低氧环境对椎间盘自发性吸收的影响及其作用机制

目的探讨低氧环境对椎间盘自发性吸收的影响及其作用机制。方法取SPF级成年日本大耳兔9只,雌雄不限,平均体质量2 kg。将兔处死后取脊柱髓核组织,经消化、分离、培养后获得传代髓核细胞,将生长良好的髓核细胞制成细胞悬液。根据不同时效的低氧环境将细胞分为5组对照组(常氧浓度下培养6 h)、低氧6 h组(2%O2浓度下培养6 h)、低氧12 h组(2%O2浓度下培养12 h)、低氧24 h组(2%O2浓度下培养24 h)和低氧48 h组(2%O2浓度下培养48 h)。采用实时聚合酶链反应法检测缺氧诱导因子(HIF)-1α、3型酸敏感离子通道(ASIC3)及水通道蛋白3(AQP3)mRNA表达水平,采用流式细胞仪检测各组髓核细胞凋亡情况。采用SPSS 24.0软件对数据进行分析。结果与对照组比较,低氧各组细胞凋亡率均明显升高,差异均有统计学意义(均P<0.01);与低氧12、24和48 h组比较,低氧6 h组细胞凋亡率最高,差异均有统计学意义(均P<0.01)。与对照组比较,低氧各组细胞HIF-1α和ASIC3 mRNA表达水平均明显上升,AQP3 mRNA表达水平均明显下降,差异均有统计学意义(均P<0.01);与低氧12、24和48 h组比较,低氧6 h组HIF-1α和ASIC3 mRNA表达水平最高,差异均有统计学意义(均P<0.01)。结论短时间低氧环境可以促进髓核细胞凋亡,从而加速椎间盘突出组织自发性吸收进程,其机制可能与HIF-1α和ASIC3的表达增加及AQP3的表达下降有关。     

Fas ligand plays an important role for the production of pro‐inflammatory cytokines in intervertebral disc nucleus pulposus cells

It is suggested that pro‐inflammatory cytokines, which are produced by interaction of the intervertebral nucleus pulposus cells and macrophages, may be linked to the cause of pain of the intervertebral disc herniation. This study carries out the in vitro experiments to examine the mechanism, with the use of the co‐culture of an immortalized cell line of nucleus pulposus of the human intervertebral disc and the macrophage cell line. As a result, it is found that the production of pro‐inflammatory cytokines is significantly larger at the co‐culture group than at the independent culture group. Also, at the co‐culture group of macrophages and intervertebral nucleus pulposus cells with over‐expression of fas ligand (FasL), the production of pro‐inflammatory cytokines is found to be far larger. Furthermore, it is found that these pro‐inflammatory cytokines are produced mainly by the intervertebral nucleus pulposus cells with over‐expression of FasL, and that the expression of a disintegrin and metalloproteinase (ADAM) 10, which controls the expression of FasL and activates reverse signaling inside cells, also increases. From these findings, it is suggested that FasL and ADAM10 play an important role in the production of pro‐inflammatory cytokines coming from interaction of the intervertebral nucleus pulposus cells and macrophages. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 608–615, 2013     

白介素-1β对髓核细胞MMP-1、2、9、13表达的影响

目的探讨IL-1β对人椎间盘髓核细胞表达基质金属蛋白酶MMP-1、2、9、13的作用。方法分离人椎间盘髓核细胞进行单层培养并利用甲苯胺蓝、番红O染色和Ⅱ型胶原免疫细胞化学染色进行鉴定,而后分别用10ng／ml和50ng／ml重组人IL—1β刺激体外培养的髓核细胞,RT-PCR检测基质金属蛋白酶-1、2、13的表达,定量PCR检测基质金属蛋白酶-9的表达。结果10ng／ml和50ng／ml重组人IL-1β均可促进髓核细胞基质金属蛋白酶-1、2、9、13的表达（P〈0．05）;基质金属蛋白酶-9、13表达随IL-1β浓度升高而升高（P〈0．05）。结论IL—1β可以促进人椎间盘髓核细胞表达基质金属蛋白酶-1、2、9、13,其加速了椎间盘基质分解的作用亦可能通过上述细胞因子的介导。     

Activation of rat nucleus pulposus cells by coculture with whole bone marrow cells collected by the perfusion method

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor‐β in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [³⁵S]‐sulfate and [³H]‐thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF‐β secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:222–228, 2009     

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。     

Synergistic effect of low‐intensity pulsed ultrasound on growth factor stimulation of nucleus pulposus cells

Low‐intensity pulsed ultrasound (LIPUS) has been reported to stimulate the activity of various cells. We have reported that the capacity of human intervertebral nucleus pulposus cell line to synthesize proteoglycan (PG) was increased by exposure to LIPUS, and postulated that one of the mechanisms underlying this response was an increase in expression of the transforming growth factor‐β type I receptor gene (TGFβR1). Therefore, the present study was conducted to assess the synergistic effect of LIPUS and TGF‐β on nucleus pulposus cells harvested from canines. The cells were cultured under four different sets of conditions: control group (Group A), LIPUS group (Group B), TGF‐β1 group (Group C), and LIPUS + TGF‐β1 group (Group D). They were evaluated by measuring cell proliferation, PG synthesis, PG content, gene expression of TGFβR1, and TGF‐β1 concentration. There were no significant differences in proliferation during culture. However, PG synthesis and endogenous TGF‐β1 production increased and demonstrated a synergistic effect between LIPUS and TGF‐β. Because LIPUS is safe and noninvasive, the results of the present study suggest that it would be a promising new therapy for prevention of intervertebral disc degeneration, which is said to be one of the primary causes of low back pain. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1574–1581, 2007     

MMP-3在退变腰椎间盘组织中的表达及意义

目的研究MMP-3在退变腰椎间盘髓核和纤维环组织中的表达及其临床意义。方法用半定量RT—PCR和免疫组织化学法检测实验组1（30例退变腰椎间盘髓核）、实验组2（30例退变腰椎间盘纤维环）和对照组（10例创伤腰椎间盘髓核）中MMP3mRNA和蛋白表达。结果实验组1MMP-3mRNA和蛋白的表达均显著高于对照组（P均〈0．01）,实验组1与2之间MMP-3mRNA和蛋白的表达没有显著差异（P均〉0．05）。结论MMP-3的表达增加可能参与腰椎间盘退变的进程。     

乌司他丁对兔髓核细胞中诱导型一氧化氮合酶和基质金属蛋白酶表达的影响

目的 探讨乌司他丁对白介素-1β(interleukin-1β,IL-1β)诱导的兔椎间盘髓核细胞中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)和MMP-3表达的影响.方法 用酶消化法...     

Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations

ObjectivesSexual dimorphism does occur in intervertebral disc (IVD) degeneration. The involvement of estrogen on IVD health has been well reported in recent years. The estrogen receptors (ER) are the main mediators of estrogen action. ER might play specific roles in the sexual variations of the IVD degeneration.MethodsThirty-six elderly patients with lumbar disc degeneration were selected and graded using Pfirrmann's system based on MRI images. Differences of ERα and ERβ immunoreactivity staining in nucleus pulposus of each sex and degeneration degree were recorded and compared.ResultsBoth cytoplasmic and nuclear staining of ERα and ERβ immunoreactivity were observed in the nucleus pulposus cells. ERα and ERβ expression significantly decreased along with the aggravation of IVD degeneration both in males and females. Expression of ERα and ERβ protein in nucleus pulposus of males was significantly higher than that of females.ConclusionsGender-specific expression of ER might play a part in sexual dimorphism of IVD degeneration. Gender and degeneration condition differences should be taken into account when the effects of estrogen on IVD metabolism are studied further.     

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。     

Hypoxia suppresses serum deprivation‐induced degradation of the nucleus pulposus cell extracellular matrix through the JNK and NF‐κB pathways

Intervertebral disc (IVD) degeneration is associated with the imbalance between anabolism and catabolism of the nucleus pulposus (NP) extracellular matrix (ECM). Serum deprivation (SD) has been reported to exacerbate IVD degeneration; however, the effect of SD on ECM metabolism is not fully understood. Hypoxia plays important roles in maintaining the physiological functions of IVD cells; however, whether hypoxia has any effect on NP ECM production under conditions of SD is still unclear. In the current study, we established an in vitro SD model by exposing NP cells to serum‐free medium. SD decreased the expression of aggrecan and collagen II, as well as the production of sulfated glycosaminoglycan (sGAG) in a time‐dependent manner. However, hypoxia abolished SD‐mediated down‐regulation of aggrecan and collagen II expression via JNK1/2 activation. Moreover, hypoxia abolished SD‐induced MMP‐3 and MMP‐13 expression by inhibiting NF‐κB activation, p65 translocation, and MMP‐3 and MMP‐13 promoter activity. These results indicated that, hypoxia maintained ECM production under conditions of SD. This effect was elicited in part through JNK1/2‐mediated up‐regulation of matrix gene expression and down‐regulation of MMP expression, through the inhibition of NF‐κB. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2059–2066, 2017.

     

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p < 0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1769–1775, 2015.     

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     

Prolonged down regulation of specific gene expression in nucleus pulposus cell mediated by RNA interference in vitro.

To investigate the efficacies and the longevity of RNA interference in nucleus pulposus cells from rat and human, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids were cotransfected with siRNA targeting Firefly luciferase to the nucleus pulposus cells extracted from Sprague Dawley rats and scoliosis patients. The inhibitory effects were evaluated by dual luciferase assay for 3 weeks. Proliferation activity of fibroblast-like cells extracted from the subcutaneous tissue of Sprague Dawley rats and the nucleus pulposus cells were measured by proliferation assay (WST-8 assay) every 2 days after plating. The expression of Firefly luciferase was drastically inhibited both in rats (94.7%) and in humans (93.7%). The inhibitory effects were maintained for 2 weeks and had disappeared completely by 3 weeks. The proliferation activity of nucleus pulposus cells was significantly lower than fibroblast-like cells. We have shown, for the first time, siRNA-mediated gene silencing in rat and human disc cells for a relatively sustained period, probably due to the stability of the nucleus pulposus cells in terms of cell proliferation. The demonstration of this study may allow further exploration of the use of siRNA for scientific research and the treatment of disc degenerative diseases.