流体剪切力对大鼠成骨样细胞生成NO的调节作用

机械应力在骨改建中起着重要的作用。本研究试图探讨机械应力刺激调节成骨细胞生理功能过程中一氧化氮 (NO)的作用机制。通过流室系统对体外培养的大鼠成骨样细胞施加 12 dyn/ cm2的流体剪切力 ,采用 NO荧光检测试剂盒检测细胞受力 5、10、15、30、6 0、12 0 min后不同时段的 NO的表达。结果表明 ,大鼠成骨样细胞受力后生成的 NO明显高于空白对照组 (P<0 .0 5 )。受力细胞在受力后 6 0 min内 ,NO的生成在各时段无明显提高 ,但在6 0 m in后开始明显增加 (P<0 .0 5 )。而空白对照组各时段 NO的生成无显著性差异 (P>0 .0 5 )。机械应力作用下 ,成骨样细胞早期释放 NO,可能是骨组织细胞将机械应力刺激转导入细胞 ,促进骨形成的生化信号分子。流体剪切力刺激诱导的反应早期可能是直接通过激活 c NOS的生化通道来实现 ,在后期可能是通过 i NOS的途径     

低渗刺激对成骨样细胞MG63功能的影响

目的本试验拟通过研究低渗透压的静牵张作用,对成骨样细胞MG63的增殖能力、碱性磷酸酶活性以及[Ca2 ]i的影响,探讨该应力形式下成骨样细胞MG63的力学响应特征。方法采用人成骨肉瘤来源的成骨样细胞MG63作为细胞源进行细胞培养传代。用不同渗透压的低渗透液,对成骨样细胞MG63分别进行2 h、4 h、8h、12 h和24 h持续牵张作用后,用免疫组化法检测ALP表达的情况,用ALP试剂盒检测ALP活性的变化,用钙试剂盒检测[Ca2 ]i含量波动情况。结果成骨样细胞MG63的ALP免疫细胞化学染色为阳性。随着作用时间的延长,成骨样细胞MG63在277 mOsm和240 mOsm低渗透液的持续性膨胀作用下,其细胞的[Ca2 ]i、ALP活性缓慢增高,但240 mOsm组ALP活性始终低于对照组(p<0.01);而细胞在163 mOsm低渗液作用下,8 h时出现[Ca2 ]i急剧升高(11.383±0.111),ALP活性(0.326±0.002)明显高于其对照组(p<0.01)。结论结果提示不同水平的低渗膨胀对MG63的增殖分化、ALP活性以及Ca2 -ATPase都有一定的影响作用,且Ca2 内流与ALP活性之间存在一定的相关性。低渗膨胀法作为一种应力形式有一定的实验意义。     

Transwell小室环境下兔成骨样细胞与骨髓基质干细胞的共培养及成骨分化

背景：以往在体外采用地塞米松、生长因子或用成骨样细胞与骨髓间充质干细胞按1∶1混合培养诱导成骨均存在种种局限。 目的：观察在Transwell小室环境下成骨样细胞与骨髓基质干细胞体外共培养及成骨样细胞定向诱导骨髓基质干细胞向成骨细胞分化的可行性。 方法：取第3代乳兔成骨样细胞与第3代兔骨髓基质干细胞接种共培养于Transwell小室内,成骨样细胞接种于培养板底层,骨髓基质干细胞接种于Transwell膜内膜上作为实验组。以骨髓基质干细胞单独接种于Transwell小室内,下层为基础培养液作为对照组。 结果与结论：实验组共培养骨髓基质干细胞明显向成骨细胞分化,细胞碱性磷酸酶活性显著高于对照组(P < 0.05)。实验组骨髓基质干细胞茜素红染色强阳性,可见呈红色结节,经PT-PCR扩增后,可见成骨启动基因核心结合因子α1的表达;对照组未见矿化结节。说明应用Transwell小室可实现成骨样细胞与骨髓基质干细胞体外共培养,并能定向诱导骨髓基质干细胞向成骨细胞分化。关键词：成骨样细胞;Transwell小室;骨髓基质干细胞;共培养;成骨分化;兔 doi:10.3969/j.issn.1673-8225.2012.19.004     

维甲酸诱导小型猪牙周膜干细胞的体外成骨

背景：近期研究发现维甲酸对胚胎干细胞及多种成体干细胞具有成骨方向诱导的作用。 目的：观察维甲酸对小型猪牙周膜干细胞体外成骨作用的影响。 方法：采用组织块法获得小型猪牙周膜细胞,有限稀释法纯化小型猪牙周膜干细胞,免疫荧光法检测STRO-1、免疫细胞化学法检测波形蛋白、角蛋白鉴定小型猪牙周膜干细胞。CCK8法测定小型猪牙周膜干细胞增殖曲线,检测小型猪PDLSC克隆形成率。使用维甲酸对第3代小型猪牙周膜干细胞进行成骨诱导,茜素红染色检测矿化结节,免疫细胞化学方法检测成骨相关基因骨桥蛋白、骨钙素、Ⅰ型胶原、Ⅲ型胶原的表达。 结果与结论：有限稀释法分离纯化小型猪牙周膜干细胞STRO-1,波形蛋白阳性表达,角蛋白阴性表达,克隆形成率为2.8%,维甲酸诱导14 d后,碱性磷酸酶染色阳性,诱导21 d后茜素红染色阳性,免疫细胞化学法检测骨桥蛋白、骨钙素、Ⅰ型胶原阳性表达,Ⅲ型胶原阴性表达。结果表明小型猪牙周膜干细胞能够被维甲酸诱导向成骨样细胞分化。     

从骨髓和脐血造血干细胞中诱导成骨样细胞

骨髓有2个重要的功能:造血和成骨。骨髓中存在造血干细胞(hem atopoietic stem cells,HSCs)(CD34 细胞)和间充质干细胞(m esenchym al stem cells,MSCs)。这2种干细胞共同存在于骨髓腔中,MSC对HSC不仅有空间位置的机械支持作用,还分泌多种造血因子支持其造血功能,有助于HSC未分     

成纤维样滑膜细胞原代培养方法的优化

目的 对优化佐剂型关节炎(AA)大鼠成纤维样滑膜细胞(FLSs)的原代培养方法并进行鉴定.方法 制备AA大鼠模型,采用酶消化法分离得到滑膜细胞,再经多次消化后重贴壁得到FLSs,并通过免疫荧光技术鉴定.结果 多次实验均成功培养出FLSs,且经鉴定的所有细胞几乎全为vimentin阳性.结论 本实验方法成功培养出高纯度的FLSs,为以滑膜为靶点的类风湿性关节炎(RA)的研究奠定了基础.     

悬浮组织块法分离培养兔成纤维样滑膜细胞

目的:建立兔成纤维样滑膜细胞悬浮组织块分离培养法。方法:取正常兔膝关节滑膜组织,用悬浮组织块法分离培养兔成纤维样滑膜细胞,观察细胞生长状况及形态特征,取第3 代对数期细胞,CCK鄄8 法绘制其生长曲线,免疫荧光法检测波形蛋白的表达情况,并与组织块贴壁法进行比较。结果:两种方法体外培养得到的兔成纤维样滑膜细胞形态为长梭形纤维样,细胞生长力较旺盛,生长曲线为典型的“S冶形,免疫荧光检测显示第3 代细胞强表达波形蛋白。结论:悬浮组织块法可以分离培养出兔成纤样维滑膜细胞,方法简便,效率高,为体外分离培养兔成纤维样滑膜细胞提供了一种新的方法。     

降钙素基因相关肽联合重组人骨形态发生蛋白对兔成骨样细胞增殖和碱性磷酸酶活性的影响

目的研究降钙素基因相关肽(CGRP)及重组人骨形成蛋白-2(rhBMP-2)在促进兔骨髓来源成骨样细胞增殖和分化方面是否有协同作用。方法取经诱导第三代兔骨髓基质细胞获得的兔成骨样细胞以2×10~6/ml的浓度接种于96孔板,分为6组,用含有不同条件的培养基进行培养。包括①A组:空白对照组,②B组:0.05 ng/ml CGRP,③C组:5 ng/ml CGRP,④D组:100 ng/ml rhBMP-2,⑤E组:5 ng/ml CGRP+100ng/ml rhBMP- 2,⑥F组:0.05ng/ml CGRP+100 ng/ml rhBMP-2。用四唑盐比色法(MTT)法测定细胞增殖情况和碱性磷酸酶( ALP)染色法检测其对ALP活性的影响。结果①100ng/ml rhBMP-2组、0.05ng/ml CGRP+100ng/ml rhBMP-2组、5ng/ml CGRP+100ng/ml rhBMP-2组细胞增殖OD值比对照组显著增强(P<0.05),其中0.05ng/ml CGRP+100ng/ml rhBMP-2组最强(P<0.01)。②100ng/ml rhBMP-2组、0.05ng/ml CGRP+100ng/ml rhBMP-2组、5ng/ml CGRP+100ng/ml rhBMP-2组,3组之间的ALP表达无明显差异(P>0.05).但3组与对照组相比显著增强( P<0.05)。结论CGRP和rhBMP-2合用对兔骨髓来源成骨样细胞的增殖有一定的协同作用,但对ALP无协同作用,与单用rhBMP-2的效果相当。     

1，25双羟维生素D3对成骨样细胞骨架和基因表达的影响

用荧光免疫法研究了１，２５双羟基维生素Ｄ３对大鼠成骨样细胞（ＲＯＳ１７／２．８）细胞骨架的影响和用斑点杂交技术研究了１，２５（ＯＨ）２Ｄ３对人成骨样细胞（ＯＳ－７３２）基因表达的影响。结果表明：在１０＾－７ｍｏｌ／Ｌ１，２５（ＯＨ）２Ｄ３连续作用４ｄ后，大鼠ＲＯＳ１７／２．８细胞的微管蛋白和波形纤维蛋白明显改善，纤粘蛋白荧光明显增强。而在１，２５（ＯＨ）２Ｄ３作用前后ＯＳ－７３２细胞ｃ－ｍｙｃ和ｍ     

骨形态发生蛋白9体外诱导牙囊细胞的成骨分化

背景：有研究表明骨形态发生蛋白9能促进多种干细胞的成骨分化,但其是否具有诱导牙囊细胞成骨向分化的能力尚不清楚。目的：探讨骨形态发生蛋白9对大鼠牙囊细胞成骨分化的诱导作用。方法：以纯化的第3代大鼠牙囊细胞为研究对象,感染骨形态发生蛋白9腺病毒后,检测牙囊细胞中碱性磷酸酶活性、钙盐沉积以及矿化相关因子基因和蛋白的表达变化。结果与结论：感染骨形态发生蛋白9的牙囊细胞碱性磷酸酶活性持续增强,钙盐沉积明显增强。Real time PCR检测结果显示感染骨形态发生蛋白9的牙囊细胞中矿化相关因子碱性磷酸酶、骨钙素、骨涎蛋白、骨桥蛋白和核心结合因子mRNA表达增强。Western blot检测结果显示感染骨形态发生蛋白9的牙囊细胞中骨桥蛋白的表达增强。以上结果表明骨形态发生蛋白9可诱导牙囊细胞向成骨方向分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

伊班膦酸钠预防与治疗糖皮质激素诱导的骨质疏松症

目的 探讨伊班膦酸钠(IBA)在糖皮质激素引起的家兔骨质丢失中的预防与治疗作用.方法 60只家兔随机分为:对照组1和2(注射生理盐水)、DX6组(注射地塞米松,连续6周)、预防组(注射地塞米松前注射1次IBA)、DX12组(注射地塞米松,连续12周)和治疗组(注射地塞米松6周后,注射1次IBA).6周末,将对照组1、DX6组和预防组家兔处死;12周末将剩余组家兔处死.结果 预防组家兔腰椎骨小梁面密度、厚度、数量比DX6组分别增加了100.00%、40.55%和45.66%(P<0.01),治疗组家兔比DX12组分别增加了73.34%、23.87%和39.02%(P<0.05).预防组家兔腰椎压缩和股骨3点弯曲最大负荷及肱骨扭转最大扭矩比DX6组分别增加了24.19%、29.91%和37.24%(P<0.01),治疗组家兔比DX12组分别增加了54.36%、21.38%和105.75%(P<0.05).结论 IBA可有效预防和治疗糖皮质激素引起的家兔骨质丢失和骨强度的下降.     

人胰岛素样生长因子1的真核细胞表达及其鉴定

目的 构建人胰岛素样生长因子１（ＩＧＦ－１）的真核细胞表达质粒。方法 用ＰＣＲ方法从人的肝细胞ｃＤＮＡ文库中克隆出ＩＧＦ－１ｃＤＮＡ,然后定向插入真核细胞表达载体ｐｃＤＮＡ３中,并用脂质体方法转染ＣＯＳ７细胞。用ＥＬＩＳＡ法和人胚肺纤维母细胞以及ＮＩＨ３Ｔ３纤维细胞增殖法分别测定转染细胞上清液中ＩＧＦ－１的含量和生物活性。结果 重组的真核细胞表达质粒ｐｃＤＮＡ３－ＩＧＦ－１所含的ＩＧＦ－１ｃＤＮＡ序列和插入方向均正确,其转染的ＣＯＳ７细胞分泌较高浓度的ＩＧＦ－１,并且具有明显促进纤维细胞增殖的能力。结论 本实验所构建的重组真核细胞表达质粒ｐｃＤＮＡ３－ＩＧＦ－１能够高效表达有活性的ＩＧＦ－１,对进一步研究ＩＧＦ－１体内表达的生理和病理作用有一定意义。     

Intrathyroidal epithelial thymoma/carcinoma showing thymus‐like differentiation; comparison with thymic lymphoepithelioma‐like carcinoma and a possibility of development from a multipotential stem cell

The purpose of our article is to describe the immunohistochemical findings of intrathyroidal epithelial thymoma/carcinoma showing thymus‐like differentiation (ITET/CASTLE) of the thyroid in detail, to clarify the difference between ITET/CASTLE and thymic lymphoepithelioma‐like carcinoma (LELC), and to discuss the pathogenesis of ITET/CASTLE. We immunohistochemically examined five ITET/CASTLE and eight LELC cases. All of ITET/CASTLE cases were strongly positive for CD5, P63, high‐molecular‐weight cytokeratin and B‐cell CLL/lymphoma‐2. Carcinoembryonic antigen‐positive carcinoma cells were found in four ITET/CASTLE cases. Neuroendocrine marker‐positive carcinoma cells were scattered in all cases. Immunohistochemical findings in thymic LELC were essentially similar to those in ITET/CASTLE, but the sensitivity was different. There is a possibility that ITET/CASTLE and thymic LELC are not the quite same disease entity. We think that ITET/CASTLE is derived from ectopic thymus, but not related to solid cell nests.     

Prognostic role of insulin‐like growth factor receptor‐1 expression in small cell lung cancer

Chang MH, Lee J, Han J, Park YH, Ahn JS, Park K, Ahn M‐J. Prognostic role of insulin‐like growth factor receptor‐1 expression in small cell lung cancer. APMIS 2009; 117: 861–9. Insulin‐like growth factor receptor‐1 (IGFR‐1) is a cellular membrane receptor which is overexpressed in many tumors and seems to play a critical role in anti‐apoptosis. The insulin‐like growth factor binding protein‐3 (IGFBP‐3) is known as a growth suppressor in multiple signaling pathways. The aim of this study was to determine IGFR‐1 and IGFBP‐3 expression in small‐cell lung cancer (SCLC) and analyze the prognostic value in patients with SCLC. We analyzed IGFR‐1 and IGFBP‐3 expression in 194 SCLC tissues by immunohistochemical staining. Correlative analyses between IGFR‐1 and IGFBP‐3 expression in SCLC and clinicopathologic factors were performed. A total of 117 patients had extensive disease (ED) (60.3%) and 77 had limited disease (39.7%). With the median follow‐up duration of 49.5 months (24–82 months), the median progression‐free survival (PFS) and overall survival (OS) were 7.2 months [95% confidence interval (CI): 6.4–8.0 months] and 14.4 months (95% CI: 12.7–16 months), respectively. IGFR‐1 expression was observed in 154 of the 190 tumor tissues, whereas there was no IGFBP‐3 expression. Multivariate analysis showed that stage (p < 0.001), response rate (p < 0.001), and lactate dehydrogenase (LDH) levels (p < 0.001) were the independent prognostic factors for PFS, and age (p = 0.014), LDH level (p < 0.001), and stage (p < 0.001) for OS. The IGFR‐1 positivity was not associated with PFS or OS in the entire cohort. Subgroup analysis revealed that OS was significantly longer in patients with IGFR‐1‐positive tissue than IGFR‐1‐negative tissue in SCLC‐ED (p = 0.034). These results suggest that IGFR‐1 expression may be useful as a prognostic marker in patients with SCLC‐ED.     

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.     

两种小鼠胸腺基质细胞对胸腺细胞凋亡的不同作用

采用两种体外建系的小鼠胸腺基质细胞（ＴＳＣ）系，即上皮样ＴＳＣ（ＭＴＥＣ１）和树突状ＴＳＣ（ＭＴＳＣ４），观察其对胸腺细胞凋亡的影响。小鼠胸腺细胞在体外培养过程中，可自发地出现细胞凋亡的特征，表现为ＤＮＡ呈梯度断裂片段，细胞经ＦＡＣＳ分析出现亚二倍体ＤＮＡ波峰，以及Ｆｅｕｌｇｅｎ′ｓ染色镜检所见的ＤＮＡ凝聚和断裂。胸腺细胞在与ＴＳＣ共育后，在ＭＴＥＣ１组可见其凋亡过程受到抑制和存活率的增加；在ＭＴＳＣ４组，仅在共育１２至１８小时时，见到胸腺细胞凋亡加强，而其存活率不受影响。结果提示在胸腺细胞发育过程中，其阴性选择作用的主要机制之一的ＰＣＤ过程受不同来源的胸腺基质细胞的调节。     

Plasma cell toll‐like receptor (TLR) expression differs from that of B cells,and plasma cell TLR triggering enhances immunoglobulin production

Toll‐like receptors (TLRs) are key receptors of the innate immune system and show cell subset‐specific expression. We investigated the messenger RNA (mRNA) expression of TLR genes in human haematopoietic stem cells (HSC), in naïve B cells, in memory B cells, in plasma cells from palatine tonsils and in plasma cells from peripheral blood. HSC and plasma cells showed unrestricted expression of TLR1–TLR9, in contrast to B cells which lacked TLR3, TLR4 and TLR8 but expressed mRNA of all other TLRs. We demonstrated, for the first time, that TLR triggering of terminally differentiated plasma cells augments immunoglobulin production. Thus, boosting the immediate antibody response by plasma cells upon pathogen recognition may point to a novel role of TLRs.     

Restoration of dysregulated CC chemokine signaling for monocyte/macrophage chemotaxis in head and neck squamous cell carcinoma patients by neem leaf glycoprotein maximizes tumor cell cytotoxicity

Previous studies have shown that the CC chemokine receptor CCR5 is downregulated on monocyte/macrophage (MO/Mφ) surfaces in head and neck squamous cell carcinoma (HNSCC) patients (stage IIIB). Ligands (RANTES, MIP-1α and MIP-1β) of this chemokine receptor were also secreted in lesser quantity from MO/Mφ of HNSCC patients in comparison with healthy individuals. In an aim to restore this dysregulated receptor–ligand signaling, we have used neem leaf glycoprotein (NLGP), a novel immunomodulator reported from our laboratory. NLGP upregulated CCR5 expression, as evidenced from studies on MO/Mφ of peripheral blood from HNSCC patients as well as healthy individuals. Expression of RANTES, MIP-1α and MIP-1β was also upregulated following NLGP treatment of these cells in vitro. Interestingly, NLGP has little effect on the expression of CCR5 and the ligand RANTES in oral cancer cells. This restored CCR5 receptor–ligand signaling seen in MO/Mφ was reflected in improved CCR5-dependent, p38 mitogen-activated protein kinase (MAPK)-mediated migration of MO/Mφ after NLGP treatment to a standard chemoattractant. NLGP also induces better antigen presentation and simultaneous costimulation to effector T cells by MO/Mφ by upregulating human leucocyte antigen (HLA)-ABC, CD80 and CD86. In addition, NLGP-treated MO/Mφ-primed T cells can effectively lyse tumor cells in vitro. The effects of NLGP on monocyte migration and T cell-mediated oral tumor cell killing were further demonstrated in transwell assays with or without CCR5 neutralization. These results suggest a new approach in cancer immunotherapy by modulating dysregulated CCR5 signals from MO/Mφ.