氯离子通道5在模拟失重下磷代谢异常致骨丧失的作用机制初步研究

目的 探讨氯离子通道5（chloride channel,CLC5）在模拟失重下磷代谢异常致骨丧失的作用机制,为失重性骨代谢异常防治提供数据参考。方法 ①利用回转器对骨细胞模拟失重,将骨细胞分为模拟失重组(MG)和对照组(CON),2天后实时定量检测DMP1和CLC5在IDG-SW3骨细胞的表达情况;②利用尾部悬吊法对小鼠模拟失重,将1月龄C57雌性小鼠随机分为悬尾组(SUS)和对照组(CON),每组5只。4周后两组胫骨石蜡切片进行CLC5免疫组织化学染色;③2月龄C57 DMP1敲除鼠和DMP1转基因鼠制备胫骨石蜡切片,进行CLC5免疫组织化学染色。结果 实时定量检测结果显示三维回转后DMP1和CLC5在IDG-SW3骨细胞的表达量均升高（P＜0.05）;免疫组织化学染色显示CLC5在对照组和悬尾组胫骨骨细胞胞质和基质中均有表达,且悬尾组表达量明显高于对照组（P＜0.05）。此外,CLC5在DMP1基因敲除鼠和转基因鼠骨细胞中CLC5的表达也较正常对照组升高（P＜0.05）。结论 CLC5为力学敏感分子;模拟失重下CLC5参与了DMP1对磷代谢的调控,但其具体作用机制有待于进一步研究。     

低磷食物对模拟失重小鼠后肢骨骨质丢失防治作用的探讨

目的 初步探讨低磷食物对模拟失重下小鼠骨丢失防治的可能性及防治效果,为后期可能的临床应用提供实验依据.方法 利用尾部悬吊法模拟失重.将24只C57BL/6雌性小鼠随机分为四组,即对照(CON)组、对照低磷(CON-LP)组、悬尾(SUS)组及悬尾低磷(SUS-LP)组,每组6只.4周实验期满后取小鼠后肢骨标本,通过Micro-CT扫描分析比较各组小鼠股骨的骨体积分数(bone volume frac-tion,BV/TV)、骨小梁密度(trabecular density,Tb.Density)等骨密度参数,骨小梁厚度(trabecular thickness,Tb.Th)、平均骨小梁数(trabecular number,Tb.N)等骨形态学参数.通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色、碱性磷酸酶(alkaline phosphatase,ALP)染色和丽春红三色(Ponceau S)染色对各组小鼠胫骨生长发育和改建情况进行组织形态学观察.结果 Micro-CT结果显示,CON-LP组与CON组比较:骨密度参数BV/TV和Tb.Density减小(P<0.05),形态学参数Tb.Th增高和Tb.N减少(P<0.05).SUS-LP组与SUS组比较:骨密度参数BV/TV和Tb.Density减小(P<0.05),形态学参数Tb.Th和Tb.N减少(P<0.05).染色结果显示:悬尾的小鼠(SUS组、SUS-LP组)与地面的小鼠(CON组、CON-LP组)比较,骨小梁出现明显的骨吸收,Tb.N减少、骨小梁间距变大.CON-LP组与CON组比较,Tb.Density减低、Tb.N减少.SUS-LP组与SUS组比较,骨小梁变细、Tb.N减少.结论 低磷食物对模拟失重下小鼠后肢骨骨丢失防治效果不明显,甚至可能产生不利的后果.     

实验性骨质疏松模型骨组织中核因子表达特征及其意义

目的 探讨核因子NF-KBP50、NF-KBP65在骨质疏松发病过程中的意义。方法选用4个月龄雌性BALB／C小鼠随机分假手术对照组（CON组）和去卵巢骨质疏松模型组（OVX组）,术后第12周测量全身骨密度后处死,取股骨下端用ABC免疫组织化学、原位杂交方法测定骨组织中核因子的蛋白含量及表达水平并进行细胞定位。结果OVX组与CON组相比,骨密度下降,股骨骨小梁稀疏、成骨细胞数目减少而破骨细胞数目增多;免疫组织化学染色结果显示,NF-kB P50、NF-kB P65蛋白在模型组和对照组均有阳性表达,主要位于骨小梁周边成骨细胞、骨细胞及骨髓细胞胞浆中,OVX组蛋白含量均高于CON组,且与骨密度呈负相关（P〈0．01或P〈0．05）;原位杂交结果表明,OVX组NF-kB P50 mRNA、NF-kB P65 mRNA表达显著高于CON组（P〈0．05）。结论骨质疏松骨组织及骨髓中核因子的蛋白含量及表达水平升高,在骨质疏松骨微环境中的表达异常并与骨密度明显相关。     

小剂量地塞米松对生长期小鼠骨微结构及骨代谢的影响

目的 观察小剂量地塞米松对生长期小鼠骨微结构及骨代谢的影响。方法 24只4周龄雌性小鼠随机分两组（n=12）：地塞米松组（DEX,1mg/kg,肌肉注射,2次/周）,对照组。4w后处死小鼠,检测血清Ⅰ型前胶原N端肽(PINP)和骨Ⅰ型胶原交联C端肽（β-CTX）蛋白的表达水平;检测胫骨干骺端骨保护素（OPG）、核因子-κ B受体活化因子配体（RANKL）表达;抗酒石酸酸性磷酸酶（TRAP）染色方法检测破骨细胞;一侧胫骨行显微CT扫描分析。结果 DEX组小鼠胫表观骨密度和骨体积分数明显高于对照组（P<0.05）,骨小梁数量高于对照组,结构模型指数低于对照组,但无统计学意义。DEX组小鼠血清PINP及β-CTX浓度显著下降（P<0.05）。DEX组小鼠胫骨干骺端OPG表达明显增多,而RANKL表达无明显变化,相对于对照组,OPG/RANKL比值升高。TRAP染色示DEX组小鼠胫骨干骺端破骨细胞数量较对照组减少。结论 小剂量地塞米松间断给药可能通过上调OPG/RANKL比率维持生长期小鼠骨量。     

间断性水平位站立对尾吊大鼠胫骨骨小梁结构影响的micro-CT评价

目的 应用micro-CT研究尾吊大鼠胫骨骨小梁微结构的变化,以此评价间断性水平位站立对抗模拟失重导致的骨微结构退化的效果.方法 24只SD雄性大鼠随机等分为3组:对照组(CON组)、尾吊组(SUS组)、水平位站立对抗组(ST组).尾吊组大鼠始终保持约30°头低位及后肢自由悬垂不负重状态,水平位站立对抗组在尾吊处理的基础上,大鼠每天固定时间水平站立1 h.实验3周后处死各组大鼠,取其右侧胫骨行micro CT扫描及三维重建,对其骨微结构进行定量分析.结果 选定感兴趣区(ROI)行定量分析及三维可视化后,与CON组相比,SUS组胫骨骨体积分数(BVF)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)均显著降低,而骨表面积/体积(BS/BV)、骨小梁间隙(Tb.Sp)及骨小梁模式因子(Trabecular Pattern Factor,TPF)均显著升高;ST组的变化介于SUS组和CON组之间;三维可视化图像结果与物理参数结果趋势一致.应用One-Way ANOVA分析及多重比较LSD-t检验,三组各参数之间统计学差异显著(P＜0.05).结论 间断性水平位站立可明显减弱尾吊大鼠胫骨的微结构退化和骨质丢失,而micro CT可以有效地辨别胫骨松质骨微结构的精细变化.     

白细胞介素10和转化生长因子β1与小鼠结肠癌肝转移的关系

目的了解小鼠脾脏接种结肠癌细胞发生肝转移所需肿瘤细胞数的临界值,探讨白细胞介素10（IL-10）和转化生长因子β1（TGF-β1）在结肠癌肝转移过程中的作用及意义。方法选择6-8周雌性Balb／c小鼠36只,随机分为6组,各6只。实验组通过脾脏注射小鼠结肠癌CT26细胞建立小鼠结肠癌肝转移模型,注射细胞数分别为1×10^5个（A组）、5×10^4个（B组）、1×10^4个（C组）、5×10^2个（D组）和1×10^3个（E组）,对照组为正常小鼠。于术后21天处死小鼠,肉眼观察肝脏肿瘤转移情况并行肝脏组织学观察及IL-10和TGF-β1免疫组织化学染色。结果A、B组小鼠肝脏转移率均为100％,其他各组小鼠均未见有肝转移瘤形成。IL-10和TGF-β1表达在对照组均为阴性,但在各实验组小鼠肝脏表达均为阳性,且表达量随着脾脏注射CT26细胞数量的增加而升高,IL-10和TGF-β1的表达量在A、B、C组与D组或E组间比较,差异均有统计学意义（P=0．000）,但在D、E两组间比较,差异无统计学意义。结论5×10^4个CT26细胞脾脏注射可成功建立小鼠结肠癌肝转移模型;CT26细胞可诱导肝脏产生IL-10和TGF-β1,IL-10和TGF-β1可促使结肠癌肝转移瘤的形成。     

培养基对小鼠单核巨噬细胞RAW264.7诱导分化破骨细胞的影响

目的 探讨培养基对小鼠单核巨噬细胞RAW264.7向破骨细胞分化的影响。方法 实验分为3组：高糖DMEM培养基组（DMEM组）;高糖DMEM/α-MEM培养基组（DMEM/α-MEM组）;α-MEM培养基组（α-MEM组）。按常规方法采用上述3种培养基进行破骨细胞培养,培养5 d后,采用抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase, TRAP）染色观察各组破骨细胞的形成情况,并采用实时荧光定量PCR方法观察破骨细胞分化相关标志物NFATc-1、c-Fos和TRAF-6 mRNA的表达;培养11 d后采用甲苯胺蓝染色进行骨陷窝面积分析,观察破骨细胞骨吸收功能情况。结果 3组均可以观察到典型的TRAP+破骨细胞。与DMEM组相比,DMEM/α-MEM组、α-MEM组TRAP+破骨细胞数量明显增加（P<0.01）,但各组形态略有不同。在骨吸收功能上,与DMEM组和DMEM/α-MEM组相比,α-MEM组骨陷窝面积明显增加（P<0.01）。在破骨细胞分化相关调控因子表达上,与DMEM组相比,α-MEM组NFATc-1、c-Fos 和TRAF-6 mRNA表达显著增加（P<0.01,P<0.05）;DMEM/α-MEM组NFATc-1 mRNA表达显著增加（P<0.01）,c-Fos 和TRAF-6 mRNA表达有增加的趋势,但差异无统计学意义;α-MEM组与DMEM/α-MEM组比较差异无统计学意义。结论 高糖DMEM培养基、高糖DMEM/α-MEM培养基、α-MEM培养基均可用于小鼠单核巨噬细胞RAW264.7诱导分化破骨细胞的实验;从破骨细胞数量、状态及功能来看,α-MEM培养基更适合做为破骨细胞分化培养基。     

失重环境对大鼠肝细胞凋亡的影响

目的 探讨失重环境对大鼠肝细胞凋亡的影响.方法 成年雄性Wistar大鼠84只,按随机数字表法分为失重组和对照组,每组又分别设1～7 d共7个时相点,每时相点失重和同步对照各6只大鼠.采用尾悬吊法建立失重动物模型,应用TUNEL法原位检测失重大鼠肝组织中凋亡细胞,免疫组织化学PV-6001法检测大鼠肝组织p53表达.对结果进行方差分析.结果 各组悬尾大鼠肝组织中均可见阳性染色细胞,细胞核呈黄棕色颗粒,少数细胞质淡染;失重1～5 d组凋亡细胞指数明显高于对照组(F=77.608,P<0.05),失重6 d和7 d组细胞凋亡指数与对照组接近.p53阳性染色颗粒均位于细胞核内,呈颗粒状、弥漫性或混合型等多种表现,悬吊早期大鼠肝组织中p53表达阳性指数明显高于对照组(F=113.063,P<0.05),失重1 d组最为明显,悬吊后期和对照组标本中见少量阳性染色细胞.结论 肝细胞凋亡和p53表达变化与失重应激反应和失重耐受有密切关系.     

Smad4促进成骨分化的机制研究

目的 探讨Smad4基因促进成骨分化的作用机制。方法 采用条件性基因敲除技术Cre/loxp,制备骨细胞特异性敲除Smad4小鼠(Smad4otcko),小鼠胚胎骨骼透明染色分析胚胎期小鼠长骨生长状况;待小鼠成长至1月龄,X-ray检测突变小鼠与对照组小鼠的骨密度差异;静态骨组织形态学分析检测突变鼠及对照鼠的骨量变化、成骨细胞数量变化等差异;实时荧光定量PCR检测Smad4突变鼠股骨成骨细胞相关因子Runx2、ALP、OSX及OCN;破骨细胞TRAP染色分析Smad4突变鼠破骨细胞形态及数量变化;qPCR检测突变鼠股骨破骨吸收标志基因RANKL、OPG,并计算RANKL/OPG比率。结果 Smad4基因敲除小鼠在胚胎期未出现长骨生长异常。X线结果显示,1月龄时,与对照组小鼠相比,Smad4突变鼠的骨密度降低（P＜0.05）,静态骨组织形态学分析表明突变鼠松质骨减少,皮质骨变薄,骨小梁数量减少（P＜0.05）;Smad4突变鼠成骨细胞标志基因表达量显著降低,成骨细胞的数量明显减少（P＜0.05）;RANKL作为破骨吸收标志物表达上调、作为其拮抗剂的OPG表达量下调,RANKL/OPG比率增高（P＜0.05）。结论 Smad4基因通过促进成骨分化,降低破骨吸收从而来维持骨稳态。     

高压氧对小鼠胫骨骨折愈合过程中骨生物力学特性及胶原蛋白含量的影响

目的 探讨高压氧对小鼠胫骨骨折愈合过程中骨生物力学特性及胶原蛋白含量的影响.方法 采用简单随机抽样法将216 只昆明小鼠随机分为对照组（108 只）和高压氧组（108 只）,建立胫骨骨折模型.高压氧组予以高压氧治疗,对照组不予高压氧治疗.分别于骨折后1、2、3、4 周取材,取材前通过行为学评分和斜板实验评价两组小鼠的行为学特征,观察取材后胫骨的一般生物学特征,检测胫骨标本的抗扭力和抗折力,测定胫骨骨痂组织的胶原蛋白含量,同时采用改良Masson 染色对骨痂组织中的胶原蛋白进行组织学观察.结果 与对照组相比,高压氧组小鼠骨折后1、2 周行为学评分降低;2、3、4 周斜板实验坡度升高（P ＜0.05）.骨折后1周高压氧组小鼠右侧胫骨宽度（骨痂处）、胫骨抗折力均大于对照组,两组比较,差异有统计学意义（P ＜0.05）;骨折后2 周高压氧组小鼠右侧胫骨抗扭力高于对照组,骨折后1、2、3 周右侧与左侧胫骨抗扭力比值以及两侧胫骨抗折力比值均高于对照组,两组比较,差异有统计学意义（P ＜0.05）;高压氧组小鼠骨折后1、2、3、4 周骨痂胶原纤维含量高于对照组（P ＜0.05）,且高压氧组改良Masson染色出现较多的蓝色反应.结论 在小鼠胫骨骨折的愈合过程中,高压氧治疗提高了骨痂组织中胶原蛋白的含量,增强了骨的生物力学性能,对骨折愈合起到促进作用.     

DMP1 depletion decreases bone mineralization in vivo: an FTIR imaging analysis.

The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype. INTRODUCTION: Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls. MATERIALS AND METHODS: Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (N(total) = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by muCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined. RESULTS: The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on muCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and muCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values. CONCLUSIONS: The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca x P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.     

Activation of Wnt Signaling by Mechanical Loading Is Impaired in the Bone of Old Mice

Aging diminishes bone formation engendered by mechanical loads, but the mechanism for this impairment remains unclear. Because Wnt signaling is required for optimal loading‐induced bone formation, we hypothesized that aging impairs the load‐induced activation of Wnt signaling. We analyzed dynamic histomorphometry of 5‐month‐old, 12‐month‐old, and 22‐month‐old C57Bl/6JN mice subjected to multiple days of tibial compression and corroborated an age‐related decline in the periosteal loading response on day 5. Similarly, 1 day of loading increased periosteal and endocortical bone formation in young‐adult (5‐month‐old) mice, but old (22‐month‐old) mice were unresponsive. These findings corroborated mRNA expression of genes related to bone formation and the Wnt pathway in tibias after loading. Multiple bouts (3 to 5 days) of loading upregulated bone formation–related genes, e.g., Osx and Col1a1, but older mice were significantly less responsive. Expression of Wnt negative regulators, Sost and Dkk1, was suppressed with a single day of loading in all mice, but suppression was sustained only in young‐adult mice. Moreover, multiple days of loading repeatedly suppressed Sost and Dkk1 in young‐adult, but not in old tibias. The age‐dependent response to loading was further assessed by osteocyte staining for Sclerostin and LacZ in tibia of TOPGAL mice. After 1 day of loading, fewer osteocytes were Sclerostin‐positive and, corroboratively, more osteocytes were LacZ‐positive (Wnt active) in both 5‐month‐old and 12‐month‐old mice. However, although these changes were sustained after multiple days of loading in 5‐month‐old mice, they were not sustained in 12‐month‐old mice. Last, Wnt1 and Wnt7b were the most load‐responsive of the 19 Wnt ligands. However, 4 hours after a single bout of loading, although their expression was upregulated threefold to 10‐fold in young‐adult mice, it was not altered in old mice. In conclusion, the reduced bone formation response of aged mice to loading may be due to failure to sustain Wnt activity with repeated loading. © 2016 American Society for Bone and Mineral Research.     

The in vivo role of DMP-1 and serum phosphate on bone mineral composition

Human DMP1 mutations or Dmp1-null (KO) mice display hypophosphatemia rickets, suggesting a causative role of low phosphate (P) in development of osteomalacia. To address the direct contribution of P to the in vivo bone mineralization we analyzed the properties of femurs obtained from Dmp1 null mice and wild type (WT) mice under a normal or high phosphorous (HiP) diet using combined assays, including histological examination, micro computed tomography (μCT), X-ray absorption near edge structure (XANES) spectroscopy and Raman spectroscopy. Histology and XANES indicate that WT mice have phosphate coordinated with Ca in the form of hydroxyapatite and tricalcium phosphate, while the KO mice have poorly coordinated soluble phosphates in their structure in both the normal and HiP diets. Raman spectroscopy and XANES indicate a higher carbonate/phosphate ratio and a low mineral/matrix ratio in the osteoid clusters in the KO femurs, which was only partially improved by HiP diets. Thus, we conclude that the hypophosphatemia induced osteomalacia phenotype in Dmp1 KO mice is contributed by at least two factors: the low Pi level and the DMP1 local function in mineralization.     

Disruption of LRP6 in osteoblasts blunts the bone anabolic activity of PTH

Mutations in low‐density lipoprotein receptor‐related protein 6 (LRP6) are associated with human skeletal disorders. LRP6 is required for parathyroid hormone (PTH)‐stimulated signaling pathways in osteoblasts. We investigated whether LRP6 in osteoblasts directly regulates bone remodeling and mediates the bone anabolic effects of PTH by specifically deleting LRP6 in mature osteoblasts in mice (LRP6 KO). Three‐month‐old LRP6 KO mice had a significant reduction in bone mass in the femora secondary spongiosa relative to their wild‐type littermates, whereas marginal changes were found in femoral tissue of 1‐month‐old LRP6 KO mice. The remodeling area of the 3‐month‐old LRP6 KO mice showed a decreased bone formation rate as detected by Goldner's Trichrome staining and calcein double labeling. Bone histomorphometric and immumohistochemical analysis revealed a reduction in osteoblasts but little change in the numbers of osteoclasts and osteoprogenitors/osteoblast precursors in LRP6 KO mice compared with wild‐type littermates. In addition, the percentage of the apoptotic osteoblasts on the bone surface was higher in LRP6 KO mice compared with wild‐type littermates. Intermittent injection of PTH had no effect on bone mass or osteoblastic bone formation in either trabecular and cortical bone in LRP6 KO mice, whereas all were enhanced in wild‐type littermates. Additionally, the anti‐apoptotic effect of PTH on osteoblasts in LRP6 KO mice was less significant compared with wild‐type mice. Therefore, our findings demonstrate that LRP6 in osteoblasts is essential for osteoblastic differentiation during bone remodeling and the anabolic effects of PTH. © 2013 American Society for Bone and Mineral Research.     

Effects of knockout receptor activator of NF-κB (RANK) in podocytes on proteinuria in mice

Objective To investigate the role of RANK in injured podocyte and its effects on proteinuria. Methods PCR analysis was carried out to identify the genotypes of podocyte-specific RANK knockout mice. Six (6-8 weeks old) female RANK-/- mice were chosen as KO group. Six (6-8 weeks old) littermates and six (6-8 weeks old) C57/B6j mice were chosen as WT group and C57 group respectively. Each group was injected with LPS to create podocyte-injured proteinuria model. 24h urine was collected before and after injection. Urinary albumin, urine creatinine and UACR were detected by automatic biochemical analyzer. The mice were sacrificed after 48 h. Cortical kidney tissue samples were stained by PAS and collected for transmission electron microscopy to detect the foot process effacement. The number of podocytes was examined at different time points by immunohistochemistry .The expression of intergrin-β1, integrin-β3 and uPAR was detected by immunofluorescence and westernblot. Results (1) Comparing KO group, WT group and C57 group, it was found that there was no difference in weight, morphology, function of kidney and ACR. (2) After LPS injection, proteinuria and ACR were increased in three groups, but less significantly in KO group (23.70±9.90), compared to those in WT group (107.56±22.32) and C57 group (132.13±14.26) (P＜0.001). (3) In three groups, the number of podocytes was decreased after LPS injection and the decrease in the KO group was the slightest. (4) Compared to WT and C57 group, podocyte foot process effacement was less obvious in KO group after LPS injection in transmission electron microscopy. (5) Immunofluorescence results showed that after LPS injection, integrin-β1 was decreased in three groups, but most significantly in KO group. Integrin-β3 was increased in three groups, but less obvious in KO group. (6) Westernblot results showed consistency with immunofluorescence. The expression of uPAR protein was increased in three groups, with the increase in KO group being the slightest. Conclusions Podocyte-specific knockout of RANK reduces LPS-induced proteinuria, suggesting that RANK might be involved in the development of proteinuria in podocyt injury. Its mechanism is probably related with integrin-β1, integrin-β3 and uPAR.     

The role of Akt1 in terminal stages of endochondral bone formation: Angiogenesis and ossification

Longitudinal bone growth is the result of endochondral bone formation which takes place in the growth plate. The rate of chondrocyte proliferation and hypertrophy, vascular invasion with the formation of primary ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. We have shown a role for the PI3K signaling pathway in chondrocyte hypertrophy and bone growth in tibia explant cultures. In this current study, we aimed to investigate the role of Akt1, an important target of PI3K, in endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14), the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect bone quality in older animals.     

Increased mandibular condylar growth in mice with estrogen receptor beta deficiency

Temporomandibular joint (TMJ) disorders predominantly afflict women of childbearing age, suggesting a role for female hormones in the disease process. In long bones, estrogen acting via estrogen receptor beta (ERβ) inhibits axial skeletal growth in female mice. However, the role of ERβ in the mandibular condyle is largely unknown. We hypothesize that female ERβ‐deficient mice will have increased mandibular condylar growth compared to wild‐type (WT) female mice. This study examined female 7‐day‐old, 49‐day‐old, and 120‐day‐old WT and ERβ knockout (KO) mice. There was a significant increase in mandibular condylar cartilage thickness as a result of an increased number of cells, in the 49‐day‐old and 120‐day‐old female ERβ KO compared with WT controls. Analysis in 49‐day‐old female ERβ KO mice revealed a significant increase in collagen type X, parathyroid hormone–related protein (Pthrp), and osteoprotegerin gene expression and a significant decrease in receptor activator for nuclear factor κ B ligand (Rankl) and Indian hedgehog (Ihh) gene expression, compared with WT controls. Subchondral bone analysis revealed a significant increase in total condylar volume and a decrease in the number of osteoclasts in the 49‐day‐old ERβ KO compared with WT female mice. There was no difference in cell proliferation in condylar cartilage between the genotypes. However, there were differences in the expression of proteins that regulate the cell cycle; we found a decrease in the expression of Tieg1 and p57 in the mandibular condylar cartilage from ERβ KO mice compared with WT mice. Taken together, our results suggest that ERβ deficiency increases condylar growth in female mice by inhibiting the turnover of fibrocartilage. © 2013 American Society for Bone and Mineral Research.     

Deletion of estrogen receptor beta accelerates early stage of bone healing in a mouse osteotomy model

Summary

This study examined the role of estrogen receptor (ER) beta during mouse femoral fracture healing by employing ER knockout (KO) mice. The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification.

Introduction

This study was conducted to examine the role of ER beta during fracture healing.

Methods

Female ERbeta knockout (KO) mice (18 weeks old) and age-matched female wild-type (WT) mice underwent open osteotomy on the right femur. They were sacrificed at 1, 2, 4 and 6 weeks post-fracture. The sera and callus samples were subjected to the following analyses: micro-computed tomography (CT)-based angiography, micro-CT evaluation, histological examination, histomorphometry examination, real-time polymerase chain reaction (PCR) analysis, biochemical marker, and mechanical testing.

Results

Micro-CT-based angiography showed that the total vessel volume at the fracture site was larger in the KO group than the WT group at 1 and 2 weeks post-fracture. Micro-CT analysis revealed that the callus volume was significantly higher in the KO group from week 2 to week 4 post-fracture when compared with the WT group consistent with the histological data. Analysis of biochemical markers indicated that circulating P1NP levels in the KO mice were significantly higher than in the WT mice from week 2 to week 4 and that temporal expression of circulating C-terminal telopeptide of type I collagen (CTX) levels was also higher in the KO mice than in the WT mice. These results were consistent with quantitative real-time PCR analysis. The ultimate load, stiffness, and energy to failure were significantly higher in the KO mice than in the WT mice at week 4.

Conclusions

The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification, but not by the end of healing. Blockade of ERbeta can be considered as another therapeutic strategy for osteoporotic fracture and non-union fracture.     

PDZK1敲除对小鼠肝脏胆固醇代谢和胆囊结石形成影响的实验研究

目的探讨敲除PDZK1(Postsynaptic density-95,disks-large,ZO-1-domain K1,PDZK1)基因对小鼠肝脏胆固醇代谢调节和胆囊结石形成的影响。方法雄性成年PDZK1基因敲除小鼠(PDZK1 knockout,KO组)和野生型小鼠(wild type,WT组),每组各10只,以成石饲料分别喂养4周,观察胆囊成石情况,并收集肝脏和胆囊组织。采用蛋白印迹法测定肝脏PDZK1和清道夫受体B族1型(scavenger receptor B type 1,SRB1)表达。采用胆总管插管收集肝胆汁,测定胆汁分泌率和胆汁胆固醇含量。采用试剂盒酶法测定胆囊胆汁成分并计算胆汁胆固醇饱和指数(cholesterol saturation index,CSI)。以实时定量PCR检测肝脏脂质代谢相关基因的mRNA表达。结果成石饲料喂养4周后,WT组小鼠全部成石(10/10),KO组小鼠则为40%(4/10)成石。两组小鼠肝胆汁分泌率差异无统计学意义,但KO组小鼠肝胆汁胆固醇含量显著降低(P0.05),胆汁酸含量增加(P0.05),且CSI降低(P0.05)。KO组小鼠肝脏SRB1蛋白表达降低(P0.05),甾醇氧-酰基转移酶基因1/2mRNA表达降低(P0.05),而肝型脂肪酸结合蛋白1和胆汁酸转运相关蛋白(ATP结合盒b11)表达则显著增加(P0.05)。结论 PDZK1影响SRB1在小鼠肝脏中表达,降低对高密度脂蛋白胆固醇摄取,减少胆汁胆固醇分泌,继而降低胆囊结石形成。