Gas7在成年大鼠脊髓和脊神经节的表达

目的 研究生长休止蛋白7（Gas7）在成年大鼠脊髓和脊神经节的表达.方法 成年SD大鼠12只,采用逆转录聚合酶链反应（RT-PCR）方法、焦油紫染色以及免疫组织化学方法来观察Gas7基因核酸和蛋白在成年SD大鼠脊髓和脊神经节的表达.结果 RT-PCR结果显示,脊髓和脊神经节有较丰富的Gas7 mRNA表达.免疫组化结果显示：与焦油紫染色相对照,脊髓灰质各板层神经元均表达Gas7蛋白,与其它版层相比较,后角Ⅱ版层胶状质的小细胞和前角Ⅸ版层的运动神经元显色较深且数量较多.脊髓白质Gas7免疫阳性反应较弱且分布均匀.脊神经节内大型感觉神经元呈Gas7免疫强阳性反应,中、小型感觉神经元为弱阳性反应.结论 本文首次描述了Gas7在成年大鼠脊髓和脊神经节的表达,为进一步研究Gas7在成年神经系统再生和修复过程中的功能提供形态学基础.     

生长休止蛋白7在成年大鼠肾脏、心脏和肝脏中的差异表达

目的研究生长休止蛋白7（Gas7）在成年大鼠肾脏、心脏和肝脏的表达。方法成年SD大鼠16只,分别采用逆转录聚合酶链反应（RT-PCR）方法和免疫组织化学方法检测Gas7基因mRNA和蛋白在成年SD大鼠肾脏、心脏和肝脏的表达,并进行图像分析和统计学处理。结果RT—PCR结果显示,Gas7mRNA在肾脏高表达,在心脏的表达弱于肾脏（P〈0．05）,而在肝脏的表达最弱,基本检测不到。免疫组化结果显示,在肾脏中,Gas7免疫阳性产物在近髓肾单位的近曲小管呈强阳性反应,在集合管表达较弱,在肾小球和其余肾小管未见表达;在心脏中,Gas7免疫阳性产物均匀分布于心肌细胞,呈中等强度反应,弱于肾脏（P〈O．05）;在肝脏中,Gas7蛋白未见明显表达,与其mRNA在肝脏的表达相似。结论Gas7在大鼠肾脏、心脏和肝脏表达的不同,尤其在肾脏组织分布的差异性,提示Gas7在成年大鼠肾脏和心脏结构以及功能的维持中可能起着重要作用。     

Gas7在大鼠海马和齿状回发育过程中的动态表达

目的研究生长休止蛋白7（Gas7）在大鼠海马和齿状回不同发育阶段的表达。方法采用免疫组织化学方法观察Gas7在SD大鼠胚胎第18d（E18）、新生（P0）、生后第7d（P7）、P14、P21和成年海马和齿状回中的表达和分布。结果在大鼠脑海马和齿状回部位的冠状切片上,Gas7免疫反应阳性产物主要表达在海马的锥体细胞、齿状回的颗粒细胞和门区的多形层细胞。随着发育的进程,在海马,Gas7较早表达在CA3区,其次是CA2和CA1区;在齿状回,Gas7在外臂的表达早于内臂,在颗粒细胞层的表达是按先外层后内层的顺序。在围生期,Gas7在海马和齿状回各区的表达逐渐增强,至P14达到高峰,后逐渐降低,至P21其表达强度和分布趋于恒定至成年水平。结论 Gas7在大鼠海马和齿状回发育过程中的动态表达具有时间和空间上的特异性,提示Gas7可能参与了海马和齿状回形态形成和功能成熟的调控。     

慢性复合应激对大鼠大脑皮质生长休止蛋白7表达影响的研究

目的探讨慢性复合应激对大鼠大脑皮质神经元中生长休止蛋白7（Gas7）的表达变化及意义。方法36只大鼠随机分为两组：慢性复合应激组和正常对照组。复合应激组动物进行6w的垂直旋转、睡眠剥夺、捆绑（6h／d）和夜间光照等慢性复合性应激试验;实验结束后,所有动物采用免疫组织化学、Western-blot等方法检测大鼠海马Gas7蛋白表达的变化。结果与对照组相比,Gas7在大鼠大脑皮质表达明显增强（P〈0．05）。结论Gas7参与了慢性复合应激对大鼠大脑皮质神经元生长发育的影响。     

Gas7在大鼠梨状皮质发育过程中的表达

目的研究生长休止蛋白(Growth arrest-specific protein 7,Gas7)在大鼠梨状皮质发育过程中的表达。方法采用逆转录聚合酶链反应(RT-PCR)方法和免疫组织化学方法检测Gas7核酸与蛋白在SD大鼠胚胎第18.5天(E18.5)、E20.5、出生当天(P0)、生后第7天(P7)、P14、P21和成年(Adult)各时期梨状皮质中的表达。结果 RT-PCR结果显示Gas7核酸在大鼠梨状皮质各发育时期均有表达,在P14时表达最强;免疫组织化学方法显示梨状皮质在E18.5时即出现Gas7免疫阳性产物,至P7时出现清晰的Gas7免疫阳性细胞,至P14时细胞数达到峰值,免疫阳性反应最强,P21细胞数少于P14(P0.05),Adult细胞数少于P21(P0.05)。结论 Gas7在梨状皮质的表达具有时间上的差异性,提示Gas7可能在梨状皮质结构形成和功能成熟方面起着重要的调控作用。     

生长休止蛋白7在大鼠小脑皮质发育过程中的动态表达

目的研究生长休止蛋白7(Gas7)在大鼠小脑皮质不同发育时期的动态表达。方法采用逆转录聚合酶链反应(RT-PCR)方法检测Gas7mRNA在大鼠小脑皮质不同发育时期的表达;免疫组织化学方法观察Gas7蛋白在大鼠小脑皮质不同发育时期的表达和分布。结果 RT-PCR结果:Gas7mRNA在大鼠小脑皮质发育时期的表达呈现先增强后减弱的趋势,高峰出现在生后第21d(P21)。免疫组化实验结果:在胚胎第18.5d(E18.5)和E20.5仅Purkinje细胞层有Gas7免疫阳性产物分布;出生当天(P0)外颗粒层出现Gas7阳性神经纤维,Purkinje细胞层出现形态不规则的Gas7免疫阳性细胞;P7外颗粒层和Purkinje细胞层免疫反应增强,内颗粒层出现一些散在的Gas7强阳性细胞,胞体较小,突起清晰可见;P14小脑皮质4层均有Gas7阳性表达;P21小脑皮质3层Gas7免疫阳性反应较P14增强(P0.01);Adult(2月龄)较P21免疫反应减弱(P0.01)。结论 Gas7在大鼠小脑皮质发育过程中的动态表达呈现出时空特异性,提示Gas7基因在大鼠小脑皮质发育过程中可能起着重要的调控作用。     

Gas7在大鼠斜角带核发育过程中的表达

目的研究大鼠发育过程中,生长休止蛋白7(growth arrest-specific protein 7,Gas7)在斜角带核(diagonal band nucleus,DB)的表达。方法应用RT-PCR方法、Western blot方法和免疫组织化学方法观察胚胎期16.5天(E16.5)、E20.5、出生当天(P0)、生后7天(P7)、P14、P21和2月龄(成年)大鼠DB的Gas7表达及定位。结果 RT-PCR和Western blot检测显示,在E16.5时Gas7表达最弱,其后逐渐增强,至P21时达到高峰;免疫组织化学染色显示,斜角带核水平支(horizontal limb of the diagonal band,HDB)在E16.5和E20.5时出现Gas7免疫反应阳性产物,Gas7阳性神经元出现于P0,至P21时阳性神经元数量最多,染色最深;斜角带核垂直支(vertical limb ofthe diagonal band,VDB)至P14时Gas7阳性神经元最多,P21和成年均有所减少。结论 Gas7的表达在大鼠DB发育过程中具有时间和空间上的特异性,提示Gas7可能参与大鼠DB的发育过程。     

生长休止蛋白7在大鼠小脑皮质中的免疫细胞化学研究

目的探讨生长休止特定蛋白7(Gas7)在大鼠小脑中的表达定位。方法应用Gas7抗血清,对大鼠小脑组织切片进行免疫组织化学染色。结果在小脑皮质分子层可见大量的Gas7阳性神经纤维;蒲氏细胞层中,Gas7主要表达在神经元胞膜和部分胞质处;颗粒层中可见Gas7阳性神经纤维。结论Gas7主要在小脑神经元的定位特征可能与Gas7促进神经元和神经突起发育的调节功能有关。     

MMP-1、MMP-3 和MMP-13在慢性睡眠剥夺所致颞下颌关节损伤中的变化

目的：观察MMP-1、MMP-3 和MMP-13 在慢性睡眠剥夺所致颞下颌关节损伤中表达的变化,探讨慢性睡眠剥夺所致颞下颌 关节损伤的可能机制。方法：采用改良多平台(MMPM)建立大鼠慢性睡眠剥夺模型,将90 只成年雄性Wastar 大鼠随机分为小平 台组、网格组和对照组。小平台组和网格组大鼠接受每天18 h的睡眠剥夺和6 h间歇期(10:00-16:00),间歇期大鼠正常笼养。实验 第7、14 和21 d时分别观察动物的行为学观察、检测动物血浆皮质醇(CORT)和促肾上腺皮质激素(ACTH)水平检测,通过免疫印 迹法和实时定量聚合酶链反应(PRC)检测颞下颌关节软骨中MMP-1、MMP-3 和MMP-13 的蛋白和mRNA表达,并通过HE 染色 法观察颞下颌关节结构的变化。结果：与对照组和网格组大鼠相比,小平台组大鼠第14 d和21 d 时髁突软骨中间部位表面纤维 在出现明显的炎症、松解及脱落现象;第21天时的血浆ACTH 和CORT 水平均显著高于网格组和对照组,差异有统计学意义 (P<0.05);第7、14、21 d时关节软骨MMP-1 和MMP-13 蛋白和mRNA 的表达水平均显著上调(P<0.05)。结论：慢性睡眠剥夺所致 的颞下颌关节损伤可能与关节软骨中MMP-1、MMP-3 和MMP-13 的表达上调有关。     

高原低氧对大鼠大脑皮质生长休止蛋白7表达的影响

目的 探讨高原低氧对大鼠大脑皮质生长休止蛋白7(Gas7)表达的影响.方法 36只大鼠随机分为正常对照组和模拟高原低氧组,模拟高原低氧组大鼠进行6周缺氧,复制慢性高原低氧动物模型.实验结束后,所有动物采用免疫组织化学和免疫印迹技术检测大鼠大脑皮质中Gas7的表达.结果 与对照组相比,Gas7在模拟高原低氧组大鼠大脑皮质的表达明显增强.结论 Gas7可能参与了高原低氧对大鼠大脑皮质神经元结构和功能的影响.     

Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage.

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.     

Induction of chondrogenesis and expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors in the infrapatellar fat pad of the knee joint treated with TGF-beta1 and BMP-7

Superficial zone protein (SZP) is a key mediator of boundary lubrication of articular cartilage in joints. In this investigation, we made the unexpected discovery that SZP was expressed in infrapatellar fat pad (IFP) from bovine knee. Quantitative analysis of secreted proteins in the medium of the IFP stromal cells demonstrated a significant stimulation by TGF-β1 and BMP-7. Real-time PCR analysis revealed the SZP expression was up-regulated by TGF-β1 and BMP-7. Chondrogenically differentiated IFP progenitor cells were stimulated by TGF-β1 and BMP-7 to synthesize and secrete SZP. SZP mRNA was significantly up-regulated by chondrogenic induction for 21 days. These findings indicate that the stimulation of SZP expression by TGF-β and BMP-7 may lead to functional improvement of damaged intraarticular tissues and that IFP progenitor cells may be a potential useful source for inducing superficial zone of articular cartilage by tissue engineering for regeneration of damaged articular cartilage due to osteoarthritis     

Identification of superficial zone articular chondrocyte stem/progenitor cells

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.     

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.     

右归丸对大鼠膝骨关节炎的干预作用及其机制*

目的: 探讨右归丸对膝骨关节炎(KOA)模型鼠关节软骨组织骨诱导因子(OGN)、骨黏连蛋白(ON)和纤维蛋白原2(FBN2)的影响。方法: 将大鼠随机分为假手术组,模型组,硫酸氨基葡萄糖组(硫酸氨基葡萄糖),右归丸(高、中、低剂量)组,每组10只。采用改良Hulth法制备大鼠KOA模型,假手术组和模型组给予等体积生理盐水灌胃,右归丸高、中、低剂量组分别灌胃给予右归丸4.8,2.4,1.2 g/kg,硫酸氨基葡萄糖组灌胃给予硫酸氨基葡萄糖 0.17 g/kg,连续给药8周。干预结束24 h后取鼠膝关节软骨,采用HE染色法观察各组软骨的病理改变,并进行Mankin评分;免疫组化法检测各组关节软骨组织中OGN、ON和FBN2的表达;Western blot法检测各组关节软骨组中糖原合成酶激酶-3β(GSK-3β)的表达。结果: 与假手术组比较,模型组大鼠软骨组织Makin 评分显著升高,软骨组织FBN2蛋白表达水平上显著增加,OGN、ON和GSK-3β蛋白表达水平上的显著降低(P＜0.01);模型组关节软骨边缘严重破坏,软骨细胞排列紊乱。与模型组比较,右归丸高剂量干预组大鼠软骨组织Makin 评分和FBN2蛋白表达水平显著降低,GSK-3β蛋白表达水平上显著增加,且右归丸中、高剂量组OGN、ON蛋白表达水平均显著增加(P＜0.05或P＜0.01),软骨结构趋于正常,软骨细胞分布仅偶见不均,关节软骨表面欠光滑。结论: 右归丸能够延缓关节软骨退变,其可能机制是通过提高骨诱导因子和骨粘连蛋白的表达水平来促进关节软骨的骨化和重构。     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.