类风湿关节炎关节液来源的间充质干细胞分离鉴定及其对破骨细胞发育的影响

为了研究炎性微环境对于间充质干细胞生物学特性的影响,从类风湿性关节炎病人关节液中分离得到间充质干细胞,以流式细胞术测定其免疫学表型,以成骨和成脂肪诱导检测其多向分化能力,将其与CD14^＋的单核细胞共同培养并以抗酒石酸的酸性磷酸酶染色检测对破骨细胞的调控作用。结果表明：类风湿性关节炎病人关节液中分离得到的间充质干细胞高达CD105,CD73,CD29,CD44,CD166和HLA—ABC,低表达CD34,CD45,CD31,HLA—DR,CD80和CD86;与健康人骨髓间充质干细胞相比,其向成骨细胞和脂肪细胞分化的能力下降,而且其支持更多的破骨细胞生成（P〈0．05）。结论：类风湿性关节炎病人关节液中存在的间充质干细胞具有与骨髓间充质干细胞相似的表型,但是其多向分化能力下降,更有意义的是其支持破骨细胞生成的能力增强。本研究结果有助于了解病理微环境中的间充质干细胞生物学特性。     

姜黄素调控大鼠骨髓间充质干细胞的成骨分化

背景:姜黄素能明显降低破骨细胞的骨重吸收,诱导破骨细胞凋亡,抑制破骨细胞形成。目的:观察不同浓度姜黄素对大鼠骨髓间充质干细胞成骨分化的影响。方法:采用贴壁法分离培养大鼠骨髓间充质干细胞,传至第3代后采用成骨培养基进行成骨诱导,在诱导前3d于诱导培养基中分别加入0(对照),10,15μmol/L姜黄素。结果与结论:接种培养7d,倒置显微镜下可见分散的骨髓间充质干细胞小集落,集落细胞呈放射状生长。随培养时间延长,细胞集落增大,细胞形态为长梭形。加入成骨培养基7d后细胞形态逐渐由长梭形变为多角形,随成骨诱导时间延长形态变化更加明显。加入不同浓度姜黄素后骨髓间充质干细胞增殖无变化。姜黄素呈剂量依赖性促进骨髓间充质干细胞碱性磷酸酶活性及Runx-2、血红素单加氧酶1的表达。提示姜黄素能有效促进大鼠骨髓间充质干细胞早期成骨分化,其机制可能与提高干细胞血红素单加氧酶1的表达有关。     

小鼠骨髓内破骨细胞诱导形成的实验观察

目的:建立骨髓间充质干细胞诱导培养成为破骨细胞的培养方法并进行初步的形态观察,为破骨细胞学及与其相关疾病的发病机制的研究提供更有意义的手段。方法:采用小鼠骨髓间充质干细胞通过1,25-(OH)2D诱导分化为破骨3细胞的培养方法,并观察原代成骨细胞对该方法破骨细胞形成及其功能的影响。根据是否加原代成骨细胞,分成未加成骨细胞、加成骨细胞二组,未加成骨细胞组是骨髓间充质干细胞直接诱导培养,加成骨细胞组是骨髓间充质干细胞与原代成骨细胞共同培养。分别培养6,9,12及15d,各组细胞到培养时间后取出进行TRAP染色阳性的多核细胞计数、骨片甲苯胺蓝染色及扫描电镜观察。结果:未加成骨细胞、加成骨细胞两组的骨髓间充质干细胞经培养后均出现破骨细胞,而且随着培养时间的延长,破骨细胞及骨片吸收陷窝的数目增多。培养相同时间的未加成骨细胞、加成骨细胞两组之间的破骨细胞及骨片吸收陷窝的数目差异无显著性意义。结论:通过1,25-(OH)2D诱导骨髓中的间充质干细胞培养破骨细胞的3方法是可行的,而且骨髓间充质干细胞诱导培养成破骨细胞时加入原代成骨细胞共同培养对破骨细胞的形成和骨吸收功能并无明显促进作用。     

骨髓贴壁细胞向破骨细胞的分化

背景：一般认为采用贴壁分离法得到的小鼠骨髓中能够贴壁的细胞为间充质干细胞,并能分化成为成骨、成脂肪及成软骨细胞,其中贴壁的细胞是否有分化为破骨细胞的潜能尚不明确。 目的：检测贴壁分离法得到的小鼠骨髓中能贴壁的细胞是否能够分化为破骨细胞。 方法：采用贴壁法得到小鼠原代间充质干细胞,以及通过贴壁1-5d后得到不同时间贴壁的骨髓细胞。原代间充质干细胞及不同时间贴壁的骨髓细胞分别用普通培养基,及含m-csf和RANKL培养基,培养9 d后进行碱性磷酸酶和抗酒石酸酸性磷酸酶染色。原代间充质干细胞传代后,第2代间充质干细胞分为4组,分别用普通培养基、含m-csf培养基、含RANKL培养基及含m-csf和RANKL的培养基培养,9 d后进行碱性磷酸酶及抗酒石酸酸性磷酸酶染色。 结果与结论：贴壁法得到较均一的间充质干细胞,原代及传代贴壁细胞的联合诱导组抗酒石酸酸性磷酸酶染色均为阳性,说明原代和传代的贴壁骨髓细胞中均有可以分化为破骨细胞的细胞。不同时间贴壁的细胞诱导后碱性磷酸酶及抗酒石酸酸性磷酸酶染色有差异,说明不同时间贴壁的细胞存在分化差异。     

硅酸钙、β-磷酸三钙和羟基磷灰石三相陶瓷复合物与骨细胞活性的体外鉴定

由于羟基磷灰石陶瓷与骨无机质的化学成份相似,所以10年来研究者对羟基磷灰石陶瓷在骨修复替换中的应用进行了深入的研究.羟基磷灰石因其高生物相容性和骨传导性已被广泛的应用,但羟基磷灰石的再吸收能力差.立童对由硅酸钙、β-磷酸三钙和羟基磷灰石组成的三相陶瓷复合物进行了研究.在三相陶瓷复合物环境下,采用不同比率硅酸钙、β-磷酸三钙和羟基磷灰石对骨髓夹源间充质干细胞进行体外研究.细胞在加入和没有加入成骨细胞的情况下培养28 d.采用碱性磷酸酶活性、碱性磷酸酶基因码转录和骨唾液蛋白Ⅱ表达评估成骨分化水平.成骨细胞和破骨细胞在陶瓷复合80%羟基磷灰石材料上完成预实验.将鼠颅盖骨成骨细胞与人单核细胞共同培养以分析其向破骨细胞分化的可能性.结果显示人骨髓间充质干细胞在陶瓷复合物材料上表现出快速黏附和高度增殖率,在所有实验材料上,成骨诱导的骨髓间充质干细胞碱性磷酸酶活性增加,碱性磷酸酶基因码转录和骨唾液酸蛋白Ⅱ的表达在3种材料上同样增加.扫描电镜和聚焦激光扫描显微镜结果证明在陶瓷复合80%羟基磷灰石材料上的人单核细胞向破骨样细胞分化.结果提示制各的三相陶瓷复合材料支持人骨髓间充质干细胞的黏附、增殖和成骨分化,以及人单核细胞与成骨细胞联合培养条件下破骨细胞形成.     

成人脂肪间充质干细胞的定向成骨诱导

背景：脂肪分离可获得大量的间充质干细胞并成功向骨、软骨、脂肪、心肌等多个方向诱导分化。 目的：建立成人脂肪间充质干细胞体外分离培养、成骨分化方法,并探讨脂肪间充质干细胞作为骨组织工程的种子细胞的前景。 方法：通过胶原酶消化法从成人脂肪中分离间充质干细胞,进行体外培养,流式细胞仪检测细胞表面标记物, CCK8检测细胞活性,成骨诱导液诱导干细胞向骨细胞分化,BCIP/NBT比色法染色检测碱性磷酸酶,茜素红染色检测钙结节形成,RT-PCR检测碱性磷酸酶,骨桥蛋白表达变化。 结果与结论：利用脂肪抽吸液成功培养出脂肪间充质干细胞,且能稳定传代,增殖能力旺盛;流式细胞仪检测证实有特定的间充质干细胞表面标记物表达;成骨诱导脂肪间充质干细胞后呈典型的成骨细胞形态;碱性磷酸酶染色阳性,茜素红染色后可见钙结节形成,成骨诱导培养0,3,7,14,21,28 d,RT-PCR定量检测结果证实碱性磷酸酶、骨桥蛋白阳性表达。说明用酶消化法可以从人脂肪中分离得到脂肪间充质干细胞;脂肪间充质干细胞可向骨细胞诱导分化,阳性表达骨桥蛋白,碱性磷酸酶,是一种优良的骨组织工程的种子细胞。     

小鼠骨髓内破骨细胞诱导形成的实验观察

目的：建立骨髓间充质干细胞诱导培养成为破骨细胞的培养方法并进行初步的形态观察，为破骨细胞学及与其相关疾病的发病机制的研究提供更有意义的手段。方法：采用小鼠骨髓间充质干细胞通过1，25-(OH)2D3诱导分化为破骨细胞的培养方法，并观察原代成骨细胞对该方法破骨细胞形成及其功能的影响。根据是否加原代成骨细胞，分成未加成骨细胞、加成骨细胞二组，未加成骨细胞组是骨髓间充质干细胞直接诱导培养，加成骨细胞组是骨髓间充质干细胞与原代成骨细胞共同培养。分别培养6，9，12及15d，各组细胞到培养时间后取出进行TRAP染色阳性的多核细胞计数、骨片甲苯胺蓝染色及扫描电镜观察。结果：未加成骨细胞、加成骨细胞两组的骨髓间充质干细胞经培养后均出现破骨细胞，而且随着培养时间的延长，破骨细胞及骨片吸收陷窝的数目增多。培养相同时间的未加成骨细胞、加成骨细胞两组之间的破骨细胞及骨片吸收陷窝的数目差异无显著性意义。结论：通过1,25-(OH)2D3诱导骨髓中的间充质干细胞培养破骨细胞的方法是可行的，而且骨髓间充质干细胞诱导培养成破骨细胞时加入原代成骨细胞共同培养对破骨细胞的形成和骨吸收功能并无明显促进作用。     

C57BL/6小鼠骨髓单核细胞分离、培养、纯化及向破骨细胞的分化

背景:目前骨髓单核细胞的分离提取国内外文献报道,大多是利用RAW264.7细胞诱导分化为破骨细胞。目的:探讨分离、培养、纯化和鉴定C57BL/6小鼠骨髓单核细胞的方法,观察小鼠骨髓单核细胞体外生长特征,诱导其向破骨细胞分化。方法:无菌分离C57BL/6小鼠股骨和胫骨,取出骨髓细胞,提取过程中先用红细胞裂解液去除红细胞;骨髓细胞过夜培养后(大于16 h),第2天分离出悬浮细胞,在含有巨噬细胞集落刺激因子的培养基中继续培养获得贴壁的小鼠骨髓单核细胞。通过换液对小鼠骨髓单核细胞进行纯化和扩增培养。进行形态学观察,测定生长曲线,用流式细胞仪检测原代小鼠骨髓单核细胞细胞表面抗原,加入巨噬细胞集落刺激因子和核因子κB受体活化因子配基诱导分化为破骨细胞。结果与结论:新分离的小鼠骨髓单核细胞呈小圆形,两端伸出触角,培养5 d后,细胞成椭圆形,两端的触角更明显,增殖依赖巨噬细胞集落刺激因子。流式结果显示分离的小鼠骨髓单核细胞纯度较高,能够诱导分化为破骨细胞。利用间充质干细胞与单核细胞的贴壁能力不同及巨噬细胞集落刺激因子显著促进单核细胞贴壁增殖的特性能有效分离获得稳定生长的小鼠骨髓单核细胞。培养的小鼠骨髓单核细胞性状稳定,表型稳定均一,适于做进一步研究。     

促进小鼠骨髓间充质干细胞增殖和成骨分化的血管内皮生长因子

背景：血管内皮生长因子是骨髓间充质干细胞所处骨髓微环境中的重要调控因子,其可促进骨髓间充质干细胞的血管内皮方向分化,但尚无其对骨髓间充质干细胞增殖和成骨分化调控作用的报道。
　　目的：探讨血管内皮生长因子对骨髓间充质干细胞增殖和成骨分化的调控作用及其机制。
　　方法：分离、培养小鼠骨髓间充质干细胞,CCK8方法检测不同质量浓度血管内皮生长因子重组蛋白对骨髓间充质干细胞增殖的影响,选定适宜血管内皮生长因子重组蛋白质量浓度并检测其对骨髓间充质干细胞成骨分化的影响,分子生物学方法检测血管内皮生长因子干预下骨髓间充质干细胞中Osterix,Runx2,碱性磷酸酶、骨钙素和血红素氧化酶1的表达。
　　结果与结论：①血管内皮生长因子可以促进骨髓间充质干细胞增殖,且具有浓度依赖性,100μg/L血管内皮生长因子重组蛋白的促增殖作用最显著。②成骨诱导剂存在条件下,血管内皮生长因子促进骨髓间充质干细胞成骨标志基因Osterix、Runx2、碱性磷酸酶和骨钙素的表达;血管内皮生长因子干预组骨髓间充质干细胞成骨分化的钙结节数量较对照组增加。③血管内皮生长因子可诱导骨髓间充质干细胞中血红素氧化酶1 mRNA和蛋白水平的高表达。以上结果表明血管内皮生长因子促进骨髓间充质干细胞增殖和向成骨细胞分化的调控机制可能与骨髓间充质干细胞内血红素氧化酶1表达增加有关。     

C57小鼠脂肪及骨髓间充质干细胞生物学特性的比较

背景：研究发现,脂肪源干细胞具有和骨髓间充质干细胞一样的贴壁和形成成纤维样克隆特性,并具有向骨、脂肪、软骨等多系分化的能力。 目的：比较C57小鼠脂肪源间充质干细胞和骨髓间充质干细胞的生物学特点。 方法：在无菌的条件下分别从C57小鼠的脂肪和骨髓中获取脂肪间充质干细胞和骨髓间充质干细胞。体外分离、培养并将脂肪间充质干细胞和骨髓间充质干细胞传至第3代,进行细胞形态、表面标记、生长动力学分化潜能测定和Notch信号相关基因的检测。 结果与结论：脂肪间充质干细胞和骨髓间充质干细胞形态学相似,第3代的脂肪间充质干细胞和骨髓间充质干细胞均表达CD29、CD105、Sca-1,不表达CD34、CD133,但骨髓间充质干细胞还表达CD45;生长曲线和细胞克隆分析显示脂肪间充质干细胞的增殖速度明显比骨髓间充质干细胞快;脂肪间充质干细胞和骨髓间充质干细胞均可向成骨、成脂、成软骨诱导分化,脂肪间充质干细胞更易向成骨诱导;Notch 相关基因检测显示脂肪源干细胞的 Jagged-1表达水平明显比骨髓间充质干细胞低,而 Hes-1的表达水平脂肪源干细胞明显高于骨髓间充质干细胞的表达水平。提示脂肪间充质干细胞比骨髓间充质干细胞扩增能力更强,更易向成骨分化,可能与Hes-1表达水平有关。     

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co- cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes.     

Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL)

Osteoclasts are bone-resorbing cells that are derived from haemopoietic precursors, including cells present in peripheral blood. The recent identification of RANKL [receptor activator of nuclear factor (NF)-kappaB ligand], a new member of the tumour necrosis factor ligand superfamily that has a key role in osteoclastogenesis, has allowed the in vitro generation of osteoclasts in the absence of cells of the stromal/osteoblast lineage. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with soluble RANKL and human macrophage colony-stimulating factor form osteoclasts. However, PBMC are heterogeneous, consisting of subsets of monocytes and lymphocytes as well as other blood cells. As the CD14 marker is strongly expressed on monocytes, the putative osteoclast precursor in peripheral blood, we have selected CD14(+) cells from PBMC to examine their osteoclastogenic potential and their expression of novel members of the tumour necrosis factor superfamily involved in osteoclastogenesis. Highly purified CD14(+) cells demonstrated mRNA expression of receptor activator of NF-kappaB, but no expression of RANKL or osteoprotegerin, whereas PBMC expressed mRNAs for all three factors. CD14(+) (but not CD14(-)) cells cultured on bone slices for 21 days with human macrophage colony-stimulating factor and soluble RANKL generated osteoclasts and showed extensive bone resorption. Similar numbers of osteoclasts were generated by 10(5) CD14(+) cells and 10(6) PBMC, but there was significantly less intra-assay variability with CD14(+) cells, suggesting the absence of stimulatory/inhibitory factors from these cultures. The ability of highly purified CD14(+) cells to generate osteoclasts will facilitate further characterization of the phenotype of circulating osteoclast precursors and cell interactions in osteoclastogenesis.     

肿瘤相关巨噬细胞与HUT-78淋巴瘤细胞相互作用的实验研究

本研究旨在探究淋巴瘤细胞对单核细胞向肿瘤相关巨噬细胞（TAM）分化的影响以及TAM对淋巴瘤细胞生长的作用.通过密度梯度离心法及免疫磁珠分选获得初治淋巴瘤患者及健康志愿者外周血单核细胞,利用Transwell嵌套建立单核细胞与淋巴瘤细胞株HUT-78的共培养模型.通过流式细胞术分析TAM细胞CD68、CD163的表达,应用细胞计数法绘制淋巴瘤细胞的生长曲线,ELISA法检测共培养体系中细胞因子IL-10、VEGF的含量.结果表明,单核细胞与淋巴瘤细胞共培养后CD68、CD163、CD68＆CD163（＋）细胞的比例显著升高（P＜0.05）,在患者及健康志愿者升高程度未见显著差异,且二者的单核细胞及其分化而来的巨噬细胞在体外培养条件下对淋巴瘤细胞的生长无直接的促进或抑制作用.患者细胞共培养组IL-10、VEGF水平明显低于单核细胞和淋巴瘤细胞单独培养组之和（P＜0.05）,而在健康志愿者无此现象.结论：淋巴瘤细胞能促进外周血单核细胞向M2型巨噬细胞分化,在作用程度上,患者及健康志愿者未见显著差异.由患者单核细胞转化而来的TAM在共培养体系中的作用类似M1型巨噬细胞,能有效抑制共培养体系中总IL-10和VEGF的分泌.     

地塞米松损害间充质干细胞的免疫抑制功能

本研究探讨地塞米松用于脐带源间充质干细胞(MSC)体外扩增过程中对MSC免疫抑制功能的影响。采用BrdU方法检测地塞米松对MSC培养增殖的影响;MSC经过14 d低浓度地塞米松的培养刺激后,用流式细胞术检测其表面标记物;与外周血单个核细胞共培养后,检测外周血单个核细胞的激活增殖情况。结果表明,经过14 d低浓度地塞米松(10 nmol/L)的培养刺激,在MSC特征性细胞表型上实验组和对照组没有明显的差异;但是,在相同的MSC数情况下,经过地塞米松培养的MSC抑制淋巴细胞活化的程度与对照组相比显著地降低。地塞米松预处理的MSC经过干扰素-γ培养12 h后,MSC的免疫调节能力得到一定程度的恢复。结论:地塞米松损害MSC的免疫抑制功能,干扰素-γ能一定程度对抗或逆转地塞米松对MSC免疫抑制功能的损害。因此,在研究MSC的免疫调节能力时,培养液中应该注意或者尽量不使用地塞米松,以避免地塞米松对免疫相关研究结果的影响。     

Interleukin-11: a new cytokine critical for osteoclast development.

Stromal cells of the bone marrow control the development of osteoclasts through the production of cytokines capable of promoting the proliferation and differentiation of hematopoietic progenitors. Moreover, the deregulated production of the cytokine IL-6 in the bone marrow mediates an increase in osteoclastogenesis after estrogen loss. IL-6, however, does not influence osteoclastogenesis in the estrogen-replete state, suggesting that other cytokines might be responsible for osteoclast development under physiologic circumstances. We report here that IL-11, a newly discovered cytokine that is produced by marrow stromal cells, induced the formation of osteoclasts exhibiting an unusually high degree of ploidy in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 were capable of bone resorption, as evidenced by the formation of resorption pits, as well as the release of 45Ca from prelabeled murine calvaria. Further, an antibody neutralizing IL-11 suppressed osteoclast development induced by either 1,25-dihydroxyvitamin D3, parathyroid hormone, interleukin-1, or tumor necrosis factor; whereas inhibitors of IL-1 or TNF had no effect on IL-11-stimulated osteoclast formation. The effects of IL-11 on osteoclast development were blocked by indomethacin; more important, however, they were independent of the estrogen status of the marrow donors.     

IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.     

双膦酸盐对破骨细胞分化及抗酒石酸酸性磷酸酶的影响

背景：抗酒石酸酸性磷酸酶是破骨细胞分化及骨吸收功能的特异性标志酶,是破骨细胞分化成熟的标志。目的：观察双膦酸盐对破骨细胞分化及骨吸收功能相关因子抗酒石酸酸性磷酸酶的影响。方法：小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组：对照组开始时加入质量浓度100μg/L核因子kB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,免疫荧光检测两组抗酒石酸酸性磷酸酶表达的差异,Western blot检测抗酒石酸酸性磷酸酶蛋白表达情况。结果与结论：各组细胞均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组（P〈0.01）。免疫荧光检测显示,对照组抗酒石酸酸性磷酸酶表达均强于双膦酸盐组（P〈0.01）。Western blot检测显示,双膦酸盐组抗酒石酸酸性磷酸酶蛋白的表达低于对照组（P〈0.01）。说明双膦酸盐通过抑制抗酒石酸酸性磷酸酶蛋白的表达,阻碍破骨细胞分化生成及骨吸收功能。     

双膦酸盐对破骨细胞分化中组织蛋白酶K及骨吸收功能的影响

背景：有研究表明双膦酸盐可抑制破骨细胞的骨吸收功能,但对其骨吸收功能关键细胞因子组织蛋白酶K是否产生作用,至今少有报道。目的：观察双膦酸盐对破骨细胞分化中组织蛋白酶K及骨吸收功能影响。方法：用小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组：对照组加入质量浓度100μg/L核因子κB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,培养72 h免疫荧光检测两组组织蛋白酶K表达差异,Western blot检测组织蛋白酶K蛋白表达情况。结果与结论：两组均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组（P〈0.01）。免疫荧光检测组织蛋白酶K表达对照组强于双膦酸盐组（P〈0.01）;Western blot检测组织蛋白酶K表达双膦酸盐组低于对照组（P〈0.01）。结果证实,双膦酸盐通过抑制组织蛋白酶K因子的表达,阻碍破骨细胞的骨吸收功能。     

Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors

Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor kappaB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms(+)RANK(-)) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms(+)RANK(+)) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.