瘦素缺乏小鼠椎间盘退变的组织学观察

目的探讨瘦素在椎间盘退变中的可能作用。方法采用HE染色观察6月龄雄性ob/ob小鼠（瘦素缺乏小鼠）和野生型小鼠（C57BL小鼠）椎间盘的形态学;免疫组织化学检测Ⅱ型胶原、蛋白聚糖的表达;Real-time PCR检测Ⅱ型胶原、Ⅹ型胶原及蛋白聚糖的基因表达。结果与野生型小鼠相比,ob/ob小鼠椎间盘HE染色表现为椎间盘组织的胶原结构紊乱、髓核碎裂、椎间盘高度降低,免疫组化检测显示Ⅱ型胶原、蛋白聚糖表达减少,Real-time PCR检测显示Ⅱ型胶原、蛋白聚糖基因表达下调而Ⅹ型胶原基因表达上调,差异有统计学意义（P〈0.05）。结论活体内瘦素缺乏可能加速小鼠椎间盘退变。     

循环机械压力诱导下兔椎间盘退变器官模型的建立及意义

背景：力学因素是导致椎间盘退变（IDD）的重要诱因,建立力学相关性IDD的器官模型能为IDD机制的研究提供理想的模型基础。 目的：建立兔椎间盘器官模型,并施以循环机械压力,探究循环机械压力载荷对IDD的影响。 方法：6月龄新西兰大白兔随机分为加力组和对照组,静脉给予1.3 ml肝素（5000 U/ml）,待肝素体内循环5 min后处死。无菌条件下完整取出带部分椎骨的腰段椎间盘,放入20%胎牛血清的培养液中培养。加力组应用加力器施以0.2 MPa压力值,每日加压1次,每次30 min。经过各个时段的培养后,苏木精-伊红（HE）染色观察椎间盘大体组织形态学变化;氯化硝基四氮唑蓝（NBT）染色及4＇,6-二眯基-2-苯基吲哚（DAPI）复染检测椎间盘细胞成活率;Realtime RT-PCR和Western-blotting检测蛋白多糖（AGN）、Ⅱ型胶原（COLⅡ）的mRNA和蛋白表达。 结果：对照组和加力组培养至第7 d的组织形态学无明显变化,培养至第14 d均表现为组织形态学破坏,且以加力组表现更为明显。对照组培养至第7 d的细胞成活率及AGN、COLⅡ表达与0 d相比无明显变化;对照组培养第14 d与0 d比较,培养第7 d加力组与对照组比较,培养第14 d加力组与对照组比较,均表现为细胞成活率明显下降,AGN、COLⅡ表达下调。 结论：成功建立短周期兔椎间盘体外器官模型,并在此模型基础上阐明循环机械压力载荷可直接导致椎间盘退变样改变。     

Experimental instability in the rabbit lumbar spine

The authors performed mechanical, biochemical, and histologic analyses of changes in the rabbit lumbar spine occurring after instability had been induced by facet removal to find whether this intervention produced an experimental model for intervertebral disc degeneration. Sham operated animals and an unoperated control group were used for comparison. Half of the operated animals were housed under conditions to promote higher physical activity than the other animals housed individually in small cages. Acutely, the removal of facet joints increased the flexibility of intervertebral joints. Over the following year, this increase in flexibility was reduced to close to control levels in all groups of animals. Within the intervertebral discs, there was no significant change in proportions or solubility of collagen or proteoglycans after surgery, nor was there microscopic or macroscopic evidence of disc degeneration. The surgical procedure produced hypermobility of the spine, but there was a subsequent restabilization, and the intended disc degeneration was not produced. These findings indicate that some as yet unidentified soft tissue repair process, facilitated by activity, overcame the hypermobility created at surgery, so degenerative changes in the intervertebral discs did not result. We suggest that other animal models of disc degeneration may represent a failure of reparative response to acute injury.     

移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

目的 观察骨髓间充质干细胞(MSCs)移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞(SFs)移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白(GFP)转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态;用实时定量聚合酶链反应(PCR)检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达;免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组(P<0.05),bax mRNA的表达量与退变组差异无统计学意义(P>0.05).MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为(16.75±2.14)%和(6.86±1.08)%;Caspase-3表达率分别为[(20.34±1.03)%和(6.09±0.77)%](P<0.05),低于退变组和SFs移植对照组[细胞凋亡率分别为(31.87±4.16)%和(29.02±2.16)%;Caspase-3表达率分别为(31.50±3.78)%和(30.20±4.93)%](P<0.05).结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.     

Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study.

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.     

Regulation of apoptosis in nucleus pulposus cells by optimized exogenous Bcl‐2 overexpression

Although the etiology of intervertebral disc degeneration is poorly understood, one possible approach to regulate the process of intervertebral disc degeneration may include the inhibition of apoptosis. We investigated the anti‐apoptotic effects of bcl‐2 in nucleus pulposus cells to enhance disc cell survival. Rat nucleus pulposus cells were transfected in vitro with a codon optimized rat bcl‐2 gene. Forty‐eight hours after transfection, cells were cultured in serum‐deprived medium. After serum withdrawal, the cells were evaluated for bcl‐2 protein levels and cell apoptosis. To investigate the effects of bcl‐2 overexpression on the final apoptotic pathways and on basic genes important for nucleus pulposus homeostasis, mRNA levels of caspase‐3, type II collagen, and aggrecan were also quantified. Nucleus pulposus cells were successfully transfected with codon optimized bcl‐2 gene, which effectively reduced serum starvation‐induced cell apoptosis. Overexpression of bcl‐2 also reduced the mRNA expression level of caspase‐3. mRNA levels of type II collagen and aggrecan were significantly higher in bcl‐2 transfected groups compared to control plasmid vector groups after serum withdrawal. We firstly showed that bcl‐2 overexpression in intervertebral disc cells was effective in preventing in vitro apoptotic cell death, indicating the potential advantages of this therapeutic approach in regulating disc degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1608–1613, 2010     

生长因子与椎间盘再生

目的：追溯近阶段国内外有关生长因子与椎间盘修复研究的进展。方法：广泛渣阅近期有关生长因子与椎间盘修复的文献，综述生长因子在椎间盘突的表达，生长因子对椎间盘细胞的作用等特点，结果：多种生长因子在椎间盘生长发育和退变过程中表达，外源性生长因子能促进培养的椎间盘细胞分化增殖，合成蛋白多糖和胶原，运用腺病毒可将生长因子基因转染至椎间盘细胞。结论：生长因子在椎间盘的生长发育和退变过程中起调节作用，可望运用生长因子进行椎间盘退变治疗和修复重建。重庆     

Nucleus pulposus allograft retards intervertebral disc degeneration

Autogenous implantation of nucleus pulposus or nucleus pulposus cells that were activated by coculture retards intervertebral disc degeneration, but harvesting such grafts causes disc degeneration at the donor site. This study examined whether nucleus pulposus allografts similarly retard disc degeneration and whether such allografting induces immunologic rejection. Japanese White rabbits served as donors and recipients for allografts. Lumbar disc degeneration was induced by aspirating the nucleus pulposus. Two weeks later, intact nucleus pulposus or nucleus pulposus cells were injected and compared with a sham procedure and normal control. The recipients' discs were examined histologically and immunologically at intervals for 16 weeks. Discs receiving an intact nucleus pulposus showed the least degeneration, followed by discs receiving nucleus pulposus cells, both of which were better than no treatment. These findings correlated directly with the intensity of immunochemical staining for Type II collagen. Allogeneic grafts did not induce any appreciable host-versus-graft response. Injection of nucleus pulposus and nucleus pulposus cells retards intervertebral disc degeneration. However, injection of intact nucleus pulposus is more effective than injection of nucleus pulposus cells alone. The intercellular matrix plays an important, but poorly understood, role in preserving intervertebral discs.     

TGF-β3修饰退变髓核细胞移植修复兔退变椎间盘的效果观察

目的探讨TGF-β3基因修饰后退变髓核细胞生物学效应以及植入兔退变椎间盘后对退变椎间盘的影响。方法将重组腺病毒载体Ad-TGF-β3与第2代退变髓核细胞按10∶1比例混合培养转染(Ad-TGF-β3组),待细胞融合后传代,MTT检测转染细胞增殖活性,Western blot检测TGF-β3蛋白含量,免疫细胞化学染色观察对数生长期转染细胞Ⅱ型胶原染色阳性率;采用病毒空载体转染髓核细胞(Adv组)和未经转染髓核细胞(空白组)作为对照。取30只新西兰兔,体重3.2～3.5 kg,雌雄不限,通过针刺L3、4、L4、5和L5、6椎间盘制备椎间盘退变模型。将实验动物按照随机数字法分为3组,转染细胞组(A组,n=12)、退变细胞组(B组,n=12)和空白对照组(C组,n=6)。A、B组将100μL浓度为1×105个/mL对应细胞悬液注射入退变椎间盘,C组同法注入等量PBS。注射后6、10、14周取A、B组各4只、C组2只实验动物处死,取L3、4、L4、5和L5、6椎间盘行组织学观察,RT-PCR检测Ⅱ型胶原和蛋白多糖mRNA表达。结果 Ad-TGF-β3转染后髓核细胞活性明显改善;转染后3、7、14 d,TGF-β3在髓核细胞内表达逐渐升高;Ad-TGF-β3组髓核细胞细胞质内见棕黄色Ⅱ型胶原阳性染色,阳性率显著高于Adv组及空白组(P<0.05)。组织学观察示,A组椎间盘退变程度较B、C组明显减轻。6、10、14周A组Ⅱ型胶原和蛋白多糖mRNA表达显著高于B、C组,差异均有统计学意义(P<0.05)。结论 TGF-β3基因修饰退变髓核细胞后可明显改善细胞生物活性,转染后髓核细胞植入兔体内可明显增加退变椎间盘的基质分泌。     

Immunolocalization of bone morphogenetic protein and its receptors in degeneration of intervertebral disc.

STUDY DESIGN: Immunohistochemical study of expression and localization of bone morphogenetic protein (BMP)-2/4 and type I and II receptors on intervertebral disc. OBJECTIVES: To determine the biologic functions of BMPs and their receptors in the process of degeneration of the intervertebral disc. SUMMARY OF BACKGROUND DATA: Biologic and pathologic processes in the cell during the degeneration of the intervertebral disc are as yet poorly understood. METHODS: The cervical spines of 15 male senescence-accelerated mice aged 8, 24, or 50 weeks were used for histologic and immunohistochemical examination of BMP-2/4 and BMP receptors IA, IB, and II. Immunostaining was performed with the avidin-biotin-peroxidase complex method. RESULTS: Degenerative change was recognized within intervertebral discs of senescence-accelerated mice aged 50 weeks. BMP-2/4 and its receptors were abundant in hyaline cartilaginous cells within the endplate of the vertebrae at 8 and 24 weeks of age. However, the expression of BMP-2/4 and its receptors moved from the hyaline cartilage of the endplate of the vertebrae to fibrous cells within the anulus and to the calcified cartilage at the site of enthesis of mice aged 50 weeks. CONCLUSIONS: BMP-2/4 and its receptors may play roles in degenerative change of intervertebral disc.     

Optimization of puncture injury to rat caudal disc for mimicking early degeneration of intervertebral disc

The caudal discs of rats have been proposed as a puncture model in which intervertebral disc (IVD) degeneration can be induced and novel therapies can be tested. For biological repair, treatments for ongoing IVD degeneration are ideally administered during the earlier stages. The purpose of this study was to elucidate the optimal puncture needle size for creating a model that mimicked the earlier stages of IVD degeneration. According to the disc height index, histologic score, and MRI grading, a puncture needle sized 21G or larger induced rapid degenerative processes in rat caudal discs during the initial 2–4 weeks. The degenerative changes were severe and continued deteriorating after 4 weeks. Conversely, puncture injury induced by needles sized 25G or smaller also produced degenerative changes in rat caudal discs during initial 2–4 weeks; however, the changes were less severe. Furthermore, the degenerative process became stabilized and showed no further deterioration or spontaneous recovery after 4 weeks. In the discs punctured by 25G needles, the expression of collagen I was increased at 2–4 weeks with a gradually fibrotic transformation thereafter. The expressions of collagen II and SOX9 were enhanced initially but returned to pre‐injury levels at 4–8 weeks. The above‐mentioned findings were more compatible with earlier degeneration in discs punctured by needles sized 25G or smaller than by needles sized 21G or larger, and the appropriate timing for intradiscal administration of proposed therapeutic agents would be 4 weeks or longer after puncture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:202–211, 2018.     

椎间盘移植实验—生物化学研究

恒河猴１２只，于Ｌ＿（３￣４）行自体间盘移植手术。对术后不同时间移植间盘进行生化分析，测定了间盘组织的水、胶原和蛋白多糖含量变化，结果显示：术后２月蛋白多糖及水含量降低，胶原含量升高，髓核较纤维环变化明显。术后４月蛋白多糖及水含量进一步降低，胶原含量回升，与对照组已无统计学差异，水份和胶原含量较，４月无明显变化。提示：椎间盘移植后虽然在早朝有退变倾向，但在后期这种退变部分恢复。     

Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes

Background The intervertebral disc has been reported to be an immunologically privileged environment, possibly mediated by Fas ligand (FasL) expression. On the other hand, recent studies have shown the infiltration of host immune cells into the degenerated disc, which may indicate the failure of the immune-privilege feature of the disc with degeneration. However, the relationship between FasL expression and disc degeneration is still unclear. Therefore, the purpose of this study was to clarify the relationship between FasL expression and disc degeneration. Methods Ten human degenerated disc specimens were obtained from spondylolisthesis patients and ten nondegenerated discs from idiopathic scoliosis patients during surgical procedures. Immunohistochemical staining was performed to determine the presence of FasL in cross-sections of those discs. Parts of the disc tissues were used to examine FasL expression quantitatively with Western blot analysis. To examine whether the change in FasL expression was influenced by aging, an animal study comparing the discs from young and old rats were performed using magnetic resonance imaging (MRI) and real-time polymerase chain reaction (PCR) assessment. Results Nucleus pulposus cells showed strong positive staining for FasL in all specimens examined. Quantitative examination demonstrated a significant decrease in FasL expression in the degenerated group compared with the nondegenerated group (average 67.6%, P<0.05). MRI showed no significant differences in the grade of disc degeneration between young and old rats, and also no significant difference in FasL mRNA in real-time PCR assay. Conclusions The current results indicate that FasL and its potential mechanism of immunological privilege could influence the protection of the intervertebral disc against degeneration.     

退变腰椎间盘细胞经体外转基因培养生物学活性的实验研究

目的构建转化生长因子（transforming growth factor,TGF-β3的真核表达载体pEGFP-TGF-β3转染椎间盘髓核细胞,研究转基因TGF-β3对退变髓核细胞生物学特性的影响。方法通过手术方法制作椎间盘突变模型,从而获取原代退变椎间盘髓核细胞,通过脂质体将真核载体pEGFP-TGF-β3导入髓核细胞,然后对细胞的形态和增殖活性（MTT法）进行观察,应用Westen Blot检测TGF-β3在髓核细胞的表达含量,应用免疫细胞化学方法检测转染后髓核细胞的Ⅱ型胶原的表达。结果髓核细胞转染后,细胞活性增强,TGF-β3表达增加,并随着时间的延长而增加。Ⅱ型胶原表达增加。结论TGF-β3转染退变髓核细胞可起到维持髓核细胞表型,并在细胞传代后仍发挥调节作用。TGF-β3确实具有促进髓核细胞增殖和Ⅱ型胶原合成的能力,从而有可能延缓甚至逆转椎间盘退变。     

纤维环穿刺诱导椎间盘退变动物模型的实验研究

目的：探讨纤维环穿刺诱导椎间盘退变建立动物模型的可行性。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人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对照的椎间盘（L2／3）行MRI检查，并行免疫组化及组织学观察。结果：术后第3周到第10周，造模后的椎间盘MRI 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型胶原含量较对照间盘进行性减少（P〈0．01）。结论：纤维环穿刺法可以诱导兔椎间盘的缓慢退变，为研究椎间盘的退行性变提供有效的动物模型。     

Regulating effects of transforming growth factor-beta (TGF-beta) on gene expression of collagen type II in human intervertebral discs

OBJECTIVE: To assess the regulating effects of TGF-beta on gene expression of collagen type II in the human intervertebral discs. METHODS: In situ hybridization was used to investigate the effect of TGF-beta1 on collagen mRNA in confluent primary and passaged monolayer cell cultures of annulus fibrosus (AF) as well as nucleus pulposus (NP). The mean photodensitometry of cell smears as semi-quantitative analysis was evaluated by VIDAS software. RESULTS: In primary cultures, 1 ng/ml and 10 ng/ml TGF-beta1 inhibited the collagen type II mRNA levels by 74.6% and 60.2% respectively in AF; they also inhibited the mRNA levels by 69.6% and 55.5% respectively in NP. In passaged cultures in which the notochordal cells and chondrocytes were in dedifferentiation status, 1 ng/ml and 10 ng/ml TGF-beta1 increased the collagen type II mRNA levels by 151% and 166% respectively in AF and also increased the mRNA levels by 145% and 198% respectively in NP. CONCLUSIONS: The regulation effect of TGF-beta on collagen type II gene expression is dependent on whether the cells are fully differentiated or undergoing phenotype loss, and TGF-beta may play an important role in the repair process during early disc degeneration, especially in nucleus pulposus.     

终板下椎体缺血诱导兔椎间盘退行性变模型的建立及终板中内皮素1的表达

目的通过终板下注射无水乙醇阻碍椎体-终板营养,建立一种新型兔腰椎椎间盘退行性变模型,并观察终板退行性变过程中内皮素1(ET-1)的表达情况。方法健康4月龄新西兰兔32只,随机分成4组,每组8只,选取L5,6椎体(对应L4/L5及L5/L6椎间盘)注射300μL无水乙醇,选取L4椎体(对应L3/L4椎间盘)注射磷酸盐缓冲液(PBS)作为实验对照,L7椎体(对应L6/L7椎间盘)未注入任何物质作为正常对照。其中1组造模后1个月提取软骨终板细胞,行免疫细胞化学染色检测ET-1表达;余3组分别于造模后1、3和5个月进行椎间盘X线和MRI检查,取椎间盘组织行HE染色观察形态学改变,免疫组织化学染色观察ET-1表达。结果注射无水乙醇后,随着时间进展,X线片显示椎间隙高度显著下降、椎间隙变窄、边缘骨赘增生,MRI T2WI显示椎间盘低信号;苏木精-伊红染色(HE)显示终板的生长板厚度变薄,终板结构破损,同时软骨终板细胞退化、直至消失,髓核中细胞发生转化(由空泡细胞转变为软骨样细胞,进而形成纤维软骨样细胞)造成髓核纤维化,纤维环结构排列紊乱、纤维化程度逐步加重;免疫组织化学染色显示,发生退行性变的终板组织内有ET-1表达,但随着退行性变加剧,ET-1表达强度下降;提取的退行性变软骨终板细胞(造模后1个月)也显示细胞质内ET-1强表达。结论通过注射无水乙醇阻碍椎体-终板营养途径可成功建立兔椎间盘退行性变模型,终板退行性变过程中伴随ET-1的表达。     

退变颈椎间盘中IL-17表达与分布的研究

目的 观察退变颈椎间盘中自细胞介素-17(IL-17)的表达与分布,并探讨其与颈椎间盘退变发生发展的关系.方法 实时荧光相对定量PCR(RQ-PCR)检测30例退变颈椎间盘及10例正常对照椎间盘中IL-1β、IL-17、肿瘤坏死因子-α(TNF-α)和孤核受体(retinoid-related orphan re-ce...