关节软骨的细胞凋亡

目的 观察正常及关节炎时关节软骨是否发生细胞凋亡及其可能的意义。方法 采用光镜、电镜观察软骨细胞形态,以琼脂糖凝胶电泳检测软骨细胞特异ＤＮＡ的梯形带,以激光共聚焦显微镜观察细胞内「Ｃａ＾２＋」ｉ动脉变化。结果 骨性关节炎及风湿性关节炎患者的关节软骨细胞经体外培养３ｄ后,光镜及电镜下均可见处于正常分化期的软骨细胞,而未经培养直接分离的部分软骨细胞可见细胞凋亡早期形态学改变。此外,体外培养的牛正常软骨     

软骨细胞老化特征及机制的研究进展

软骨细胞的老化是一个极其复杂的过程，其特征包括：细胞不可逆的生长停滞于G1期；老化相关β-半乳糖苷酶的表达；端粒长度缩短；软骨细胞分化特征的改变。目前认为其机制为基因表达的程序性或减进性改变，包括肿瘤抑制基因p53和pRb等在细胞生长停滞中的作用；端粒结合蛋白和端粒酶对端粒长度的调节；细胞骨架蛋白的重组使软骨细胞形态的变化；基质降解酶类表达增加以及各种细胞因子的变化对软骨细胞代谢的影响。     

实验性骨关节病中软骨细胞的凋亡

应用兔膝关节伸直位石膏管型固定造成的实验性骨关节病模型,对其关节软骨细胞进行形态学及原位ＤＮＡ 断裂标记研究。结果显示：制动１ 周时即出现软骨表层细胞凋亡,２ 周后凋亡呈增加趋势,６周时出现全层软骨细胞大量凋亡,而正常及实验对照组很少出现凋亡细胞。提示制动可引起软骨细胞凋亡;软骨细胞凋亡可能是关节软骨出现退变的重要机制之一。     

软骨细胞老化特征及机制的研究进展

软骨细胞的老化是一个极其复杂的过程,其特征包括:细胞不可逆的生长停滞于G1期;老化相关β-半乳糖苷酶的表达;端粒长度缩短;软骨细胞分化特征的改变.目前认为其机制为基因表达的程序性或减进性改变,包括肿瘤抑制基因p53和pRb等在细胞生长停滞中的作用;端粒结合蛋白和端粒酶对端粒长度的调节;细胞骨架蛋白的重组使软骨细胞形态的变化;基质降解酶类表达增加以及各种细胞因子的变化对软骨细胞代谢的影响.     

软骨细胞上清液诱导骨髓间充质干细胞分化的软骨细胞的形态学及功能检测

摘 要］目的：应用家兔肋软骨细胞上清液诱导骨髓间充质干细胞（MSCs）向软骨细胞定向分化,并对分化的细胞进行形态学和功能检测,探讨MSCs在体外成软骨分化的条件。方法：分离家兔的MSCs和肋软骨细胞进行体外培养,收集前3代肋软骨细胞上清液作为诱导液,对第3代MSCs进行14 d的诱导培养。设置实验组、阳性对照组和阴性对照组,观察各组细胞的形态,甲苯胺蓝染色观察特异性蛋白表达、各组ALP活性,RT-PCR法检测各组细胞中的Ⅱ型胶原(ColⅡ) mRNA的表达。结果：经过14 d的诱导培养,实验组MSCs由长梭形逐渐变成多角形,甲苯胺蓝染色呈阳性;ALP活性显著升高,与其他2组比较差异有统计学意义(P＜0.01）;ColⅡ mRNA表达呈阳性。阴性对照组甲苯胺蓝染色呈阴性,ColⅡ mRNA表达呈阴性。阳性对照组甲苯胺蓝染色呈阳性,ColⅡ mRNA表达呈阳性。结论：家兔肋软骨细胞上清液能够促进MSCs向软骨细胞方向分化。     

膝关节骨关节炎软骨细胞差异表达蛋白的研究

目的 探讨骨关节炎(OA)差异表达蛋白的病理作用.方法 分别从正常膝关节提取正常软骨(NAC,n=22)和从接受膝关节表面置换的OA患者提取OA软骨(OAC,n=17).分离软骨细胞,提取总蛋白直接进行双向电泳分析.胶图导入Imagemaster 2-D platinum3.0软件筛选差异表达蛋白点,切胶消化并提交质谱分析.质谱生成的肽段指纹序列输入到MASCOT,依托NCBI蛋白数据库确立差异表达蛋白的身份.结果 NAC和OAC组双向电泳胶图分别显示约1000个蛋白点,其中35个NAC蛋白点和31个OAC蛋白点被确立为差异表达蛋白.通过质谱分析识别19个差异表达蛋白,包括6型胶原、与软骨细胞胶原代谢相关的酶、应激相关蛋白,以及功能未知蛋白.结论 差异表达蛋白的分离和识别提示了多方面的OA病理信息,为深入认识OA的分子病因学拓宽了视野.     

实验性骨关节病中软骨细胞的凋亡

The right knees of rabbits were immobilized in full extension for up to eight weeks using plaster cast. Specimens of the articular cartilage obtained from tibial plateau were studied by histopathologic and TDT-mediated fluorescein-dUTP nick-end labelling(TUNEL) techniques. The results showed that TUNEL-positive chondrocytes with apoptosis specific morphology were detected on superficial and middle layer of the articular cartilage from one to two weeks after immobilization, and these changes progressed until 4 weeks after immobilization. Six weeks after immobilization, TUNEL-positive chondrocytes were seen through the entire thickness of the articular cartilage. Our findings indicate that apoptosis of chondrocytes could be induced by immobilization and might be responsible for articular cartilage degeneration, and which is one of the pathways involved in the pathophysiological mechanism of osteoarthritis.     

猪耳廓软骨细胞体外培养

目的 为组织工程化软骨修复机体软骨缺损提供实验基础。方法 用消化组织块培养法体外培养猪耳廓软骨细胞。结果 软骨细胞在ＰＨ值为７．２０,含１０％胎牛血清的Ｆ１２培养基中生长良好,可传代４～５代,细胞数目扩增为原来的１０～２０倍。结论 消化组织块方法培养软骨细胞是一种可以扩增软骨细胞数目的确实可行的方法。     

软骨细胞与复合水凝胶的生物相容性研究

目的构建光固化壳聚糖与自固化海藻酸钠复合水凝胶体系,研究其与软骨细胞的体外生物相容性,探索其作为软骨组织工程支架的可行性。方法以三维多孔光固化壳聚糖水凝胶作为细胞的空间载体,利用自固化海藻酸钠水凝胶将软骨细胞胶封入光固化壳聚糖水凝胶支架,构成复合水凝胶-细胞复合体。采用扫描电子显微镜观察光固化壳聚糖的结构形貌以及细胞在支架内部的分布与形态;CCK8法绘制细胞增殖曲线;DAPI细胞核染色荧光显微镜观察细胞核形态变化,判断细胞活性。结果纯光固化壳聚糖水凝胶材料呈孔隙较为均匀一致的多孔结构,平均孔径为300μm左右。复合水凝胶能够模拟天然软骨细胞外基质环境,使细胞保持圆形生长状态;促进成软骨细胞的增殖,具有良好的生物相容性。结论复合水凝胶能够为软骨细胞提供一个类似天然的生活环境,并能够促进其增殖、生长,有望作为软骨组织工程支架应用于软骨缺损修复的实验研究。     

兔软骨细胞的分离与培养

目的 探讨免软骨细胞培养方法。方法采用胶原酶Ⅱ分段消化法从4周龄新西兰兔的关节软骨中分离出软骨细胞并进行原代和传代培养。每日在倒置显微镜下观察细胞形态及其生长情况。并用甲苯胺蓝异染色进行细胞表型鉴定。结果软骨细胞形成单层的时间原代培养1周左右，传代培养约4～5天，细胞以圆形或上皮样细胞形态为主。甲苯胺蓝染色证实细胞可合成蛋白多糖，异染反应主要集中在细胞集落样生长区，异染程度以原代培养最为显。结论采用本方法培养兔软骨细胞是可行的。     

Mitochondrial transfer from bone-marrow-derived mesenchymal stromal cells to chondrocytes protects against cartilage degenerative mitochondrial dysfunction in rats chondrocytes

BackgroundPrevious studies have reported that mitochondrial dysfunction participates in the pathological process of osteoarthritis (OA). However, studies that improve mitochondrial function are rare in OA. Mitochondrial transfer from mesenchymal stem cells (MSCs) to OA chondrocytes might be a cell-based therapy for the improvement of mitochondrial function to prevent cartilage degeneration. This study aimed to determine whether MSCs can donate mitochondria and protect the mitochondrial function and therefore reduce cartilage degeneration.MethodsBone-marrow-derived mesenchymal stromal cells (BM-MSCs) were harvested from the marrow cavities of femurs and tibia in young rats. OA chondrocytes were gathered from the femoral and tibial plateau in old OA model rats. BM-MSCs and OA chondrocytes were co-cultured and mitochondrial transfer from BM-MSCs to chondrocytes was identified. Chondrocytes with mitochondria transferred from BM-MSCs were selected by fluorescence-activated cell sorting. Mitochondrial function of these cells, including mitochondrial membrane potential (Δψm), the activity of mitochondrial respiratory chain (MRC) enzymes, and adenosine triphosphate (ATP) content were quantified and compared to OA chondrocytes without mitochondrial transfer. Chondrocytes proliferation, apoptosis, and secretion ability were also analyzed between the two groups.ResultsMitochondrial transfer was found from BM-MSCs to OA chondrocytes. Chondrocytes with mitochondrial from MSCs (MSCs + OA group) showed increased mitochondrial membrane potential compared with OA chondrocytes without mitochondria transfer (OA group) (1.79 ± 0.19 vs. 0.71 ± 0.12, t = 10.42, P < 0.0001). The activity of MRC enzymes, including MRC complex I, II, III, and citrate synthase was also improved (P < 0.05). The content of ATP in MSCs + OA group was significantly higher than that in OA group (161.90 ± 13.49 vs. 87.62 ± 11.07 nmol/mg, t = 8.515, P < 0.0001). Meanwhile, we observed decreased cell apoptosis (7.09% ± 0.68% vs.15.89% ± 1.30%, t = 13.39, P < 0.0001) and increased relative secretion of type II collagen (2.01 ± 0.14 vs.1.06 ± 0.11, t = 9.141, P = 0.0008) and proteoglycan protein (2.08 ± 0.20 vs. 0.97 ± 0.12, t = 8.227, P = 0.0012) in MSCs + OA group, contrasted with OA group.ConclusionsMitochondrial transfer from BM-MSCs provided protection for OA chondrocytes against mitochondrial dysfunction and degeneration through improving mitochondrial function, cell proliferation, and inhibiting apoptosis in chondrocytes. This finding may offer a new therapeutic direction for OA.     

猪耳廓软骨细胞外分泌基质的检测

目的　通过体外传代培养猪耳廓软骨细胞 ,并连续检测软骨细胞形成外分泌基质的能力 ,寻找出最适宜接种的种子细胞。方法　通过Ⅱ型胶原酶消化法获得高活性的软骨细胞 ,定量接种 6× 10 6个细胞于培养皿 ,每周传代 1次 ,连续 5周 ,留取所有培养液用阿利新蓝法测定软骨细胞外分泌基质糖胺多糖 (GAG) ;利用光镜观察原代及传代软骨细胞的生长情况。结果　第 1,2 ,3 ,4,5周软骨细胞外分泌基质GAG平均含量分别为 :612 ,12 3 5 ,14 62 ,113 8和 710 μg。 结论　体外培养的第 2代软骨细胞功能和活性最佳 ,传代第 2周是最佳接种时间。     

Serum-free media for articular chondrocytes in-vitro expansion

Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair.Classical culture conditions usually use animal serum as a medium supplement,wh...     

瘦素对骨关节炎中软骨细胞线粒体自噬状态的影响

背景 既往研究表明,瘦素可通过促进软骨细胞的分解代谢起到推动骨关节炎(osteoarthritis,OA)发展的作用;而细胞稳态维持机制——线粒体自噬受损时,可导致软骨细胞死亡,进一步发展为OA.目的 探索瘦素在线粒体自噬调节中的作用及其与软骨细胞退变的关系,阐明瘦素在OA中的调节作用.方法 体外分离、培养软骨细胞,分...     

蛇床子素对大鼠原代软骨细胞增殖的抑制作用

目的：观察蛇床子素对大鼠软骨细胞增殖的影响。方法：分离出生24h内大鼠的软骨细胞,进行原代培养。细胞扩增至第2代时观察1周内细胞增殖情况,并用细胞免疫荧光法鉴定Ⅱ型前胶原基因（type Ⅱ procollagen gene,Col2a1）的表达。将第2代软骨细胞分为对照组和不同浓度蛇床子素（6．25、12．5、25和50μmol／L）组。药物作用24和48h后,观察细胞增殖情况,蛋白质印迹法和聚合酶链反应法检测增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）和细胞周期素D1（cyclinD1）的表达。结果：第2代原代软骨细胞活力正常,Col2al表达明显。蛇床子素可以抑制软骨细胞的增殖,并随浓度的增加,抑制作用愈明显。蛋白质印迹法和聚合酶链反应法检测发现,随着蛇床子素浓度的增加,软骨细胞中PCNA和cyclinD1的表达下降。结论：蛇床子素可能通过抑制PCNA和cyclinD1的表达发挥抑制软骨细胞增殖的作用。     

Parthenolide inhibits tumor necrosis factor-alpha induced catabolism of aggrecan in chondrocytes in osteoarthritis: in vitro experiment with cultured human chondrocytes

OBJECTIVE: To investigate the effect of parthenolide (PAR) on the tumor necrosis factor (TNF)-alpha induced aggrecan catabolism of chondrocytes in ostroarthritis (OA). METHODS: Human chondrocytes were obtained from the condyles of femur of OA patients undergoing knee joint replacement during operation, cultured, and randomly divided into 4 groups: control group, TNF group (cultured in medium containing 10 ng/ml TNF-alpha) , PAR group (cultured in medium containing 10 micromol/L PAR), and PAR + TNF group (cultured in medium containing 10 micromol/L PAR and 10 ng/ml TNF-alpha). Eight days later 1, 9-dimethylmethylene blue spectrophotometric assay was used to measure the content of glycosaminoglycan (GAG) , marker of aggrecan catabolism, in the culture fluid and the cells. Using anti-aggrecan monoclonal antibodies Mab 5D4 and 3B3, ELISA was employed to detect the contents of 5D4 and 3B3, aggrecan catabolic fragments. RT-PCR was used to detect the mRNA expression of the aggrecan, ADAMTS-4 and ADAMTS-5, both aggrecanases, tissue inhibitor of metalloprotease (TIMP)-1, mitogen-activated protein kinase (MAPK)-1 and 18S, a marker. RESULTS: The GAG percentage in the culture fluid of the TNF-alpha was 57.1% +/- 2.0%, significantly higher than those of the control and TNF-alpha + PAR groups (P = 0.001 and 0.02). The 5D4 fragment level of the TNF-alpha group was 509 ng/ml +/- 32 ng/ml, significantly higher than that of the control group (166 ng/ml +/- 15 ng/ml, t = 11.60, P =0.007), and the level of 5D4 fragment of the PAR + TNF-alpha group was 333 ng/ml +/- 15 ng/ml, significantly lower than that of the TNF-alpha group (t = 7.93, P = 0.016). There was not significant difference in the 3B3 fragment level among the 4 groups (F = 1.316, P = 0.335). The aggrecan mRNA expression level of the TNF-alpha group was significantly lower than those of the other 3 groups (F = 133.7, P = 0.000), the mRNA expression levels of ADAMTS-5 and MAPK-1 of the TNF-alpha group were significantly higher than those of the other 3 groups (F = 209. 7, 117.1; P =0. 000), the ADAMTS-5 mRNA expression level of the PAR group was significantly lower than that of the control group (t = 11.1, P= 0.008) , and there was not significant differences in the mRNA expression levels of ADAMTS-4 and TIMP-1 among the 4 groups (F = 1.87, 0.73; P > 0.05) . CONCLUSION: PAR inhibits TNF-alpha induced catabolism of aggrecan in the chondrocytes of OA and reduces the mRNA expression of ADAMTS-5 and MAPK-1. PAR may be useful in the treatment of OA and other inflammatory joint diseases.     

西藏小型猪肋软骨细胞的分离培养及鉴定

目的分离培养西藏小型猪肋软骨细胞,并对其进行鉴定。方法实验采用3日龄西藏小型猪,二步消化法分离培养西藏小型猪肋软骨细胞(PCCs),同样的方法分离培养小鼠的肋软骨细胞(MCCs),观察两个物种软骨细胞的形态,并分别以西藏小型猪胚胎成纤维细胞(PEFs)和小鼠胚胎成纤维细胞(MEFs)为阴性对照,对所分离培养的软骨细胞进行甲苯胺蓝染色鉴定以及胶原蛋白Ⅱ、蛋白聚糖的免疫荧光鉴定。结果成功分离培养西藏小型猪和小鼠肋软骨细胞,两种细胞的形态有差别,所分离培养的两个物种的软骨细胞均呈甲苯胺蓝染色阳性,胶原蛋白Ⅱ、蛋白聚糖免疫荧光阳性。结论成功建立了西藏小型猪肋软骨细胞分离培养及鉴定的方法。     

体外培养人胎儿软骨细胞的生物学特性

目的 建立人胎儿软骨细胞分离培养、扩增技术体系,研究其体外单层培养条件下的生长特性,探讨胎儿软骨细胞作为软骨组织工程种子细胞的可行性。方法酶消化法获取胎儿软骨细胞,体外培养扩增,经测定细胞生长曲线、群体倍增时间及累计倍增数目,观察细胞生长动力学并确定体外生长规律。通过免疫组织化学检测Ⅱ型胶原分泌,阿利辛蓝比色法测定基质糖胺多糖(GAG)含量。P1细胞接种于聚羟乙酸(PGA)纤维支架,观察两者生物相容性及细胞体外成软骨能力。结果 关节软骨细胞形态呈典型的多角形,随着传代的次数增加,逐渐变成“成纤维细胞样”的条梭形。平均倍增时间为74 h。细胞连续培养5代,累计倍增数目为7.83±0.42。P1-P5间接免疫组化Ⅱ型胶原表达阳性,P6不表达。GAG含量P1细胞低于P0细胞,随体外培养时间延长逐渐增加。P1细胞接种于PGA纤维支架,体外培养14 d,组织学观察显示有软骨组织形成。结论 胎儿软骨细胞经常规体外培养,至第6代失去分泌软骨特异性基质的功能。P1细胞接种PGA纤维展现良好的生物相容性,初步证实胎儿软骨细胞作为软骨组织工程种子细胞的可行性。