervertebral disc degeneration.
Part 2. The expression of Fas ligand on normal and stabbed disc cells of rabbit IDD model
Objective To clarify the relation between physiologic barrier and the role of FasL,and investigate a possible pathogenesis of intervertebral disc degeneration (IDD).Methods The anular puncture model of rabbit were established using defined needle gauges and depths. The normal and the stabbed discs were harvested at each time period of 3,6,and 10 weeks after surgery. Immunohistochemical staining of these discs for Fas and FasL was performed by standard procedures. The mean apoptosis index of disc cells was analyzed by flow cytometry.Results The normal discs cells exhibited relatively weak immunopositivity, and the stabbed disc cells exhibited intense immunopositivity. A significant difference in the percentage of FasL-positive disc cells between the normal discs and the stabbed discs (P<0.001). The mean apoptosis indexes at 3 weeks, 6 weeks, 10 weeks post stab were significantly higher than normal disc (P<0.001,0.002 and 0.006, respectively). There was a significant correlation between the degree of FasL-positive expression and the degree of Fas-positive expression of the disc cells post stab (r = 0.571, P = 0.0036). Conclusions This observation indicates that the nucleus pulposus is a 'immune-privileged' sites. The immune privilege is maintained by FasL and the physiologic barrier together. When the physiologic barrier was damaged, the role of FasL changed, coexpressed with Fas induce apoptosis of disc cells. These results indicate that an autoimmune reaction may be a possible pathogenesis of IDD.
Part 3. The expression of Fas ligand mRNA on human intervertebral disc.
Objective. To clarify the existence of Fas ligand on intact intervertebral disc cells and herniated lumbar disc tissues.Methods. Sixteen herniated lumbar disc tissues (contained disc, n=8; noncontained disc, n=8)and four normal human disc tissues were examined to investigate the mRNA expression of FasL,Fas,TNF and IL by reverse transcription polymerase chain reaction method. The degeree of mRNA expression were compared.Results. The result of RT-PCR confirmed the existence of Fas ligand in normal and herniated disc tissues, but Fas,TNF and IL mRNA were expressed only in herniated disc tissues. A higher degree of FasL expression in herniated disc cells was found in noncontained discs than in contained discs ( P<0.05). Conclusion. We demonstrated the existence of Fas ligand on normal disc cells, which should play a key role in the potential molecular mechanism to maintain immune privilege of the disc. But after herniation, undergo inflammatory reaction and apoptotic cell death via autocrine or paracrine FasL mechanisms by the disc cells themselves.
目的:观察采用18G皮肤穿刺针穿刺纤维环,诱导兔椎间盘退变建立动物模型。方法:新西兰大白兔24只,用持针器夹持18G皮肤穿刺针从左前外侧刺入L3/4、L4/5、L5/6椎间盘的纤维环,深度控制在5mm。术前及术后3、6、10周对造模后的椎间盘及正常的椎间盘(L2/3)行MRI扫描,并在术后3、6、10周对其行免疫组化及组织学观察。结果:MRI结果提示,从术后第3周到第10周,造模后的椎间盘T2WI信号呈现持续减弱趋势,免疫组化及组织学观察发现髓核细胞的数量及Ⅱ型胶原含量进行性减少(p<0.01)。结论:纤维环穿刺法可以诱导兔椎间盘的缓慢退变,这一模型为研究椎间盘退变提供了有效的载体。
第二部分纤维环穿刺对兔髓核细胞中FasL表达影响的实验研究
目的了解生理学屏障与FasL的分子生物学效应之间的联系,探讨椎间盘退变的发病机制。方法采用18规格的皮肤穿刺针穿刺家兔纤维环,在术后的3、6、10周收集正常及穿刺后的椎间盘组织,免疫组织化学染色观察Fas与FasL的表达,流式细胞技术分析髓核细胞凋亡指数。结果正常髓核组织中可见少许髓核细胞胞浆中FasL呈弱阳性染色,实验组髓核细胞胞浆则呈强阳性染色。FasL阳性细胞百分比在正常组与各实验组之间的差异具有非常显著性差异(P<0.001,两两比较)。三个实验组的髓核细胞平均凋亡指数明显高于正常组(P<0.001,0.002 and 0.006,两两比较)。实验组髓核细胞FasL阳性表达百分比与Fas阳性表达百分比之间具有显著相关性(r = 0.571, P = 0.0036)。结论FasL与生理学屏障的共同作用,可能是使髓核组织产生免疫豁免效应的一个重要因素。当生理学屏障受到损伤后(如穿刺纤维环),Fas/FasL系统的作用会引起髓核细胞凋亡,这也可能是椎间盘退变的发生机制之一。
第三部分FasL mRNA在人椎间盘中表达的实验研究
目的了解FasL目的基因在正常人椎间盘及椎间盘突出症患者椎间盘组织中的表达情况。方法收集16例腰椎间盘突出症患者的椎间盘组织(破裂型与非破裂型各8例)及4例正常人椎间盘组织,通过RT-PCR技术观察FasL,Fas,TNF and IL目的基因的表达情况,并比较其表达程度。结果正常髓核组织中仅有FasL的表达,突出的椎间盘组织中存在FasL,Fas,TNF and IL的表达,而且表达指数在破裂型组与非破裂型组之间具有显著性差异(P<0.05)。结论正常髓核组织中存有FasL的表达,提示FasL是使髓核组织产生免疫豁免效应的一个重要因素。当椎间盘突出时(纤维环破裂),则会在局部激发炎症反应,而髓核细胞通过自分泌或旁分泌途径导致FasL表达增高是可能的机制之一。
Part 1. A Rabbit Model of Disc Degeneration by an Anulus Needle Puncture
Objective : To establish a slowly progressive, reproducible rabbit model of intervertebral disc degeneration by puncturing the anulus with needles of defined gauges. Method: The L3/4, L4/5 and L5/6 lumbar intervertebral discs of 24 New Zealand White rabbits were stabbed by 18-gauge hypodermic needle to a depth of 5mm in the left anterolateral anulus fibrosus. Magnetic resonance imaging scans, Immunohistochemical and histologic analyses of the stabbed discs and intact L2/3 control discs were performed preoperation or at 3, 6, 10weeks post surgery. Result: Form the magnetic resonance imaging,we detected the stabbed discs exhibited a progressive decrease in signal intensity of T2-weighted images starting at 3 weeks post stab and continuing through 10 weeks. Immunohistochemical and histologic analyses revealed progressive loss of chondrocyte-like cells and type II collagen(p<0.01).Conclusion: The Stabbing approach, resulted in a slowly progressive intervertebral disc degeneration of rabbit. This model would appear suitable for studying and testing safety and efficacy of novel treatments of intervertebral disc degeneration.
Part 2. The expression of Fas ligand on normal and stabbed disc cells of rabbit IDD model
Objective To clarify the relation between physiologic barrier and the role of FasL,and investigate a possible pathogenesis of intervertebral disc degeneration (IDD).Methods The anular puncture model of rabbit were established using defined needle gauges and depths. The normal and the stabbed discs were harvested at each time period of 3,6,and 10 weeks after surgery. Immunohistochemical staining of these discs for Fas and FasL was performed by standard procedures. The mean apoptosis index of disc cells was analyzed by flow cytometry.Results The normal discs cells exhibited relatively weak immunopositivity, and the stabbed disc cells exhibited intense immunopositivity. A significant difference in the percentage of FasL-positive disc cells between the normal discs and the stabbed discs (P<0.001). The mean apoptosis indexes at 3 weeks, 6 weeks, 10 weeks post stab were significantly higher than normal disc (P<0.001,0.002 and 0.006, respectively). There was a significant correlation between the degree of FasL-positive expression and the degree of Fas-positive expression of the disc cells post stab (r = 0.571, P = 0.0036). Conclusions This observation indicates that the nucleus pulposus is a 'immune-privileged' sites. The immune privilege is maintained by FasL and the physiologic barrier together. When the physiologic barrier was damaged, the role of FasL changed, coexpressed with Fas induce apoptosis of disc cells. These results indicate that an autoimmune reaction may be a possible pathogenesis of IDD.
Part 3. The expression of Fas ligand mRNA on human intervertebral disc.
Objective. To clarify the existence of Fas ligand on intact intervertebral disc cells and herniated lumbar disc tissues.Methods. Sixteen herniated lumbar disc tissues (contained disc, n=8; noncontained disc, n=8)and four normal human disc tissues were examined to investigate the mRNA expression of FasL,Fas,TNF and IL by reverse transcription polymerase chain reaction method. The degeree of mRNA expression were compared.Results. The result of RT-PCR confirmed the existence of Fas ligand in normal and herniated disc tissues, but Fas,TNF and IL mRNA were expressed only in herniated disc tissues. A higher degree of FasL expression in herniated disc cells was found in noncontained discs than in contained discs ( P<0.05). Conclusion. We demonstrated the existence of Fas ligand on normal disc cells, which should play a key role in the potential molecular mechanism to maintain immune privilege of the disc. But after herniation, undergo inflammatory reaction and apoptotic cell death via autocrine or paracrine FasL mechanisms by the disc cells themselves.
引文
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1 Olmarker K, Blomquist J, Stromberg J, et al. Inflammatogenic properties of nucleus pulposus[J]. Spine, 1995,120:665–669.
2 Satoh K, Konno S, Nishiyama K, et al. Presence and distribution of antigen- antibody complexes in the herniated nucleus pulposus[J]. Spine, 1999,124:1980– 1984.
3 Bellgrau D, Gold D, Selawry H, et al. A role for CD95 ligand in preventing graft rejection[J]. Nature, 1995,1377:630–632.
4 Griffith TS, Brunner T, Fletcher SM, et al. FasL-induced apoptosis as a mechanism of immune privilege[J]. Science, 1995,1270:1189–1192.
5 Nagata S, Golstein P. The Fas death factor[J]. Science 1995;267:1449–1456.
6 Suda T, Takahashi T, Golstein P, et al.Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family[J]. Cell,1993;75:1169–1178.
7 Takahashi T, Tanaka M, Brannan CI, et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[J]. Cell,1994;76:969–976.
8 Ju ST, Panka DJ, Cui H, et al. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation[J]. Nature,1995;373:444–448.
9 Bechmann I, Mor G, Nilsen J, et al. FasL (CD95L, Apo1L) is expressed in the normal rat and human brain: evidence for the existence of an immunological brain barrier[J]. Glia,1999;27:62–74.
10 Runic R, Lockwood CJ, Ma Y, et al. Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival[J]. J Clin Endocrinol Metab,1996;81:3119–3122.
11 O’Connell J, Houston A, Bennett MW, et al. Immune privilege or inflammation? Insights into the Fas ligand enigma[J]. Nat Med ,2001;7:271–274.
12 Allison J, Georgiou HM, Strasser A, et al. Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts[J]. Proc Natl Acad Sci USA ,1997;94:3943–3947.
13 Kang SM, Schneider DB, Lin Z, et al. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction[J]. Nat Med,1997;3:738–743.
14 Takeuchi T, Ueki T, Nishimatsu H, et al. Accelerated rejection of Fas ligand expressing heart grafts[J]. J Immunol,1999;162:518–522.
15 Masuda K, Aota Y, Muehleman C,et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration[J]. Spine, 2005,30:5-14.
16 Sobajima S, John F,Kompel MS, et al. A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-Ray, and histology[J]. Spine,2005,30;:15–24.
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19 Heyde CE,Tschoeke SK,Hellmuth M.Trauma induces apoptosis in human thoracolumbar intervertebral discs[J].BMC Clin Pathol ,2006;6:5.
1 Nagata S, Golstein P. The Fas death factor[J]. Science 1995;267:1449–1456.
2 Suda T, Takahashi T, Golstein P, et al.Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family[J]. Cell,1993;75:1169–1178.
3 Bellgrau D, Gold D, Selawry H, et al. A role for CD95 ligand in preventing graft rejection[J]. Nature, 1995,1377:630–632.
4 Griffith TS, Brunner T, Fletcher SM, et al. FasL-induced apoptosis as a mechanism of immune privilege[J]. Science, 1995,1270:1189–1192.
5 McCarron RF, Wimpee MW, Hudkins PG, et al. The inflammatory effect of nucleus pulposus: a possible element in the pathogenesis of low-back pain.Spine 1987; 12:760–4.
6 Takahashi T, Tanaka M, Brannan CI, et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[J]. Cell,1994;76:969–976.
7 Ju ST, Panka DJ, Cui H, et al. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation[J]. Nature,1995;373:444–448.
8 Bechmann I, Mor G, Nilsen J, et al. FasL (CD95L, Apo1L) is expressed in the normal rat and human brain: evidence for the existence of an immunological brain barrier[J]. Glia,1999;27:62–74.
9 Runic R, Lockwood CJ, Ma Y, et al. Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival[J]. J Clin Endocrinol Metab,1996;81:3119–3122.
10 Allison J, Georgiou HM, Strasser A, et al. Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilegeupon islet allografts[J]. Proc Natl Acad Sci USA ,1997;94:3943–3947.
11 Kang SM, Schneider DB, Lin Z, et al. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction[J]. Nat Med,1997;3:738–743.
12 Takeuchi T, Ueki T, Nishimatsu H, et al. Accelerated rejection of Fas ligand expressing heart grafts[J]. J Immunol,1999;162:518–522.
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