北京大学学报（医学版）
北京大學學報（醫學版）
북경대학학보（의학판）
JOURNAL OF BEIJING MEDICAL UNIVERSITY(HEALTH SCIENCES)
2010年
2期
131-136
,共6页
史祎%周业波%蔡嫣%滕旭%宋君秋%段晓辉%张炜真%齐永芬
史祎%週業波%蔡嫣%滕旭%宋君鞦%段曉輝%張煒真%齊永芬
사의%주업파%채언%등욱%송군추%단효휘%장위진%제영분
钙质沉着症%疾病模型,动物%年龄因素%胆骨化醇%尼古丁%大鼠,Sprague-Dawley
鈣質沉著癥%疾病模型,動物%年齡因素%膽骨化醇%尼古丁%大鼠,Sprague-Dawley
개질침착증%질병모형,동물%년령인소%담골화순%니고정%대서,Sprague-Dawley
Calcinosis%Disease models,animal%Age factors%Cholecalciferol%Nicotine%Rats,Sprague-Dawley
目的:比较不同年龄大鼠对维生素D_3和尼古丁诱导血管钙化的反应性,以利于血管钙化动物模型制备的合理选择.方法:不同月龄大鼠用维生素D_3肌肉注射(300 000 IU/kg)和尼古丁灌胃(25 mg/kg)处理后6周,测定各组大鼠颈动脉血压和心功能、血管钙含量及碱性磷酸酶活性;用Von Kossa染色方法检测血管钙沉积;用竞争性半定量逆转录聚合酶链反应方法测定主动脉骨标志蛋白骨桥蛋白(osteopontin,OPN)、骨保护素(osteoprotegerin,OPG)、基质Gla蛋白(matrix Gla protein,MGP)、骨形态发生蛋白2(bone morphogenetic protein-2,BMP_2)的mRNA水平;用Western blot的方法检测主动脉平滑肌肌动蛋白-α(smooth musule actin-α,α-SMA)水平.结果:2、8和16月龄钙化组大鼠收缩压(systolic blood pressure,SBP)分别升高了20.7%、29.4%和22.2%(P<0.05);左心室收缩末压(left ventricular systolic pressure,LVSP)分别升高13.6%、21.1%和16.2%(P<0.05);左心室压力时间变化率最大值(+LVdP/dt_(max))分别升高49.1%(P<0.01)、21.4%和13.1%(P<0.05),左心室压力时间变化率最小值(-LVdP/dt_(max))分别升高56.3%(P<0.01)、24.4%和11.3%(P<0.05).2、8和16月龄钙化组大鼠主动脉钙含量分别是年龄匹配对照组的2.62倍(P<0.05)、24.87倍(P<0.01)和10.01倍(P<0.05);8月龄钙化组钙含量是2月龄和16月龄钙化组钙含量的5.28倍和2.63倍(P<0.05).与对照组相比,各年龄钙化组主动脉碱性磷酸酶(alkaline phospha-tases,ALP)活性较年龄匹配对照组分别高126.6%、115.2%和227.9%(P<0.01).8月龄钙化组主动脉ALP活性比2月龄和16月龄钙化组高176%和75%(均P<0.01).Von kossa染色结果显示,各月龄钙化组的主动脉中膜均有棕黑色颗粒沉积,弹力层断裂,尤其8月龄钙化组最严重.各年龄钙化组骨调节蛋白mRNA水平较对照组均显著上调(P<0.01或P<0.05),其中8月龄钙化组OPN比2月龄和16月龄钙化组分别高3.41倍(P<0.01)和1.34倍(P<0.05).各年龄钙化组的α-SMA表达较相应对照分别低17.6%(P<0.01)、11%(P<0.05)和41.7%(P<0.01).结论:维生素D_3和尼古丁处理2、8和16月龄大鼠均能复制典型血管钙化模型,其中8月龄动物血管钙化指标变化最显著,更适于血管钙化的研究.
目的:比較不同年齡大鼠對維生素D_3和尼古丁誘導血管鈣化的反應性,以利于血管鈣化動物模型製備的閤理選擇.方法:不同月齡大鼠用維生素D_3肌肉註射(300 000 IU/kg)和尼古丁灌胃(25 mg/kg)處理後6週,測定各組大鼠頸動脈血壓和心功能、血管鈣含量及堿性燐痠酶活性;用Von Kossa染色方法檢測血管鈣沉積;用競爭性半定量逆轉錄聚閤酶鏈反應方法測定主動脈骨標誌蛋白骨橋蛋白(osteopontin,OPN)、骨保護素(osteoprotegerin,OPG)、基質Gla蛋白(matrix Gla protein,MGP)、骨形態髮生蛋白2(bone morphogenetic protein-2,BMP_2)的mRNA水平;用Western blot的方法檢測主動脈平滑肌肌動蛋白-α(smooth musule actin-α,α-SMA)水平.結果:2、8和16月齡鈣化組大鼠收縮壓(systolic blood pressure,SBP)分彆升高瞭20.7%、29.4%和22.2%(P<0.05);左心室收縮末壓(left ventricular systolic pressure,LVSP)分彆升高13.6%、21.1%和16.2%(P<0.05);左心室壓力時間變化率最大值(+LVdP/dt_(max))分彆升高49.1%(P<0.01)、21.4%和13.1%(P<0.05),左心室壓力時間變化率最小值(-LVdP/dt_(max))分彆升高56.3%(P<0.01)、24.4%和11.3%(P<0.05).2、8和16月齡鈣化組大鼠主動脈鈣含量分彆是年齡匹配對照組的2.62倍(P<0.05)、24.87倍(P<0.01)和10.01倍(P<0.05);8月齡鈣化組鈣含量是2月齡和16月齡鈣化組鈣含量的5.28倍和2.63倍(P<0.05).與對照組相比,各年齡鈣化組主動脈堿性燐痠酶(alkaline phospha-tases,ALP)活性較年齡匹配對照組分彆高126.6%、115.2%和227.9%(P<0.01).8月齡鈣化組主動脈ALP活性比2月齡和16月齡鈣化組高176%和75%(均P<0.01).Von kossa染色結果顯示,各月齡鈣化組的主動脈中膜均有棕黑色顆粒沉積,彈力層斷裂,尤其8月齡鈣化組最嚴重.各年齡鈣化組骨調節蛋白mRNA水平較對照組均顯著上調(P<0.01或P<0.05),其中8月齡鈣化組OPN比2月齡和16月齡鈣化組分彆高3.41倍(P<0.01)和1.34倍(P<0.05).各年齡鈣化組的α-SMA錶達較相應對照分彆低17.6%(P<0.01)、11%(P<0.05)和41.7%(P<0.01).結論:維生素D_3和尼古丁處理2、8和16月齡大鼠均能複製典型血管鈣化模型,其中8月齡動物血管鈣化指標變化最顯著,更適于血管鈣化的研究.
목적:비교불동년령대서대유생소D_3화니고정유도혈관개화적반응성,이리우혈관개화동물모형제비적합리선택.방법:불동월령대서용유생소D_3기육주사(300 000 IU/kg)화니고정관위(25 mg/kg)처리후6주,측정각조대서경동맥혈압화심공능、혈관개함량급감성린산매활성;용Von Kossa염색방법검측혈관개침적;용경쟁성반정량역전록취합매련반응방법측정주동맥골표지단백골교단백(osteopontin,OPN)、골보호소(osteoprotegerin,OPG)、기질Gla단백(matrix Gla protein,MGP)、골형태발생단백2(bone morphogenetic protein-2,BMP_2)적mRNA수평;용Western blot적방법검측주동맥평활기기동단백-α(smooth musule actin-α,α-SMA)수평.결과:2、8화16월령개화조대서수축압(systolic blood pressure,SBP)분별승고료20.7%、29.4%화22.2%(P<0.05);좌심실수축말압(left ventricular systolic pressure,LVSP)분별승고13.6%、21.1%화16.2%(P<0.05);좌심실압력시간변화솔최대치(+LVdP/dt_(max))분별승고49.1%(P<0.01)、21.4%화13.1%(P<0.05),좌심실압력시간변화솔최소치(-LVdP/dt_(max))분별승고56.3%(P<0.01)、24.4%화11.3%(P<0.05).2、8화16월령개화조대서주동맥개함량분별시년령필배대조조적2.62배(P<0.05)、24.87배(P<0.01)화10.01배(P<0.05);8월령개화조개함량시2월령화16월령개화조개함량적5.28배화2.63배(P<0.05).여대조조상비,각년령개화조주동맥감성린산매(alkaline phospha-tases,ALP)활성교년령필배대조조분별고126.6%、115.2%화227.9%(P<0.01).8월령개화조주동맥ALP활성비2월령화16월령개화조고176%화75%(균P<0.01).Von kossa염색결과현시,각월령개화조적주동맥중막균유종흑색과립침적,탄력층단렬,우기8월령개화조최엄중.각년령개화조골조절단백mRNA수평교대조조균현저상조(P<0.01혹P<0.05),기중8월령개화조OPN비2월령화16월령개화조분별고3.41배(P<0.01)화1.34배(P<0.05).각년령개화조적α-SMA표체교상응대조분별저17.6%(P<0.01)、11%(P<0.05)화41.7%(P<0.01).결론:유생소D_3화니고정처리2、8화16월령대서균능복제전형혈관개화모형,기중8월령동물혈관개화지표변화최현저,경괄우혈관개화적연구.
Obiective:To explore the effect of age on vascular calcification induced by vitamine D_3 and nicotine.Methods:Vascular calcification in rats was induced by adminstration of vitamine D_3 plus nico-tine(VDN treatment).After six weeks,Von Kossa staining,calcium content,alkaline phosphatase ac-tivity,phosphorus and calcium content in plasma were assayed.Carotid blood pressue,cardiac function and the relative amounts of osteopontin(OPN)、osteoprotegerin(OPG)、matrix Gla protein(MGP)、bone morphogenetic protein-2(BMP2) mRNA level and smooth musule actin-α(α-SMA)protein level were measured.Results:Compared with control group,the systolic blood pressure(SBP)of the rats of 2,8 and 16 months with vascular calcification respectively increased by 20.7%,29.4%and 22.2%(P<0.05);the left ventricular systolic pressure(LVSP)respectively increased by 13.6%,21.1% and 16.2%(P<0.05);+LVdP/dt_(max) respectively increased by 49.1%(P<0.01),21.4%and 13.1%(P<0.05);-LVdP/dt_(max) respectively increased by 56.3%(P<0.01),24.4%and 11.3%(P<0.05).Aortic calcium contents of the 2-,8- and 16- month calcified rats were respectively 2.62-fold(P<0.05),24.87-fold(P<0.01)and 10.01-fold(P<0.05)of the age-matched control group.As com-pared with the aortic calcium contents of calcified groups at different ages,the calcification group of 8 months had higher aortic calcium content than those of 2 and 16 months,which were respectively,5.28-fold and 2.63- fold(P<0.05).Compared with the control groups,alkaline phosphatases activity(ALP)of calcification groups increased respectively by 126.6%,115.2%and 227.9%(P<0.01)in the 2-,8- and 16- month rats.As compared with the ALP activity of calcified groups at difierent ages,ALP acti-vity of aortic calcification group of the 8-month-old rats was higher than that of the 2-month-old and 16-month-old rats,which increased by 176%and 75%respectively(all P<0.01).Von kossa staining for calcification showed positive staining as black/brown areas within the main,large,nodular structures as shown in extracellular matrix and cytoplasma in VDN groups at different ages,especially in the 8-month-old VDN group,with the most dispersed calcific nodules deposited and a few of the elastic fibers of the medial layer collapse.The mRNA expressions of OPN,OPG,MGP,BMP,were up-regulated(P<0.01 or P<0.05)and protein levels of α-SMA were down-regulated in difierent calcification groups(P<O.05).The mRNA levels of OPN in 8-month-old calcification group increased by 3.41-fold(P<0.01) and 1.34-fold(P<0.05)respectively compared with the 2-month-old and 16-month-old calcification groups.And the α-SMA protein expression levels were lower at calcification groups in different ages,which were respectively equivalent to 17.6%of the 2-month-old control group(P<0.01),11% of the 8-month-old control group(P<0.05)and 41.7% of 16-month-old control group(P<0.01).Conclusion:SD rats of 2.8 and 16 months can all be used to duplicate vascular calcification model induced by vitamine D_3 plus nicotine and the 8-month-old rat has the most sensitivity to the calcification treatment,which means that the 8-month-old rat may be the most appropriate age for the study of vascular calcification.
