燃煤型氟中毒大鼠脑组织神经型尼古丁受体mRNA及蛋白表达水平改变

目的 观察燃煤型氟中毒大鼠学习记忆能力变化,测定大鼠脑组织神经型尼古丁受体(nAChR)mRNA和蛋白表达水平,探讨大鼠学习记忆能力改变的发生机制.方法 健康SD大鼠24只,体质量100～120 g,按体质量随机分为3组,每组8只.对照组饲以常规饲料,低氟组和高氟组以燃煤型氟中毒重病区燃煤烘烤的当地玉米为主要饲料(含氟量分别为11.30、104.20 mg/kg)来复制慢性氟中毒大鼠模型,染氟时间为6个月.染氟结束后,用Morris水迷宫方法检测大鼠行为学变化,处死动物取脑,用匀浆-氟离子选择电极法测定脑组织含氟量,实时荧光定量PCR法检测nAChR mRNA水平,蛋白印迹法测定nAChR蛋白表达水平.结果 低氟组和高氟组大鼠逃避潜伏期时间[(12.42±8.03)、(17.48±8.05)s]较对照组[(7.04±3.29)s]显著延长(P均<0.05),高氟组第7天穿过平台次数[(1.62±0.87)次]和逗留平台象限时间[(16.70±5.02)s]较对照组[(3.53±1.67)次、(23.33±5.35)s]降低(P均<0.05).低氟组和高氟组大鼠脑组织含氟量[(1.14±0.04)、(1.79±0.04)mg/kg]显著高于对照组[(0.52±0.05)mg/kg,P均<0.05],且高氟组大鼠脑组织含氟量高于低氟组(P<0.05).高氟组大鼠脑组织nAChR α3、α4、α7亚单位mRNA水平(1.51±0.20、1.45±0.06、1.63±0.08)较对照组(1.79±0.11、1.66±0.14、1.83±0.06)显著降低(P均<0.05),而低氟组(1.65±0.17、1.59±0.09、1.71±0.03)与对照组比较无明显改变(P均>0.05).低氟组和高氟组大鼠脑组织nAChR α3、α4、α7亚单位蛋白表达水平(0.58±0.13、0.16±0.03、1.41±0.38和0.56±0.23、0.08±0.02、0.51±0.16)较对照组(1.48±0.42、0.57±0.21、2.56±0.26)显著降低(P<0.05或<0.01).结论 燃煤型氟中毒大鼠学习记忆能力降低可能与脑组织nAChR蛋白表达及mRNA水平降低有关,nAChR表达改变可能是引起动物学习记忆能力降低的主要机制.

Abstract：

Objective To observe the learning and memory changes in coal-burning type of fluorosis rats, detect the expressions of neuronal nicotinic acetylcholine receptor(nAChR) at mRNA and protein levels in rat brains and to reveal the mechanism of changed learning and memory ability. Methods Twenty-four healthy SD rats, weighting 100 - 120 g, were randomly divided into three groups(8 in each). Control group was fed with normal diet, and low- and high-dose fluoride groups were fed with corn polluted with high fluoride (fluoride were 11.30,104.20 mg/kg, respectively) during drying processes with local burning-coal from the areas of endemic fluorosis to established rat model of chronic fluorosis. After exposed to fluoride for 6 months, behavioral changes were measured by Morris water maze. Animals were sacrificed, the brain was taken, after homogenizing the fluoride content of brain tissue was determined by fluoride ion selective electrode. The α3, α4 and α7 nAChR subunits at mRNA and protein levels were analyzed by real-time PCR and Western blotting, respectively. Results For rats in low- and high-fluoride groups, the escape latency time[(12.42 ± 8.03),(17.48 ± 8.05)s] was significantly longer than that in the control[(7.04 ± 3.29)s, all P< 0.05]. For rats in high-fluoride group, the numbers of crossing the platforms (1.62 ± 0.87) and the time of staying at the platforms[(16.70 ± 5.02)s] were significantly decreased as compared to that of control[3.53 ± 1.67, (23.33 ± 5.35)s, all P < 0.05]. The fluoride content in rat brain tissue in low- or high-fluoride groups [(1.14 ± 0.04), (1.79 ± 0.04)mg/kg] was significantly higher than that of control [ (0.52 ± 0.05) mg/kg, all P < 0.05]; in addition, the amount of fluoride in brain tissue of high-fluoride group was significantly higher than that of low-fluoride group(P < 0.05). In high-fluoride group, the mRNA expressions of α3, α4 and α7 nAChR subunits in rat brains(1.51 ± 0.20,1.45 ± 0.06,1.63 ± 0.08) were significantly lower as compared to controls (1.79 ± 0.11,1.66 ± 0.14,1.83 ± 0.06, all P< 0.05); whereas there were no significant changes in mRNA levels of these receptor subunits of the rat brains between low-fluoride group(1.65 ± 0.17,1.59 ± 0.09,1.71 ± 0.03) and controls (all P > 0.05). Furthermore, the protein levels of α3, α4 and α7 nAChR subunits in rat brains of highfluoride group(0.58 ± 0.13,0.16 ± 0.03,1.41 ± 0.38) and low-fluoride group(0.56 ± 0.23,0.08 ± 0.02,0.51 ± 0.16) were significantly lower than those of controls( 1.48 ± 0.42,0.57 ± 0.21,2.56 ± 0.26, P<0.05 or < 0.01). Conclusions Decreased ability of learning and memory in coal-burning type of fluorosis rats may be associated with declined expressions of nAChR at proteins and mRNA levels, which might be the main mechanism of the behavior change.     

慢性氟中毒性肝损伤及其发病机制

地方性氟中毒是一种在特定地理环境中发生的生物地球化学性疾病,除了引起氟斑牙和氟骨症外,还引起机体多个器官和组织发生病理损害[1-3].     

慢性氟中毒对大鼠脑组织Phospho-Elk-1表达的影响

目的 观察慢性氟中毒大鼠脑组织中细胞外调节蛋白激酶(ERK1/2)信号转导通路下游作用底物三元复合物因子Phospho-Elk-1的表达和分布,探讨慢性氟中毒所致学习记忆损害的发生机制.方法 SD 大鼠72只,体质量100～120 g,按体质量随机分为3组,每组24只,雌雄各半.对照组饮用自来水(含氟量<0.5 mg/L),低氟组和高氟组饮用加入氟化钠的自来水(P质量浓度分别为5.0、50.0 mg/L).6个月后,称取大鼠体质量,观察氟斑牙发生情况,用氟离子选择电极法检测大鼠尿氟及骨氟;用Morris水迷宫方法的定向航行实验检测大鼠学习能力,空间探索实验检测大鼠记忆能力;用免疫组织化学方法检测大鼠脑组织中Phospho-Elk-1在蛋白水平的表达和分布.结果 低氟组和高氟组大鼠体质量[(449.2±77.1)、(312.8 ±89.7)g]较对照组[(635.5±76.2)g]显著下降(P均<0.05),出现不同程度氟斑牙(x2=7.83,P<0.05),尿氟[(2.56±0.91)、(5.73±3.14)mg/L]及骨氟[(709.2±37.4)、(1306.3 ±102.4)mg/kg]较对照组[(0.92±0.30)mg/L、(348.5 ±89.2)mg/kg]明显升高(P均<0.05).低氟组和高氟组大鼠逃避潜伏期[(7.4±4.1)、(12.2±5.7)s]较对照组[(4.8±2.7)s]明显延长(P均<0.05),第1次穿越平台区时同[(4.18±1.10)、(5.89±0.56)s]较对照组[(1.17±0.75)s]显著延长(P均<0.05),均以高氟组尤为明显(P均<0.05).低氟组和高氟组大鼠海马CA1区(167.4±8.3、163.2±9.4)、CA2区(175.7±5.0、183.3±4.2)、CA3区(165.2±11.6、162.9±4.4)、CA4 区(168.7±6.9、169.5±5.3)、齿状回(185.2 ±4.0、193.1±6.1)及尾壳核(181.4±3.8、179.8±5.5)神经细胞Phospho-Elk-1表达水平较对照组(142.4±8.1、144.9±8.4、143.6±5.8、116.8±9.1、140.2±7.8、163.1±13.1)显著增加(P均<0.05).结论 慢性氟中毒可引起大鼠脑组织海马及尾壳核区域Pbospho-Elk-1表达水平升高,这种改变可能与大鼠学习记忆能力下降机制有一定关系.

Abstract：

Objective To investigate the expression and distribution of the downstream substrate of extracellular regulated protein kinase(ERK1/2) pathway, ternary complex factor phospho-Elk-1, in rat brains with chronic fluorosis, and reveal the mechanism of the impaired learning and memory ability caused by chronic fluorosis. Methods Seventy-two SD rats, weighing 100 - 120 g, were randomly divided into 3 groups, 24 in each group (half male and half female). The rats in control group were fed with tap water (fluoride < 0.5 mg/L); low- and high-dose fluoride groups were fed with tap water with different concentrations of NaF(5.0,50.0 mg/L F-, respectively). After 6 months, body weight was weighed, dental fluorosis was determined by observation and urinary fluoride and bone fluoride were detected by fluorine ion-selective electrode; the learning ability of rats was measured by navigation test of Morris water maze, and memory ability by spatial probe test in Morris water maze; the expression and distribution of phospho-Elk-1 in different brain regions were detected by immunohistochemistry method. Results In low- and high-fluoride groups, the body weight of rat[(449.2 ± 77.1), (312.8 ± 89.7)g] was significantly decreased than that of control [(635.5 ± 76.2 )g, all P< 0.05], the varying degrees of dental fluorosis were observed(x2 = 7.83, P<0.05), urinary fluoride[(2.56 ±0.91),(5.73 ±3.14)mg/L] and bone fluoride[(709.2 ± 37.4) ,(1306.3 ± 102.4) mg/kg] were significantly higher than those in controls[(0.92 ± 0.30)mg/L,(348.5 ± 89.2)mg/kg, all P< 0.05]. The escape latency of low- and high-fluoride groups[ (7.4 ± 4.1), (12.2 ± 5.7)s] was longer than that of control [(4.8 ± 2.7 )s, all P < 0.05] and the escape latency in high-fluoride group was significantly longer than that in other groups (all P < 0.05); in spatial probe test, the time of first crossing platform was longer in rats with fluorosis [(4.18 ± 1.10),(5.89 ± 0.56)s] as compared to control[(1.17 ± 0.75)s, all P< 0.05]. Expressions of phospho-Elk-1 in the hippocampus CA1(167.4 ± 8.3,163.2 ± 9.4), CA2(175.7 ± 5.0,183.3 ± 4.2), CA3(165.2 ± 11.6,162.9 ± 4.4), CA4(168.7± 6.9,169.5 ±5.3), fascia dentate (185.2 ±4.0,193.1 ±6.1) and caudate putamen( 181.4 ± 3.8, 179.8 ± 5.5) in low- and high-fluoride groups were higher than those of controls(142.4 ± 8.1,144.9 ± 8.4,143.6 ± 5.8, 116.8 ± 9.1,140.2 ± 7.8,163.1 ± 13.1, all P< 0.05). Conclusion Chronic fluorosis can cause increased expression of phospho-Elk-1 in the hippocampus and caudate putamen region of rat brains, which might be related to the mechanisms of decreased learning and memory ability of rats overexposed to fluoride.     

灯盏乙素对痴呆大鼠脑组织神经炎性反应的干预作用

目的:观察灯盏乙素对痴呆模型大鼠脑组织中几种炎性因子表达的影响,探讨其可能的抗炎治疗机制.方法:Wistar大鼠42只,随机分为5组,正常对照组、假手术组、学习记忆损伤模型组、灯盏乙素处理组和脑复康处理组.用双侧脑室注射β淀粉样蛋白(Aβ25-35)联合ip D-半乳糖方法建立痴呆大鼠模型;造模后次日分别用28 mg· kg-1灯盏乙素和365 mg·kg-1脑复康连续ig 20 d;实时定量聚合酶链法(RT-PCR)测定大鼠脑组织核因子(nuclear factor,NF)-κB p65表达的变化;免疫组织化学方法检测大鼠脑组织白介素-1β(IL-1β),白介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)的表达.结果:与正常组和假手术组相比,模型组大鼠脑组织中NF-κB p65 mRNA表达水平上升了27％和31％ (P ＜0.05),IL-1β,IL-6,TNF-α表达的阳性细胞分值正常组(1.3±0.5,1.6±0.5,1.6±0.69)和假手术组(1.5±0.5,1.6±0.7,2.0±0.7).模型组明显上升(2.9±0.7,3.2±0.8,3.0 ±0.82),P＜0.05.灯盏乙素处理后NF-κB p65的mRNA表达水平下调了20％ (P ＜0.05),IL-1β,IL-6和TNF-α表达的阳性细胞分值明显下降(2.1±0.67,2.3±0.70,2.2±0.53),P＜0.05.结论:灯盏乙素可阻断痴呆大鼠脑组织中NF-κB p65的活化,抑制炎性因子释放,改善学习记忆能力,通过减轻神经炎症损伤起到神经保护的作用.     

贵州少数民族人群乙肝流行率及流行模式分析

摘要:目的　了解贵州少数民族人群乙肝流行率及流行模式,为制定乙肝防治策略提供依据。方法　采用多阶段整群随机抽样方法,于2013年11-12月抽取贵州省3个少数民族自治州中的2个县8个村共1629名常住居民,进行问卷调查同时采集血样,用时间分辨免疫荧光法（TRFIA）检测血清中乙肝表面抗原（HBsAg）、乙肝表面抗体（HBsAb）、乙肝核心抗体（HBcAb）,分析乙肝流行模式。结果　1629名调查对象中,HBV 感染者825例,总感染率为 50.6%;共有 6种血清标志物模式组合,按照血清标志物模式分布特征将乙肝流行模式分为三类,其中以易感模式为主占45.9%、免疫模式占31.9%、感染模式占22.2%;不同民族、一起生活的人有无表面抗原阳性、不同婚姻状态、不同年龄、家庭HBV感染人数、是否接种乙肝疫苗、家庭人口数、是否饮酒、文化程度在3组流行模式间差异均有统计学意义（P＜0.05）;是否外出打工过、性别、是否共用牙刷、过去1年家庭总收入不同、是否吸烟在3组流行模式间差异均无统计学意义（P＞0.05）。结论　贵州少数民族人群乙肝流行模式以易感模式（3项全阴）为主,不同特征人群流行模式存在差异;易感模式流行的人群应加强免疫接种,提高该人群的乙肝特异性免疫力;对感染模式流行人群加强健康宣教,减少其乙肝的传播。     

慢性氟中毒脑损伤机制探讨

慢性氟中毒是一种严重危害人类健康的地球化学性疾病,长期摄入过多的氟除了引起氟骨症和氟斑牙外,还可造成其他系统的损害[1].氟能透过血脑屏障蓄积在脑组织,影响大脑神经细胞的形态和功能[2-3].近年来,人们较为关注的是地方性氟中毒病区患者识认功能改变和病区儿童智力降低的情况,有关慢性氟中毒脑损伤的机制较为复杂,主要有以下几个方面的认识.     

氟对中枢神经系统钙/钙调素依赖性蛋白激酶Ⅱ α亚单位mRNA和蛋白表达的影响

目的 观察氟对人神经母细胞瘤细胞(SH-SY5Y细胞)及发育期大鼠海马钙/钙调素依赖性蛋白激酶Ⅱα亚单位(α-CaMKⅡ)mRNA及蛋白表达水平的影响.方法 ①离体细胞实验:体外培养SH-SY5Y细胞,在培养液中加入NaF,使培养液中氟离子(F-)终浓度分别为0(对照)、0.05、0.50、2.00、5.00 mmol/L.培养48 h后,用qRT-PCR和Western blot技术检测细胞α-caMKⅡmRNA和蛋白水平.②动物实验:12只SD大鼠按雌∶雄=3∶1自然交配,待仔鼠出生后1 d,仔鼠及其哺乳母鼠-并按体质量随机分为3组.哺乳母鼠分别饮用含F-为0(对照)、2、3 mmol/L的蒸馏水,仔鼠通过乳汁摄入氟.每组有8只仔鼠在出生第14天处死,应用Western blot技术检测海马α-CaMKⅡ蛋白水平;另8只仔鼠自出生后21 d起,直接饮用与其哺乳母鼠相同染氟剂量的水溶液,至第28天处死.检测海马α-CaMKⅡ蛋白水平.结果 ①离体细胞实验:随着染氟剂量的增加,SH-SY5Y细胞中α-CaMKⅡ mRNA和蛋白水平依次降低.0、0.05、0.50、2.00、5.00 mmol/L组SH-SY5Y细胞中α-CaMKⅡ mRNA水平分别为1.00±0.00、0.77±0.18、0.40±0.11、0.22±0.06、0.15±0.03,蛋白水平分别为100.00±0.00、76.17±2.08、59.16±2.12、48.52±2.71、43.51±2.57,任意两组比较,差异有统计学意义(P<0.05或<0.01).②动物实验:2、3 mnlo/L组出生第14天和第28天仔鼠海马α-CaMKⅡ蛋白水平(75.02±2.88、73.83±3.88和81.00±2.54、45.70±2.34)低于对照组(100.00±0.00、100.00±0.00,P均<0.01),3 mmol/L组出生第28天仔鼠海马α-CaMKⅡ蛋白水平低于2 mmol/L组(P<0.01).结论 氟能降低神经细胞及海马中α-CaMKⅡ mRNA和蛋白表达水平,这可能是氟中毒引起学习记忆损害的机制之一.

Abstract：

Objective To investigate the effect of fluoride on the expression of a subunit of calcium/calmodulin-dependent protein kinase- Ⅱ (α-CaMK Ⅱ ) at both mRNA and protein levels in human neuroblastoma cells were cultured in DMEM with final concentrations of NaF 0(control) ,0.05,0.50,2.00,5.00 mmol/L, respectively, for 48 hours. Then quantitative RT-PCR and Western blot were performed to detect the expression level of α-CaMK Ⅱ P1 (postnatal day 1) pups together with their mothers were randomly divided into three groups. Lactating rats were given drinking water containing NaF at concentrations 0(control) ,2,3 mmol/L. And pups were exposed to NaF through milk. In each group, 8 pups were sacrificed on day 14 after birth. In post-weaning period, another 8 pups in each group were given drinking water with the same dose of fluoride as their mother's 21 day after birth. After then, these pups were killed on day 28, and hippocampus was dissected immediately and Western blot was conducted mRNA and protein levels were decreased. When NaF concentrations were 0,0.05,0.50,2.00,5.00 mmol/L, the mRNA relative ratios of α-CaMKⅡ in SY5Y cells were 1.00 ± 0.00,0.77 ± 0.18,0.40 ± 0.11,0.22 ± 0.06 and 0.15 ± 0.03, and protein levels of α-CaMK Ⅱ were 100.00 ± 0.00,76.17 ± 2.08,59.16 ± 2.12,48.52 ± 2.71 and 43.51 ± 2.57, any mmol/L group, hippocampus α-CaMK Ⅱ protein levels on day 14 and 28(75.02 ± 2.88,73.83 ± 3.88 and 81.00 ± 2.54,45.70 ± 2.34) were significantly lower than that of control groups(100.00 ± 0.00,100.00 ± 0.00, all P < 0.01). In 3 mmol/L group, hippocampus α-CaMKⅡ protein level on day 28 was lower than that of 2 mmol/L group (P < 0.01). Conclusion Fluoride can decrease mRNA and protein levels of α-CaMK Ⅱ in nerve cells and hippocampus, which may be one of the mechanisms of learning and memory impairment by fluorosis.     

应力环境下破骨细胞碳酐酸酶Ⅱ的基因表达

目的 研究流体切应力条件下大鼠破骨细胞碳酐酸酶Ⅱ（CaⅡ）mRNA的表达变化,探讨应力环境下大鼠破骨细胞的功能活动情况。方法 采用1,25-（OH） 2D3/地塞米松诱导SD大鼠骨髓细胞,抗酒石酸酸性磷酸酶染色和扫描电镜鉴定破骨细胞,0.25%胰蛋白酶/0.02%乙二胺四乙酸（EDTA）进行细胞纯化。对破骨细胞施加不同力值和作用时间的流体切应力,采用实时荧光定量巢式逆转录聚合酶链反应（RT-PCR）检测CaⅡ mRNA的表达。结果 随着力值的增加和作用时间的延长,破骨细胞CaⅡ mRNA的表达量分别呈下降趋势（P＜0.05）。结论 在一定范围的流体切应力作用下,破骨细胞CaⅡ mRNA的表达受到抑制,表达水平与力值大小和加载时间分别存在负相关关系。     

适用于天麻AFLP分析用的DNA提取及纯化方法

扩增片段多态性(amplified fragment length polymorphisms,AFLP)是上世纪90年代发展起来的一种检测DNA多态性的新方法,其基本原理是选择性扩增基因组DNA的酶切片段,由于不同材料的DNA酶切片段存在差异,因而便产生了扩增产物的多态性.     

天麻ITS序列的聚合酶链反应扩增

目的:为天麻核糖体DNA中的内转录间隔区(internal transcribed spacer ,ITS)序列分析提供可靠、特异的聚合酶链反应(PCR)条件.方法:选择ITS序列通用引物对天麻ITS序列进行PCR扩增,对一系列PCR条件参数进行摸索,同时对PCR产物进行纯化后直接测序.结果:天麻ITS序列PCR扩增产物约为750 bp,不同来源样本存在有一定的差异.经测序后与Genebank中相关序列比对,天麻ITS序列总长度为598 bp,其中ITS1为235 bp,ITS2为209 bp,5.8S为154 bp,G C(%)为52%.结论:扩增天麻ITS序列的PCR反应条件可以得到可靠、特异的结果.