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。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：     

Roles of osteoclasts and bone-derived IGFs in the survival and growth of human breast cancer cells in human adult bone implanted into nonobese diabetic/severe combined immunodeficient mice

Human breast cancer frequently metastasizes to bone, and effective therapies for patients with bone metastasis are required. However, the molecular mechanism for the bone metastasis of human breast cancer has not yet been fully elucidated. The present study aimed to evaluate the importance of active osteoclasts and bone-derived insulin-like growth factors (IGFs) for the survival and growth of breast cancer cells in bone. Human breast cancer cell line MCF-7 cells were injected into human adult bone (HAB) implanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The mice were then treated with recombinant human osteoclastogenesis inhibitory factor/osteoprotegerin (rhOCIF/OPG), a decoy receptor for receptor activator of NF-kappaB ligand (RANKL), or an anti-human IGF monoclonal antibody. Histomorphometric analyses revealed that both treatments significantly decreased the tumor area of MCF-7 cells in cross-sections of the implanted HAB to about 30% of the tumor area in control mice, but had no effect on the growth of subcutaneously injected MCF-7 cells. Consistent with the results for the tumor area in HAB, there were fewer osteoclasts in the implanted HAB in rhOCIF/OPG-treated mice than in vehicle-treated mice. However, treatment with the anti-human IGF monoclonal antibody had no effect on the number of osteoclasts in HAB. The results indicate that the active osteoclasts induced by RANKL and the IGFs released as a result of bone resorption by these osteoclasts play crucial roles in the survival and growth of human breast cancer cells in bone and suggest that neutralization of bone-derived IGFs will be effective in preventing the development of bone tumors in breast cancer patients.     

The analgesic drug buprenorphine inhibits osteoclastic bone resorption in vitro,but is proinflammatory in rat adjuvant arthritis

We have examined the effect of the -opioid analgesic buprenorphine on osteoclastic bone resorption in vitro and in the rat adjuvant arthritis model. In the bone slice assay buprenorphine inhibited osteoclastic bone resorption with an IC₅₀ of 1 M. This effect was not mimicked by the -opioid agonist ([D-Ala,N-Me-Phe, Gly-ol]-enkephalin and was not prevented by the -opioid antagonist naloxone. Since other agents that inhibit osteoclastic bone resorption, such as bisphosphonates and calcitonin prevent bone erosion in the rat adjuvant arthritis model, we also examined the effect of buprenorphine in this model. Surprisingly, buprenorphine exacerbated inflammation measured by paw volume and increased joint destruction assessed by X-ray scores, in the injected paws and particularly in the non-injected paws. These studies also show that attempts to ameliorate animal suffering in this chronic model by using centrally acting analgesics such as buprenorphine may lead to complications in interpreting screening results obtained with novel, potential anti-arthritic compounds.accepted by I. Ahnfelt-Rønne     

人骨髓间质干细胞向造血细胞分化潜能的实验研究

目的:在体研究人骨髓间质干细胞(hBMMSCs)向造血细胞分化的潜能。方法:将hBMMSCs经尾静脉注射给环磷酰胺处理的严重联合免疫缺陷(SCID)小鼠,利用流式激活细胞分析系统(FACS)检测hBMMSCs输注后存活35d的SCID小鼠外周血、骨髓和脾脏中人源性造血细胞的表型和水平。结果:hBMMSCs输注组外周血(PB)、骨髓(BM)和脾脏(spleen)中可检测到人CD45⁺/H-2D^d-、CD34⁺/H-2D^d-细胞,而对照组PB、BM和脾脏均未检测到上述表型的人造血细胞。结论:hBMMSCs具有向造血细胞分化的潜能。     

人骨髓间充质干细胞体外分离培养及向血管内皮样细胞分化的研究

目的:研究人骨髓间充质干细胞(hMSCs)的体外分离培养方法及在特定微环境下分化为血管内皮样细胞的潜能,可能为银屑病研究提供实验基础.方法:密度梯度离心法结合贴壁法分离人骨髓间充质干细胞(hMSCs),体外扩增培养并进行鉴定.加入血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)定向诱导hMSCs向血管内皮细胞分化,流式细胞仪进行细胞表型鉴定,Dil-ac-LDL摄取实验鉴定血管内皮细胞功能.结果:经流式细胞仪测定分离培养的hMSCs CD71、CD44阳性表达,CD54、CDl06、CD45弱阳性表达,CD34、CD31、VWF、KDR、HLA-DR阴性表达;经bFGF、VEGF诱导后的hMSCs可见类似内皮细胞样改变,经流式细胞术检测,与对照组相比内皮细胞特异表面标志CD34、CD31、VWF、KDR表达转为阳性(P＜0.01),而HLA-DR、CD54、CDl06、CD45表达明显上调(P＜0.01);诱导分化后的内皮样细胞具有摄取Dil-ac-LDL的能力.结论:密度梯度离心法结合贴壁法可获得较纯的MSCs;在bFGF、VEGF诱导作用下,hMSCs具有向内皮细胞分化潜能,产生功能性内皮细胞.     

人骨髓间充质干细胞的分离培养及血管内皮生长因子对其体外诱导分化作用

目的：分离和培养人骨髓间充质干细胞(mcsenchymal stem cells，MSCs)并检测其免疫学表型，探讨血管内皮生长因子(vascular endothelial growth factor，VECF)对其体外诱导分化作用。方法：利用Pcreoll梯度分离、贴壁筛选法及单克隆培养法分离、培养、扩增MSCs；采用免疫荧光和流式细胞术检测MSCs免疫学表型；通过VFGF-165基因转染分析MSCs的表型变化。结果：体外分离、培养出高度同源性的MSCs；MSCs具有独特的细胞免疫学表型，即CD44，CD29，c-kit阳性，CD34，CD31，CD54阴性；VEGF-165诱导后CD44表达明显降低，CD31显著升高，MSCs向内皮分化。结论：成功建立人MSCs分离培养方法，探讨了MSCs细胞免疫学表型及VEGF对它的内皮诱导分化作用，为MSCs的应用提供理论基础。