骨髓间充质干细胞、骨形态发生蛋白和骨发生潜力与髋关节置换后假体的稳定性

背景：骨髓微环境的成骨潜力与全髋关节置换后假体长期稳定性之间的关系尚不清楚。作者提出一个观点,通过骨髓成骨潜力的分析可以预测假体的契合强度。 目的：以线性回归分析评估全髋关节置换患者骨髓间充质干细胞活力、成骨蛋白信号通路相关基因表达与成骨发生在假体稳定性中的相关性。 方法：选择12例接受全髋关节置换的患者,置换过程中取股骨近端的骨髓组织在体外扩增至子一代,取出细胞进行流式细胞学或RNA检测,以线性回归分析评估成骨潜力各项指标之间的相关性。 结果与结论：患者骨髓间充质干细胞中Stro-1阳性细胞的数量、碱性磷酸酶活性以及成骨相关基因的表达情况具有较大的个体差异。Stro-1阳性细胞比率与骨形成蛋白受体1a,MSX2,Runx2、碱性磷酸酶活性存在显著相关性(P < 0.05),细胞经成骨培养后碱性磷酸酶活性与Runx2表达也存在显著相关性(P < 0.05)。提示在全髋关节置换患者中骨髓微环境的成骨潜力具有较大的个体差异,而骨髓间充质干细胞各项指标对于全关节置换长期稳定性的潜在影响有待进一步深入研究。     

股骨近端Stro-1+细胞与骨形态发生蛋白及其受体的相关性

背景：髋关节置换后假体的融合率与假体周围的骨重建密切相关,这个过程中骨髓间充质干细胞的募集、向成骨细胞分化的途径受骨形态发生蛋白调控。 目的：分析股骨近端Stro-1⁺细胞与骨形态发生蛋白7、骨形态发生蛋白受体的相关性。 方法：32例患者初次进行髋关节置换,取术中股骨矩开槽时骨块,体外细胞培养14 d后检测骨髓间充质干细胞数量,Stro-1⁺标记干细胞的总量,骨形态发生蛋白7、骨形态发生蛋白受体1a、骨形态发生蛋白受体2的表达。 结果与结论：Stro-1⁺、骨形态发生蛋白7、骨形态发生蛋白受体1a、骨形态发生蛋白受体2的表达在不同年龄组有轻微差异,但差异无显著性意义,在不同性别组差异也无显著性意义;股骨近端Stro-1⁺细胞数量(即代表具有向成骨细胞分化功能的细胞)与骨形态发生蛋白7表达呈负相关关系,与骨形态发生蛋白受体1a表达呈正相关关系,其可以影响髋关节假体的骨融合与生存率。     

酪蛋白激酶2相互作用蛋白1调控骨质疏松大鼠骨髓间充质干细胞的成骨能力北大核心

背景:目前对酪蛋白激酶2相互作用蛋白1(casein kinase 2-interaction protein-1,CKIP-1)分子机制的体外研究主要集中在基因敲除小鼠来源成骨细胞或骨髓间充质干细胞,鲜见报道骨质疏松模型大鼠来源骨髓间充质干细胞中CKIP-1表达的研究。目的:探讨下调CKIP-1基因前后骨质疏松状态骨髓间充质干细胞成骨分化能力的变化。方法:用维甲酸诱导雌性SD大鼠骨质疏松模型,采用全骨髓贴壁法体外培养骨质疏松组、正常组大鼠骨髓间充质干细胞。成骨诱导后进行茜素红染色、碱性磷酸酶染色及实时定量RT-PCR检测骨桥蛋白、Runx2 mRNA的相对表达,实时定量RT-PCR检测成骨诱导过程中2组细胞中CKIP-1的动态表达;通过基因转染沉默CKIP-1基因,成骨诱导后进行茜素红染色、碱性磷酸酶染色及实时定量RT-PCR检测骨桥蛋白、Runx2 mRNA的相对表达。结果与结论:①与正常组相比,骨质疏松组骨髓间充质干细胞茜素红染色钙结节定量、碱性磷酸酶活性及骨桥蛋白、Runx2的mRNA水平降低(P<0.05),骨质疏松组骨髓间充质干细胞中CKIP-1基因动态表达水平总体偏高;②与未下调CKIP-1的骨质疏松组骨髓间充质干细胞相比,下调CKIP-1基因表达后,骨质疏松组骨髓间充质干细胞茜素红染色钙结节定量、碱性磷酸酶活性及骨桥蛋白、Runx2的mRNA水平明显升高(P<0.05);③结果表明,维甲酸诱导的骨质疏松大鼠骨髓间充质干细胞成骨能力降低,下调CKIP-1基因可以部分提高其成骨分化能力。     

腺病毒介导的人骨形态发生蛋白2基因转染骨髓间充质干细胞

背景：骨髓间充质干细胞作为骨、软骨创伤缺损及退变修复的种子细胞越来越受到关注。 目的：分析人骨形态发生蛋白2基因转染对白色封闭群大鼠(SD大鼠)骨髓间充质干细胞的影响。 方法：分离纯化SD大鼠骨髓间充质干细胞并体外扩增,通过腺病毒载体介导人骨形态发生蛋白2基因转染骨髓间充质干细胞,分别通过荧光显微镜观察荧光表达情况及蛋白质水平来测定转染后人骨形态发生蛋白2的表达,碱性磷酸酶定量测定鉴定成骨活性及MTT法评估人骨形态发生蛋白2转染对骨髓间充质干细胞的影响。 结果与结论：从SD大鼠骨髓提取物中分离培养的细胞形态为梭形,呈铺路石状、漩涡状生长,经流式细胞仪检测及多项分化能力鉴定符合骨髓间充质干细胞的特征;经转染人骨形态发生蛋白2基因后,骨髓间充质干细胞表达人骨形态发生蛋白2、碱性磷酸酶;MTT法检测转染人骨形态发生蛋白2基因后,骨髓间充质干细胞增殖能力明显增强(P < 0.05)。说明人骨形态发生蛋白2基因转染骨髓间充质干细胞后可以持续、高效表达人骨形态发生蛋白2和碱性磷酸酶,在体外明显促进骨髓间充质干细胞的增殖。     

股骨近端微量元素Zn与骨形态发生蛋白7及Stro-1~+细胞的相关性:预测髋关节假体寿命

背景:假体周围微量元素Zn的含量与骨形态发生蛋白7、Stro-1+细胞相关性可能影响髋关节假体的骨融合与生存率。目的:分析股骨近端微量元素Zn与骨形态发生蛋白7、Stro-1+细胞的相关性。方法:24例初次髋关节置换患者,取术中股骨矩开槽时废弃骨块,提取细胞后体外培养骨髓间充质干细胞。培养14 d采用流式细胞仪检测骨髓间充质干细胞中的Stro-1+细胞,酶联免疫吸附测定试剂盒检测培养细胞离心上清液中骨形态发生蛋白7的表达,放射免疫荧光法检测培养细胞离心上清液中Zn含量。结果与结论:Zn含量在不同年龄组及不同性别组差异无显著性意义;Zn与Stro-1+、骨形态发生蛋白7正相关联系。关于股骨近端微量元素Zn的进一步研究可以用来预测和干预髋关节假体的寿命。     

人骨形成蛋白4基因腺相关病毒及绿色荧光蛋白基因腺相关病毒体外诱导成骨的比较

背景:由于骨形成蛋白的释放速度与新骨生长速度不匹配,单纯应用骨形成蛋白的效果尚不理想,因此,应有合适的载体来调节骨形成蛋白的释放速度。目的:观察重组人骨形成蛋白4基因腺相关病毒及绿色荧光蛋白基因腺相关病毒诱导兔骨髓间充质干细胞向成骨方向分化的作用。方法:全骨髓法培养兔骨髓间充质干细胞,分别应用人骨形态形成蛋白4基因腺相关病毒载体及绿色荧光蛋白基因腺相关病毒转染兔骨髓间充质干细胞,设定感染复数值为5×104,观察两组细胞形态改变,分别行碱性磷酸酶染色、Von Kossa染色、茜素红染色及碱性磷酸酶含量测定,比较两组成骨活性差异。结果与结论:人骨形态形成蛋白4基因腺相关病毒组转染骨髓间充质干细胞后,细胞形态呈现典型的成骨改变,碱性磷酸酶染色及Von Kossa染色、茜素红染色均出现成骨的特征性改变。绿色荧光蛋白基因腺相关病毒组未观察到上述改变。人骨形态形成蛋白4基因腺相关病毒组碱性磷酸酶含量高于绿色荧光蛋白基因腺相关病毒组(P0.01)。提示人骨形态形成蛋白4基因腺相关病毒转染骨髓间充质干细胞后,骨髓间充质干细胞表现出更加明显的成骨活性改变。     

携带低氧诱导因子1α慢病毒感染大鼠骨髓间充质干细胞后成骨基因的表达

背景:人低氧诱导因子1α可调控其下游成骨及成血管基因的表达,具有提高成骨活性的作用。目的:观察携带人低氧诱导因子1α慢病毒感染的大鼠骨髓间充质干细胞中成骨基因表达情况。方法:通过RT-PCR方法从Hela细胞中获得低氧诱导因子1α,构建携带低氧诱导因子1α的慢病毒表达质粒Lenti-HIF-1α-eGFP,与LentiPac HIV混合包装质粒共包装293Ta细胞,获得病毒;采用全骨髓直接贴壁法分离培养大鼠骨髓间充质干细胞,使用流式细胞仪对骨髓间充质干细胞进行鉴定。分别在慢病毒感染骨髓间充质干细胞1,4,7,14 d后,用实时荧光定量PCR检测骨髓间充质干细胞中成骨基因骨形态发生蛋白2、骨钙蛋白、骨桥蛋白、碱性磷酸酶的表达水平。结果与结论:Lenti-HIF-1α-eGFP有效地感染骨髓间充质干细胞,实时荧光定量PCR检测结果显示成骨基因骨形态发生蛋白2、骨钙蛋白、骨桥蛋白、碱性磷酸酶在Lenti-HIF-1α-eGFP感染后第4天开始明显过表达,且持续至14 d。结果表明低氧诱导因子1α可以提高骨髓间充质干细胞的成骨活性。     

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1mRNA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。     

龟鹿二仙胶含药血清介导大鼠骨髓间充质干细胞的成骨分化

背景:在研究骨髓间充质干细胞增殖的基础上揭示其分化机制是防治骨质疏松症研究的热门课题之一。目的:探讨龟鹿二仙胶含药血清诱导骨髓间充质干细胞成骨分化以防治骨质疏松症的分子机制与激活细胞外信号调节激酶1/2/E26转录因子1信号通路因子之间的相关性。方法:购买商品化第3代SD大鼠骨髓间充质干细胞,根据加入不同成分培养基分为胎牛血清组、空白血清组、龟鹿二仙胶含药血清组、经典诱导组以及通路抑制剂组、龟鹿二仙胶含药血清+通路抑制剂组,根据分组条件进行成骨诱导分化,每3 d换液1次,干预7 d时采用碱性磷酸酶活性检测试剂盒测定前4组骨髓间充质干细胞的成骨分化水平;干预14 d时采用茜素红染色观察前4组骨髓间充质干细胞的成骨矿化水平,实时荧光定量PCR检测前4组骨髓间充质干细胞中碱性磷酸酶、Runt家族相关转录因子2、过氧化物酶体增殖物激活受体γ和E26转录因子1的基因表达水平,Western blot法检测6组骨髓间充质干细胞中Ⅰ型胶原、碱性磷酸酶、Runt家族相关转录因子2、过氧化物酶体增殖物激活受体γ、E26转录因子1、细胞外信号调节激酶1/2和磷酸化细胞外信号调节激酶1/2蛋白表达。结果与结...     

肥胖小鼠骨髓间充质干细胞的成骨分化

背景:骨髓间充质干细胞分化受到多种因素的影响,肥胖小鼠骨髓间充质干细胞具有更佳的成骨分化能力。目的:探究正常饮食小鼠与高脂饮食诱导的肥胖小鼠骨髓间充质干细胞成骨分化的差异。方法:将4-6周龄Balb/c小鼠随机分为正常饮食组和高脂饮食组,饲养20周后,通过全骨髓培养法得到骨髓间充质干细胞,成骨诱导7 d后进行碱性磷酸酶染色以及qRT-PCR检测成骨相关基因的表达,成骨诱导14 d后进行茜素红染色。高脂饲养20周后,Micro-CT分析股骨骨量的变化,小鼠股骨切片进行苏木精-伊红染色。结果与结论:(1)与正常饮食组小鼠相比,高脂饮食饲养20周后小鼠体质量显著增加;(2)与正常饮食组小鼠骨髓间充质干细胞相比,高脂饮食组小鼠骨髓间充质干细胞中碱性磷酸酶阳性表达和钙结节数目显著增加,成骨相关基因碱性磷酸酶、骨桥蛋白和RUNT相关转录因子2表达水平升高;(3)Micro-CT重建和骨形态参数量化结果显示,与正常饮食组小鼠相比,高脂饮食组小鼠骨量显著增加,其中骨密度、骨体积分数、骨面积与骨体积比值、骨小梁厚度和骨小梁数目均显著增加,而骨小梁分离度显著减小,苏木精-伊红染色显示高脂饮食组小鼠股骨内含...     

The effect of TNFα secreted from macrophages activated by titanium particles on osteogenic activity regulated by WNT/BMP signaling in osteoprogenitor cells

Wear particles are the major cause of osteolysis associated with failure of implant following total joint replacement. During this pathologic process, activated macrophages mediate inflammatory responses to increase osteoclastogenesis, leading to enhanced bone resorption. In osteolysis caused by wear particles, osteoprogenitors present along with macrophages at the implant interface may play significant roles in bone regeneration and implant osteointegration. Although the direct effects of wear particles on osteoblasts have been addressed recently, the role of activated macrophages in regulation of osteogenic activity of osteoblasts has scarcely been studied. In the present study, we examined the molecular communication between macrophages and osteoprogenitor cells that may explain the effect of wear particles on impaired bone forming activity in inflammatory bone diseases. It has been demonstrated that conditioned medium of macrophages challenged with titanium particles (Ti CM) suppresses early and late differentiation markers of osteoprogenitors, including alkaline phosphatase (ALP) activity, collagen synthesis, matrix mineralization and expression of osteocalcin and Runx2. Moreover, bone forming signals such as WNT and BMP signaling pathways were inhibited by Ti CM. Interestingly, TNFα was identified as a predominant factor in Ti CM to suppress osteogenic activity as well as WNT and BMP signaling activity. Furthermore, Ti CM or TNFα induces the expression of sclerostin (SOST) which is able to inhibit WNT and BMP signaling pathways. It was determined that over-expression of SOST suppressed ALP activity, whereas the inhibition of SOST by siRNA partially restored the effect of Ti CM on ALP activity. This study highlights the role of activated macrophages in regulation of impaired osteogenic activity seen in inflammatory conditions and provides a potential mechanism for autocrine regulation of WNT and BMP signaling mediated by TNFα via induction of SOST in osteprogenitor cells.     

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.     

Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics

In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.     

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.     

Bone morphogenetic protein 7-transduced human dermal-derived fibroblast cells differentiate into osteoblasts and form bone in vivo

Background: Human dermal-derived fibroblast cells (hDDFCs) are multipotent. Bone morphogenetic proteins (BMPs) are a group of cytokines that promote different developmental processes, including the formation of bone. BMPs can promote hDDFC osteogenesis, but the role of BMP7 in hDDFC osteogenesis in vitro and bone formation in vivo has not been investigated in depth. Materials and Methods: hDDFCs were stably transfected with a human BMP7 recombinant adenovirus and osteogenic differentiation was examined by alkaline phosphatase staining and calcium accumulation. In addition, we measured the expression of osteoblast-related genes. To examine osteogenesis in vivo, we injected C57BL/6 nude mice with adenovirus-transfected hDDFCs in a calcium alginate hydrogel and examined bone formation using soft X-ray, histological, and immunohistochemical analyses. Results: Our findings showed that adenovirus-mediated BMP7 expression promoted osteogenic differentiation of hDDFCs and enhanced expression of osteoblast-related genes in vitro. Cells infected with BMP7 adenoviruses showed enhanced bone formation and osteoblast-related gene expression in vivo after the injection of hDDFC–hydrogel mixture. Conclusions: Taken together, our data indicate that BMP7 significantly promotes hDDFC osteogenesis, and confirm that infecting hDDFCs with BMP7-expressing adenoviruses is a useful tool for bone tissue engineering.     

微渠多孔羟基磷灰石支架诱导骨髓间充质干细胞成骨分化的miRNA表达谱分析

文题释义： 羟基磷灰石：是目前最为理想的生物活性材料,具有生物相容性、骨传导性和骨诱导性,植入人体后对组织无刺激和排斥作用,能与骨形成很强的化学结合,用作骨缺损的充填材料,能为新骨的形成提供支架,发挥骨传导作用,是理想的硬组织替代材料。 MicroRNA(miRNA)：是一类内生的、长度为20-24个核苷酸的单链小分子RNA,由具有发夹结构的70-90个碱基大小的单链RNA前体经过Dicer酶加工后生成,其可以通过几个miRNAs的组合在转录后水平精细调控基因的表达。 背景：多孔羟基磷灰石支架具有良好的体内外成骨效能,但其所涉及的miRNAs复杂调控机制相关研究较少。 目的：探讨多孔羟基磷灰石支架材料介导大鼠骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化。 方法：体外分离、培养和鉴定大鼠骨髓间充质干细胞,将骨髓间充质干细胞与多孔羟基磷灰石支架共培养为实验组,骨髓间充质干细胞单独培养为空白对照组,分别进行成骨诱导7 d,运用miRNA高通量测序技术分析两组骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化并进行GO分析,筛选出两组中表达差异明显的miRNA分子并进行qRT-PCR验证。 结果与结论：①与空白对照组比较,成骨诱导7 d时实验组BMP2、ALP、Runx2 mRNA表达上调,其中BMP2上调明显(P < 0.05);②microRNA高通量测序结果显示miR-210-3p、miR-146a-5p等13个miRNAs明显上调;let-7c-3p、let-3615等17个miRNAs明显下调;③GO分析上调的miRNA靶基因主要参与生物学调节、细胞基因表达、基因表达调节等,包括NF-κB、Toll样受体9、细胞间黏附、白细胞介素1调节、血管生成、Hippo等信号通路;④实时荧光定量qPCR验证结果显示miRNA-210在实验组上调15倍,miR-146a-5p在实验组上调10倍(P < 0.05);⑤结果表明,新型微渠多孔羟基磷灰石支架可以通过上调骨髓间充质干细胞miRNA-210-3p和miR-146a表达,促进骨髓间充质干细胞的成骨分化。 ORCID: 0000-0002-8722-1548(郑佳俊) 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     

17β-雌二醇对骨髓基质细胞骨形态发生蛋白受体ⅠA、B mRNA表达的影响

目的:研究骨髓基质细胞在向成骨细胞分化的介质中,17β-雌二醇(E₂)对其骨形态发生蛋白受体(BMPR)ⅠA,ⅠBmRNA表达的影响,探讨雌二醇对成骨细胞生成的作用。方法:用1,25(OH)₂D₃和地塞米松(DEX)诱导大鼠骨髓基质细胞向成骨细胞分化,应用半定量RT-PCR技术,观察不同浓度E₂对骨髓基质细胞分化过程中BMPR-ⅠA,ⅠBmRNA表达的影响,以α-磷酸奈酚为底物测定细胞碱性磷酸酶的活性,VanGieson染色法显示Ⅰ型胶原的含量。结果:E₂能明显抑制骨髓基质细胞分化过程中BMPR-ⅠAmRNA的表达,且呈剂量依赖性,随E₂浓度增加,BMPR-ⅠAmRNA从(23.7±1.7)%降至(16.3±1.5)%(P<0.05)和(8.3±1.2)%(P<0.01);BMPR-ⅠBmRNA随E₂浓度增加而增加,从(1.3±0.6)%增至(5.7±2.0)%(P<0.05)和(15.3±3.0)%(P<0.01)。ALP活性随E₂浓度增加而降低,从(42.6±2.5)U·L^-1·g^-1protein降至(10.8±1.5)U·L^-1·g^-1protein和(3.6±1.7)U·L^-1·g^-1protein(P<0.01);细胞Ⅰ型胶原的含量随E₂浓度增加而减少。结论:E₂能明显抑制体外培养的骨髓基质细胞分化过程中BMPR-ⅠAmRNA的表达,降低成骨细胞生成。