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。     

Effects of cyclic dynamic tensile strain on previously compressed inner annulus fibrosus and nucleus pulposus cells of human intervertebral disc—an in vitro study

Our objective was to investigate whether dynamic tensile strain on previously compressed human intervertebral disc (IVD) cells can restore the biosynthetic effects of collagen and glycosaminoglycan. Inner annulus fibrosus (AF) and nucleus pulposus (NP) tissues of adolescent idiopathic scoliosis cases undergoing thoracoscopic discectomy and fusion were cultured on compressive plates. Compressive stress was applied using 0.4 MPa at 1 Hz, for 2 h twice a day for 7 days, to the inner AF and NP tissues, followed by equibiaxial cyclic tensile strain to deform the released cells onto the plate's flexible bottom. With 10% elongation at a rate of 1 Hz, for 2 h twice a day for 7 days, a significant increase in the level of collagen and glycosaminoglycan of the previously compressed inner AF, as well as the level of glycosaminoglycan of the previously compressed NP cells were found. The DNA content and number of endoplasmic reticulum under transmission electron micrograph of the previously compressed inner AF and NP cell were also significantly increased. The results suggested that equibiaxial cyclic tensile strain at a rate of 1 Hz with 10% tensile strain was capable of increasing collagen and glycosaminoglycan synthesis of previously compressed inner AF cells, and glycosaminoglycan synthesis of previously compressed NP cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:503–509, 2010     

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.     

Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells.

The pathogenesis of vibration-induced disorders of intervertebral disc at the cellular level is largely unknown. The objective of this study was to establish a method to investigate the ranges of constructive and destructive hydrostatic loading frequencies and amplitudes in preventing or inducing extracellular disc matrix degradation. Using a hydraulic chamber, normal rabbit intervertebral disc cells were tested under dynamic hydrostatic loading. Monolayer cultures of disc outer annulus cells and 3-dimensional (3-D) alginate cultures of disc nucleus pulposus cells were tested. Effects of different loading amplitudes (3-D culture, 0-3 MPa; monolayer, 0-1.7 MPa) and frequencies (1-20 Hz) on disc collagen and protein metabolism were investigated by measuring 3H-proline-labeled proteins associated with the cells in the extracellular matrix and release of 3H-proline-labeled molecules into culture medium. High frequency and high amplitude hydrostatic stress stimulated collagen synthesis in cultures of outer annulus cells whereas the lower amplitude and frequency hydrostatic stress had little effect. For the same loading duration and repetition, neither treatment significantly affected the relative amount of protein released from the cell layers, indicating that protein degradation and stability were unaffected. In the 3-D nucleus culture, higher amplitude and frequency increased synthesis rate and lowered degradation. In this case, loading amplitude had a stronger influence on cell response than that of loading frequency. Considering the ranges of loading amplitude and frequency used in this study, short-term application of high loading amplitudes and frequencies was beneficial in stimulation of protein synthesis and reduction of protein degradation.     

兔腰椎间盘成分抗原性差异的研究

目的 初步了解兔椎间盘不同成分的免疫源性差别,进一步提示人体内不同椎间盘细胞的抗原性差别.方法 根据生物基因相似性原理及在椎间盘不同部位髓核成分含量存在差异的基础上,采用手术切取实验动物不同区域椎间盘组织并埋植于术野中邻近的椎旁肌内,从而建立髓核糖蛋白埋植组(n=20)和纤维环胶原蛋白埋植组(n=20),并且建立埋植自体肌肉的空白对照组(n=20).分别于第1、2、4、6、8周分批处死动物取标本进行HE染色,观察新生血管化和淋巴细胞浸润;进行免疫组织化学染色,观察CD4、CD8 T淋巴细胞阳性率.结果 HE染色显示糖蛋白埋植组标本在术后1周可见淋巴细胞浸润,4周可见典型新生血管化,并且持续至第8周;胶原蛋白埋植组标本在术后4周才可见少量淋巴细胞浸润,不典型新生血管化;肌肉埋植组在各个时间点均未见典型淋巴细胞浸润和新生血管化形成.各组标本总新生血管化率差异有统计学意义.各时间点所取标本进行免疫组织化学染色,计数CD4+和CD8+T淋巴细胞,发现糖蛋白埋植组CD4+和CD8+淋巴细胞计数均高于胶原蛋白埋植组和肌肉埋植组,胶原蛋白埋植组CD4+和CD8+淋巴细胞计数高于肌肉埋植组,差异有统计学意义.结论 兔腰椎间盘成分中髓核糖蛋白相比纤维环胶原蛋白更易诱导T淋巴细胞分化为CD4+和CD8+细胞,进而推论糖蛋白的自身免疫源性强于胶原蛋白,而胶原蛋白强于肌肉组织,具有弱抗原性.     

兔腰椎间盘成分抗原性差异的研究

目的 初步了解兔椎间盘不同成分的免疫源性差别,进一步提示人体内不同椎间盘细胞的抗原性差别.方法 根据生物基因相似性原理及在椎间盘不同部位髓核成分含量存在差异的基础上,采用手术切取实验动物不同区域椎间盘组织并埋植于术野中邻近的椎旁肌内,从而建立髓核糖蛋白埋植组(n=20)和纤维环胶原蛋白埋植组(n=20),并且建立埋植自体肌肉的空白对照组(n=20).分别于第1、2、4、6、8周分批处死动物取标本进行HE染色,观察新生血管化和淋巴细胞浸润;进行免疫组织化学染色,观察CD4、CD8 T淋巴细胞阳性率.结果 HE染色显示糖蛋白埋植组标本在术后1周可见淋巴细胞浸润,4周可见典型新生血管化,并且持续至第8周;胶原蛋白埋植组标本在术后4周才可见少量淋巴细胞浸润,不典型新生血管化;肌肉埋植组在各个时间点均未见典型淋巴细胞浸润和新生血管化形成.各组标本总新生血管化率差异有统计学意义.各时间点所取标本进行免疫组织化学染色,计数CD4+和CD8+T淋巴细胞,发现糖蛋白埋植组CD4+和CD8+淋巴细胞计数均高于胶原蛋白埋植组和肌肉埋植组,胶原蛋白埋植组CD4+和CD8+淋巴细胞计数高于肌肉埋植组,差异有统计学意义.结论 兔腰椎间盘成分中髓核糖蛋白相比纤维环胶原蛋白更易诱导T淋巴细胞分化为CD4+和CD8+细胞,进而推论糖蛋白的自身免疫源性强于胶原蛋白,而胶原蛋白强于肌肉组织,具有弱抗原性.     

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。     

Viscoelastic properties of intervertebral disc cells. Identification of two biomechanically distinct cell populations

STUDY DESIGN: A combined experimental and theoretical biomechanical study to quantify the mechanical properties of living cells of the porcine intervertebral disc. OBJECTIVES: To quantify zonal variations in the mechanical properties and morphology of cells isolated from the intervertebral disc. SUMMARY OF BACKGROUND DATA: Cellular response to mechanical stimuli is influenced by the mechanical properties of cells and of the extracellular matrix. Significant zonal variations in intervertebral disc matrix properties have been reported. No information is currently available on the corresponding regional variations in the mechanical properties of intervertebral disc cells, despite evidence of significant differences in cellular phenotype and biologic response to loading. METHODS: The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of cells isolated from the anulus fibrosus, transition zone, and nucleus pulposus. RESULTS: Intervertebral disc cells exhibited viscoelastic solid behaviors. Highly significant differences were observed in the morphology, cytoskeletal arrangement, and biomechanical properties of the nucleus pulposus cells as compared with anulus fibrosus or transition zone cells. Cells of the nucleus pulposus were approximately three times stiffer and significantly more viscous than cells of the anulus fibrosus or transition zone. CONCLUSIONS: The findings of this study provide new evidence for the existence of two biomechanically distinct cell populations in the intervertebral disc. These differences in mechanical behavior may be related to observed differences in the cytoskeletal architecture between these cells, and may further play an important role in the development, maintenance, and degeneration of the intervertebral disc.     

Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis.

To establish cell culture of the nucleus pulposus and anulus fibrosus of rat intervertebral disc, the effects of culture conditions on the growth of cells and the synthesis of DNA, proteoglycan, and collagen were studied. For cell culture of the nucleus pulposus, the use of 3-week-old rats and a medium adjusted to pH 7.0 was optimal. There was almost no difference in growth between cells in Ham's F12 medium and those in Dulbecco's Modified Eagle Medium. In cells isolated from the anulus fibrosus, a medium adjusted to pH 7.0-7.6 was preferable, but irrespective of rat age. Culture cells of the nucleus pulposus were composed of large cells with vacuoles and small polygonal cells. These cells had a slight growth activity and a fair capability of proteoglycan and collagen synthesis. Culture cells of the anulus fibrosus were composed of polygonal and spindle-shape cells, and the growth was more vigorous with the potentials for proteoglycan and collagen synthesis than the nucleus cells.     

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。     

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     

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.     

Cyclic Tensile Stretch Stimulates the Release of Reactive Oxygen Species from Osteoblast-like Cells

It is known that the excessive generation of reactive oxygen species (ROS) is a significant factor in tissue injury observed in many disease states. To determine whether extreme levels of mechanical stress applied to osteoblasts enhances ROS synthesis, we loaded cyclic tensile stretch on osteoblast-like HT-3 cells. Cyclic tensile stretch loaded on these cells clearly enhanced ROS synthesis in a time- and magnitude-dependent fashion. Cyclic tensile stretch also enhanced superoxide dismutase (SOD) activity. The disruption of microfilaments with cytochalasin D abolished the stress-induced ROS synthesis. Rotenone, an inhibitor of the mitochondrial electron transport chain, enhanced stress-induced ROS synthesis. These data suggest that actin filament and mitochondria are involved in this action.     

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.     

Changes in the nucleus pulposus of the intervertebral disc in bipedal mice. A light and electron microscopic study

Bipedal mice were produced by clipping the forelimbs and tails of mice within one week of birth. Using light and electron microscopy, the nucleus pulposus of the lumbar intervertebral disc in the bipedal mice was compared with that in normal mice at three, six, and 12 months of age. In normal neonatal mice, the nucleus pulposus is composed of densely packed notochordal cells, which undergo degenerative changes and decrease in number with age. In the bipedal mice, degenerative changes in the nucleus pulposus were accelerated, and herniation of the nucleus pulposus occurred frequently. At the same time, active chondrocytes associated with cartilage matrix appeared in the nucleus pulposus. This sequence of morphologic changes in the nucleus pulposus of the bipedal mice resembles the age-related changes that occur in the nucleus pulposus of the human intervertebral disc. These morphologic changes can be accelerated by creating abnormal mechanical stress. Chondrocytes in the nucleus pulposus may develop from surrounding cartilaginous tissue--cartilage plates and annulus fibrosus.     

Nucleus pulposus glycosaminoglycan content is correlated with axial mechanics in rat lumbar motion segments.

The unique biochemical composition and structure of the intervertebral disc allow it to support load, permit motion, and dissipate energy. With degeneration, both the biochemical composition and mechanical behavior of the disc are drastically altered, yet quantitative relationships between the biochemical changes and overall motion segment mechanics are lacking. The objective of this study was to determine the contribution of nucleus pulposus glycosaminoglycan content, which decreases with degeneration, to mechanical function of a rat lumbar spine motion segment in axial loading. Motion segments were treated with varying doses of Chondroitinase-ABC (to degrade glycosaminoglycans) and loaded in axial cyclic compression-tension, followed by compressive creep. Nucleus glycosaminoglycan content was significantly correlated (p < 0.05) with neutral zone mechanical behavior, which occurs in low load transition between tension and compression (stiffness: r = 0.59; displacement: r = -0.59), and with creep behavior (viscous parameter eta(1): r = 0.34; short time constant tau(1): r = 0.46). These results indicate that moderate decreases in nucleus glycosaminoglycan content consistent with early human degeneration affect overall mechanical function of the disc. These decreases may expose the disc to altered internal stress and strain patterns, thus contributing through mechanical or biological mechanisms to the degenerative cascade.     

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。     

兔椎间盘髓核细胞体外生物学特性的实验研究

目的 对兔椎间盘髓核细胞进行体外培养,观察细胞的形态、表型及超微结构改变,研究其体外生物学特性.方法 取2周龄健康新西兰白兔椎间盘髓核组织,在含有15%灭活FBS的DMEM/F12培养液中培养,倒置相差显微镜下观察原代和传代细胞形态.分别在取材后、原代、第1代、第2代细胞培养期间,进行髓核细胞活力测定;爬片培养后进行甲苯胺蓝、HE、聚集蛋白聚糖番红O、Ⅰ型及Ⅱ型胶原免疫组织化学染色观察;MTT法绘制髓核细胞生长曲线,并行原代及第2代细胞透射电镜观察,对体外细胞的生物学特性进行研究.结果 倒置相差显微镜见原代髓核细胞呈类圆形,折光性较强;5 d开始有细胞贴壁,细胞呈多角形或短梭形;6～8 d细胞生长进入指数生长期;约17 d时,细胞长满瓶壁,可进行传代;随传代次数增加,细胞形态逐渐由多角形、短梭形向长梭形改变.髓核细胞活力测定在刚分离完成后细胞活力为95%～97%,原代培养期间为98%～100%,第1代培养期间仍能维持为100%,第2代细胞活力下降较为明显,为75%～80%.髓核细胞甲苯胺蓝染色呈强阳性:HE染色见细胞核、细胞质着色明显.第1代髓核细胞Ⅰ型胶原免疫组织化学染色呈阴性,Ⅱ型胶原免疫组织化学染色呈阳性,聚集蛋白聚糖番红O染色呈阳性;第2代细胞Ⅰ型胶原免疫组织化学染色呈阳性,Ⅱ型胶原免疫组织化学染色呈弱阳性,聚集蛋白多糖番红O染色着色较浅.MTT生长曲线与体外细胞培养时生长过程相符.透射电镜显示原代髓核细胞内线粒体少,胞质内有大量糖原颗粒,随传代次数增加,糖原颗粒减少,线粒体数量增多,细胞器开始肿胀.结论 明确了兔髓核细胞体外生物学特性变迁,为组织工程髓核的种子细胞研究提供了实验依据.     

The effect of hydrostatic pressure on intervertebral disc metabolism.

STUDY DESIGN: By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in an alginate. OBJECTIVE: To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells. SUMMARY OF BACKGROUND DATA: The influence of compression (both hydrostatic and mechanical) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc, was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate. METHODS: Fresh cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 1 MPa and the other at atmospheric pressure. The effects of 1 MPa were compared against atmospheric pressure by measuring the incorporation of [3H]-proline and [35S]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and II collagen. RESULTS: Comparisons with atmospheric pressure yielded the following findings: 1) In the incorporation studies, the nucleus and anulus cells exhibited a differential response to a hydrostatic pressure of 1 MPa. Collagen and proteoglycan syntheses were stimulated in the nucleus cells and inhibited in the anulus cells. 2) There was no significant increase in cell proliferation, as measured by DNA content, at 1 MPa for either the anulus or nucleus cells. 3) The mRNA levels of collagen (Col 1A1 and Col 2A1) and aggrecan increased at 1 MPa in both the nucleus and anulus cells. CONCLUSIONS: Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells.