Wistar大鼠成骨细胞粘弹性与相对增殖指数关系的实验研究

本文利用微管吸吮技术(Micropipetteaspirationtechnique),测量不同应变水平作用下Wistar大鼠成骨细胞的粘弹性.发现在细胞相对增殖指数最大时,细胞的粘弹性大于控制组,从细胞生物力学的角度证实细胞骨架参与细胞的增殖与分化过程.     

Wistar大鼠成骨细胞粘弹性与相对增殖指数关系的实验研究

本文利用微管吸吮技术（Micropipette aspiration technique），测量不同应变水平作用下Wistar大鼠成骨细胞的粘弹性。发现在细胞相对增殖指数最大时，细胞的粘弹性大于控制组，检细胞生物力学的角度证实细胞骨架参与细胞的增殖与分化过程。     

Comparison of gene expressions in the 2-cell embryos produced in vitro from blocking and non-blocking strain mice

目的:检测阻滞品系昆明小鼠和非阻滞品系B6C3F1小鼠体外发育2-细胞胚内基因表达水平的差异,探讨昆明小鼠植入前胚体外发育阻滞与哪些基因表达调控有关.方法:分别收集昆明小鼠和B6C3F1小鼠体外培养2-细胞胚,运用基因芯片分析和Real-time PCR验证.结果:基因芯片检测共筛出差异表达基因303个,其中昆明小鼠2-细胞胚表达上调基因有168个；下调基因有135个.昆明小鼠2细胞胚表达上调的基因以信号转导、细胞周期、细胞结构和运动、细胞增殖和分化以及发育进程等为主；而下调基因与电子转移、蛋白质代谢和修饰、氨基酸代谢以及蛋白质靶向运输和定位等相关.结论:胚内能量供应不足和蛋白质合成障碍可能是昆明小鼠植入前胚体外发育出现2-细胞阻滞的重要原因.     

大鼠、小鼠颌下腺内副交感神经节神经肽的免疫组织化学观察

Wistar大鼠 10只 ,昆明小鼠 4只 ,取颌下腺置于 0 .4%苯醌或 Bouin固定液固定 ,作冰冻切片或石蜡包埋。采用免疫组织化学 ABC方法 ,分别对大鼠、小鼠颌下腺副交感神经节细胞内 P物质 (SP)、血管活性肠肽(VIP)、神经肽 Y(NPY)、生长抑素 (SOM)、降钙素基因相关肽 (CGRP)等神经肽进行定性观察。结果显示 :在颌下腺小叶间结缔组织中 ,除可见有较大的血管、小叶间导管外 ,还可见有副交感神经节。神经节内神经元胞体较大 ,呈圆形、椭圆形或不规则形 ,可见有神经元胞体发出的短突起或细长突起 ,与邻近神经元似有联系。在大鼠标本中 ,神经…     

突触结合蛋白I和Ⅳ在小鼠背海马表达的区域差异及年龄相关性变化

目的:探讨昆明小鼠背海马突触前囊泡蛋白(Syt)Ⅰ和Ⅳ的分布差异及其年龄相关性变化.方法:选取3个年龄段昆明小鼠22月龄17只、11月龄22只和6月龄28只作为研究对象,采用免疫组织化学技术检测其背海马Syt Ⅰ和Syt Ⅳ含量.结果:SytⅠ与Syt Ⅳ在不同年龄组小鼠背海马锥体细胞和颗粒细胞中均有表达,其中SytⅠ在CA3区透明层和齿状回(DG)门区呈高表达,而Syt Ⅳ在锥体细胞和颗粒细胞胞膜呈高表达.22月龄鼠在海马CA1、 CA3和DG中Syt Ⅰ的相对含量高于11月龄和6月龄鼠,而Syt Ⅳ的相对含量低于6月龄鼠.结论:相对于中、青年鼠而言,老年昆明小鼠背海马Syt Ⅰ水平升高而Syt Ⅳ水平降低;Syt Ⅰ的优势表达可能在苔藓纤维及其与CA3区锥体细胞顶树突形成的轴-树或树-树型突触,Syt Ⅳ的优势表达可能在锥体细胞和颗粒细胞的轴-体或树-体型突触.     

携带 EGFPC3 质粒的重组减毒鼠伤寒沙门氏菌在小鼠体内的表达

目的 构建重组减毒鼠伤寒沙门氏菌X8786/pEGFP-C3,检测增强型绿色荧光蛋白C3(EGFP-C3)在小鼠体内荧光表达.方法 将质粒pEGFP-C3电转化到减毒鼠伤寒沙门菌SL8786,构建重组减毒鼠伤寒沙门菌X8786(pEGFP-C3),分别灌胃饲服昆明种小鼠,流式细胞仪检测脾脏细胞荧光表达,利用荧光显微镜检测小鼠组织荧光表达.结果 在体外稳定试验中,重组菌经LB固体及液体培养基连续传15代后,单个菌落和菌液均呈绿色;流式细胞仪结果证实,体外情况下,减毒鼠伤寒沙门菌可以将pEGFP-C3转入小鼠腹腔灌洗巨噬细胞,并在其中表达.在体内稳定试验中,经昆明种小鼠连续传代10次,从肝脏、脾脏中分离的细菌经LB固体培养仍为绿色,证明构建的重组减毒鼠伤寒沙门氏菌是稳定的.用免疫剂量的重组细菌接种昆明种小鼠后,观察1个月未见有异常现象,同时剖检后也未见脏器有眼观病变.免疫一定时间后,在小鼠肝脏、脾脏、肾脏、胸腺、十二指肠、肌肉等组织有荧光表达.结论 表达EGFP-C3的重组鼠伤寒沙门氏菌的成功构建为减毒沙门氏菌作为活载体的基因工程疫苗研制提供一个优良模型.     

Wistar大鼠成骨细胞粘弹性与相对增殖指数关系的实验研究

目的利用微管吸吮技术（Micropipetteaspirationtechnique）,测量不同应变水平作用下Wistar大鼠成骨细胞粘弹性,从细胞生物力学的角度证实细胞骨架参与细胞的增殖与分化过程。方法用自制的细胞四点弯曲单向交变等应变加载装置,对Wistar大鼠成骨细胞加载,基底应变加载水平为500＆＃61549;＆＃61541;、1000＆＃61549;＆＃61541;、1500＆＃61549＆＃61541;三档,加载时间仍为12h,24h,36h,加载频率为O．8HZ。实验组和对照组细胞取自同代,同源细胞,接种培养时间相向。当实验组细胞加载后,把实验组和对照组细胞同时采用胰蛋白酶…     

增殖细胞核抗原在大小鼠肠、肝和肾的表达

目的探讨增殖细胞核抗原(PCNA)在大小鼠肠、肝、肾的表达及其在大小鼠同一器官的表达差异。方法分别取体重为(111.6±16.0)gSD大鼠6只和(17.1±2.0)g昆明小鼠7只的肠、肝、肾,常规石蜡切片,苏木精伊红(HE)染色观察形态,SABC法检测PCNA的表达。结果PCNA在大小鼠肠绒毛固有层、肠腺细胞、肝细胞、肾小球和肾小管中有不同程度的表达,PCNA免疫反应阳性物质在肠、肝、肾的积分光密度在大小鼠同一器官的差异没有统计学意义(P>0.05)。结论正常情况下大小鼠肠、肝、肾均有一些细胞处于增殖状态,同一器官中PCNA的表达差异不明显。     

实验性应激对STZ昆明小鼠血糖水平影响的对照研究

目的 :了解慢性实验性应激对 STZ昆明小鼠血糖水平的影响和作用特点。方法 :取 STZ鼠 4 0只 ,正常昆明小鼠 2 0只 ,按血糖、性别配对后分为四组。实验性应激方法为限制、旋转和拥挤 ,为时六周 ,实验后每两周复查一次血糖。结果 :慢性应激可使雄性 STZ昆明小鼠血糖明显升高。药物、应激与性别单一因素对血糖的作用均不明显 ,药物与应激的交互作用对第 4、 6周血糖作用明显 ,药物、应激与性别三者的交互作用对第 6周血糖作用明显。结论 :慢性实验性应激刺激能使 STZ鼠血糖显著升高 ;雄鼠对应激的血糖反应性比雌鼠敏感。     

不同种鼠对实验脑型疟发生的影响研究

目的比较昆明小鼠和C57BL/6小鼠作为种鼠对实验脑型疟模型的影响。方法分别以伯氏疟原虫ANKA株感染C57BL/6小鼠和昆明小鼠作为传代用种鼠,当种鼠原虫率为5%～15%时接种子代C57BL/6小鼠,观察两组小鼠原虫率、脑型疟发生率以及死亡率。同时,通过脑组织切片和脑部淋巴细胞的流式检测,观察两组发生脑型疟小鼠的脑部微血管中感染疟原虫红细胞和CD8＋T细胞的粘附情况,另外,通过感染CD8＋TKO小鼠,证实CD8＋T细胞在两组小鼠发生脑型疟中的作用。结果用昆明小鼠作为种鼠的实验组的原虫率和脑型疟发生率均明显高于用C57BL/6小鼠作为种鼠的实验组,发生脑型疟小鼠的脑部组织切片发现,脑部微血管可见明显的感染疟原虫红细胞的粘附和CD8＋T淋巴细胞浸润;而用昆明小鼠作为种鼠感染CD8＋T细胞缺失的C57BL/6小鼠并不能诱导实验脑型疟的发生。结论与C57BL/6小鼠相比,昆明小鼠作为种鼠的实验组的脑型疟发病率更高,而且感染疟原虫红细胞和CD8＋T细胞在脑部微血管内的粘附也是该脑型疟发生的主要因素,因此,更适合用于实验脑型疟模型的建立及其机制的探讨。     

Collagen-phagocytosing ability of periodontal osteoblasts at the bone surface

The collagen-phagocytosing activity of osteoblasts at the alveolar bone-ligament interface of rat mandibular first molars was investigated both histologically and histochemically. Alveolar bones of male Wistar rats (6 months old) were used in this study. Collagen-containing phagosomes appeared in cuboidal osteoblasts aligned on the bone surface. The 5.7% of the osteoblasts exhibiting alkaline phosphatase activity revealed collagen-containing phagosomes, and the collagen fibrils within the phagosomes were at various stages of degradation. In addition, acid phosphatase activity and the immunocytochemical distribution of cathepsin B were found in these collagen-containing phagosomes at similar locations. The presence of both enzymes in the phagosomes suggests that an intracellular degradation of collagen occurs. Therefore, in addition to the osteoblastic functions of synthesizing and secreting bone matrices, osteoblasts are also capable of phagocytosis and the intracellular disintegration of collagen. Our findings suggest that osteoblasts at the alveolar bone-periodontal ligament interface have a collagen-phagocytosing ability and play an important role in the physiological remodeling and metabolic breakdown of collagen fibrils of periodontal ligament without osteoclastic bone remodeling.     

辣根过氧化物酶标记的纯视细胞层移植

目的　利用HRP标记Wistar鼠的视细胞 ,观察移植的纯视细胞在受体的分布。　方法　 30 %的HRP标记Wistar鼠视细胞 ,经外路途径移入皇家外科学院鼠 (RoyalCollegeofSurgeonRat,RCS)的视网膜下腔 ,术后 2周取RCS鼠眼做冰冻切片 ,组织化学染色法染色 ,普通光学显微镜下观察。　结果　在视细胞移植术后 2周 ,移植视细胞存活 ,HRP标记的Wistar鼠纯视细胞层在RCS鼠视网膜内呈褐色 ,排列平覆。　结论　在RCS鼠视网膜内可以观察到移植的HRP标记的Wistar鼠纯视细胞     

Increased proliferation of osteoblastic cells expressing the activating Gs alpha mutation in monostotic and polyostotic fibrous dysplasia.

We studied the osteoblastic abnormalities resulting from activating mutation of the Gs alpha gene in two patients with McCune-Albright syndrome and one patient with monostotic fibrous dysplasia. Histomorphometric analysis of dysplastic lesions showed a low number of differentiated osteoblasts along the bone surface and numerous immature alkaline phosphatase-positive mesenchymal cells actively depositing a woven bone matrix. Osteoblastic cells isolated from dysplastic bone lesions expressed a missense mutation in the Gs alpha gene in position 201 and showed increased intracellular basal cyclic adenosine 3',5'-monophosphate levels compared with normal cells isolated from a noninvolved area in the same patient. Cell proliferation evaluated by DNA synthesis was two-fold to threefold greater in osteoblastic cells expressing the mutation compared with normal cells from the same patient and was greater in cells isolated from more severe than less severe fibrotic lesions. In contrast, the synthesis of osteocalcin, a marker of mature osteoblasts, was lower in osteoblastic cells expressing the Gs alpha mutation compared with normal cells from the same patient and was lower in cells isolated from severe compared with less severe fibrotic lesions, indicating that the increased growth in mutated osteoblastic cells was associated with reduced cell differentiation. The results show that activating mutation of Gs alpha in osteoblastic cells leads to constitutive activation of adenylate cyclase, increased cell proliferation, and inappropriate cell differentiation, resulting in overproduction of a disorganized fibrotic bone matrix in polyostotic and monostotic fibrous dysplasia.     

新型改性聚乳酸与成骨细胞相容性的研究

探讨了成骨细胞与新型生物可降解材料乙二胺改性聚乳酸(EMPLA)的细胞相容性。在PLA、EMPLA及玻璃(对照组)上培养成骨细胞,采用细胞形态学观察法和细胞增殖法,在相差显微镜下观察细胞在上生长情况;分别于1、2、4、6d用MTT法记数,绘制生长曲线图。实验结果表明EMPLA组的成骨细胞比PLA组和对照组的形态好,增殖快,说明EMPLA比PLA表现出更好的细胞相容性,EMPLA在生物医学,特别是在组织工程领域存在着广泛的应用性。     

Ablation of Wntless in endosteal niches impairs lymphopoiesis rather than HSCs maintenance

Osteoblasts and perivascular stromal cells constitute essential niches for HSC self‐renewal and maintenance in the bone marrow. Wnt signaling is important to maintain HSC integrity. However, the paracrine role of Wnt proteins in osteoblasts‐supported HSC maintenance and differentiation remains unclear. Here, we investigated hematopoiesis in mice with Wntless (Wls) deficiency in osteoblasts or Nestin‐positive mesenchymal progenitor cells, which presumptively block Wnt secretion in osteoblasts. We detected defective B‐cell lymphopoiesis and abnormal T‐cell infiltration in the bone marrow of Wls mutant mice. Notably, no impact on HSC frequency and repopulation in the bone marrow was observed with the loss of osteoblastic Wls. Our findings revealed a supportive role of Wnts in osteoblasts‐regulated B‐cell lymphopoiesis. They also suggest a preferential niche role of osteoblastic Wnts for lymphoid cells rather than HSCs, providing new clues for the molecular nature of distinct niches occupied by different hematopoietic cells.     

Cl-通道活动对自发性高血压大鼠血管张力的影响

目的：观察自发性高血压大鼠(SHR)血管平滑肌细胞Ca2+激活Cl-通道［ICl（Ca）］的活动。 方法： 测定离体肠系膜血管床灌注压和离体尾动脉肌条血管张力, 以其对去甲肾上腺素(NE)收缩反应的变化作为舒缩活动的指标。 结果： （1）SHR肠系膜动脉和尾动脉对NE收缩反应显著大于Wistar大鼠。（2）硝呋咪酸可显著抑制NE诱发的肠系膜动脉和尾动脉收缩反应，并具有浓度依赖性，SHR肠系膜动脉和尾动脉收缩活动受硝呋咪酸的抑制程度明显小于Wistar大鼠。（3）SHR肠系膜动脉对低氯缓冲液的反应显著大于Wistar大鼠。 结论： SHR肠系膜动脉和尾动脉血管平滑肌的Ca2+激活Cl-通道的活动增强，并导致血管对NE的反应性增高。这可能是在高血压的发生过程中引起和维持较高血管张力和外周血流阻力的因素之一。     

Efficacy and cytotoxicity of cationic-agent-mediated nonviral gene transfer into osteoblasts

Ex vivo gene transfer into osteoblastic cells is an advantageous strategy for bone tissue engineering. This study investigated the efficacy and cytotoxicity of in vitro cationic-agent-mediated nonviral gene transfer into osteoblasts. Various cationic agents, lipid, gelatin, and polyethylenimine (PEI) were tested. Each was formulated in various concentrations to form a complex with plasmid DNA encoding red fluorescent protein. The cationic agent/DNA complexes were transfected into human fetal osteoblastic cell line and rat bone-marrow-derived primary osteoblasts, as well as NIH 3T3 fibroblast controls. Rat primary osteoblasts were transfected more with cationic lipid and PEI agents than with gelatin carrier, yielding transfection efficacy up to 18.1% and 12.7 %, respectively. In contrast, human fetal osteoblastic cell line was transfected more with cationic lipid and gelatin than with PEI. There was a positive correlation between the lipid and PEI doses and cytotoxicity. When the lipid and PEI were used to transfect the rat primary osteoblasts in a dose that yielded the highest transfection efficacy, cell survival rates decreased as low as 40%. When their transfection efficacies into primary osteoblasts were compromised at two thirds of the highest value, that is, 12.6% and 8.3% for the lipid and PEI, respectively, the cell survival rate was nearly 80%. Cationic gelatin was associated with cell survival rates over 60 % in any cell type, regardless of the doses tested. These results suggest that different types of osteoblastic cells may possess different ability to the uptake and expression of cationic-agent-bound DNA. There seemed to be agent-specific threshold doses that dropped the cell survival rate. Cationic-agent-mediated nonviral gene transfer into osteoblastic cells may be successful when the agent- and dose-dependent transfection efficacy and cytotoxicity are optimized.     

Overexpression of the transcriptional factor Runx2 in osteoblasts abolishes the anabolic effect of parathyroid hormone in vivo

There is convincing evidence that Runx2 could be a regulator of the anabolic action of parathyroid hormone (PTH) in bone. We therefore decided to determine how Runx2 overexpression in osteoblasts affects the anabolic response to PTH. Transgenic osteoporotic female mice overexpressing Runx2 (TG) and their wild-type littermates (WT) were treated with PTH (100 microg/kg/day, 7 days a week) or with the vehicle for 6 weeks. Unexpectedly, Runx2 overexpression blunted the increase in the mineral density and volume of bone induced by intermittent PTH in WT mice. Our findings also indicate that PTH failed to increase bone formation in TG mice overexpressing Runx2. This abolition of the effect of PTH by Runx2 overexpression was attributable to a decrease in the differentiation of osteoblastic cells both in vivo and in vitro. Finally, we showed that less cAMP was induced by PTH and that there were fewer PTH binding sites in TG than WT osteoblasts. In conclusion, our findings demonstrate that in vivo a high level of Runx2 abolishes the anabolic effect of PTH, probably via a decrease in the sensitivity of TG osteoblasts to PTH, and that the level of expression of Runx2 is critical if PTH is to produce its anabolic effect on bone in vivo.