慢性氟中毒大鼠脑组织中Nrf2和HO1蛋白表达

目的：观察慢性氟中毒时大鼠脑组织中核因子E2相关因子2（nuclear factor-E2-related factor 2,Nrf2）和血红素氧合酶1（heme oxygenase-1,HO1）蛋白水平表达改变,探讨氟中毒性脑损伤机制.方法：SD大鼠90只,随机分为正常对照组、染氟组及维生素E（VitE）拮抗组,实验期10个月;观察氟斑牙情况,用氟离子选择电极法测定大鼠尿氟及骨氟含量,用免疫组织化学方法检测脑组织中Nrf2和HO1蛋白表达.结果：染氟组和VitE拮抗组大鼠出现不同程度的氟斑牙,染氟组重于VitE拮抗组,差异有统计学意义（P＜0.05）;染氟组与VitE拮抗组大鼠尿氟和骨氟含量均高于对照组,染氟组高于VitE拮抗组,差异有统计学意义（P＜0.05）;免疫组化显示染氟组和VitE拮抗组大鼠脑组织中Nrf2及HO1阳性表达较对照组升高,VitE拮抗组升高程度不如染氟组,差异都有统计学意义（P＜0.05）.结论：慢性氟中毒可导致脑组织中Nrf2及HO1表达水平升高,增强细胞抗氧化能力,可能与慢性氟中毒脑损伤的机制有关.     

燃煤型氟中毒对大鼠脑组织NO含量及NOS mRNA和蛋白表达的影响

目的观察燃煤型氟中毒对大鼠脑组织NO含量及NOS mRNA和蛋白表达的影响。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,低氟组和高氟组以氟病区燃煤烘烤的玉米为主要饲料(含氟量分别为11.30、104.20 mg/kg),来复制氟中毒大鼠模型,染氟时间为3个月。用氟离子选择电极法检测动物尿氟、骨氟、脑氟含量,观察大鼠海马CA1区神经元病理变化,用Biomias 2000图像分析系统测试大鼠海马CA1区神经元胞体平均面积、周长及平均灰度值,比色法测定脑组织NOS活性,硝酸还原酶法测定脑组织NO含量,蛋白印迹方法测定NOS蛋白水平;实时荧光定量PCR方法测定NOS mRNA水平。结果低、高氟组大鼠尿氟为(2.61±0.11)(4.39±0.13) mg/L,骨氟(2 734±137)(4 323±203) mg/kg,脑氟(1.00±0.08)(1.14±0.02) mg/kg;与对照组尿氟(1.59±0.10) mg/L、骨氟(1 399±152) mg/kg、脑氟(0.41±0.06) mg/kg比较,差异有统计学意义(P0.05或P0.01);尼氏染色显示,与对照组大鼠神经元平均灰度值(119.0±9.7)比较,染氟组大鼠均明显增加[低氟(153.0±8.9),高氟(159.4±2.9)];低氟组、高氟组大鼠脑组织总NOS活性[(8.57±2.40)、(11.49±3.10) kU/g]较对照组(16.80±3.20) kU/g显著性下降(P0.05),高氟组大鼠脑组织NO含量(2.05±0.25)μmol/L较对照组(4.68±1.78)μmol/L显著性降低(P0.05)。低氟组、高氟组大鼠脑组织NOS蛋白表达水平[(0.53±0.24),(0.82±0.28)]较对照组(0.17±0.02)显著增加(P0.05/0.01),低氟组、高氟组大鼠脑组织NOS mRNA水平[(1.37±0.07),(1.38±0.08)]均较对照组(1.53±0.17)显著下降(P0.05)。结论低剂量氟具有一定神经毒性,表现为食用燃煤型氟中毒病区燃煤烘烤的粮食低剂量、早期可导致动物脑氟含量增高,使大鼠海马CA1区神经元蛋白合成功能下降,脑组织NOS活性、NO含量、NOS蛋白及mRNA表达改变。     

贵州省地方性氟中毒地区人群GSTP1基因Ile105Val位点多态性分析

Objective To investigate plasma glutathione S-transferase(GSTs) activity and GSTP1 gene Ile105Val polymorphism in Bijie City, Guizhou Province, a coal-burning fluorosis endemic area. Methods One hundred and sixty villagers from Yachi Twon using non-improved cooking stoves were selected as the non-intervened group in Bijie City, Guizhou Province where coal-burning fluorosis was prevailing; 153 villagers as the intervented group were chosen from Changchun Twon, where cooking stoves were improved; 151 villagers were served as the control group from Baiyunshan Twon, Changshun County without endemic fluorosis. The activity of GSTs was tested by colorimetric analysis with spectrophotometer. The genotype of the GSTP1 gene Ile105Val polymorphism, presenting as either homozygous wild-type (AA), or heterozygous mutation type (AG), or homozygous mutation type (GG), was detected through the PCR-RFLP procedure. Results The activity of GSTs in plasma of non-intervened group [(12.44±4.97) kU/L]was significantly lower than that of intervened group (P < 0.05), and that of intervened group[(20.78±6.20)kU/L]was significantly lower than that of control group[(24.30±6.27)kU/L, P< 0.05]. The difference of the enzyme activity of three groups were statistically significant (F = 51.71, P < 0.05), but this enzyme activity did not vary significantly in each sex of each grnup(P > 0.05). Compared intervened group [AA:67.3%(103/153), AG:29.4%(45/153),GG:3.3%(5/153)]and non-intervened group[AA:66.9%(107/160), AG:30%(48/160), GG:3.1%(5/160)]with control group[AA:74.8%(113/151), AG:25.2%(38/151), GG:0 (0/151)], the Ile105Val polymorphism site of GSTP1 gene had significant difference(χ2= 6.04,6.07, both P< 0.05), but not significant between intervened and non-intervened groups(χ2 = 0.02, P>0.05). Conclusions Fluorosis can decrease the activity of GSTs and introduce the GSTP1 gene Ile105Val polymorphism, intervention with the fluorine intake will improve the effect of fluoride on the body.     

贵州省地方性氟中毒地区人群GSTP1基因Ile105Val位点多态性分析

Objective To investigate plasma glutathione S-transferase(GSTs) activity and GSTP1 gene Ile105Val polymorphism in Bijie City, Guizhou Province, a coal-burning fluorosis endemic area. Methods One hundred and sixty villagers from Yachi Twon using non-improved cooking stoves were selected as the non-intervened group in Bijie City, Guizhou Province where coal-burning fluorosis was prevailing; 153 villagers as the intervented group were chosen from Changchun Twon, where cooking stoves were improved; 151 villagers were served as the control group from Baiyunshan Twon, Changshun County without endemic fluorosis. The activity of GSTs was tested by colorimetric analysis with spectrophotometer. The genotype of the GSTP1 gene Ile105Val polymorphism, presenting as either homozygous wild-type (AA), or heterozygous mutation type (AG), or homozygous mutation type (GG), was detected through the PCR-RFLP procedure. Results The activity of GSTs in plasma of non-intervened group [(12.44±4.97) kU/L]was significantly lower than that of intervened group (P < 0.05), and that of intervened group[(20.78±6.20)kU/L]was significantly lower than that of control group[(24.30±6.27)kU/L, P< 0.05]. The difference of the enzyme activity of three groups were statistically significant (F = 51.71, P < 0.05), but this enzyme activity did not vary significantly in each sex of each grnup(P > 0.05). Compared intervened group [AA:67.3%(103/153), AG:29.4%(45/153),GG:3.3%(5/153)]and non-intervened group[AA:66.9%(107/160), AG:30%(48/160), GG:3.1%(5/160)]with control group[AA:74.8%(113/151), AG:25.2%(38/151), GG:0 (0/151)], the Ile105Val polymorphism site of GSTP1 gene had significant difference(χ2= 6.04,6.07, both P< 0.05), but not significant between intervened and non-intervened groups(χ2 = 0.02, P>0.05). Conclusions Fluorosis can decrease the activity of GSTs and introduce the GSTP1 gene Ile105Val polymorphism, intervention with the fluorine intake will improve the effect of fluoride on the body.     

贵州省3个少数民族 HLA-G基因14 bp插入/缺失的多态性研究

目的：了解贵州省3个少数民族（布依族、水族、苗族）群体人类白细胞抗原-G（ HLA-G）基因14 bp插入/缺失多态性的分布规律及其与遗传背景之间的关系。方法：采用PCR扩增、电泳及测序的方法对来自贵州地区的布依族、水族、苗族共344例健康个体DNA进行HLA-G基因14 bp插入/缺失多态性检测,并与文献报道的仡佬族、壮族等13个民族群体的HLA-G基因14 bp插入/缺失多态性分布数据进行对比。结果：布依族、水族和苗族人群的+14 bp 等位基因频率分别为23.7%、26.0%和38.1%,+14 bp/+14 bp 基因型频率分别为9.40%、10.4%和15.9%,14 bp插入频率比较示布依族和苗族差异有统计学意义（χ2=11.345,P=0.001）,苗族与水族差异有统计学意义（χ2=6.125,P=0.009）而布依族和水族间比较差异无统计学意义（ P>0.017）;与其他人群数据比较,苗族群体的14 bp插入/缺失分布与其他人群相似,而作为壮侗语族的布依族、水族则有自己独特的14 bp插入/缺失分布规律。结论：布依族、水族人群的14 bp插入/缺失分布相似,但是与苗族人群的分布存在一定的差异。     

贵州省江口县土家族葡萄糖-6-磷酸脱氢酶基因突变分析

目的 了解贵州省江口县土家族葡萄糖-6-磷酸脱氢酶（G6PD）缺陷症的基因频率、基因突变类型特点及分布特征，从分子水平揭示G6PD基因多态性。方法 采集世居当地土家族男性血液样本227例，用四氮唑蓝（NBT）纸片定性法及G6PD／6PGD比值法做G6PD缺陷症筛查。确诊者用错配引物介导的聚合酶链反应／限制性内切酶酶切分析法（PCR-RE）进行中国人常见9种G6PD基因突变型分析，突变特异性扩增系统法（ARMS）验证其中3种突变。结果227名土家族男性中检出17例G6PD缺陷者，总检出率7．49％，G6PD缺陷症基因频率0．0749。基因分析检出cDNA1388（G→A）12例，在G6PD缺陷者中的基因频率为0．706，未知突变5例，基因频率为0．294。结论 G6PD缺陷症在贵州土家族人群有着较高的基因频率，其主要突变类型为G6PD基因cDNA1388（G→A）。     

神经型尼古丁乙酰胆碱受体在阿尔茨海默病中的神经保护作用

老年性痴呆主要分为三种:一种叫阿尔茨海默病(AD),其最为常见,大约占68%;第二种叫血管性痴呆(VD),是一种由于血供不足引起的不同大脑区域损伤所致的渐进性认知障碍;第三种是以上两种病症并存,叫混合性痴呆。AD是一种主要在老年期发生的以进行性痴呆为主要特征的神经元退行性变     

SH-SY5Y神经细胞中α7神经型尼古丁受体基因过表达对CaMK Ⅱ和CREB的影响

目的研究SH-SY5Y神经细胞中α7 nAChR基因过表达对CaMKⅡ和CREB的影响。方法复苏稳定转染α7 nAChR-pc DNA3.1质粒及空载质粒的SH-SY5Y神经细胞后,用含G418的培养液进行筛选培养;应用实时荧光定量PCR法和蛋白印迹法(Western blot)检测α7 nAChR基因过表达组、空载质粒组和正常对照组细胞中CaMKⅡ、CREB mRNA及蛋白表达水平的变化。结果与对照组相比,α7 nAChR基因过表达细胞组的CaMKⅡ、CREB mRNA表达水平分别增加了116.8%和114.7%(P0.01);及CaMKⅡ、CREB蛋白表达水平分别增加了8.7%(P0.05)和41.4%(P0.05)。结论α7 nAChR的神经保护作用可能与上调细胞CaMKⅡ、CREB水平有关。     

α7神经型尼古丁受体激动剂PNU282987对APP/PS1小鼠海马组织中DYN-Ⅰ蛋白表达的影响

目的研究特异性激动APP/PS1转基因小鼠脑组织中α7神经型尼古丁受体(α7 nAChR)水平后对海马组织DYN-Ⅰ蛋白的影响;探讨α7 nAChR在阿尔茨海默病(Alzheimer disease,AD)发病机制中的神经保护作用机制。方法实验动物分为对照组(Control)、野生加PNU282987组(WP)、APP/PS1转基因组(APP/PS1)和APP/PS1加PNU282987组(AP)各8只,WP和AP组给予α7 nAChRs特异性激动剂PNU-282987,另两组给予生理盐水,给药方式为小鼠24 w龄时1 mg/kg腹腔注射PNU-282987,连续5 d。采用Real-time PCR法和蛋白免疫印迹(Western Blot)法分别测定小鼠海马组织中发动蛋白Ⅰ(DYN-Ⅰ)mRNA和蛋白表达水平的变化。结果与control组相比,APP/PS1组海马组织中发动蛋白Ⅰ(DYN-Ⅰ)mRNA和蛋白水平下降(P0.05,P0.01);而特异性激动α7 nAChR水平后,与对照组相比WP组中发动蛋白Ⅰ(DYN-Ⅰ)mRNA和蛋白水平升高(P0.05,P0.01);与APP/PS1组相比AP组小鼠大脑海马组织中发动蛋白Ⅰ(DYN-Ⅰ)mRNA和蛋白水平明显升高(P0.01,P0.01)。结论特异性激动APP/PS1转基因小鼠海马组织中α7 nAChR水平能够使网格蛋白内吞调节蛋白DYN-Ⅰ表达升高。这可能提示了α7 nAChR对突触有一定的保护作用,进一步说明α7 nAChR在阿尔茨海默病的发病中起着重要作用。     

SH-SY5Y细胞α7尼古丁受体蛋白抑制对tau蛋白及p38 MAPK通路的影响及其与AD发病机制的关系

目的研究α7尼古丁受体(nAChR)蛋白抑制对SH-SY5Y细胞tau蛋白磷酸化水平的影响及其与p38 MAPK通路的关系,探讨α7 nAChR调节tau蛋白磷酸化的相关机制。方法用α7 nAChR阻断剂MLA阻断SH-SY5Y细胞α7 nAChR蛋白的活化及其表达,用p38 MAPK阻断剂SB203580阻断SH-SY5Y细胞p38 MAPK信号通路蛋白的活化及其表达,Western blotting方法测定tau蛋白、p-tau(S404)、p-tau(S214)、α7 nAChR、p38 MAPK及p-p38 MAPK(Thr180/Tyr182)蛋白表达水平。结果细胞经MLA处理后,p-tau(S404)和p-tau(S214)蛋白水平明显升高(P0.01),p-p38 MAPK和α7 nAChR蛋白水平明显降低(P0.01),tau蛋白和p38 MAPK蛋白水平保持不变;经SB203580处理后,SB203580及MLA共同处理后均引起p-tau(S404)、p-tau(S214)、p-p38 MAPK和α7nAChR蛋白水平显著降低(P0.01),tau蛋白和p38 MAPK蛋白水平无变化。结论α7 nAChR可通过阻断p38MAPK信号传导通路抑制tau蛋白过度磷酸化。