缺氧缺糖/复氧复糖对大鼠皮质及海马神经元ClC-3氯离子通道表达的影响

目的:研究原代培养大鼠前额叶皮质和海马神经元缺氧缺糖/复氧复糖后电压门控氯离子通道3(voltage-gated chloride channel 3,ClC-3)在mRNA和蛋白水平的表达。方法:取原代培养8 d的大鼠皮质和海马神经元,随机分为对照组和模型组。模型组缺氧缺糖30 min后再复氧复糖,于复氧复糖后6、24、48、72 h采用反转录酶-聚合酶链锁反应(RT-PCR)、Western blot技术检测ClC-3 mRNA及蛋白水平的表达。结果:原代培养大鼠皮质和海马神经元ClC-3 mRNA及蛋白水平呈低水平表达。缺氧缺糖/复氧复糖24 h后培养皮质神经元ClC-3mRNA及蛋白水平开始上调,48 h仍然高表达,72 h后下降(P<0.05)。海马神经元ClC-3 mRNA在缺氧缺糖/复氧复糖6 h后即开始升高,高峰持续从24～48 h(P<0.05),72 h下降至略高于正常水平(P<0.05)。海马神经元ClC-3蛋白在24 h后表达开始逐渐升高,至72 h仍高表达(P<0.05)。结论:C1C-3通道表达上调可能增强复氧复糖后细胞对氧化应激、炎性反应的同时也促进细胞凋亡。     

骨形态计量学观察睾酮对雄性去势大鼠皮质骨的影响

目的通过骨形态计量方法观察雄激素替代疗法对去睾丸大鼠皮质骨代谢的影响。方法30只4月龄SD雄性大鼠,随机分成基础对照组(A组、实验开始时处死),年龄对照组(B组)、去睾丸组(C组)和去睾丸加睾丸酮组(D组),B组和C组生理盐水5ml·kg-1·d-1,D组甲基睾丸酮片1.8mg·kg-1·d-1,灌胃90d。实验结束,处死全部大鼠,取胫骨中段进行不脱钙骨制片,用计算机全自动图象分析系统进行骨组织形态计量学分析。结果去睾丸后皮质骨静态参数如截面总面积、髓腔面积等无明显变化,动态参数骨外膜骨形成降低(P<0.05),内膜骨形成和吸收均有增加趋势。睾酮则使去睾丸大鼠皮质骨的静态参数有增加趋势,促使骨外膜形成增加,减少内膜骨吸收(P<0.05),对内膜骨形成影响不大。结论睾酮补充治疗短期内能对抗去睾丸引起的大鼠皮质骨内外膜的代谢变化,维持正常的皮质骨结构。     

沉默ClC-3氯通道基因对HeLa细胞周期分布的影响

目的:探讨沉默HeLa细胞的ClC-3氯通道基因后细胞周期分布的变化及其作用机制。方法:依照siRNA设计原则构建沉默ClC-3基因的ClC-3 siRNA并转染HeLa细胞;实验分为空白对照组(control组)、转染试剂对照组(Lipo组)、阴性对照组(negative siRNA组)和ClC-3 siRNA组。采用real-time PCR检测ClC-3 siRNA的沉默效率;流式细胞术检测细胞周期分布情况;Western blot检测ClC-3蛋白及相关细胞周期蛋白(cyclin)D1、细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)4、CDK6、P21和P27等表达。结果:CIC-3 siRNA成功沉默HeLa细胞的ClC-3基因。和其它组相比,ClC-3 siRNA组的细胞周期被阻抑在G_0/G_1期。CIC-3 siRNA组的cyclin D1、CDK4和CDK6蛋白表达水平明显下降,P21和P27蛋白表达水平明显上升。结论:沉默HeLa细胞ClC-3氯通道基因可影响cyclin D1、CDK4、CDK6、P21和27蛋白的表达水平胆抑HeLa细胞周期停滞在G_0/G_1期。     

ClC-3氯通道过表达对小鼠甲状腺结构及功能的影响

目的:研究过表达Cl C-3基因对小鼠甲状腺结构及功能的影响。方法:以3月龄FVB小鼠为实验对象,研究野生型(WT)小鼠和Cl C-3转基因小鼠甲状腺结构及功能的差异。用q PCR、Westren blot和免疫荧光技术观察小鼠甲状腺Cl C-3的表达与分布;对小鼠日常活动进行行为学监测;ELISA检测小鼠血清总三碘甲状腺原氨酸(TT3)、总甲状腺素(TT4)和促甲状腺激素(TSH)的浓度。结果:与WT组小鼠相比:Cl C-3转基因组小鼠甲状腺Cl C-3表达明显增多(P0.05);甲状腺表现为明显的增生,滤泡明显增大;体重减轻但摄食量增加(P0.05),毛发无光泽,行为活跃,易激惹;TT3、TT4明显增高(P0.05),但TSH变化不明显。结论:Cl C-3过表达可导致甲状腺组织增生,甲状腺激素分泌增多。本研究提示Cl C-3很可能参与了甲状腺激素的合成。     

ClC-3在神经病理性痛大鼠脊髓背角及背根神经节的表达及其参与痛行为调控的研究

目的:观察电压门控性氯通道(voltage-gated chloride channel,ClC)3型在腓总神经结扎神经病理性痛模型大鼠脊髓背角(spinal dorsal horn,SDH)和背根神经节(dorsal root ganglion,DRG)内的表达变化及阻断氯离子通道后痛行为的改变。方法:应用免疫组织化学染色法、蛋白印迹法以及痛行为检测观察ClC-3在神经病理性痛大鼠SDH和DRG的变化和作用。结果:在正常大鼠,ClC-3主要位于DRG神经元胞膜;在SDH,ClC-3阳性纤维主要位于Ⅰ层。在腓总神经结扎大鼠,1周内结扎侧背角Ⅰ层及DRG的ClC-3表达增加,2～4周表达逐渐减少,在DRG也观察到相同的现象。给予氯离子通道阻断剂后,腓总神经结扎大鼠的痛阈下降。结论:ClC-3在神经病理性痛早期表达上调,随病程发展逐渐下降;阻断ClC-3可使大鼠痛阈下降。     

敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 目的: 探讨ClC-3氯通道是否为IK1钾通道的调节靶点,重点研究鼻咽癌细胞IK1钾通道对ClC-3氯通道功能及蛋白表达的影响。方法: 采用siRNA转染技术抑制低分化鼻咽癌上皮细胞(CNE-2Z) IK1 基因的表达;real-time PCR技术检测ClC-3 mRNA的表达;Western blot检测ClC-3的蛋白表达;细胞免疫荧光结合激光共聚焦显微镜技术检测ClC-3和IK1蛋白在细胞内分布;全细胞膜片钳记录细胞氯电流。结果: IK1 siRNA可以成功转染CNE-2Z细胞,有效抑制鼻咽癌细胞IK1钾离子通道的表达;用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后, ClC-3的mRNA表达上调而ClC-3蛋白却表达减少:在低分化鼻咽癌上皮细胞,低渗刺激可激活氯通道,产生一个较大的氯电流,在成功转染IK1 siRNA的细胞,此氯电流明显减弱。结论: 敲低IK1钾离子通道可抑制ClC-3氯离子通道的表达和功能。     

活性维生素D对卵巢切除大鼠骨转换与骨质量的影响

目的　研究１αＯＨＤ３ 对骨质疏松的预防治疗效果和作用机理。　方法　将４０ 只雌性Ｗｉｓｔａｒ大鼠分为４组,以切除卵巢大鼠为模型,分别用０．０４ μｇ／ｋｇ·ｄ 和０．５ μｇ／ｋｇ·ｄ １αＯＨＤ３ 灌胃给药１２周。测定各组大鼠的血、尿生化指标,骨密度,生物力学和形态计量学等参数,以及骨粘连蛋白基因的表达水平。　结果　切除卵巢大鼠骨吸收和骨形成显著加强,骨密度明显降低,骨小梁结构恶化,骨生物力学性质显著降低。１αＯＨＤ３ 使大鼠骨量恢复,骨密度提高,骨小梁相对体积（ＴＢＶ）增大,并使骨生物力学性质明显提高。０．５ μｇ／ｋｇ·ｄ １αＯＨＤ３ 对骨生物力学的效果优于０．０４ μｇ／ｋｇ·ｄ １αＯＨＤ３ 的给药量。不同浓度１αＯＨＤ３ 对骨形成与骨吸收的作用不同：０．５ μｇ／ｋｇ·ｄ１αＯＨＤ３ 明显抑制骨转换;０．０４ μｇ／ｋｇ·ｄ １αＯＨＤ３ 则促进骨转换。１αＯＨＤ３ 的浓度对骨的矿化速率无显著影响。大鼠去卵巢后,骨粘连蛋白（ｏｓｔｅｏｎｅｃｔｉｎ, ＯＮ）ｍ ＲＮＡ表达水平降低,０．５ μｇ／ｋｇ·ｄ １αＯＨＤ３ 处理可恢复并促进ＯＮｍ ＲＮＡ 表达。　结论　１αＯＨＤ３ 有明显增加骨密度和骨强度的作用。高浓度１αＯＨ     

大鼠DRG内ClC-3敲减导致的TNF-α/IL-10失衡对电压门控性钠通道表达和机械痛敏的影响

目的:研究敲减大鼠背根神经节(DRG)内ClC-3氯通道/反向转运体对神经元电压门控性钠通道表达和大鼠机械痛敏的影响及机制。方法:成年SD大鼠鞘内注射ClC-3 shRNA腺相关病毒(AAV-ClC-3 shRNA)以敲减DRG内ClC-3的表达,RT-qPCR、免疫荧光染色和Western blot从mRNA和蛋白水平检测ClC-3、细胞因子和电压门控性钠通道表达的变化,用up-down方法检测机械痛敏。结果:鞘内注射AAV-ClC-3 shRNA后DRG组织ClC-3的表达下调,大鼠出现机械触诱发痛。敲减DRG组织ClC-3的表达可致电压门控性钠通道Nav1. 3、Nav1. 7、Nav1. 8和Nav1. 9的表达增加。敲减DRG组织ClC-3的表达可提高肿瘤坏死因子α(TNF-α)水平,降低白细胞介素10(IL-10)水平。结论:大鼠DRG内ClC-3下调导致TNF-α/IL-10失衡,进而增加电压门控性钠通道Nav1. 3、Nav1. 7、Nav1. 8和Nav1. 9表达,增强神经组织兴奋性,最终导致机械痛敏。     

强骨康疏胶囊对去卵巢骨质疏松大鼠骨细微结构及骨代谢的影响

目的:观察强骨康疏胶囊对去卵巢骨质疏松大鼠的骨密度、OPG及RANKL蛋白表达、骨组织形态计量学参数及骨组织细微结构的影响。方法:制备去卵巢骨质疏松大鼠模型后,分组:正常对照组、模型空白组、中药低剂量预防组、中药高剂量预防组、雌激素预防组。给药1月后,检测各组股骨骨密度值,显微镜下观察股骨骨小梁的结构变化,并检测骨组织形态计量学参数。采用免疫组织化学染色法检测大鼠股骨OPG及RANKL蛋白表达。结果:模型空白组大鼠股骨骨密度减少,骨小梁厚度、面积、面积百分数均减少,骨小梁间距增大,股骨OPG蛋白平均光密度值显著降低,RANKL蛋白平均光密度值明显增高;雌激素预防组、中药低剂量组、中药高剂量组对上述指标均有明显改善。结论:强骨康疏胶囊能有效提高骨量,维持骨小梁立体空间结构,改善大鼠股骨远端松质骨的显微结构,能够提高骨OPG蛋白表达及抑制RANKL蛋白表达。     

骨折合并脑损伤对骨愈合和骨代谢的影响

背景：骨折周围神经损伤能够有效抑制破骨细胞活动,促进骨折早期愈合。目的：观察了大鼠肢体骨折合并脑损伤对骨密度、骨微结构、骨生物力学特征和骨代谢影响。 方法：63只大鼠随机分为假手术组、单纯骨折组和脑损伤合并骨折组。在术后3周、6周和3个月分批麻醉处死动物保存骨骼和血清标本,检测骨密度、骨微结构和生物力学性能以及血清Ⅰ型胶原氨基末端肽和骨钙素水平的变化。 结果与结论：与单纯骨折组相比,在造模3周和6周后,脑损伤合并骨折组胫骨近端的骨密度、松质骨微结构骨体积分数、骨小梁厚度、胫骨皮质骨截面总面积和骨髓腔面积、胫骨极限载荷和极限应力、血清原氨基末端肽和骨钙素水平均显著增高(P < 0.05),造模后3个月,3组间上述指标均差异无显著性意义。结果证实,脑损伤可增加骨折局部骨密度,改善骨微结构,提高生物力学性能,以此促进骨折局部的骨愈合和骨代谢。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

泼尼松对大鼠松质骨结构的影响

目的：探讨泼尼松对松质骨结构的影响。方法：用泼尼松４．５ｍｇ／ｋｇ,给３月龄ＳＤ雄性大鼠灌胃,每周２次,持续９０ｄ。扫描电镜观察大鼠腰椎松质骨结构的改变。结果：与正常组比较,泼尼松组的大鼠骨小梁变少、变细、断裂、连接不紧密,表现常见骨吸收形成的陷窝。结论：糖皮质激素可造成松质骨三维结构的损害,使力学强度降低,增加骨折的危险性。     

Effects of voluntary wheel running on goserelin acetate-induced bone degeneration

A common treatment option for many breast and prostate cancer patients is the use of a luteinizing hormone-releasing hormone agonist such as goserelin acetate (GA) which reduces sex hormone levels. This treatment, however, is associated with bone degeneration, and exercise has been suggested as a means of preventing this side effect. Little is known about the effects of low intensity, low volume exercise on GA-induced bone loss. The purpose of this study, therefore, was to investigate the effects of voluntary wheel running on bone architecture in growing male (M) and female (F) rats receiving GA treatment. Rats received an 8-week GA treatment or placebo (CON) and were either housed in cages equipped with voluntary running wheels (WR) or remained sedentary (SED) in standard cages throughout the experimental period. Following treatments, tibiae were excised and analyzed for cortical bone (cross-sectional volume, cortical volume, marrow volume, cortical thickness) and cancellous bone (bone volume/total volume, trabecular number, trabecular thickness, trabecular spacing) using micro-computed tomography. Treatment with GA resulted in a significant reduction in running wheel distances in both sexes throughout the study period (P < 0.05). GA treatment had no effect on cortical bone architecture in neither sex (P > 0.05). Cancellous bone degeneration, however, was observed in M and F SED + GA (P < 0.05). No significant differences were observed in M WR + GA animals in bone volume/total volume, trabecular number and trabecular spacing when compared to M SED + CON (P > 0.05). In F WR + GA, trabecular thickness did not differ from that of F SED + CON (P > 0.05), and trabecular spacing was found to be significantly lower than F SED + GA (P < 0.05). The current report indicates that 8 weeks of GA treatment promotes cancellous bone degeneration, and voluntary wheel running provides no clear osteoprotection in growing male and female rats.     

ClC-3氯通道蛋白磷酸化及其功能的研究进展

细胞容积调节广泛参与细胞的各种生理病理过程,如上皮细胞物质转运、物质代谢、细胞兴奋性、激素释放、细胞迁移、细胞增殖及细胞坏死凋亡等[1, 2].在低渗环境刺激下,容积激活性氯通道参与细胞容积调节,对维持细胞容积起着重要调节作用.     

Ultrasonic characterisation in determining elastic modulus of trabecular bone material

The pulse transmission ultrasonic technique is used to characterise the actual pathway and the wavelength dependence in relation to the bone specimen and microstructural dimensions. The average velocity through individual trabecular bone is 2901 m s⁻¹ (SD 161), and the mean velocity through cylindrical cancellous bone specimens is 2717 m s⁻¹ (SD 171). Thus, the velocity through the cylindrical cancellous bone specimens is underestimated by as much as 6.4% of that through individual trabeculae. There is statistically significant difference in the ultrasonic velocity between individual trabeculae and cylindrical cancellous bone specimens (p=0.0012).     

Skeletal alterations in hypophysectomized rats: I. A histomorphometric study on tibial cancellous bone

Background: Hypophysectomy (HX) results in a cessation of bone growth and a decrease in bone metabolism. The purpose of this study is to examine the effect of HX on the static and dynamic histomorphometry of cancellous bone in the secondary spongiosa of the proximal tibial metaphysis in rats. Methods: Female rats, at 2 or 3 months of age, were HX and sacrificed at 0, 5 days, 2 and 5 weeks after the surgery. Age-matched intact rats served as controls. Cancellous bone histomorphometry was performed on doublefluorescent labeled, 30-um-thick sections of the proximal tibia. Tartrateresistant acid phosphatase histomorphometry was performed at 5 days on HX and control rats to evaluate the resorption in the metaphyseal bone. Results: Although the intact rats gained in body weight, tibial length, tibial weight, and density after 5 weeks, these changes did not occur following HX. As compared to the basal group, HX resulted in a decrease in the density and dry weight of the metaphysis. The histomorphometric data showed that the cancellous bone volume and trabecular number of the secondary spongiosa were decreased and the separation was increased in the HX rats. The dynamic results showed that HX significantly decreased longitudinal growth rate and tissue-based bone formation and resorption. However, the bone surface-based eroded surface, labeled surface, the mineral apposition rate, and the bone formation rate did not differ between the intact and the HX rats at either the 2 or 5 weeks study. Five days after HX, the bone surface and tissue-based osteoclast surfaces were significantly lower in the HX than in the intact rats. Conclusions: Pituitary hormone deficiency results in cancellous bone loss. The bone loss is due primarily to the suppression of longitudinal growth-dependent bone gain and the inhibition of tissue-based bone turnover with a lower bone formation relative to bone resorption. The surfacebased bone turnover is not affected. © 1995 Wiley-Liss, Inc.     

Quantitative peripheral computed tomodensitometric study of cortical and trabecular bone mass in relation with menopause

Changes in total, cortical and trabecular bone mass were studied using quantitative peripheral computed tomodensitometry on the forearm of 58 normal eugonadal premenopausal women and 116 normal postmenopausal women to evaluate the evolution of bone components with age. In premenopausal women, no changes were seen in any bone component. In postmenopausal women, only trabecular bone mass diminished in the first 5 years after menopause (P < 0.05). It continued to decrease in the next 5 years (P < 0.05), but not later. Cortical bone mass experienced a significant loss 6–10 years after menopause (P < 0.001), and more than 15 years after menopause (P < 0.0005). These results are similar to those obtained with other techniques, and document the differing behavior of the cortical and trabecular bone components with years of menopause.     

人体松质骨矿质密度与弹性模量关系

目的 测量人体多部位松质骨矿质密度、轴向弹性模量,建立矿质密度与轴向弹性模量相关关系的本构方程,为国人有限元材料属性赋值提供依据。方法 采取10例成人新鲜尸体作为样本源,选取胫骨近端、大转子、股骨颈、肱骨头和椎体5个部位的松质骨,加工成直径约6 mm、长约30或40 mm的准试样。测量尺寸并计算体积,CT扫描试样骨矿质密度。对松质骨试样进行力学性能测试,分析不同部位松质骨弹性模量。对矿质密度与轴向弹性模量关系进行线性与幂次回归分析。结果 测试成功的试样来自5个部位,共169枚,其中胫骨近端52枚,大转子31枚,股骨颈15枚,肱骨头17枚,椎体54枚;5个部位松质骨矿质密度、轴向弹性模量均有所差异,线性相关性均较好（0.850>r2>0.785）,3个部位（胫骨近端、大转子、椎体）的幂次相关性较好（0.871>r2>0.825）,2个部位（肱骨头、股骨颈）的幂次相关性较弱(0.671>r2>0.643)。结论 各个部位骨矿质密度与轴向弹性模量的线性和幂次回归的相关性均较高,且同部位两种回归的r2值之间无明显差异;可应用于体外检测患者的骨骼质量,准确分辨骨质变化的部位,配合有限元建模能够预测骨折的风险。     

仙珍骨宝抗泼尼松致大鼠松质骨结构破坏的作用

目的观察仙珍骨宝对糖皮质激素引起松质骨结构破坏的防治作用。方法选用3月龄SD雄性大鼠24只,随机分为对照组、激素组和治疗组。激素组用泼尼松4.5mg/kg灌胃,每周2次。治疗组给予100%仙珍骨宝5ml/kg,每周6次,持续90d。用骨组织形态计量学方法测算胫骨近端骨小梁的静态指标,并在扫描电镜下观察大鼠腰椎松质骨结构的改变。结果与正常组比较,激素组的大鼠胫骨的骨小梁的面积减少72.43%,骨小梁数目减少74.09%。腰椎的骨小梁变少,变细,断裂,连接不紧密,表面常见骨吸收形成的陷窝。治疗组大鼠胫骨骨小梁的面积比激素组增加179.70%,骨小梁数目增多187.60%。腰椎的骨小梁较粗大,排列整齐,连接紧密。结论仙珍骨宝能有效防止糖皮质激素所引起的松质骨三维结构的损害,保持骨的正常力学强度,避免骨折。     

电压门控氯通道3（ClC-3）的生物学作用及其与疾病的关系

ClC-3属于电压门控氯通道家族,存在于细胞膜和细胞质,以及某些肿瘤细胞的细胞核内,高表达于中枢神经系统、肾脏和肠道。ClC-3参与离子转运、容积调节、免疫应答、细胞迁移、增殖、分化、凋亡等生理生物学活动,近年发现ClC-3与肿瘤、糖尿病等疾病的发生密切相关。