氟中毒,牙%大鼠%线粒体融合基因Mfn1%神经系统
氟中毒,牙%大鼠%線粒體融閤基因Mfn1%神經繫統
불중독,아%대서%선립체융합기인Mfn1%신경계통
Fluorosis,dental%Rats%Mito-fusion1 mfn1%Nervous system
目的 研究慢性氟中毒对大鼠神经细胞线粒体融合基因Mfn1表达及线粒体形态的影响.方法 将60只1月龄清洁级SD大鼠以单纯随机法分为3组,每组20只,雌雄各半,分笼喂养.以饮水中加入氟剂量的不同,将大鼠分为对照组、低氟组、高氟组,染毒剂量分别为0、10和50 mg/L,分别在饲养3、6个月后,观察氟斑牙程度.采用透射扫描电子显微镜观察线粒体形态.用westernblotting和免疫组织化学方法检测线粒体融合基因Mfn1表达.结果 染氟组大鼠出现不同程度的氟斑牙,3个月时低氟组以Ⅰ度氟斑牙为主(16只),高氟组发生Ⅰ度、Ⅱ度氟斑牙分别为11、9只；6个月时低氟组发生Ⅰ度、Ⅱ度、Ⅲ度氟斑牙分别为14、6、0只,高氟组为6、13、1只；对照组均未出现.低氟组、高氟组在3个月时尿氟分别为(3.30±1.18)、(5.10 ±0.35)mg/L(F =3.18,P ＜0.05)；6个月时分别为(4.16±1.39)、(5.70±1.70)mg/L(F=3.17,P ＜0.05).3个月时,对照组大鼠大脑皮质神经细胞线粒体形态变化不明显,染氟组大鼠神经细胞线粒体均出现球状空泡状改变.Mfn1的蛋白免疫组织化学强阳性表达神经细胞数发生了改变,与对照组[(53.0±4.54)个]相比较,低氟组[(51.09±6.25)个]的改变无统计学意义(t=1.7,P＞0.05),而高氟组[(59.71 ±5.64)个]增加,差异有统计学意义(t=2.1,P＜0.05)；western-blotting法测得蛋白印迹平均吸光度值也具有相同的改变,与对照组(1.21±0.18)相比,低氟组(1.22±0.26)的改变无统计学意义(t=1.1,P＞0.05),高氟组(1.66 ±0.20)的水平升高,差异有统计学意义(t=2.1,P＜0.05).6个月时低氟组、高氟组大鼠大脑皮质神经细胞线粒体出现嵴消失、球状空泡状改变,对照组无明显改变.Mfn1的蛋白免疫组织化学强阳性表达神经细胞数发生了改变,与对照组[(36.43±4.04)个]相比较,低氟组[(20.05±4.55)个]、高氟组[(17.10±3.86)个]均降低,差异有统计学意义(t值分别为2.1,2.2,P值均＜0.05)；western-blotting法蛋白印迹平均吸光度值也有改变,与对照组(1.00 ±0.13)相比,低氟组(0.64±0.08)、高氟组(0.39±0.06)的水平降低,差异有统汁学意义(t值分别为2.2,2.2,P值均＜0.05).结论 摄入过量的氟可造成融合基因Mfn1表达改变和线粒体膜网络结构断裂,慢性氟中毒神经细胞损伤可能与线粒体融合功能障碍有关.
目的 研究慢性氟中毒對大鼠神經細胞線粒體融閤基因Mfn1錶達及線粒體形態的影響.方法 將60隻1月齡清潔級SD大鼠以單純隨機法分為3組,每組20隻,雌雄各半,分籠餵養.以飲水中加入氟劑量的不同,將大鼠分為對照組、低氟組、高氟組,染毒劑量分彆為0、10和50 mg/L,分彆在飼養3、6箇月後,觀察氟斑牙程度.採用透射掃描電子顯微鏡觀察線粒體形態.用westernblotting和免疫組織化學方法檢測線粒體融閤基因Mfn1錶達.結果 染氟組大鼠齣現不同程度的氟斑牙,3箇月時低氟組以Ⅰ度氟斑牙為主(16隻),高氟組髮生Ⅰ度、Ⅱ度氟斑牙分彆為11、9隻；6箇月時低氟組髮生Ⅰ度、Ⅱ度、Ⅲ度氟斑牙分彆為14、6、0隻,高氟組為6、13、1隻；對照組均未齣現.低氟組、高氟組在3箇月時尿氟分彆為(3.30±1.18)、(5.10 ±0.35)mg/L(F =3.18,P ＜0.05)；6箇月時分彆為(4.16±1.39)、(5.70±1.70)mg/L(F=3.17,P ＜0.05).3箇月時,對照組大鼠大腦皮質神經細胞線粒體形態變化不明顯,染氟組大鼠神經細胞線粒體均齣現毬狀空泡狀改變.Mfn1的蛋白免疫組織化學彊暘性錶達神經細胞數髮生瞭改變,與對照組[(53.0±4.54)箇]相比較,低氟組[(51.09±6.25)箇]的改變無統計學意義(t=1.7,P＞0.05),而高氟組[(59.71 ±5.64)箇]增加,差異有統計學意義(t=2.1,P＜0.05)；western-blotting法測得蛋白印跡平均吸光度值也具有相同的改變,與對照組(1.21±0.18)相比,低氟組(1.22±0.26)的改變無統計學意義(t=1.1,P＞0.05),高氟組(1.66 ±0.20)的水平升高,差異有統計學意義(t=2.1,P＜0.05).6箇月時低氟組、高氟組大鼠大腦皮質神經細胞線粒體齣現嵴消失、毬狀空泡狀改變,對照組無明顯改變.Mfn1的蛋白免疫組織化學彊暘性錶達神經細胞數髮生瞭改變,與對照組[(36.43±4.04)箇]相比較,低氟組[(20.05±4.55)箇]、高氟組[(17.10±3.86)箇]均降低,差異有統計學意義(t值分彆為2.1,2.2,P值均＜0.05)；western-blotting法蛋白印跡平均吸光度值也有改變,與對照組(1.00 ±0.13)相比,低氟組(0.64±0.08)、高氟組(0.39±0.06)的水平降低,差異有統汁學意義(t值分彆為2.2,2.2,P值均＜0.05).結論 攝入過量的氟可造成融閤基因Mfn1錶達改變和線粒體膜網絡結構斷裂,慢性氟中毒神經細胞損傷可能與線粒體融閤功能障礙有關.
목적 연구만성불중독대대서신경세포선립체융합기인Mfn1표체급선립체형태적영향.방법 장60지1월령청길급SD대서이단순수궤법분위3조,매조20지,자웅각반,분롱위양.이음수중가입불제량적불동,장대서분위대조조、저불조、고불조,염독제량분별위0、10화50 mg/L,분별재사양3、6개월후,관찰불반아정도.채용투사소묘전자현미경관찰선립체형태.용westernblotting화면역조직화학방법검측선립체융합기인Mfn1표체.결과 염불조대서출현불동정도적불반아,3개월시저불조이Ⅰ도불반아위주(16지),고불조발생Ⅰ도、Ⅱ도불반아분별위11、9지；6개월시저불조발생Ⅰ도、Ⅱ도、Ⅲ도불반아분별위14、6、0지,고불조위6、13、1지；대조조균미출현.저불조、고불조재3개월시뇨불분별위(3.30±1.18)、(5.10 ±0.35)mg/L(F =3.18,P ＜0.05)；6개월시분별위(4.16±1.39)、(5.70±1.70)mg/L(F=3.17,P ＜0.05).3개월시,대조조대서대뇌피질신경세포선립체형태변화불명현,염불조대서신경세포선립체균출현구상공포상개변.Mfn1적단백면역조직화학강양성표체신경세포수발생료개변,여대조조[(53.0±4.54)개]상비교,저불조[(51.09±6.25)개]적개변무통계학의의(t=1.7,P＞0.05),이고불조[(59.71 ±5.64)개]증가,차이유통계학의의(t=2.1,P＜0.05)；western-blotting법측득단백인적평균흡광도치야구유상동적개변,여대조조(1.21±0.18)상비,저불조(1.22±0.26)적개변무통계학의의(t=1.1,P＞0.05),고불조(1.66 ±0.20)적수평승고,차이유통계학의의(t=2.1,P＜0.05).6개월시저불조、고불조대서대뇌피질신경세포선립체출현척소실、구상공포상개변,대조조무명현개변.Mfn1적단백면역조직화학강양성표체신경세포수발생료개변,여대조조[(36.43±4.04)개]상비교,저불조[(20.05±4.55)개]、고불조[(17.10±3.86)개]균강저,차이유통계학의의(t치분별위2.1,2.2,P치균＜0.05)；western-blotting법단백인적평균흡광도치야유개변,여대조조(1.00 ±0.13)상비,저불조(0.64±0.08)、고불조(0.39±0.06)적수평강저,차이유통즙학의의(t치분별위2.2,2.2,P치균＜0.05).결론 섭입과량적불가조성융합기인Mfn1표체개변화선립체막망락결구단렬,만성불중독신경세포손상가능여선립체융합공능장애유관.
Objective To observe the mitochondrial fragmentation and the expression of mitofusion 1 gene in the cortical neurons of rats with chronic fluorosis,and to reveal their roles in mitochondria damage to neurons due to chronic fluorosis.Methods SD rats were divided randomly into three groups of 20 each (a half females and a half males housed individually in stainless-steel cages),and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride,and low-fluoride and high supplemented with 10 and 50 mg/L fluoride,respectively).After 3 or 6 months exposure,the mitochondrial morphology of the neurons in rat brains were observed by transmission electron microscopy (TEM),then the exprcssion of mitochondrial fusion gene,Mfn1,were detected by immunohistochemistry and western-blotting,respectively.Results Dental fluorosis was obvious in the rats exposed to excessive fluoride in their drinking water,that is,(16 rats out of 20) numbers of Ⅰ ° detal fluorosis in the low-flouride group,and (11 rats out of 20) numbers of Ⅰ ° and (9 rats out of 20) numbers of Ⅱ ° detal fluorosis in the high-flouride group were observed after 3 months exposure.Moreover,(14 rats out of 20) numbers of Ⅰ ° and (6 rats out of 20) numbers of Ⅱ ° detal fluorosis in the low-flouride group and (6rats out of 20) numbers of Io,(13 rats out of 20) numbers of Ⅱ °,and (1 rats out of 20) numbers of Ⅲ°detal fluorosis in the high-flourde group were observed after 6 months exposure.And both of untreated controls without detal fluorosis were also observed.The urinary level of fluoride in the low-flouride group (3.30 ± 1.18) mg/L and in the high-fluoride group (5.10 ±0.35) were observed after 3 months exposure (F =3.18,P ＜ 0.05).Moreover,the urinary level of fluoride in the low-flouride group (4.16 ± 1.39) mg/Land in the high-fluoride group (5.70 ± 1.70) mg/L were also observed after 6 months exposure (F =3.17,P＜ 0.05).The normal mitochondrial morphology of neurons in rats without fluorosis was observed after 3 and 6 months,while the abnormal mitochondrial morphology of neuros with fluorosis was shown,presenting mitochondrial fragmentation with swollen cristae and even the fragmented,shortened or stacked punctuate membranes (section observation of three bullous mitochondrial-mitochondrial fission process) by TEM.As compared with controls (53.0±4.54 and 1.21 ± 0.18) at the experiment period of 3 months,Mif1 protein analysis with immunocytochemical (the numbers of positive cells:51.09 ± 6.25) and western-blotting (1.22 ±0.26) were no significant difference for low fluoride group(t =1.7,1.1,P ＞ 0.05) ; Mif1 protein analysis with immunocytochemical (the numbers of positive cells:59.71 ± 5.64) and western-blotting (1.66 ±0.20) were significantly increasing for high fluoride group(t =2.1,2.1,P ＜ 0.05).As compared with controls (36.43 ±4.04 and 1.00 ± 0.13) at the experiment period of 6 months,Mif1 protein analysis with immunocytochemical (the numbers of positive cells 20.05 ± 4.55 and 17.10 ± 3.86) and westernblotting (0.64 ± 0.08 and 0.39 ± 0.06) were significantly decreasing for the two fluoride group (t =2.1,2.2 ;2.2,2.2 respectively,all P value were ＜ 0.05).Conclusions Taking excessive amount of fluoride might result in the mitochondrial fragmentation for the changed expression of Mfn1,and the neurons damage from the chronic fluorosis might be associated with the dysfunction of mitochondrial fusion.
