过氧化氢酶基因-262C/T及-21A/T多态性与燃煤型氟中毒相关性的研究

目的 探讨过氧化氢酶(CAT)基因启动子区-262C/T、-21A/T多态性与燃煤型氟中毒之间的关系.方法 2007年,在贵州省毕节市鸭池镇燃煤型氟中毒非改灶村抽取150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150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150名村民作对照组,应用聚合酶链反应限制性片段长度多态性(PCR-RFLP)分析方法,分别检测各组CAT-262C/T、-21A/T多态的基因型.结果 非干预组、干预组和对照组CAT-262C/T、CAT-21A/T多态的基因型频率分布符合Hardy-weinberg遗传平衡定律(P＞0.05).在研究人群中检测到CAT-262C/T多态的两种基因型CC和CT,未检测到TT基因型;检测到CAT-21A/T多态的三种基因型AA、AT和TT.对照组、干预组和非干预组CAT基因-262C/T多态位点的基因型(CC、CT)频率分别为89.33%(134/150)、10.67%(16/150)、88.67%(133/150)、11.33%(17/150)、9333%(140/150)、6.67%(10/150);对照组、干预组和非干预组C等位基因频率分别为94.67%(284/300)、94.33%(283/300)、96.67%(290/300);T等位基因频率分别为5.33%(16/300)、5.67%(17/300)、3.33%(10/300).对照组,干预组和非干预组CAT基因-21A/T多态位点基因型(AA、AT、TT)频率分别为48.67%(73/150)、46.00%(69/150)、5.33%(8/150)、52.67%(79/150)、38.00%(57/150)、9.33%(14/150)、51.33%(77/150)、38.00%(57/150)、10.67%(16/150);对照组、干预组和非干预组A等位基因频率分别为71.67%(215/300)、71.67%(215/300)、70.33%(2ll/300);T等位基因频率分别为28.33%(85/300)、28.33%(85/300)、29.67%(89/300).CAT-262C/T和CAT-21A/T基因型频率及等位基因频率在对照组,干预组和非干预组中的分布,组间比较差异均无统计学意义(x2=0.331、0.336,P均＞0.05).结论 CAT基因-262C/T、-21A/T多态性与燃煤型氟中毒无明显的相关性.

Abstract：

Objective To explore the relationship between -262C/T and -21A/T polymorphisms of catalase(CAT) gene and coal-burning borne fluorosis. Methods In 2007, 150 villagers were taken as a nonintervention group in Bijie city from the village of coal-burning borne fluorosis areas with unchanged cooking stoves;150 villagers were taken as the intervention group from the town of Changchun county where cooking stoves changed; 150 villagers were taken as control from non-endemic fluorosis areas in Baiyun town of Changshun county.PCR-restriction fragment length polymorphism were employed to detect genotypes of CAT-262C/T and CAT-21A/T polymorphism of CAT gene. Results The genotypic frequencies of CAT-262C/T and CAT-21A/T in nonintervention group,intervention group and control group were in line with Hardy-Weinberg equilibrium law (P＞ 0.05 ).The genotypes of CC and CT were detected while no TT were detected for CAT-262C/T polymorphism; the genotypes of AA, AT and TT were detected for CAT-21A/T. The genotype frequencies of CAT-262 CC, CT in control group, intervention group and non-intervention group were (89.33%(134/150), 10.67%(16/150); 88.67%(133/150), 11.33% (17/150),93.33% (140/150),6.67% (10/150), respectively. The gene frequency of C in control group, intervention group and non-intervention group were (94.67% (284/300), 94.33% (283/300),96.67%(290/300), respectively. The gene frequency of T in control group, intervention group and non-intervention group were 5.33%(16/300), 5.67%(17/300), 3.33%(10/300), respectively. The genotype frequencies of CAT-21 AA,AT and TT in control group, intervention group and non-intervention group were 48.67%(73/150),46.00%(69/150),5.33%(8/150) ,52.67%(79/150) ,38.00%(57/150) ,9.33% (14/150) ,51.33%(77/150) ,38.00%(57/150), 10.67%(16/150), respectively. The gene frequency of A in control group, intervention group and non-intervention group were 71.67%(215/300),71.67%(215/300),70.33%(211/300), respectively. The gene frequency of T in control group, intervention group and non-intervention group were 28.33% (85/300),28.33% (85/300),29.67% (89/300),respectively. CAT-262C/T and CAT-21A/T genotype and allele frequencies in the control group, the intervention group and non-intervention group showed no significant differences in the distribution(x2= 0.331,0.336, all P ＞0.05 ). Conclusion CAT-262C/T and CAT-21A/T polymorphism is not associated with coal-burning borne fluorosis.     

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.     

阿尔茨海默病患者的神经型尼古丁受体α7亚单位基因多态性

目的：探讨散发性阿尔茨海默病（Spordic alzheimer disease,SAD）患者神经型尼古丁受体α7亚单位（CHRNA7）基因多态性情况。方法：用聚合酶链式反应-温度梯度凝胶电泳（PCR-TGGE）和DNA测序技术,分析12例SAD病人CHRNA7基因全部10个外显子及其两侧的部分内含子基因序列。结果：在CHRNA7基因上发现1个新的突变位点,即在外显子7附近的内含子7上的117643＋GTG三碱基插入突变,未发现任何已报道的多态性位点。结论：SAD患者的CHRNA7基因多态性与其它人群可能有差异。     

不同浓度氟对大鼠肝、肾功能及氧化应激的影响

目的:研究不同浓度氟对大鼠肝、肾功能及氧化应激的影响.方法:在饮水中加入不同剂量的氟化钠喂饲大鼠4个月后,检测血清内反映肝、肾功能的指标,测定血清、肝和肾的丙二醛(MDA)含量及超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)活性.结果:与对照组比较,不同浓度的氟使大鼠血清、肝和肾的MDA含量逐渐升高,SOD、GR活性逐渐下降;但氟对大鼠的肝、肾功能的影响不明显.结论:慢性氟中毒可损伤机体的抗氧化功能,但对大鼠肝、肾功能的损害不明显.     

Alzheimer''s病与神经型尼古丁胆碱能受体α4基因多态性关联研究

目的 探讨Alzheimer's 病(AD)与神经型尼古丁胆碱能受体α4亚单位(CHRNA4)基因所有外显子基因多态性的关联关系.方法 用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和DNA测序技术分析23例AD病人及30例正常人的CHRNA4基因所有外显子基因序列.结果 CHRNA4的外显子3上发现3个新的多态性位点,这3个多态位点在病例组与对照组之间存在差异显著性.CHRNA4的外显子5上发现3个多态性位点,病例组与对照组之间没有差异.CHRNA4的外显子6上发现7个多态性位点,病例组与对照组之间没有差异.结论 在CHRNA4外显子3上发现的3个新的多态性位点与AD发病可能存在相关关系.因此 CHRNA4基因外显子3多态性可能与AD发病有关联.     

报告基因研究及其应用进展

报告基因是现代分子生物学研究领域中用于分析结构基因旁侧区域潜在的顺式元件(如启动子、增强子和沉默子等)和反式作用因子相互作用关系的一种重要工具,通过它的表达产物便捷、可靠、灵敏地解析基因时空表达调控的内在规律.报告基因技术广泛应用于基因表达调控、信号转导、转基因、启动子分析、受体功能鉴定、基因治疗以及药物筛选等领域,该文将对报告基因的主要类型、特点及其应用研究进展进行综述.     

α7尼古丁受体mRNA表达抑制对SH-SY5Y细胞氧化应激水平的影响

目的探讨通过RNA干扰技术抑制神经母细胞瘤细胞(SH-SY5Y细胞)中α7尼古丁乙酰胆碱受体(nAChR)基因表达后细胞氧化应激水平的变化,以了解该受体的神经保护作用。方法 SH-SY5Y细胞转染针对α7 nAChR的小分子干扰RNA,Real-time PCR法检测转染细胞中α7 nAChR mRNA水平蛋白质印迹方法检测蛋白表达水平;并用Aβ1～42处理培养细胞,比色法测定细胞脂质过氧化产物水平、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性。结果转染siRNA后,α7 nAChR mRNA水平和蛋白表达水平分别降低了81.7%和76.9%;细胞脂质过氧化水平升高38.4%;SOD和GSH-PX活性分别降低30.3%和21.1%。用Aβ1～42处理细胞后氧化应激水平升高,且转染siRNA后能增强Aβ的神经毒性作用。结论α7 nAChR基因表达抑制后能增强细胞氧化应激水平,同时增强Aβ的神经毒性作用。