灯盏细辛对神经细胞尼古丁受体表达的影响及其神经保护作用研究

目的研究中药灯盏细辛对神经母细胞瘤（SH-SYSY）细胞神经型尼古丁受体表达的影响及其对抗β淀粉样蛋白（Aβ）神经毒性作用机制。方法采用比色法测定细胞MTT还原率筛选药物浓度安全范围,选取一定浓度的灯盏细辛乙醇提取物醋酸乙酯部位处理SH-SY5Y细胞后,再加入Aβ1-42处理,测定MTT还原率,蛋白质印迹法（Western blotting）方法测定神经型尼古丁受体α3、α7蛋白表达的变化;采用改进的Ellman比色法测定细胞培养基中乙酰胆碱酯酶（Acetylcholinesterase,AChE）及丁酰胆碱酯酶（Butyrylcholinesterase,BuChE）活性。结果灯盏细辛乙醇提取物醋酸乙酯部位能提高细胞α3及α7尼古丁受体亚单位蛋白质表达水平;降低细胞乙酰胆碱酯酶活性;并能对抗Aβ引起的MTT还原率降低。结论灯盏细辛乙醇提取物醋酸乙酯部位能上调尼古丁受体亚单位蛋白表达水平,降低细胞乙酰胆碱酯酶活性,且能对抗Aβ的神经毒性作用,在阿尔茨海默氏病的治疗中可能起到一定作用。     

贵州省从江县侗族人群葡萄糖-6-磷酸脱氢酶基因突变型的检测

目的了解贵州省从江县侗族葡萄糖-6-磷酸脱氢酶(Glucose-6-phosphate dehydrogenase,G6PD) 缺乏症的发生率、基因突变类型及特点。方法对贵州省从江县侗族524人采用四氮唑蓝定性法进行G6PD缺乏症初筛、G6PD／6PGD比值法验证．再经自然引物及错配引物介导的聚合酶链反应／限制性酶切分析法检测中国人常见的9种基因突变型,对于未定型采用变性梯度凝胶电泳法(DGGE)检查外显子2、8、9、12基因突变情况。结果 G6PD缺乏症34例,检出率为6．49％,其中检出G1388A突变4例、C592T突变18例。未定型12例经DGGE检测外显子突变情况,未发现突变,有待于进一步对其余外显子进行研究。结论贵州省从江侗族是G6PD缺乏症的高发区。592 C→T突变型为该地该民族常见突变型,而不是中国人常见的G1376T、G1388A或A95G突变型。此次基因突变型调查为了解贵州省少数民族G6PD缺乏症的分布特征提供了原始数据。     

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.     

血管性认知障碍患者脑微出血的评价及其诊断价值初探

目的 通过对脑小血管病(CSVD)认知障碍患者脑微出血(CMBs)的部位和数量进行评价,探讨CMBs在血管性认知障碍(VCI)诊断中的可能价值.方法 头颅磁敏感加权成像(SWI)显示微出血灶31例,其中符合CS-VD伴有认知障碍患者10例(VCI组),符合CSVD但无认知障碍患者21例作为对照组,按照CMBs分布的解剖区域对其数量和严重程度分级进行评价,比较两组脑叶型、深部脑组织型和幕下型CMBs的数量和严重程度的差别.结果 VCI组脑叶型CMBs的数量和严重程度明显高于对照组,二者差别具有统计学意义(P＜0.05);VCI组深部脑组织型和幕下型CMBs的数量和严重程度与对照组的差别无统计学意义(P＞0.05).结论 脑叶型CMBs及其数量对脑小血管病患者认知功能损害可能起到关键作用,对CMBs的部位和严重程度进行评价有助于VCI的诊断.     

过氧化氢酶基因多态性与疾病相关性的研究

过氧化氢酶(catalase,CAT)是人体内很重要的一种抗氧化酶,它与超氧化物歧化酶(superoxide dismutase,SOD),谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)共同构成人体重要的抗氧化酶系统,三者协同,减少了活性氧自由基的产生,防止脂质过氧化及其中间代谢产物对机体的损害,维持体内氧化-抗氧化平衡.过氧化氢酶的基因多态性可能与该酶的活性及转录活性有关,从而影响个体间对疾病的易感性不同.     

燃煤污染型氟中毒大鼠学习记忆能力及胆碱酯酶活性改变

Objective To observe the influence of coal burning fluorosis on learning and memory ability in rats and reveal its possible mechanisms. Methods Healthy 48 SD rats were divided into control, low-fluoride and high-fluoride group. All rats in fluoride exposed groups were fed with the eom polluted by drying processes with burning coal containing high level of fluoride obtained from the endemic fluorosis area to produce the animal model of fluorosis. The experiment period were 3,6 mouths, respectively. The ability of leaning and memory was measured by Morris test and cholinesterase activity detected by photometric method at 3 or 6 month after experiment, respectively. Results Fluoride contents signifieantlly influenced the escape latency, the numbers of crossing the platforms and the time of staying the platforms(the value of F was 29.29,6.47,6.50, respectively, P<0.01).In addition, the numbers of crossing the platforms and the time of staying the platforms were influenced by the exposed time(the value of F was 16.11,45.59, P<0.01). Furthermore, the fluoride contents and the exposed time had an interaction between the numbers of crossing the platforms and the time of staying the platforms (the value of F was 4.67,5.68, P<0.05 or<0.01). Three months after the experiment, the mean values of escape latency [(14.71± 4.85)s] of rats in highly fluoride exposed group were significantly prolonged as compared with controls [(9.28±4.22)s]; 6 month after the experiment, the mean values of escape latency[(12.42±8.03)s, (17.48± 8.05)s] of rats in both groups exposed to fluoride were significantly prolonged as compared to controls [(7.04± 3.29)s, P<0.05]. The decreased numbers of crossing the platforms[(1.62±0.87)number] and the declined time of staying the platforms[(16.70±5.02)s] were found in the rats exposed to high fluoride as compared to controls [(3.53±1.67 )number, (23.33±5.35)s, P<0.05]. The fluoride contents obviously influenced the activities of acetylcholinesterase and butylcolinesterase (the value of F was 12.83,13.27, P<0.01). On the other hand, the times of breeding also influnced the activities of butylcolinesterase (the value of F was 16.26, P<0.01). In 3 months of the experiment, the activities of butylcolinesterase [(0.55±0.12)kU/g] in low fluoride exposed group were significantly decreased in comparison with controls[(0.73±0.10)kU/g, P<0.05]. The activities of acetylcholinesterase[(0.62±0.42)kU/g] and butylcolioesterase[(0.58±0.10)kU/g] in high fluoride group were significantly decreased as compared to eontrois[(1.41±0.52), (0.73±0.10)kU/g, P<0.05]. The correlation analysis showed that there was a negative correlation between the cholinesterase and the escape latency(r=-0.68, P< 0.01), and a positive correlation between the cholinesterase and the time of staying the platforms(r=0.57, P< 0.01). Conclusions The ability of learning and memory in rats with coal buring fluorosis was decreased, which might be connected to the decreased activity of cholinesterase in a dose-effect correlation.     

α3神经型尼古丁受体基因上调对SH-SY5Y细胞突触素水平的影响

目的构建使α3nAChR基因上调的重组质粒α3nAChR-pcDNA3.1并转染至神经母细胞瘤细胞(SH-SY5Y),研究α3nAChR基因上调对细胞突触素(SYP)水平的影响。方法设计α3nAChR上下游引物,通过逆转录PCR的方法获取人α3nAChR特异核苷酸序列,并将其克隆到质粒载体pcDNA3.1上,构建重组质粒α3nAChR-pcDNA3.1;瞬时转染至SH-SY5Y细胞后,用Real-time法和蛋白免疫印迹法分别α3nAChR及蛋白水平,并检测上调细胞中SYPmRNA和蛋白水平的变化。结果成功构建了使α3nAChR基因上调的重组质粒α3nAChRpcDNA3.1;将α3nAChR-pcDNA3.1质粒转染到SH-SY5Y细胞后,与空载质粒组及正常对照组相比,α3nAChRmRNA及蛋白表达水平分别增加了422%和106%(P0.05);SYPmRNA及蛋白表达水平分别增加了115%和43%(P0.05)。结论α3nAChR表达的上调可使细胞SYP的表达增加,说明了α3nAChR与SYP密切相关,在AD的发生中可能起着重要作用。     

慢性氟中毒大鼠脑组织中c-Jun氨基末端激酶表达水平观察

目的 观察慢性氟中毒大鼠脑组织中c-Jun氨基末端激酶(JNK)信号转导激酶表达变化,进一步揭示慢性氟中毒神经损伤的分子机制.方法 SD大鼠随机分为3组:对照组、低氟组、高氟组,每组24只,饮用水含氟量分别为＜0.5和5.0、50.0 mg/L,实验期为6个月.用氟离子选择电极法测定大鼠尿氟及血氟,用Western blotting和免疫组织化学方法检测脑组织中JNK信号转导激酶的表达和分布,并分析血氟与活化的JNK激酶的相关关系.结果低氟组和高氟组大鼠尿氟[(2.56±0.91)、(5.73±3.14)mg/L]和血氟[(0.36±0.14)、(0.50±0.18)mg/L]均较对照组[(0.92±0.30)、(0.12±0.07)mg/L]升高(P均＜0.05).高氟组(1.74±0.69)脑组织phospho-JNK表达高于对照组(1.00±0.37)和低氟组(1.20±0.28,P均＜0.05);total-JNK蛋白表达水平3组间比较,差异无统计学意义(F=0.046,P＞0.05).phospho-JNK、total-JNK阳性表达神经元主要集中在皮质、海马和背侧丘脑,其中高氟组大鼠phospho-JNK在顶叶皮质(119.3±14.1)、枕叶皮质(112.7±5.4)、海马CA3区(100.6±8.9)、背侧丘脑(117.8±10.4)及橄榄核(112.6±5.9)中阳性表达较对照组(104.1±8.9、106.6±9.6、106.6±9.7、108.9±6.4、100.3±8.4)和低氟组(96.7±17.1、102.5±8.3、106.4±6.5、110.2±9.3、102.4±4.7、102.5±9.8)明显增高(P均＜0.05),而total-JNK在各组大鼠脑组织中阳性表达分布未见明显改变(P均＞0.05).相关分析结果发现,随大鼠血氟升高,脑组织中phospho-JNK表达呈增高趋势,二者存在正相关关系(r=0.677).结论慢性氟中毒导致脑组织中磷酸化JNK表达改变,并与机体中氟蓄积量存在相关关系,这些改变可能与慢性氟中毒导致的神经损伤有关系.     

氟中毒人群过氧化氢酶和谷胱甘肽S-转移酶的活力

目的：探讨燃煤型氟中毒地区人群过氧化氢酶（CAT）、谷胱甘肽S-转移酶（GSTs）活力与氧化应激的关系。方法：于贵州省毕节市鸭池镇燃煤型氟中毒非改灶村抽取245名村民作为非干预组,于该市长春镇燃煤型氟中毒改灶村抽取240名村民作为干预组,于非地方性氟中毒长顺县白云山镇抽取247名村民作对照组,利用比色法分别测定CAT、GSTs的活力。结果：非干预组血浆CAT活力和GSTs活力低于干预组和对照组,干预组低于对照组,各组间差异有统计学意义（P〈0.05）;各组间CAT、GSTs活力无性别差异（P〉0.05）。结论：过量氟可能使体内自由基代谢失衡,推测通过人为干预氟的摄入,有助于机体恢复氧化平衡。     

灯盏细辛活性部位对抗β-淀粉样蛋白引起的细胞凋亡

目的：研究灯盏细辛醇提物不同活性部位对β-淀粉样蛋白（Aβ）引起的神经母细胞瘤细胞（SH-SY5Y）凋亡的拮抗作用,探讨其是否在阿尔茨海默病（Alzheimer’s disease,AD）治疗中有神经保护作用。方法：选用灯盏细辛醇提物不同活性部位及Aβ25-35处理SH-SY5Y细胞,用比色法测定MTT还原率,Annexin V-FITC试剂盒测定细胞凋亡水平。结果：安全浓度的灯盏细辛醇提物不同活性部位能对抗Aβ引起的MTT还原率降低和细胞凋亡。结论：灯盏细辛醇提物不同活性部位可对抗Aβ的神经毒性作用。