体外诱导大鼠破骨样细胞形成及促进骨吸收的实验

目的:通过大鼠四肢长骨骨髓细胞的体外诱导培养,观察1.25-(OH)2D3对破骨样细胞形成的影响。方法:4周龄SD大鼠长骨骨髓细胞悬液接种于预置盖玻片或牙本质片的24孔培养板内,试验组加入诱导剂1.25-(OH)2D3(1×10-8mol/L)而对照组不加,每3d换液1次,培养2周。结果:培养1周左右光镜下可见有多核破骨样细胞形成,胞体大,抗酒石酸酸性磷酸酶(TRAP)染色阳性,牙本质片上有骨吸收陷窝形成。证明所形成多核细胞为破骨样细胞。结论:1.25-(OH)2D3(1×10-8mol/L)可诱导大鼠骨髓破骨样细胞的形成。     

体外诱导大鼠破骨样细胞形成及促进骨吸收的实验

目的：通过大鼠四肢长骨骨髓细胞的体外诱导培养，观察1．25-(OH)2D3对破骨样细胞形成的影响。方法：4周龄SD大鼠长骨骨髓细胞悬液接种于预置盖玻片或牙本质片的24孔培养板内，试验组加入诱导剂1.25-(OH)：D2(1&;#215;10^mol／L)而对照组不加，每3d换液1次，培养2周?结果：培养1周左右光镜下可见有多核破骨样细胞形成，胞体大，抗酒石酸酸性磷酸酶(TRAP)染色阳性，牙本质片上有骨吸收陷窝形成：证明所形成多核细胞为破骨样细胞。结论：1.25-(OH)2D3(1&;#215;10^-8mol／L)可诱导大鼠骨髓破骨样细胞的形成。     

香豆雌酚对体外破骨细胞分化和骨吸收活性的影响

目的:观察植物雌激素香豆雌酚对破骨细胞的影响,分析其抗骨质疏松的作用方式。方法:实验于2005-07/2006-03在东南大学医学院临床实验中心和分子影像实验室、南京师范大学物理实验中心完成。以1,25(OH)2D3诱导大鼠骨髓细胞形成破骨样细胞,通过相差显微镜下的形态观察,抗酒石酸酸性磷酸酶染色和骨片上骨吸收陷窝的形成来鉴定破骨细胞。分别加入0,10-9,10-8,10-7,10-6,10-5mol/L香豆雌酚,以10-9mol/L17β-雌二醇为阳性对照,于培养3,6和9d分别计数抗酒石酸酸性磷酸酶阳性细胞个数,磷酸苯二钠法测定破骨细胞抗酒石酸酸性磷酸酶的活性,于培养12d利用图像分析系统对骨吸收陷窝的面积进行测量分析,原子吸收光谱法测定破骨细胞和骨片联合培养的上清中钙的浓度。结果:①利用1,25(OH)2D3成功诱导出破骨样细胞。②香豆雌酚抑制破骨细胞的形成和分化:随香豆雌酚浓度增加,每孔抗酒石酸酸性磷酸酶染色阳性的破骨细胞数量呈剂量依赖性减少(香豆雌酚10-9,10-8,10-7,10-6,10-5mol/L组分别为357±8,347±11,303±17,275±18,268±6,空白对照组为360±15,17β-雌二醇组为200±12,P<0.05)。③破骨细胞噬骨活性:骨吸收陷窝面积香豆雌酚各浓度组与空白对照组相比均有显著下降,香豆雌酚10-9与10-8,10-7,10-6,10-5mol/L组之间,香豆雌酚10-8,10-7mol/L组与10-6,10-5mol/L组之间差异均有显著性。④破骨细胞和骨片联合培养的上清中钙的浓度:应用香豆雌酚10-8～10-5mol/L处理后共培养体系中的钙浓度与空白对照组相比分别下降了24.7%,36.2%,36.9%,39.1%。结论:香豆雌酚可通过抑制破骨细胞的形成、抗酒石酸酸性磷酸酶的活性和骨吸收作用,抑制破骨细胞分化和骨吸收活性,从而具有抗骨质疏松作用。     

葛根素抑制1,25-二羟基维生素D3促破骨细胞骨吸收功能的最佳剂量

背景:近年来国内外学者对葛根素的骨形成研究较多,但对于葛根素抑制1,25-(OH)2D3促破骨细胞骨吸收功能尚未见报道。目的:观察不同浓度葛根素在体外对1,25-(OH)2D3促破骨细胞骨吸收功能的抑制作用。方法:收集3周龄小鼠骨髓细胞并接种到24孔培养板中。实验组培养液为1,25-(OH)2D3+α-MEM完全培养基+不同质量浓度的葛根素,对照组不加葛根素。结果与结论:不同浓度组葛根素处理的培养体系均诱导出破骨样细胞且形态差异不明显;除100μg/L葛根素组在第3天时碱性磷酸酶表达显著增高外,各组之间培养液上清中碱性磷酸酶水平无显著性差异(P>0.05);10μg/L葛根素组培养液上清Ca2+水平在第3,5,12天时较对照组显著降低(P<0.05～0.01),50μg/L葛根素组培养液上清Ca2+水平在第12天时较对照组显著降低(P<0.01),100μg/L葛根素组培养液上清Ca2+水平在所有检测点较10μg/L葛根素组显著上升(P<0.05)。提示葛根素最佳抑制剂量在10～50μg/L之间。     

香豆雌酚对体外破骨细胞分化和骨吸收活性的影响

目的：观察植物雌激素香豆雌酚对破骨细胞的影响，分析其抗骨质疏松的作用方式。 方法：实验于2005-07／2006-03在东南大学医学院临床实验中心和分子影像实验室、南京师范大学物理实验中心完成。以1，25（OH）2D3诱导大鼠骨髓细胞形成破骨样细胞，通过相差显微镜下的形态观察，抗酒石酸酸性磷酸酶染色和骨片上骨吸收陷窝的形成来鉴定破骨细胞。分别加入0，10^-9，10^-8，10^-7，10^-6，10^-5mol／L香豆雌酚，以10^-9mol／L 17 β-雌二醇为阳性对照，于培养3,6和9d分别计数抗酒石酸酸性磷酸酶阳性细胞个数，磷酸苯二钠法测定破骨细胞抗酒石酸酸性磷酸酶的活性，于培养12d利用图像分析系统对骨吸收陷窝的面积进行测量分析，原子吸收光谱法测定破骨细胞和骨片联合培养的上清中钙的浓度。 结果：①利用1，25（OH）2D3成功诱导出破骨样细胞。②香豆雌酚抑制破骨细胞的形成和分化：随香豆雌酚浓度增加，每孔抗酒石酸酸性磷酸酶染色阳性的破骨细胞数量呈剂量依赖性减少（香豆雌酚10^-9,10^-8，10^-7，10^-6,10^-5 mol／L组分别为357&;#177;8，347&;#177;11，303&;#177;17，275&;#177;18，268&;#177;6，空白对照组为360&;#177;15，17 β-雌二醇组为200&;#177;12，P＜0．05）。③破骨细胞噬骨活性：骨吸收陷窝面积香豆雌酚各浓度组与空白对照组相比均有显著下降，香豆雌酚10^-9与10^-8，10^-7，10^-6,10^-5 mol／L组之间，香豆雌酚10^-8，10^-7mol／L组与10^-6，10^-5mol／L组之间差异均有显著性。④破骨细胞和骨片联合培养的上清中钙的浓度;应用香豆雌酚10^-8～10^-5mol/L处理后共培养体系中的钙浓度与空白对照组相比分别下降了24．7％，36.2％，36．9％，39．1％。 结论：香豆雌酚可通过抑制破骨细胞的形成、抗酒石酸酸性磷酸酶的活性和骨吸收作用，抑制破骨细胞分化和骨吸收活性，从而具有抗骨质疏松作用。     

骨保护素对破骨细胞分化和骨吸收活性的抑制作用

背景骨保护素(osteoprotegerin,OPG)可抑制破骨细胞的分化,抑制成熟破骨细胞的骨吸收活性并诱导其凋亡,在骨质疏松、类风湿性关节炎、癌症骨转移的领域有潜在的应用价值.目的鉴定在CHO细胞中表达的人骨保护素生物学活性.设计随机对照的实验研究.地点和对象实验在解放军第四军医大学西京医院骨科研究所完成,研究对象人骨肉瘤细胞株MG63、中国仓鼠CHO细胞株、克隆质粒pUC19及真核表达质粒pcDNA3为本室保存,12只6～8周龄BABL/c雄性小鼠由本校动物中心提供.干预RT-PCR法获得人OPG编码区cDNA并构建真核表达载体,在脂质体介导下转染CHO细胞,经Western blot鉴定筛选稳定表达OPG的细胞系.获取含有OPG蛋白的条件培养基浓缩液,实验组体外培养的鼠破骨细胞培养基中加入含有OPG蛋白的条件培养基浓缩液.对照组1只加入转染pcDNA3空载体的CHO细胞培养基的浓缩液.对照组2只加入完全培养基.主要观察指标观察人OPG对破骨细胞的分化和骨吸收的影响.结果转染人OPG编码区基因的CHO细胞能分泌表达OPG.小鼠骨髓细胞在1α,25(OH)2D3(1×10-8mol/L)的存在下可稳定分化出具有骨吸收功能的破骨细胞样细胞,在该培养体系中加入含有OPG的CHO细胞培养上清浓缩液,TRAP染色阳性细胞数明显减少(t=5.547,P＜0.01),骨吸收陷窝的数量显著减少(t=3.409,P＜0.01).结论人骨保护素可在CHO细胞中分泌表达,并对体外培养状态下的破骨细胞的分化和骨吸收功能有抑制作用.     

1，25-二羟维生素D3诱导人成骨样细胞模型基质金属蛋白酶-1表达

目的研究1,25-二羟维生素D3[1α,25-dihydroxyvitamin D3,1α,25(OH)2D3]对人成骨细胞膜型基质金属蛋白酶-1(membrane-type matrix metalloproteinase-1,MT1-MMP)表达的影响,探讨1α,25(OH)2D3调节骨代谢作用机制.方法人成骨细胞用1α,25(OH)2D3干预.Western杂交检测MT1-MMP蛋白质表达.MMP-2活性用ELISA检测.结果观察到1α,25(OH)2D3诱导成骨细胞MT1-MMP表达呈剂量依赖性(均P＜0.05).1α,25(OH)2D3干预12～24h促进MT1-MMP表达(均P＜0.05).1α,25(OH)2D3促进MMP-2激活呈剂量依赖性(均P＜0.05).结论由于MT1-MMP在骨重建过程中起着维持骨形成的关键作用,1α,25(OH)2D3可通过诱导成骨细胞MT1-MMP表达促进骨形成.此可能为1α,25(OH)2D3治疗骨质疏松症机理之一.     

Tim-3对单核细胞诱导破骨样细胞生成和骨吸收功能的影响

目的探讨T细胞免疫球蛋白及粘蛋白结构域3(T cells immunoglobulin domain and mucin domain 3,Tim-3)对单核细胞诱导破骨样细胞生成和骨吸收功能的影响。方法流式细胞术检测RA患者和体检健康者外周血单核细胞上Tim-3的表达水平;利用人外周血单核细胞体外诱导破骨样细胞;实时定量PCR法检测破骨样细胞形成过程中RANK、CTSK和MMP9 mRNA表达水平;瑞氏染色和肌动蛋白染色观察细胞形态,TRAP染色计数细胞形成数量;采用骨吸收功能试验检测破骨样细胞骨吸收陷窝的数量和面积。结果 RA患者外周血单核细胞上Tim-3表达水平[(77.31±10.66)%]明显高于健康人对照组[(51.72±16.69)%],差异有统计学意义(t=7.593,P0.01),不同Tim-3水平的单核细胞在诱导为破骨样细胞时, Tim-3高水平组骨吸收陷窝面积[(1.054±0.085)S/mm~2]明显低于中间水平组[(1.889±0.053)S/mm~2]和低水平组[(2.763±0.066)S/mm~2],差异有统计学意义(F=9.318,P0.05)。结论 Tim-3可能对破骨样细胞的骨吸收功能具有负向调控作用。     

巨噬细胞集落刺激因子/核因子κB受体激活物配体体外诱导培养高纯度破骨细胞:最佳剂量探讨

背景:巨噬细胞集落刺激因子(macrophage colony-stlimulating factor,M-CSF)/核因子_kB受体激活物配体(receptor activatorof nuclear kappa B ligand,RANKL)两种细胞因子协同诱导骨髓干细胞形成破骨细胞是一种较新的,可以获取较高纯度和数量破骨细胞的诱导培养法,但尚缺乏统一的培养标准.目的:建立有效的M-CSF/RANKL诱导小鼠骨髓干细胞诱导分化破骨细胞的培养体系.方法:分离小鼠四肢骨获取骨髓干细胞.将小鼠骨髓干细胞在含有M-CSF的α-MEM培养基中培养24 h,调整细胞浓度为10~7,10~8,10~9L~(-1).然后在培养基中同时加入10μg/LM-CSF和不同质量浓度(20,50,100μg/L)RANKL.行抗酒石酸酸性磷酸酶染色观察干细胞向破骨细胞的转变过程及细胞形态和染色情况,并对各组染色阳性的破骨细胞进行计数,比较不同诱导条件对破骨样细胞数量的影响. 结果与结论:培养3 d后可见少量破骨样细胞,胞质内含许多红色抗酒石酸酸性磷酸酶染色阳性颗粒,细胞内可见淡染的双核;培养6 d后可见大量染色阳性破骨样细胞;培养9 d后出现多核巨型染色阳性破骨样细胞,细胞明显增大,细胞核可达到3个以上.在固定细胞接种浓度条件下,RANKL质量浓度为100 μg/L时诱导分化形成的破骨样细胞数量较另两质量浓度增多(P<0.05);在固定RANKL质量浓度条件下,细胞浓度为10~5 L~(-1)时诱导分化形成的破骨样细胞数量较另两细胞接种浓度增多(P<0.05):细胞接种浓度为10~8L~(-1),RANKL质量浓度为100 μg/L时诱导分化形成的破骨样细胞数量高于其他条件组合(P<0.05).说明在M-CSF/RANKL诱导小鼠骨髓干细胞分化培养破骨细胞的体系中,RANKL最佳质量浓度为100 μg/L,最佳细胞接种浓度为10~8L~(-1).     

两种方法培养大鼠破骨细胞的噬骨能力比较

背景：原代培养的破骨细胞数量少,而诱导培养产生的破骨样细胞数量多,能够满足一些研究骨代谢实验的要求.但是两种细胞在特异酶及噬骨能力方面是否具有相同的效果,到目前为止,还没有详尽的数据来支持.目的：比较大鼠原代分离的破骨细胞及诱导形成的破骨样细胞噬骨能力上的差异.方法：取24 h内新生Wistar大鼠四肢长骨,酶消化法分离培养破骨细胞,将破骨细胞培养过程中消化下来的骨髓单核细胞加入1,25（OH）2 D 3结果与结论：诱导第9天,破骨样细胞数目为破骨细胞数目的11倍,其形态与原代消化获得的细胞形态相同.抗酒石酸酸性磷酸酶染色呈阳性.甲苯胺蓝染色显示两组破骨细胞在骨片上培养时产生骨陷凹面积及深度差异无显著性意义.结果显示破骨样细胞的形态、特异酶及噬骨能力与破骨细胞无差异.诱导生成破骨样细胞.苏木精-伊红染色、抗酒石酸酸性磷酸酶（TRAP）染色鉴定.用甲苯胺蓝染色共培养骨片比较两组破骨细胞噬骨能力.     

Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.     

1,25（OH）2D3对大鼠肾小球系膜细胞凋亡的影响

目的探讨1,25一二羟基维生素D3[1,25(OH)2D3]对大鼠系膜细胞增殖与凋亡及其相关基因Fas表达的影响。方法体外培养的大鼠系膜细胞,分为正常对照组(0 mol/L)和不同浓度1,25(OH)2D3干预组(10-10mol/L;10-8mol/L;10-6mol/L),采用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪,检测不同作用时间(24 h、48 h)下,系膜细胞增殖及凋亡情况,免疫荧光染色法观察干预24 h后,凋亡相关基因Fas的表达情况并测定各组荧光强度。结果与正常对照组相比,1,25(OH)2D3干预组,可明显抑制系膜细胞增殖,促进凋亡,并呈作用时间依赖性。同时免疫荧光染色证实干预浓度越大,系膜细胞内Fas表达增多,荧光强度增高,差异有统计学意义(P<0.01)。结论 1,25(OH)2D3干预大鼠肾小球系膜细胞,能抑制细胞增殖,促进细胞凋亡,Fas的表达增多,呈浓度和时间依赖性。     

Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells

Severe deficiency of osteoclasts, monocytes, and peritoneal macrophages in osteopetrotic (op/op) mutant mice is caused by the absence of functional macrophage colony-stimulating factor (M-CSF). To clarify the role of M-CSF in the osteoclast differentiation, we established a clonal stromal cell line OP6L7 capable of supporting hemopoiesis from newborn op/op mouse calvaria. Although very few macrophages appeared in the cocultures of bone marrow cells and OP6L7 cells, a 50-fold larger number of macrophages was detected in the day 7 cocultures when purified recombinant human M-CSF (rhM-CSF) was exogenously supplied. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive cells appeared only when bone marrow cells were cultured in contact with OP6L7 cells and both rhM-CSF and 1 alpha, 25 (OH)2D3 were added. The TRACP-positive cells became multinucleated with increasing time in culture and expressed the c-fms/M-CSF receptor. These results indicate that both contact with stromal cells and M-CSF are requisite for osteoclast differentiation under physiological conditions.     

妊娠期肝内胆汁淤积症血清25-(OH)D3水平与总胆汁酸、甘胆酸水平及母婴结局的相关性分析

目的探讨妊娠期肝内胆汁淤积症(ICP)血清25-(OH)D3水平与总胆汁酸、甘胆酸水平及母婴结局的相关性。方法回顾性选取2018年12月至2020年4月间在监利市人民医院确诊的孕晚期ICP孕妇90例作为ICP组,选择同期进行产检的孕晚期正常孕妇100例作为正常对照组。检测2组患者的血清25-(OH)D3水平,根据ICP组患者25-(OH)D3水平的中位数将其分为高25-(OH)D3组、低25-(OH)D3组各45例。比较不同血清25-(OH)D3水平的ICP患者总胆汁酸、甘胆酸水平和母婴结局的差异。结果ICP组孕妇血清25-(OH)D3水平为(26.38±4.90)μg/m L,低于正常对照组孕妇[(35.12±6.45)μg/m L],差异有统计学意义(P<0.05)。低25-(OH)D3组孕妇血清中总胆汁酸、甘胆酸的水平为(15.94±2.31)、(411.64±49.72)μmol/L,均高于高25-(OH)D3组孕妇[(11.76±1.34)、(254.65±32.81)μmol/L],差异有统计学意义(P<0.05)。低25-(OH)D3组孕妇妊娠终止时间为(36.02±3.15)周,短于高25-(OH)D3组[(37.54±4.87)周],新生儿体质量为(2.71±0.48)kg,小于高25-(OH)D3组[(3.08±0.41)kg],产后出血量为(290.72±34.18)m L,大于高25-(OH)D3组[(348.56±50.71)m L],剖宫产(71.11%)、肝功能损伤(17.78%)、早产(42.22%)发生率均高于高25-(OH)D3组(44.44%、4.44%、22.22%),差异均有统计学意义(P<0.05)。结论ICP患者血清25-(OH)D3水平异常降低,与总胆汁酸、甘胆酸水平及母婴结局直接相关。     

瘦素对RAW264.7细胞肿瘤坏死因子α表达的影响

背景:瘦素是脂肪组织分泌的一种多肽激素,研究显示瘦素在动脉粥样硬化形成中发挥了一定重要的作用。目的:观察瘦素对鼠源性巨噬细胞系RAW264.7细胞肿瘤坏死因子α表达的影响,并从核转录因子κB活性变化角度探讨其可能机制。设计:对照观察实验。单位:华中科技大学同济医学院生物化学及分子生物学系。材料:实验于2005-04/2006-02在华中科技大学同济医学院生物化学及分子生物学系及附属协和医院普外科实验室完成。将培养的RAW264.7细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)、IkappaB激酶抑制剂组及空白对照组。每组3瓶,重复实验3次。方法:将鼠源性巨噬细胞株RAW264.7细胞以1×109L-1密度接种于6孔板中,用含体积分数为0.1的小牛血清的RPMI-1640培养基培养。待RAW264.7细胞生长至80%时,换用无血清培养基Opti-MEM继续培养24h后,将细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)及空白对照组,瘦素孵育4h后采用反转录-聚合酶链反应检测肿瘤坏死因子α在mRNA水平的表达。上述分组细胞经瘦素分别孵育1,3,6和9h后采用双抗夹心酶联免疫吸附实验检测肿瘤坏死因子α在蛋白水平的表达。上述分组细胞经瘦素孵育不同时间后采用凝胶迁移率实验检测细胞核内核转录因子κB活性。将RAW264.7细胞分为以下4组:空白对照组、IkappaB激酶特异性抑制剂PS1145(10μmol/L)处理组、瘦素(50μg/L)处理组、瘦素(50μg/L) PS1145(10μmol/L)组,各组孵育时间均为6h,分别检测细胞核内核转录因子κB活性及肿瘤坏死因子α在mRNA水平的表达。主要观察指标:①不同浓度瘦素对RAW264.7细胞肿瘤坏死因子α:mRNA表达水平的影响;蛋白分泌的影响。②不同浓度瘦素对RAW264.7细胞核内核转录因子κB活性的影响。③抑制IkappaB激酶活性对瘦素诱导RAW264.7细胞肿瘤坏死因子α的影响。结果:①RAW264.7细胞经不同浓度的瘦素处理后,肿瘤坏死因子α在mRNA水平呈瘦素剂量依赖性增加,50μg/L瘦素处理组达峰值。②蛋白水平的表达呈瘦素剂量时间依赖性增加,50μg/L瘦素处理6h即可达峰值。③核转录因子κB的活性亦与瘦素浓度正相关,50μg/L瘦素处理6h后核转录因子κB活性最高(P<0.05)。④抑制IkappaB激酶活性可部分抑制肿瘤坏死因子α的表达。结论:瘦素可直接促进RAW264.7细胞肿瘤坏死因子α的表达和分泌,并呈剂量时间依赖性,其机制可能与瘦素激活核转录因子κB有关。这可能是瘦素致动脉粥样硬化的机制之一。     

雌二醇对骨髓基质细胞核结合因子α1基因及成骨细胞生成的影响

目的研究骨髓基质细胞在向成骨细胞分化的条件培养体系中, 17β-雌二醇( E2)对其核结合因子α 1 (Cbfα 1 )基因表达的影响,探讨雌二醇对成骨细胞生成的作用.方法取自 1只 3月龄雌性 SD大鼠骨髓基质细胞在生长培养基中传代后,用 1,25(OH)2D3和地塞米松( DEX)诱导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核结合因子α 1mRNA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, Van Gieson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 Cbfα 1 mRNA的表达,且呈剂量依赖性.E2浓度为 0,1× 10- 10, 1× 10- 8和 1× 10- 6mmol/L时, Cbfα 1 mRNA的表达量分别为 23.4%± 1.8%, 21.8%± 2.0%, 15.8%± 1.5% ( t=6.3, P< 0.01)和 5.8%± 0.8%( t=17.8, P< 0.01).Northern blot 结果显示 Cbfα 1 mRNA的表达量分别为 4.22± 0.11, 2.51± 0.12( t=21, P< 0.01), 1.88± 0.10( t=31, P< 0.01)和 1.17± 0.09( t=41, P< 0.01).ALP活性随 E2浓度增加而降低,在上述 E2浓度时, ALP活性分别为 (42.6± 2.5)U/g,(17.9± 2.0)U/g( t=15, P< 0.01) ,(10.8± 1.5) U/g( t=21, P< 0.01)和 (3.6± 0.7)U/g( t=29, P< 0.01).细胞Ⅰ型胶原的含量随 E2浓度增加而降低.结论 E2能明显抑制骨髓基质细胞分化过程中 Cbfα 1mRNA的表达,减少成骨细胞的生成.     

脂氧素A4对Aβ_(25-35)所致N2a细胞损伤的保护作用初探

目的:初步探讨脂氧素A4(LXA4)对β-淀粉样蛋白25-35(Aβ_(25-35))损伤N2a细胞的保护作用及其机制。方法:用不同浓度Aβ_(25-35)处理N2a细胞,建立阿尔茨海默病(AD)细胞损伤模型,通过细胞形态学观察、CCK-8法和Hoechst 33258染色来评价细胞模型是否构建成功。细胞分为对照组、模型组(20μmol/L Aβ_(25-35))和LXA4保护组(50、100、200 nmol/L)。采用CCK-8法检测N2a细胞的活性;荧光酶标仪检测N2a细胞内活性氧(ROS)水平;ELISA法检测细胞培养上清中白介素-10(IL-10)和肿瘤坏死因子-α(TNF-α)。结果:20μmol/L Aβ_(25-35)处理N2a细胞24 h可建立细胞损伤模型。LXA4保护组的细胞存活率均较模型组显著升高。与模型组相比,200 nmol/L LXA4保护组细胞内ROS水平显著降低(P0.01);200 nmol/L LXA4保护组IL-10水平显著高于模型组(P0.01);200 nmol/L LXA4保护组的TNF-α水平显著低于模型组(P0.01)。结论:LXA4对Aβ_(25-35)损伤N2a细胞具有保护作用,其机制可能是抑制ROS水平以及调节炎症因子的表达。     

Vitamin D compounds. Effect on clonal proliferation and differentiation of human myeloid cells.

We examined the effect of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and a variety of vitamin D analogs on proliferation and differentiation of normal and leukemic myeloid clonogenic cells. Only cells from myeloid leukemic lines that contained relatively mature cells (HL-60, U937, THP, HEL, M1) were induced to differentiate and were inhibited in their clonal growth by exposure to 1 alpha,25(OH)2D3 (50% inhibition, 3 X 10(-8)-8 X 10(-10) M). A fluorinated analog of vitamin D was 5-10-fold more potent than 1 alpha,25(OH)2D3. Cells from a human myeloblast line (KG-1) and normal human granulocyte-monocyte stem cells (GM-CFC), both of which depend on colony-stimulating factor (CSF) for clonal growth, were stimulated in their clonal proliferation by 1 alpha,25(OH)2D3 in the presence of suboptimal concentrations of CSF. Leukemic cells from 10 of 14 patients with myeloid leukemia, but not normal GM-CFC from 12 patients in remission, were markedly inhibited in their clonal proliferation by 1 alpha,25(OH)2D3. Our results suggest that 1 alpha,25(OH)2D3 may be a cofactor in hematopoiesis and that vitamin D analogs may have a differential effect on normal versus leukemic growth.     

Effect of 1,25-dihydroxyvitamin D3 and 13-cis-retinoic acid on in vitro hematopoiesis in the myelodysplastic syndromes

The effect of 2 X 10(-10) to 2 X 10(-7) mol/L 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) and 10(-9) to 10(-6) mol/L 13-cis-retinoic acid on in vitro differentiation and proliferation of marrow cells from patients with myelodysplastic syndrome (MDS) was assessed. Cells from 17 patients were studied by the semisolid technique, and cells from seven patients by both liquid and semisolid cultures. After incubation in liquid culture with 1,25(OH)2D3, in six of seven patients evaluated an increasing number of myeloid cells (185% to 470%) acquired the morphologic appearance of mature monocyte-macrophages, and a decrease in the number of immature myeloid cells (26% to 75%) was observed. Phagocytosis and killing of Candida albicans by monocyte-macrophages incubated with 1,25(OH)2D3 were normal and similar to those processes in untreated cells. 1,25(OH)2D3 increased the percentage of monocytes that phagocytosed C. albicans in three patients. Thirteen of 17 patients showed reduced myeloid cloning, and eight showed increased cluster formation. Cloning efficiency was significantly lower in patients with refractory anemia with excess of blasts and chronic myelomonocytic leukemia. Concentrations of 2 X 10(-9) to 2 X 10(-8) mol/L 1,25(OH)2D3 and 10(-8) to 10(-7) mol/L retinoic acid had a stimulatory effect on myeloid colony growth in five of the six patients with sideroblastic and refractory anemia, but in only two of the 11 patients with refractory anemia with excess of blasts and chronic myelomonocytic leukemia. The results indicate that the differentiation pattern of myeloid precursor cells from a subset of patients with MDS was altered by exposure to 1,25(OH)2D3.     

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype

Osteoclasts (OCLs) in Paget's disease are markedly increased in number and size, have increased numbers of nuclei per multinucleated cell, and demonstrate increased resorption capacity and increased sensitivity to 1,25-(OH)(2)D(3), the active form of vitamin D. These cells also contain nuclear inclusions, reminiscent of those seen in paramyxovirus-infected cells, which cross-react with antibodies to measles virus nucleocapsid (MVNP) antigen. To elucidate the role of MV in the abnormal OCL phenotype of Paget's disease, we transduced normal OCL precursors with retroviral vectors expressing MVNP and the MV matrix (MVM) genes. The transduced cells were then cultured with 1,25-(OH)(2)D(3) for14 or 21 days to induce formation of OCL-like multinucleated cells. The MVNP-transduced cells formed increased numbers of multinucleated cells, which contained many more nuclei and had increased resorption capacity compared with multinucleated cells derived from empty vector-transduced (EV-transduced) and MVM-transduced or normal bone marrow cells. Furthermore, MVNP-transduced cells showed increased sensitivity to 1, 25-(OH)(2)D(3), and formed OCLs at concentrations of 1, 25-(OH)(2)D(3) that were 1 log lower than that required for normal, EV-transduced, or MVM-transduced cells. These results demonstrate that expression of the MVNP gene in normal OCL precursors stimulates OCL formation and induces OCLs that express a phenotype similar to that of pagetic OCLs. These results support a potential pathophysiologic role for MV infection in the abnormal OCL activity and morphology that are characteristic of pagetic OCLs.