维药买朱尼含药血清影响大鼠关节软骨细胞基质金属蛋白酶1及其组织抑制因子1的表达

背景：基质金属蛋白酶1及其组织抑制因子1的失衡是骨关节炎软骨降解的关键。 目的：观察维药买朱尼对白细胞介素1β诱导的大鼠关节软骨细胞基质金属蛋白酶1及其组织抑制因子1表达的影响。 方法：从1周龄SD大鼠关节中分离培养原代软骨细胞,鉴定后选用第2代细胞随机分为正常对照组、模型组、买朱尼组。模型组和买朱尼组用10 μg/L的白细胞介素1β干预24 h后,买朱尼组在此基础上加入体积分数10%的买朱尼含药血清,模型组加入正常大鼠血清,继续培养12,24,48 h进行实验。 结果与结论：各组细胞培养48 h,RT-PCR检测发现体积分数10%的买朱尼含药血清可上调软骨细胞基质金属蛋白酶组织抑制因子1 mRNA的表达同时抑制基质金属蛋白酶1 mRNA的表达,但此作用在细胞培养的24,36 h时不明显。同时免疫荧光细胞化学染色也显示买朱尼含药血清可促进软骨细胞基质金属蛋白酶组织抑制因子1的表达。说明买朱尼可以调节白细胞介素1β诱导的大鼠关节软骨细胞基质金属蛋白酶1及其组织抑制因子1的表达,且此作用具有时间依赖性。     

骨关节炎时内源性细胞因子及基质金属蛋白酶抑制因子1对关节软骨的影响*

背景：研究发现,细胞因子通过影响关节软骨基质的分解代谢和合成代谢的平衡来参与骨关节炎的形成,其中白细胞介素1β、基质金属蛋白酶抑制因子1在其中起重要作用。 目的：观察内源性细胞因子白细胞介素1β及基质金属蛋白酶抑制因子1与骨关节炎关节软骨退变的关系。 方法：纳入2006-06/2009-09于哈尔滨医科大学附属第五医院就诊的原发性骨关节炎患者37例,在患者行关节镜检时抽取关节液及分离平滑光亮的滑膜组织。另取10例正常关节液标本及平滑光亮的滑膜组织标本作为对照。 结果与结论：ELISA法检测结果显示骨关节炎患者关节液和滑膜组织中白细胞介素1β、基质金属蛋白酶抑制因子1水平均显著高于正常水平,且患者骨关节炎越严重,关节液和滑膜组织中白细胞介素1β、基质金属蛋白酶抑制因子1的水平越高。说明关节液中白细胞介素1β、基质金属蛋白酶抑制因子1水平与骨关节炎、关节软骨退变有关。关键词：基质金属蛋白酶抑制因子1;骨关节炎;白细胞介素1β;关节液;滑膜组织;关节软骨退变 缩略语注释：TIMP: tissue inhibitors of metalloproteinase, 基质金属蛋白酶抑制因子;IL-1β: interleukin-1β,白细胞介素1β doi:10.3969/j.issn.1673-8225.2012.15.002     

细胞外基质因子在兔软骨细胞损伤模型中的表达变化

目的:研究细胞外基质因子在兔关节软骨细胞损伤后不同时间点的表达变化水平。方法:通过机械划伤制备关节软骨细胞损伤模型,观察软骨细胞在划伤后的形态变化。采用实时荧光定量PCR检测软骨细胞在划伤前与划伤后1、3、5 d和7 d共5个时间点的基质金属蛋白酶13(MMP13)及基质金属蛋白酶抑制物1(TIMP1)、透明质酸和Ⅱ型胶原(ColⅡ)基因mRNA表达水平。结果:成功建立了兔关节软骨细胞损伤模型。与划伤前相比,MMP13基因水平在细胞损伤第1天表达明显下降,随后出现升高趋势,第5天达到最高后逐渐下降。TIMP1基因表达量损伤后先上升至第1天然后下降,在第3天降至最低,随后呈现升高趋势。透明质酸和Col-Ⅱ基因水平第1天表达明显降低,随后呈上升趋势,透明质酸mRNA表达量至第5天达到最高,然后逐渐下降。结论:MMP13、TIMP1、透明质酸和Col-Ⅱ基因在细胞损伤后的表达规律相异,为软骨细胞外基质因子表达与软骨细胞之间关系提供实验依据。     

急性脊髓损伤模型大鼠白细胞介素1β和核因子κB的表达

背景：在脊髓损伤后的继发性损伤过程中,白细胞介素1β参与刺激其他细胞因子和损伤介质的合成。 目的：观察白细胞介素1受体拮抗剂对急性脊髓损伤模型大鼠损伤脊髓白细胞介素1β与核因子κB表达的影响。 方法：采用改良Allen法建立SD大鼠急性脊髓损伤模型,造模后分别在损伤处敷含白细胞介素1受体拮抗剂或仅有生理盐水的明胶海绵,于脊髓损伤1,48,72 h取损伤段脊髓标本,免疫组织化学染色检测白细胞介素1β与核因子κB的表达。 结果与结论：经白细胞介素1受体拮抗剂治疗后,损伤脊髓组织白细胞介素1β和核因子κB的表达均显著降低。说明白细胞介素1受体拮抗剂可通过抑制白细胞介素1β和核因子κB的表达,减轻局部炎症反应,对急性脊髓损伤大鼠损伤段脊髓发挥保护作用。     

成骨细胞旁分泌影响软骨细胞中MMPs与TIMPs的表达

背景：细胞之间体外共培养能最大限度的模拟体内真实的微环境,细胞划痕实验及炎症因子白细胞介素1β刺激后基质金属蛋白酶及基质金属蛋白酶抑制剂之间的平衡可能破坏,从而导致关节软骨细胞外基质的降解,软骨细胞功能的失调,关节软骨的退变。目的：在成骨细胞上清液与软骨细胞体外共培养下,观察炎症因子白细胞介素1β对体外培养的软骨细胞的迁移、基质金属蛋白酶及组织金属蛋白酶抑制剂表达的影响。方法：实验分为软骨细胞单培养组﹑软骨细胞与成骨细胞上清液共培养组和软骨细胞与成骨细胞上清液共培养+白细胞介素1β组,划痕实验观察3组24 h软骨细胞的迁移变化;半定量PCR实验分析以上3组24 h软骨细胞中基质金属蛋白酶1,2,3,9及组织金属蛋白酶抑制剂1,2,3,4的变化情况。结果与结论：与单培养组比较,共培养组和共培养+白细胞介素1β组细胞迁移率显著增加(P < 0.01);与单培养组比较,共培养组中基质金属蛋白酶1,2,3,9基因表达明显增高(P < 0.05),共培养+白细胞介素1β组基质金属蛋白酶1,3,9基因表达明显增高(P < 0.01);与单培养组比较,共培养组和共培养+白细胞介素1β组中组织金属蛋白酶抑制剂1基因表达明显升高(P < 0.01),组织金属蛋白酶抑制剂3,4基因表达明显下降(P < 0.05)。提示成骨细胞上清液与软骨细胞共培养促进软骨细胞的迁移,增强软骨细胞中基质金属蛋白酶1,2,3,9的基因表达且调节组织金属蛋白酶抑制剂家族的基因表达。白细胞介素1β抑制共培养的软骨细胞迁移及组织金属蛋白酶抑制剂家族的基因表达。        中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

白细胞介素1β对软骨细胞miR-27b和基质金属蛋白酶13蛋白表达的影响

BACKGROUND: Matrix metalloproteinase-13 is most active in the degradation of collagen type II in the extracellular matrix of cartilage. Interleukin-1 (IL-1) is thought to be the inducer of matrix metalloproteinases, and participates in the degradation and degeneration of articular cartilage. OBJECTIVE: To study the influence of IL-1β on microRNA-27b (miR-27b) and matrix metalloproteinase-13 expression of chondrocytes in rats. METHODS: Chondrocytes isolated from seven male Wistar rats were cultured and divided into IL-1β stimulation group and control group. No stimulus was given in the control group; 10 μg/L of serum free medium was used to culture rat chondrocytes in the IL-1β stimulation group. Cell growth was observed at 0, 24, and 48 hours under an inverted microscope. miR-27b and matrix metalloproteinase-13 expression in the cultured chondrocytes were detected. RESULTS AND CONCLUSION:The relative expression of matrix metalloproteinase-13 in rat chondrocytes was gradually increased when induced by IL-1β at 0, 24, and 48 hours (P < 0.05). Expression of miR-27b and miR-31 in rat chondrocytes at 24 and 48 hours induced by IL-1β gradually decreased (P < 0.05); conversely, expression of MiR-26a, miR-26b, miR-23, and miR-204 gradually increased (P < 0.05). After 48 hours of IL-1β induction, expression of miR-27b was the lowest in rat chondrocytes (P < 0.05). These findings suggest that IL-1β inhibits miR-27b expression, strengthens the expression of matrix metalloproteinase-13, and damages chondrocytes, contributing to both the onset and progression of osteoarthritis. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

粉防己碱减轻IL-1β诱导的体外人关节软骨细胞损伤

目的 粉防己碱(Tet)对白细胞介素1β(IL-1β)诱导原代人关节软骨细胞损伤的影响。方法 将人关节软骨细胞分为对照组、IL-1β组、低氧诱导因子-1α(HIF-1α)抑制剂[二甲氧基雌二醇(2-ME2)]组、Tet低、中、高浓度组,每组设置6个重复。MTT法检测细胞增殖;流式细胞仪检测细胞凋亡;ELISA检测细胞中炎性相关因子肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶3(MMP-3)、诱导性一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)的水平以及抗氧化因子超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)的活性;Western blot检测细胞中HIF-1α、VEGF蛋白的表达。结果 与对照组相比,IL-1β组细胞的凋亡率、TNF-α、MMP-3、iNOS、COX-2的水平以及HIF-1α、VEGF蛋白表达均升高,细胞存活率、SOD和GPx活性均降低,差异有统计学意义(P<0.05);与IL-1β组相比,2-ME2组和Tet低、中、高浓度组细胞的凋亡率、TNF-α、MMP-3、iNOS、COX-2的水平以及HIF-1α、VEGF蛋白表达均降低,细胞存活率、SOD...     

氨基葡萄糖对白细胞介素1β诱导体外培养人骨关节炎软骨细胞蛋白聚糖代谢的影响

背景：蛋白聚糖和胶原纤维的降解是骨关节炎发病的主要生理和病理学基础,氨基葡萄糖不仅可以减轻骨关节炎疼痛症状,同时可以延缓骨关节炎的病理改变。 目的：了解氨基葡萄糖对白细胞介素1β诱导骨关节炎软骨细胞蛋白聚糖代谢的影响。 方法：取骨关节炎患者的软骨细胞,分阶段酶消化法进行体外原代培养。在培养液中加入白细胞介素1β诱导剂,设立不含药兔血清对照组、白细胞介素1β对照组和加入不同浓度兔氨基葡萄糖含药血清的实验组。 结果与结论：各浓度氨基葡萄糖含药血清组体外培养软骨细胞释放入培养液中糖胺聚糖百分比均值明显低于对照组(P < 0.01),随氨基葡萄糖浓度的增加,糖胺聚糖百分比逐渐降低;蛋白聚糖合成标记物3B3含量的均值较对照组明显增高(P < 0.01),与氨基葡萄糖浓度呈正相关;而蛋白聚糖降解标记物5D4含量的均值则正相反;氨基葡萄糖可以增加骨关节炎患者软骨细胞蛋白聚糖mRNA表达,减低基质金属蛋白酶1,3 mRNA表达。表明氨基葡萄糖可以抑制白细胞介素1β对骨关节炎患者软骨细胞蛋白聚糖代谢的促进作用,达到保护软骨,防止骨关节炎的目的。     

双醋瑞因干预体外培养关节软骨结缔组织生长因子的表达

背景：在骨关节炎软骨退变中结缔组织生长因子作为一种重要的效应分子在软骨细胞的增殖、分化方面发挥重要作用。临床应用双醋瑞因治疗骨关节炎已取得了良好的效果,但其治疗的确切机制尚不清楚。 目的：观察不同浓度双醋瑞因对体外白细胞介素1β诱导下软骨细胞中结缔组织生长因子的影响。 方法：体外培养SD大鼠关节软骨细胞,重组人白细胞介素1β刺激软骨细胞制备体外骨关节炎模型。实验分组：正常对照组不给予任何处理因素;模型对照组给予重组人白细胞介素1β;实验组给予不同浓度双醋瑞因+10 μg/L重组人白细胞介素1β。利用MTT比色法观察软骨细胞的增殖情况,Western Blot法检测结缔组织生长因子的表达。以上实验均重复3次。 结果与结论：MTT结果显示,与正常对照组比较,双醋瑞因能促进软骨细胞MTT增殖活性,以浓度为10-5 mol/L更明显(P < 0.01),白细胞介素1β作用后各实验组软骨细胞增殖能力下降(P < 0.05);但与正常对照组比较,无论是否加白细胞介素1β,浓度为10-4 mol/L和10-5 mol/L双醋瑞因仍能促进软骨细胞MTT增殖活性(P < 0.05)。Western Blot检测结果显示,白细胞介素1β能够降低结缔组织生长因子的表达(P < 0.01),浓度为    10-5 mol/L双醋瑞因能够显著促进白细胞介素1β诱导下结缔组织生长因子的表达,显著高于模型对照组(P < 0.01)。提示双醋瑞因可以促进白细胞介素1β诱导下结缔组织生长因子的高表达,其可能是双醋瑞因促进软骨细胞的分化增殖,治疗骨关节炎的作用机制之一。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

白芍总苷对骨关节炎软骨细胞炎症及退变的影响

背景:软骨退变是骨关节炎的病理表现之一,在骨关节炎发病进展中起着重要作用.白芍总苷是白芍的有效成分,有很强的抗炎、止痛、免疫调节作用,然而其对骨关节炎软骨细胞炎症及退变的影响仍不明确.目的:观察白芍总苷对骨关节炎软骨细胞中白细胞介素1β、肿瘤坏死因子α及基质金属蛋白酶13水平的影响.方法:应用胶原酶消化法从膝关节置换手...     

Age-related changes in rabbit articular chondrocytes

Cell culture techniques have been used extensively in the study of the aging process at the cellular level. The "senescent" articular chondrocyte seems to be a good model to examine the responses to aging in osteoarthritis, one of the most frequent diseases of old age. Thus in vitro chondrocyte "senescence", established by weekly subculture was characterized by a declining proliferation rate during late passages, from a rapid growth rate in early subculture to a complete loss of the proliferation capacity after 8 +/- 1 passages. Flow cytometric analysis show a time course decrease in the fraction S and G2 + M during the subculture, and a concomitant enhancement in protein content related to the increase of cell size. The immunocytochemistry assays revealed an appearance of a rigid cytoarchitecture with an increase in the number, and organization, of three cytoskeletal components: actin, tubulin and vimentin. The cultured chondrocytes therefore undergo in vitro aging analogous to that described for diploid fibroblasts, and could constitute a cellular model for pharmacological and toxicological assays.     

同种异体血清体外培养兔膝关节软骨细胞的增殖*◆

背景：兔关节软骨细胞体外单层培养采用胎牛血清培养基易去分化,有必要寻找合适培养基来提高兔关节软骨细胞培养质量。 目的：观察同种异体兔血清对体外培养的兔膝关节软骨细胞增殖能力的影响。 方法：以0.4%链霉蛋白酶和0.025%Ⅱ型胶原酶分离成年兔膝关节软骨细胞,将获得的软骨细胞随机分为实验组和对照组。实验组以体积分数10%异体兔血清+DMEM/F12培养;对照组以体积分数10%胎牛血清+DMEM/F12培养,传代培养至4代。 结果与结论：实验组前4代软骨细胞增殖较对照组慢,但软骨细胞形态未发生明显改变而对照组软骨细胞出现去分化现象。提示体积分数10%异体兔血清培养利于维持软骨细胞增殖和形态的稳定,是较好的获取大量优良软骨细胞的体外培养方式。      

成人关节软骨细胞增殖与人参皂甙Rg1的促进作用

背景：人参总皂甙的主要有效成分为人参皂甙Rg1,有促进细胞增殖、抗衰老、抗细胞凋亡、抗炎及抗自由基等作用,但以往实验对象多为鼠、兔等动物的关节软骨细胞。 目的：体外培养成人软骨细胞,观察人参皂甙Rg1对体外培养的成人关节软骨细胞增殖的促进作用。 方法：实验分2组,Rg1组使用含质量浓度为40 mg/L人参皂甙Rg1的DMEM培养液培养,对照组使用未加人参皂甙的DMEM培养液培养,每日应用倒置相差显微镜观察细胞生长情况至第7天,并进行细胞计数。同时培养至第6天检测软骨细胞中Ⅱ型胶原的表达。 结果与结论：对照组自培养第2天起细胞进入对数增殖期,到第7天细胞数约为接种时的8倍。Rg1组自培养第2天起细胞增殖能力较对照组增强(P < 0.05),到第7天细胞数约为接种时的18倍(P < 0.05)。免疫组织化学SABC法检测结果显示,Rg1组与对照组细胞胞浆均可见Ⅱ型胶原表达,两组差异无显著性意义(P > 0.05)。说明人参皂甙Rg1可促进成人软骨细胞的增殖。     

Poly(hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro

The present investigation describes the production of extracellular matrix of rabbit articular cartilage chondrocytes grown on scaffolds of polyhydroxybutyrate (PHB) blended with poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for up to 7 days. The mRNA level of type II collagen of chondrocytes seeded on all scaffolds consisting of PHBHHx were obviously higher than that of PHB-only scaffold throughout the culture period, suggesting the positive effect of PHBHHx on extracellular matrix production. Second-harmonic generation (SHG) imaging technique, combined with confocal fluorescence microscopy (CFM) revealed that PHBHHx in PHB scaffold provided better surface properties for anchoring type II collagen filaments and their penetration into internal layers of the scaffolds. Glycosaminoglycan (GAG), a major composition of extracellular matrix, showed a sharp increase in construct of 1:2 PHB/PHBHHx scaffold after 7 day cultivation, while only a small increase was observed in all other tested scaffolds. At the same time, total collagen contents in all scaffolds containing PHBHHx increased with time, with the maximum collagen production of 742.1+/-99.2mg/g dry weight observed in construct of 1:2 PHB/PHBHHx scaffold inoculated for 7 days, this was almost 4-fold higher than that in scaffold of PHB only. It appears that the presence of right proportion of PHBHHx in the composite system of PHB/PHBHHx highly favored the production of extracellular matrix of articular cartilage chondrocytes.     

Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production

Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.     

Thioredoxin gene expression in rat knee articular cartilage after full-thickness injury

To study the expression of the antioxidative protein thioredoxin (TRX) in intact and injured articular cartilage, we examined the presence of trx mRNA in rat knee joints by in situ hybridization. Our results showed that in the intact knee, most cells, including articular cartilage chondrocytes, expressed trx mRNA. We examined joints at 1, 7, 14, and 28 days after the infliction of full-thickness cartilage injuries on distal femoral condyles. At 1 day after injury, no significant changes were observed in the wound or in trx expression pattern. However, at 7 to 28 days after injury, the wound became filled with repair tissue. Also, trx expression was detected in differentiating mesenchymal cells in the deeper zones of the wound but not in fibroblast-like cells in the upper part of the repair tissue, toward the joint cavity. This lack of TRX expression in the fibroblast-like cells may underlie the susceptibility of the repair tissue fibrocartilage to oxidative stress.     

软骨损伤后基质细胞衍生因子1的表达

背景：研究表明基质细胞衍生因子1对骨髓间充质干细胞的迁移、聚集有影响。 目的：观察软骨损伤后不同时间损伤修复区组织基质细胞衍生因子1的表达以及与骨髓间充质干细胞迁移的关系。 方法：建立兔软骨损伤模型,分别于建模后2,5,7,10,14,28 d取损伤灶及边缘区组织,检测基质细胞衍生因子1表达和体外细胞迁移实验,观察基质细胞衍生因子1对骨髓间充质干细胞、软骨细胞的迁移影响。 结果与结论：基质细胞衍生因子1的表达呈现出时间变化的趋势,软骨损伤后第7天达到高峰(P < 0.05)。体内移植的骨髓间充质干细胞主要聚集在软骨损伤灶周围,但在封闭CXC趋化因子受体4后,此聚集现象逐渐减弱(P < 0.05)。结果证实,局部组织中基质细胞衍生因子1的表达在软骨损伤早期明显升高,对骨髓间充质干细胞向软骨损伤修复区迁移有重要作用。     

Integrative repair of cartilage with articular and nonarticular chondrocytes

Articular chondrocytes can synthesize new cartilaginous matrix in vivo that forms functional bonds with native cartilage. Other sources of chondrocytes may have a similar ability to form new cartilage with healing capacity. This study evaluates the ability of various chondrocyte sources to produce new cartilaginous matrix in vivo and to form functional bonds with native cartilage. Disks of articular cartilage and articular, auricular, and costal chondrocytes were harvested from swine. Articular, auricular, or costal chondrocytes suspended in fibrin glue (experimental), or fibrin glue alone (control), were placed between disks of articular cartilage, forming trilayer constructs, and implanted subcutaneously into nude mice for 6 and 12 weeks. Specimens were evaluated for neocartilage production and integration into native cartilage with histological and biomechanical analysis. New matrix was formed in all experimental samples, consisting mostly of neocartilage integrating with the cartilage disks. Control samples developed fibrous tissue without evidence of neocartilage. Ultimate tensile strength values for experimental samples were significantly increased (p < 0.05) from 6 to 12 weeks, and at 12 weeks they were significantly greater (p < 0.05) than those of controls. We conclude that articular, auricular, and costal chondrocytes have a similar ability to produce new cartilaginous matrix in vivo that forms mechanically functional bonds with native cartilage.     

Three-dimensional (3-D) imaging of chondrocytes in articular cartilage: growth-associated changes in cell organization

Three-dimensional (3-D) imaging and analysis techniques can be used to assess the organization of cells in biological tissues, providing key insights into the role of cell arrangement in growth, homeostasis, and degeneration. The objective of the present study was to use such methods to assess the growth-related changes in cell organization of articular cartilage from different sites in the bovine knee. Three-dimensional images of fetal, calf, and adult cartilage were obtained and processed to identify cell nuclei. The density of cells was lower with growth and with increasing depth from the articular surface. The cell organization, assessed by the angle to the nearest neighboring cell, also varied with growth, and reflected the classical organization of cells in adult tissue, with neighboring cells arranged horizontally in the superficial zone (average angle of 20 degrees) and vertically in the deep zone (60 degrees). In all other regions and growth stages of cartilage, the angle was approximately 32 degrees, indicative of an isotropic organization. On the contrary, the nearest neighbor distance did not vary significantly with growth or depth. Together, these results indicate that cartilage growth is associated with distinctive 3-D arrangements of groups of chondrocytes.