24,25-(OH)2D3 Regulation of Matrix Vesicle Protein Kinase C Occurs Both During Biosynthesis and in the Extracellular Matrix

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCα) and PKCζ, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)₂D₃ [14]. In the present study, we examined whether the effect of 24,25-(OH)₂D₃ on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10⁻⁹–10⁻⁷ M 24,25-(OH)₂D₃. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)₂D₃-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)₂D₃-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)₂D₃-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)₂D₃ on PKC involved phospholipase A₂ (PA₂). Quinacrine (a PA₂ inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)₂D₃, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA₂ plays a role in the nongenomic regulation of PKC by 24,25-(OH)₂D₃. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)₂D₃ modulated the distribution of PKCα, β, and ζ between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24,25-(OH)₂D₃ regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation. Received: 11 October 1996 / Accepted: 25 April 1997     

三种软骨细胞分离培养方法对细胞骨架的影响比较

目的 比较分离培养人类软骨细胞的3种方法，探讨各种方法的特点及对细胞骨架的影响。方法 分别采用酶消化法、组织贴块法、酶消化结合组织贴块法分离软骨细胞，测量每种方法分离的软骨细胞瓶底汇合形成单层时间，进行比较，以甲苯氨蓝染色、Ⅱ型胶原免疫组织化学评价细胞的生物特性，并用磁驱动原子力显微镜（MAC mode AFM）对二代细胞表面形态及细胞骨架特性进行检测。结果 MAC mode AFM检测显示，酶消化结合组织贴块法分离软骨细胞表面形态、细胞骨架分布更接近生理状态，甲苯氨蓝、Ⅱ型胶原免疫组织化学染色显示强阳性，进行方差分析及q检验，3种方法分离的软骨细胞瓶底汇合形成单层时间差异有统计学意义（P〈0．05）。结论 酶消化结合组织贴块法细胞分离效率高、生长速度快、细胞骨架特性保持更好、细胞纯度高，在细胞骨架研究中可优先选用。     

马钱子碱对兔软骨细胞增殖的影响

目的：观察马钱子碱对体外培养兔软骨细胞增殖的影响。方法：分离、培养兔的软件细胞，采用四甲基偶氮唑盐比色法检测细胞增殖。结果：马钱子碱高、中、低剂量组细胞增殖率均高于空白对照组(P＜0．05)，一氧化氮(NO)能抑制软骨细胞的增殖，马钱子碱能拮抗NO对软骨细胞增殖的抑制作用。结论：马钱子碱能有效促进软骨细胞增殖。     

同种异型骨组织细胞软骨组织细胞种植修复股骨头坏死的实验研究

目的：利用同种异型骨细胞，软骨细胞群种植修复兔的坏死股骨头，观察种植细胞生长状况，方法：采用酶消化1-3d龄的新西兰乳兔的颅盖骨及干骺端，分别提取骨细胞，软骨细胞群，混合注射入成年家兔的股骨头坏死区。结果：与对照侧相比，种植术后2-8周，50%的种植区表现为活跃的原始结缔组织增生及膜性化骨，术后8周观察到1例软骨化骨，术后12周，坏死区的骨修复差别消化。结论：原始的骨细胞，软骨细胞群，具有低免疫原性及旺盛分裂增殖特点，在同种异型种植移植时，其迅速合成的其质可包埋保护自身，降低免疫排异，获得生存。     

自由基在大骨节病病理过程中的作用 Ⅲ.自由基及黄腐酸对软骨细胞的损伤

本文利用软骨细胞的体外培养技术研究了活性氧自由基及黄腐酸对软骨细胞的损伤效应。结果表明:在一定浓度的自由基和黄腐酸作用下,软骨细胞的生长受到了抑制,细胞形态及其合成分泌基质的能力也发生了明显的变化。本文对黄腐酸的自由基性质及大骨节病与软骨细胞损伤的关系进行了探讨。     

生长因子在关节软骨损伤修复中的应用进展

关节是人体重要的运动器官,其组织结构特殊,成熟关节软骨组织没有血供,一旦损伤难以自愈.临床上常用的关节软骨损伤修复手段有微骨裂术、关节软骨移植、关节置换术、软骨组织工程等,但这些方法的修复效果都不理想.生长因子是体内组织分泌的一种具有生物活性的物质,可促进细胞生长、增殖、迁移和分化.软骨发育过程中有许多生长因子参与,如成纤维细胞生长因子(FGF)、骨形态发生蛋白(BMP)、胰岛素样生长因子(IGF)等.研究显示,在关节软骨修复过程中加入外源性生长因子可有效促进关节软骨损伤的修复.对目前应用于关节软骨损伤修复研究中的生长因子进行综述,讨论这些生长因子在关节软骨发育及其在关节软骨修复中的作用,分析生长因子在关节软骨修复应用中存在的问题.     

A mechanical refractory period of chondrocytes after dynamic hydrostatic pressure

Purpose: Mechanical stimulation, a crucial factor for maintaining the cartilaginous phenotype and promoting the chondrogenesis, has been widely used in autologous chondrocyte transplantation. This study was designed to investigate a novel concept of mechanical refractory period of chondrocytes after dynamic hydrostatic pressure (dHP).

Materials and methods: dHP protocols (0.1?Hz, 2?MPa) were applied. The variation in type II collagen (Col II) expression induced by each dHP unit was measured. The dynamic remodeling of F-actin during the mechanical protocols was observed morphologically and mechanically by laser confocal microscopy and optical magnetic twisting cytometry (OMTC), respectively. About 20?ng/ml VEGF was used to stabilize the F-actin and restrain the mechanical refractory period.

Results: Compared with the remarkable increase of Col II (16-fold) induced by the initial dHP unit, the chondrocytes entered a mechanical refractory period and the second unit hardly elevated Col II expression (only 2.9-fold). This refractory period recovered partially within 2?h. The uniform, parallel, and coarse fibers of F-actin before dHP became thin, sparse, and disordered, and the cell stiffness decreased concomitantly. The variations in both the morphology and the mechanical property of F-actin were highly synchronous to the mechanical refractory period and recovered in a time-dependent manner. VEGF postponed the appearance of this refractory period and maintained the high expression of Col II by VEGF/p38/MAPKAPK-2/LIMK/cofilin pathway.

Conclusion: A mechanical refractory period of chondrocytes has been discovered and defined in this study. The F-actin depolymerization is the putative mechanism, and this refractory period can be postponed by VEGF-induced F-actin stabilization.     

筋骨痹痛汤药物血清对白细胞介素-1β诱导的兔软骨细胞凋亡的保护作用

目的 观察筋骨痹痛汤药物血清对白细胞介素-1β（IL-1β）诱导的兔软骨细胞Bcl-2、Bax表达及细胞凋亡的影响,探讨筋骨痹痛汤治疗骨性关节炎的机制。
方法 随机取雄性成年新西兰兔8只分成4组,分别灌服生理盐水、补肝肾中药（骨碎补、续断和淫羊霍）、独活寄生汤和筋骨痹痛汤,制得空白血清和药物血清。分离制备幼兔软骨细胞,采用甲苯胺蓝染色和Ⅱ型胶原免疫荧光法鉴定软骨细胞。取第3代软骨细胞,随机分5组实验：空白组（A组）、模型组（B组）、补肝肾中药对照组（C组）、独活寄生汤对照组（D组）、筋骨痹痛汤组（E组）。A组用含10%空白血清的DMEM培养36h,其余各组均先采用含IL-1β的DMEM诱导培养12h,然后B、C、D、E组分别给予含10%空白血清、10%补肝肾中药药物血清、10%独活寄生汤药物血清、10%筋骨痹痛汤药物血清的DMEM培养24h。实时定量聚合酶链反应（Real-time PCR)和免疫印迹法检测兔软骨细胞Bcl-2和Bax的mRNA和蛋白表达,流式细胞术检测细胞凋亡比例。结果 经鉴定分离制备的细胞为软骨细胞。软骨细胞培养24h后,C、D、E组Bcl-2 mRNA和蛋白表达高于B组,D、E组高于C组,E组高于D组,差异均有统计学意义（P＜0.05）;Bax mRNA和蛋白表达,以及软骨细胞凋亡率,C、D、E组低于B组,D、E组低于C组,E组低于D组,差异均有统计学意义（P＜0.05）。结论 筋骨痹痛汤药物血清对IL-1β诱导的兔软骨细胞凋亡具有明显保护作用,其机制可能是通过促进Bcl-2表达和抑制Bax表达,调节Bcl-2与Bax的比例来实现。     

芹菜素对实验室兔膝关节软骨缺损的影响观察

目的 研究芹菜素对于转骨形态发生蛋白(BMP-7)软骨细胞修复关节软骨缺损的作用.方法 IL-1β体外诱导兔软骨细胞分泌白细胞介素-8(IL-8)和可溶性细胞黏附因子-1(sICAM-1).在培养系统中加入不同浓度芹菜素(10 μmol/L、25 μmol/L和50 μmol/L),采用酶联免疫吸附法检测芹菜素对诱导兔软骨细胞分泌IL-8和sICAM-1的影响.将软骨细胞种植于Matrigel胶支架上,将体外培养出的软骨膜块植入家兔关节软骨缺损模型中,采用随机数字表法分为3组(n=4):假手术组、转BMP-7组和转BMP-7+芹菜素组.5周后进行组织学观察和Wakitani评分.结果 10 μmol/L、25 μmol/L和50 μmol/L芹菜素能显著抑制IL-1β诱导兔软骨细胞分泌IL-8和sICAM-1的作用[(6803.63±162.31) ng/g、(6005.74±201.49) ng/g、(5202.34 ±271.67) ng/g比(10011.84±239.29)ng/g,P=0.00],并促进软骨细胞增殖.转BMP-7组和转BMP-7+芹菜素组的Wakitani评分显著低于假手术组[(3.68±0.86)比(8.25 ±0.90),P=0.00;(3.21 ±0.78)比(8.25 ±0.90),P=0.00].另外,还发现芹菜素可减少修复过程中的炎症反应发生.结论 芹菜素可促进转BMP-7软骨细胞膜块构建以及关节软骨缺损的修复.     

体外培养大鼠软骨细胞老化的机制初探

目的对体外传代培养的大鼠软骨细胞进行相关因子检测，初步探讨其复制性老化（传代培养出现的老化表现）的机制。方法对连续培养的第二、三、四代软骨细胞进行实验，采用免疫细胞化学检测p16、pRb、E2F、CyclinD、CDK4等因子表达水平，TRAP—ELISA检测端粒酶活性，观察软骨细胞老化过程中各因子的变化。结果随着细胞复制性老化p16、pRb、CyelinD表达水平显著上升（P〈0．01），E2F、CDK4、端粒酶表达水平显著下降（P〈0．01）。结论体外培养软骨细胞老化过程与p16、pRb、CyclinD表达增加，E2F、CDK4、端粒酶表达下降密切相关。