Nrf2基因敲除抑制应力负荷下骨形成

目的培养核转录因子2(Nrf2)基因敲除小鼠原代成骨细胞,研究Nrf2基因在应力所致骨形成中的作用。方法选取同窝杂交出生的幼鼠分为Nrf2基因敲出组(KO)及野生组(WT),在颅骨中分离出初级2成骨细胞,在平板流动腔中进行60 min的震荡流体剪切应力实验(12dynes/cm,Fluid Shear Stress FSS),后提取RNA,进行实时定量PCR分析Nrf2 m RNA。结果震荡60 min使野生组Nrf2m RNA的表达水平增加,但基因敲出组没增加。NAD(P)H醌氧化还原酶1(NQO1)m RNA和Wnt5a m RNA在Nrf2基因敲出组成骨细胞中的表达水平较野生组显著减少,而RANK配体m RNA表达水平显著上调。FSS也刺激WT组成骨细胞中Wnt3a和Wnt5a m RNA的表达,但是在Nrf2 KO组没有作用。结论 Nrf2基因敲出抑制成骨细胞的增长和活性,提示Nrf2基因促进应力负荷下成骨细胞的生长     

烟碱型乙酰胆碱受体α7在Fmr1基因敲除小鼠中的表达及意义

目的观察烟碱型乙酰胆碱受体α7在Fmr1基因敲除小鼠前额皮层及海马的表达,探讨其在Fmr1基因敲除小鼠学习记忆中的作用。方法将4周龄FVB小鼠分为WT组和KO组,用跳台实验分别检测KO及WT小鼠学习记忆的能力,利用Western blotting和免疫组化方法检测KO及WT小鼠前额皮层及海马α7 nAChR的表达。结果跳台实验结果显示,与WT空白组比较,KO空白组第一天及第二天的潜伏期均显著缩短,错误次数明显增多,差异均具有统计学意义(P0.05);免疫组化结果显示,与WT空白组相比,KO空白组前额皮层和海马区α7 nAChR免疫阳性细胞数均显著减少(P0.05);Western Blotting结果显示,KO空白组前额皮层及海马区α7 nAChR表达量均较WT空白组减少,海马区更为显著,差异均具有统计学意义(P0.05)。结论 Fmr1基因敲除小鼠前额皮层、海马区的α7 nAChR表达量较野生型小鼠减少,提示α7 nAChR参与了Fmr1基因敲除小鼠学习记忆障碍的发病机制。     

微管相关蛋白1B在FMR1基因敲除小鼠睾丸间质细胞中的表达

目的对不同周龄的Fmr1基因敲除和野生型雄性小鼠睾丸组织微管相关蛋白1B的表达进行分析比较,探讨Fmr1基因敲除小鼠的睾丸间质细胞微管相关蛋白1B表达的差异。方法采用不同周龄(4、6、8、10周)的FMR1基因敲除型(KO)和野生型(WT)各6只,先采用聚合酶链式反应(PCR)技术对KO小鼠和WT小鼠进行基因型鉴定,之后所有小鼠麻醉取睾丸组织、石蜡包埋切片进行HE染色对比观察Fmr1小鼠睾丸组织的形态,最后用免疫组织化学染色技术对KO小鼠和WT小鼠睾丸微管相关蛋白1B的表达进行检测并作对比分析。结果微管相关蛋白1B在4～10周小鼠睾丸间质细胞阳性表达,8、10周为强阳性表达,且KO小鼠在睾丸的阳性表达均高于WT小鼠。结论微管相关蛋白1B在同周龄FMR1基因敲除小鼠睾丸间质细胞的表达均显著高于WT小鼠,提示微管相关蛋白1B可能参与脆性X综合征巨睾症的发病过程。     

Fmr1基因敲除小鼠生后不同发育阶段的附睾iNOS的变化

目的对不同周龄的Fmr1基因敲除和野生型雄性小鼠附睾组织诱导型一氧化氮合酶(iNOS)的表达进行分析比较,探讨Fmr1基因敲除小鼠的附睾组织iNOS表达的差异,为脆性综合征的研究提供背景资料。方法本研究采用不同周龄(4、6、8、10周)的Fmr1(fragile X mental retardation 1)基因敲除型(knockout,KO)和野生型(wild-type,WT)各6只,先采用聚合酶链式反应(PCR)技术对KO小鼠和WT小鼠进行基因型鉴定,之后所有小鼠麻醉取附睾组织、石蜡包埋切片进行免疫组织化学染色技术对KO小鼠和WT小鼠附睾iNOS的表达进行检测并作对比分析。结果 iNOS在4周小鼠附睾阳性表达,在6、8和10周小鼠呈强阳性表达,且KO小鼠附睾的阳性表达均弱于WT小鼠。结论 Fmr1基因敲除小鼠在缺失Fmr1蛋白(fragile X retardation-1 protein,FMRP)后的附睾iNOS的表达降低。     

去势大鼠5种骨转换生化指标的动态变化

目的： 对去势大鼠骨质疏松动物模型形成过程中5种骨转换指标的变化及其相关性进行研究。方法: 将3月龄SD雌性大鼠分为3组：切除卵巢组(OVX)，假手术组(sham)和对照组(control)，术前和术后分别于1、1.5、2、2.5、3和4月检测血清骨钙素(OC)、碱性磷酸酶(ALP)、 骨碱性磷酸酶(BALP)、抗酒石酸酸性磷酸酶(TRAP)和羟脯氨酸(HYP)水平，并作大鼠胫骨病理切片检查。结果: OVX组血清OC、ALP、BALP、TRAP和HYP水平均明显高于sham组，其变化顺序依次为：TRAP/HYP→OC→ALP/BALP；5种指标之间呈显著正相关；术后3月OVX组大鼠胫骨小梁结构有病理改变。结论: 去势大鼠属于高转换型骨质疏松；在模型形成过程中，骨吸收指标的变化早于骨形成指标的改变；骨转换指标是反映绝经后早期骨量丢失的灵敏指标。     

Fmr1小鼠海马α7 nAChR与痛觉敏感性的关系

目的观察4周龄Fmr1基因敲除小鼠海马烟碱型乙酰胆碱受体α7的表达变化及热刺激缩足反射潜伏期的变化。方法取4周龄FVB WT和KO小鼠各20只,应用PL-200热刺痛仪测定小鼠后肢热痛阈值,使用免疫印迹检测KO及WT小鼠海马α7 nAChR的表达变化。结果 4周龄KO鼠的痛阈均比WT鼠的痛阈增高,差异有统计学意义(P0.05)。免疫印迹结果显示,KO空白组海马区α7 nAChR表达量均较WT空白组减少,差异有统计学意义(P0.05)。结论 Fmr1基因敲除小鼠海马区的α7 nAChR表达量减少可能与Fmr1基因敲除小鼠的痛觉敏感性的改变相关,可能是FXS患者自残行为的发病基础。     

miR-23b通过靶基因Cxcl12调控肾性高血压

目的:研究微小RNA-23b(miR-23b)是否通过靶基因Cxcl12调控肾性高血压。方法:通过miR-23b基因敲除(miR-23b KO)小鼠,运用RT-qPCR、ELISA、HE染色、PAS染色、免疫组化、彩色多普勒血流成像(CDFI)、肾皮质磁共振成像(MRI)等方法,观察分析miR-23b对肾脏的影响,通过RNA测序(RNA-Seq)技术分析miR-23b靶基因的变化,萤光素酶报告基因实验测定靶基因。结果:从临床中发现肾病综合征患者miR-23b表达水平降低(P0.01),为了探讨miR-23b与肾病综合征的相关性,对miR-23b KO小鼠的研究发现,miR-23b基因敲除后,小鼠出现尿微量白蛋白显著升高(P0.05)。通过CDFI和肾皮质MRI测得miR-23b基因敲除后小鼠肾脏血管阻力指数显著升高(P0.01),肾皮质变薄(P0.05)。通过ELISA确定miR-23 KO小鼠血清中肾素和血管紧张素蛋白表达显著增高(P0.05)。对miR-23b KO小鼠肾脏组织的RNA-Seq发现,肾脏总体变化基因与TargetScan数据库中的miR-23b靶基因有交集,GO Term分析发现这些交集的基因主要富集于血流。RT-qPCR结果显示,与健康人群相比,肾病综合征患者Cxcl12基因表达水平显著升高(P0.01),同时miR-23b KO小鼠Cxcl12基因表达水平也较WT小鼠显著升高(P0.05)。体外萤光素酶报告基因实验表明,miR-23b过表达可以显著抑制Cxcl12萤光素酶活性(P0.01),从而确定miR-23b通过调控靶基因Cxcl12发挥其生物学功能。结论:miR-23b可通过靶基因Cxcl12参与肾性高血压的发生与发展。本研究发现miR-23b在肾性高血压中的重要作用,miR-23b可能成为肾性高血压的一种崭新、有效的治疗靶点。     

Fmr1小鼠的跳台实验和海马哺乳动物雷帕霉素靶蛋白的关系

目的观察Fmr1基因敲除(KO)小鼠跳台实验行为及哺乳动物雷帕霉素靶蛋白(mTOR)的变化,探讨mTOR与KO小鼠学习记忆能力的关系。方法利用PCR技术鉴定20只4周龄FVB近交系KO及20只野生型(WT)小鼠,随机分为行为学前WT组、行为学后WT组、行为学前KO组和行为学后KO组。通过避暗实验测试行为学WT及KO组小鼠的学习记忆能力,Western blot检测各组海马区p-mTOR及mTOR表达的变化。结果跳台实验第1天KO鼠的潜伏期较WT鼠短,错误次数较WT鼠多。跳台实验第2天KO鼠的潜伏期较WT鼠短,错误次数较WT鼠多。Western blot检测显示:KO鼠空白组mTOR表达较WT鼠空白组增高(P0.05);KO鼠空白组PmTOR与WT鼠空白组相比表达增高(P0.05)。结论 Fmrp对mTOR信号通路具有负性调节作用;Fmr1基因敲除小鼠学习记忆能力障碍与mTOR信号通路受损有关。     

Fmr1基因敲除小鼠耳蜗GAD的表达

目的对4周龄Fmr1基因敲除小鼠的耳蜗的GAD表达进行观察,探讨耳蜗GAD的表达是否受FMRP的影响。方法使用PCR技术对Fmr1基因敲除小鼠鉴定后对4周龄的Fmr1基因敲除小鼠和野生型小鼠各15只进行耳蜗的苏木精-伊红(HE)染色和GAD免疫组织化学的表达观察,数据采用多因素方差分析处理。结果耳蜗HE染色结果:4周龄组KO鼠较WT鼠形态学观察无差异。4周龄KO小鼠的耳蜗中GAD表达的平均阳性细胞数均高于WT小鼠,P<0.01,差异具有统计学意义。结论 GAD表达的改变可能与FMR1基因KO小鼠听源性惊厥发病有关。     

白细胞相关免疫球蛋白样受体-1在破骨细胞发育中的作用北大核心CSCD

目的研究白细胞相关免疫球蛋白样受体-1(LAIR-1)在破骨细胞(OC)发育过程中的表达规律及影响。方法 PCR检测小鼠LAIR-1(mLAIR-1)分子在体外诱导OC生成过程中mRNA水平的变化;重酒石酸盐抗性酸性磷酸酶(TRAP)染色观察OC的数量;Corning Osteo Assay surface检测OC骨吸收陷窝的形成;ELISA检测类风湿性关节炎(RA)患者血清可溶型LAIR-1(sLAIR-1)与疾病的炎症状态和活动度是否相关。结果 PCR结果显示在OC形成过程中,LAIR-1 mRNA显著降低;TRAP染色和骨吸收陷窝实验均显示加入mLAIR-1单克隆抗体后,OC数量明显减少。RA病人类风湿因子高低水平组之间的血清sLAIR-1水平存在显著差异。结论 LAIR-1分子可能参与了OC的发育过程,并且与RA疾病的炎症状态有一定关系。     

Inflammatory Bone Loss in Experimental Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Interleukin-1 Receptor Antagonist Knockout Mice

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.     

p-p38 MAPK在A2AR敲除新生小鼠缺氧缺血脑区的表达及意义

 目的：探讨p-p38 MAPK在腺苷A2A受体敲除(A2AR-/-)新生小鼠缺氧缺血脑区的表达及意义。方法：采用改良Rice法建立新生小鼠缺氧缺血性脑损伤（HIBD）模型。A2AR-/-(KO)及同期野生型(A2AR+/+,WT)C57/BL6新生小鼠（7日龄）分别按照完全随机分组方法分成假手术组及模型组,模型组按HIBD后取标本时间不同又分为造模后1 d组、3 d组和7 d组,共8个实验动物组,每组取小鼠8只,共64只。采用TUNEL结合尼氏染色检测神经细胞凋亡,采用免疫组织化学法检测活化caspase-3及p-p38 MAPK表达。同时,KO及同期WT小鼠分别取假手术组（SKO和SWT, n=10)及模型组(MKO和MWT,n=30)于HIBD后1 d同时进行新生鼠短期神经行为学评定。结果：（1）缺氧缺血后皮层及海马CA1区神经细胞凋亡、活化caspase-3及p-p38 MAPK表达均增加,造模后1 d为高峰;（2）A2AR敲除导致神经细胞凋亡及活化caspase-3表达增加,与同一时点的WT小鼠相比均有显著差异（P<0.01）;p-p38 MAPK表达增加,其中,1 d及3 d后2种基因型小鼠间均有显著差异（P<0.01）;（3）Peason相关分析显示,活化caspase-3表达与p-p38 MAPK表达呈显著正相关(皮层：r=0.957, P<0.01;海马CA1区：r= 0.939, P<0.01)。结论：p-p38 MAPK可能参与了A2AR敲除增加新生小鼠脑缺氧缺血后神经细胞凋亡、加重脑损害的过程。     

Loss of Smad3 gives rise to poor soft callus formation and accelerates early fracture healing

Smad3 is an intracellular signaling molecule in the transforming growth factor β (TGF-β) pathway that serves as a regulator of chondrogenesis and osteogenesis. To investigate the role of the TGF-β/Smad3 signaling in the process of fracture healing, an open fracture was introduced in mouse tibiae, and the histology of the healing process was compared between wild-type (WT) and Smad3-null (KO) mice. In KO mice, the bone union formed more rapidly with less formation of cartilage in the callus and eventually the fracture was repaired more rapidly than in WT mice. Alkaline phosphatase staining showed that osteoblastic differentiation in the fracture callus was promoted in KO mice. Additionally, TRAP staining and the TUNEL assay revealed that the induction of osteoclasts and apoptotic cells was significantly promoted in the healing callus of KO mice. Sox9 expression clearly decreased at both mRNA and protein levels in the early stage of fracture in KO mice. In contrast, the expression of genes for osteogenesis and osteoclast formation increased from day 5 until day 14 post-fracture in KO mice compared to WT mice. From these results, we concluded that the loss of TGF-β/Smad3 signaling promoted callus formation by promoting osteogenesis and suppressing chondrogenesis, which resulted in faster fracture healing.     

氯化锂对FMR1基因敲除小鼠的高架十字迷宫行为的干预作用

目的 探讨糖原合成酶激酶3（GSK3）抑制剂氯化锂对FMR1基因敲除（KO）小鼠的高架十字迷宫行为的干预作用及机制。方法 给90只30日龄FMR1基因敲除小鼠连续5d腹腔注射不同剂量的氯化锂，用药第6天进行高架十字迷宫行为学实验，通过录像机录像，然后用Smart软件分析录像，观察能否改善KO鼠的高架十字迷宫的表型；同时通过免疫印迹技术检测KO及野生型（WT）鼠的海马和皮层中GSK3β和磷酸化GSK3β（p-GSK3β）的变化。结果 在高架十字迷宫实验中，与WT组小鼠比较，KO组小鼠的运动性、兴奋性、探索性明显增强，且KO组小鼠在开放区域的活动时间、次数以及路程均明显高于WT组。KO鼠用氯化锂后，在开放区域的活动时间、次数以及路程均减低，差异具有统计学意义（P ＜0.05）。免疫印迹实验结果显示，KO鼠p GSK3β表达比WT鼠少；用氯化锂后，KO鼠p-GSK3β表达增加。WT鼠用氯化锂后，开放区域活动时间、次数以及路程有较少改善，p-GSK3β表达也有增加。未使用氯化锂的KO鼠与WT鼠相比，总GSK3β表达无明显差异，KO鼠和WT鼠使用氯化锂后，总GSK3β无明显改变，P ＞0.05。结论 GSK3β的抑制剂氯化锂能改善KO鼠的高架十字迷宫表型，对KO鼠有治疗作用，其机制可能与氯化锂导致的p-GSK3β表达增加有关。     

Vitamin C deficiency accelerates bone loss inducing an increase in PPAR-γ expression in SMP30 knockout mice

Senescence marker protein (SMP) 30 knockout (KO) mice display symptoms of scurvy, including spontaneous bone fractures, and this was considered to be induced by a failure of collagen synthesis owing to vitamin C deficiency. However, low bone mineral density is also known to be associated with spontaneous bone fracture. Therefore, we investigated the effects of vitamin C deficiency on the balance between osteoblasts and osteoclasts in SMP30 KO mice as evidenced by histopathology. All mice were fed a vitamin C‐free diet, and only one group (KV) mice were given water containing 1.5 g/l of vitamin C, whereas wild‐type (WT) and KO mice were given normal drinking tap water without vitamin C for 16 weeks. After 16 weeks, all femur samples were removed for histopathological examination. The femurs of KO mice showed significantly reduced bone area and decreased number of osteoblasts compared with those of WT mice and KV mice. KO mice also exhibited the lowest level of alkaline phosphatase (ALP) expression in their femurs. However, KO mice showed the most elevated expression of the receptor activator of nuclear factor kappa‐B ligand (RANKL). Moreover, KO mice had the strongest peroxisome proliferator‐activated receptor (PPAR)‐γ expression level in their osteoblasts and the highest number of TUNEL‐positive bone marrow cells. Therefore, we concluded that vitamin C deficiency plays an important role in spontaneous bone fracture by inhibiting osteoblast differentiation and promoting transition of osteoblasts to adipocytes, and this could in turn be related to the increased PPAR‐γ expression.     

Selective cognitive dysfunction in mice lacking histamine H1 and H2 receptors

Previous pharmacological experiments provide conflicting findings that describe both facilitatory and inhibitory effects of neuronal histamine on learning and memory. Here, we examined learning and memory and synaptic plasticity in mice with a null mutation of gene coding histamine H1 or H2 receptor in order to clarify the role of these receptors in learning and memory processes. Learning and memory were evaluated by several behavioral tasks including object recognition, Barnes maze and fear conditioning. These behavioral tasks are highly dependent on the function of prefrontal cortex, hippocampus or amygdala. Object recognition and Barnes maze performance were significantly impaired in both H1 receptor gene knockout (H1KO) and H2 receptor gene knockout (H2KO) mice when compared to the respective wild-type (WT) mice. Conversely, both H1KO and H2KO mice showed better auditory and contextual freezing acquisition than their respective WT mice. Furthermore, we also examined long-term potentiation (LTP) in the CA1 area of hippocampus in H1KO and H2KO mice and their respective WT mice. LTP in the CA1 area of hippocampus was significantly reduced in both H1KO and H2KO mice when compared with their respective WT mice. In conclusion, our results demonstrate that both H1 and H2 receptors are involved in learning and memory processes for which the frontal cortex, amygdala and hippocampus interact.     

应用破骨细胞-颅骨联合培养体系研究先天性成骨不全的骨再建过程

目的　采用先天性成骨不全(OI)小鼠,oim/oim为动物模型,应用破骨细胞-颅骨联合培养体系研究OB和OC两种细胞在OI骨再建过程中的功能改变和相互作用。 方法　实验采用小鼠颅骨（CAL）组织培养,实验设两组：WTCAL-WTOC组：联合培养对照组颅（WTCAL） 与对照破骨细胞（WTOC）;OICAL-OIOC组：联合培养OI颅骨（OICAL）与OI破骨细胞（OIOC）。以免疫组化染色方法　-TRAP识别破骨细胞,ALP免疫组化染色方法识别成骨细胞。破骨细胞骨吸收活性为骨吸收陷窝占颅骨表面百分比。单位OC吸收面积为总骨吸收陷窝除以破骨细胞数。 结果　于7d,OICAL-OIOC组破骨细胞数低于WTCAL-WTOC组;OICAL-OIOC组的OC/OB比例低WTCAL-WTOC组;OICAL-OIOC组单位破骨细胞吸收能力高于WTCAL-WTOC组。 结论　OI的小鼠模型骨再建中骨量丢失一方面由于其成骨细胞功能异常,另一方面也可能因为其破骨细胞的代偿性功能活跃。