中华地方病学杂志
中華地方病學雜誌
중화지방병학잡지
Chinese Journal of Endemiology
2014年
4期
357-362
,共6页
陈静宏%曹峻岭%王治伦%马天有%王梦莹%何颖%杨占田%陈晨
陳靜宏%曹峻嶺%王治倫%馬天有%王夢瑩%何穎%楊佔田%陳晨
진정굉%조준령%왕치륜%마천유%왕몽형%하영%양점전%진신
大骨节病%基质金属蛋白酶%T-2毒素%硒
大骨節病%基質金屬蛋白酶%T-2毒素%硒
대골절병%기질금속단백매%T-2독소%서
Kashin-Beck disease%Matrix metalloproteinases%T-2 toxin%Selenium
目的：观察基质金属蛋白酶（Matrix metalloproteinases，MMPs）在大骨节病（Kashin-Beck disease， KBD）儿童软骨和低硒条件下T-2毒素中毒大鼠软骨中的表达，以及T-2毒素对软骨细胞MMP-13启动子的激活作用，探讨MMPs与KBD发病的关系。方法以5例尸检KBD儿童（KBD组）和5例因车祸死亡或畸形手术的正常儿童（对照组）手指节软骨为研究对象，采用免疫组织化学染色的方法，检测软骨中MMP-1和MMP-13的表达。雄性健康SD大鼠32只，按体质量采用随机数字表法分为常规饲料组、低硒饲料组，每组各16只。常规饲料硒含量为101.500μg/kg，低硒饲料硒含量为1.118μg/kg。大鼠低硒动物模型建造成功后，常规饲料组分为常规组和常规+T-2组，低硒饲料组分为低硒组和低硒+T-2组，每组8只。 T-2毒素（100μg·kg-1·d-1）灌胃饲养，30 d后处死大鼠，取左侧膝关节，用免疫组织化学染色方法检测大鼠关节软骨细胞MMP-1和MMP-13的表达水平。体外培养人软骨细胞系C28/I2细胞，分成空载体组、MMP-13启动子载体组、MMP-13启动子载体+T-2毒素20μg/L组、MMP-13启动子载体+T-2毒素40μg/L组，将-1602/+20-MMP13-Luc启动子重组荧光素酶报导质粒以及空载体瞬时转染C28/I2细胞，24 h后，加入20μg/L和40μg/L T-2毒素，继续培养24 h，用荧光素酶报告基因检测T-2毒素对MMP-13启动子的作用。结果 KBD组儿童关节软骨表层和中层MMP-1表达阳性率[（29.73±10.12）%、（28.27±0.91）%]均高于对照组[（2.47±0.11）%、（0.00±0.00）%，P均<0.05]，KBD组儿童关节软骨表层和中层 MMP-13表达阳性率[（13.21±4.32）%、（41.85±6.32）%]明显高于对照组[（5.72±0.31）%、（0.00±0.00）%，P均<0.05]。低硒+T-2毒素组大鼠关节软骨MMP-13在表层和中层表达水平[（13.21±4.32）%、61.85±8.68）%]明显高于常规组和低硒组[（2.43±0.22）%、（5.89±0.69）%，（3.03±0.29）%、（25.99±0.57）%，P均<0.05]。 T-2毒素可以激活外源性MMP-13启动子在C28/I2细胞的表达，MMP-13启动子载体+T-2毒素20μg/L组、MMP-13启动子载体+ T-2毒素40μg/L组的MMP-13启动子表达活性（0.08278±0.00840、0.10335±0.01319）高于空载体组、MMP-13启动子载体组（0.02419±0.00096、0.04032±0.00356，P均<0.05）。结论 MMPs在KBD儿童以及低硒条件下T-2毒素中毒大鼠的关节软骨过量表达，T-2毒素对软骨基质的降解作用与T-2毒素上调软骨细胞MMP-13启动子活性有关，MMPs过表达参与KBD软骨破坏及其软骨细胞死亡过程。
目的：觀察基質金屬蛋白酶（Matrix metalloproteinases，MMPs）在大骨節病（Kashin-Beck disease， KBD）兒童軟骨和低硒條件下T-2毒素中毒大鼠軟骨中的錶達，以及T-2毒素對軟骨細胞MMP-13啟動子的激活作用，探討MMPs與KBD髮病的關繫。方法以5例尸檢KBD兒童（KBD組）和5例因車禍死亡或畸形手術的正常兒童（對照組）手指節軟骨為研究對象，採用免疫組織化學染色的方法，檢測軟骨中MMP-1和MMP-13的錶達。雄性健康SD大鼠32隻，按體質量採用隨機數字錶法分為常規飼料組、低硒飼料組，每組各16隻。常規飼料硒含量為101.500μg/kg，低硒飼料硒含量為1.118μg/kg。大鼠低硒動物模型建造成功後，常規飼料組分為常規組和常規+T-2組，低硒飼料組分為低硒組和低硒+T-2組，每組8隻。 T-2毒素（100μg·kg-1·d-1）灌胃飼養，30 d後處死大鼠，取左側膝關節，用免疫組織化學染色方法檢測大鼠關節軟骨細胞MMP-1和MMP-13的錶達水平。體外培養人軟骨細胞繫C28/I2細胞，分成空載體組、MMP-13啟動子載體組、MMP-13啟動子載體+T-2毒素20μg/L組、MMP-13啟動子載體+T-2毒素40μg/L組，將-1602/+20-MMP13-Luc啟動子重組熒光素酶報導質粒以及空載體瞬時轉染C28/I2細胞，24 h後，加入20μg/L和40μg/L T-2毒素，繼續培養24 h，用熒光素酶報告基因檢測T-2毒素對MMP-13啟動子的作用。結果 KBD組兒童關節軟骨錶層和中層MMP-1錶達暘性率[（29.73±10.12）%、（28.27±0.91）%]均高于對照組[（2.47±0.11）%、（0.00±0.00）%，P均<0.05]，KBD組兒童關節軟骨錶層和中層 MMP-13錶達暘性率[（13.21±4.32）%、（41.85±6.32）%]明顯高于對照組[（5.72±0.31）%、（0.00±0.00）%，P均<0.05]。低硒+T-2毒素組大鼠關節軟骨MMP-13在錶層和中層錶達水平[（13.21±4.32）%、61.85±8.68）%]明顯高于常規組和低硒組[（2.43±0.22）%、（5.89±0.69）%，（3.03±0.29）%、（25.99±0.57）%，P均<0.05]。 T-2毒素可以激活外源性MMP-13啟動子在C28/I2細胞的錶達，MMP-13啟動子載體+T-2毒素20μg/L組、MMP-13啟動子載體+ T-2毒素40μg/L組的MMP-13啟動子錶達活性（0.08278±0.00840、0.10335±0.01319）高于空載體組、MMP-13啟動子載體組（0.02419±0.00096、0.04032±0.00356，P均<0.05）。結論 MMPs在KBD兒童以及低硒條件下T-2毒素中毒大鼠的關節軟骨過量錶達，T-2毒素對軟骨基質的降解作用與T-2毒素上調軟骨細胞MMP-13啟動子活性有關，MMPs過錶達參與KBD軟骨破壞及其軟骨細胞死亡過程。
목적：관찰기질금속단백매（Matrix metalloproteinases，MMPs）재대골절병（Kashin-Beck disease， KBD）인동연골화저서조건하T-2독소중독대서연골중적표체，이급T-2독소대연골세포MMP-13계동자적격활작용，탐토MMPs여KBD발병적관계。방법이5례시검KBD인동（KBD조）화5례인차화사망혹기형수술적정상인동（대조조）수지절연골위연구대상，채용면역조직화학염색적방법，검측연골중MMP-1화MMP-13적표체。웅성건강SD대서32지，안체질량채용수궤수자표법분위상규사료조、저서사료조，매조각16지。상규사료서함량위101.500μg/kg，저서사료서함량위1.118μg/kg。대서저서동물모형건조성공후，상규사료조분위상규조화상규+T-2조，저서사료조분위저서조화저서+T-2조，매조8지。 T-2독소（100μg·kg-1·d-1）관위사양，30 d후처사대서，취좌측슬관절，용면역조직화학염색방법검측대서관절연골세포MMP-1화MMP-13적표체수평。체외배양인연골세포계C28/I2세포，분성공재체조、MMP-13계동자재체조、MMP-13계동자재체+T-2독소20μg/L조、MMP-13계동자재체+T-2독소40μg/L조，장-1602/+20-MMP13-Luc계동자중조형광소매보도질립이급공재체순시전염C28/I2세포，24 h후，가입20μg/L화40μg/L T-2독소，계속배양24 h，용형광소매보고기인검측T-2독소대MMP-13계동자적작용。결과 KBD조인동관절연골표층화중층MMP-1표체양성솔[（29.73±10.12）%、（28.27±0.91）%]균고우대조조[（2.47±0.11）%、（0.00±0.00）%，P균<0.05]，KBD조인동관절연골표층화중층 MMP-13표체양성솔[（13.21±4.32）%、（41.85±6.32）%]명현고우대조조[（5.72±0.31）%、（0.00±0.00）%，P균<0.05]。저서+T-2독소조대서관절연골MMP-13재표층화중층표체수평[（13.21±4.32）%、61.85±8.68）%]명현고우상규조화저서조[（2.43±0.22）%、（5.89±0.69）%，（3.03±0.29）%、（25.99±0.57）%，P균<0.05]。 T-2독소가이격활외원성MMP-13계동자재C28/I2세포적표체，MMP-13계동자재체+T-2독소20μg/L조、MMP-13계동자재체+ T-2독소40μg/L조적MMP-13계동자표체활성（0.08278±0.00840、0.10335±0.01319）고우공재체조、MMP-13계동자재체조（0.02419±0.00096、0.04032±0.00356，P균<0.05）。결론 MMPs재KBD인동이급저서조건하T-2독소중독대서적관절연골과량표체，T-2독소대연골기질적강해작용여T-2독소상조연골세포MMP-13계동자활성유관，MMPs과표체삼여KBD연골파배급기연골세포사망과정。
Objective To investigate the expressions of matrix metalloproteinases(MMPs) in Kashin-Beck disease(KBD) cartilage as well as in a KBD rat model of T-2 toxin poisoning under selenium deficient conditions, and to investigate the effect of T-2 toxin on MMP-13 expression in human chondrocytes in vitro in order to determine a possible mechanism underlying KBD. Methods Samples of articular cartilage were divided into 2 groups:controls(samples from 5 normal children, traffic accident or operation), and KBD(samples from 5 children with KBD, auctopsy). Thirty-two Sprague-Dawley rats were divided into two groups by body weight using random number table: normal diet group(n = 16) and selenium-deficient diet group(n=16). The selenium level in normal diet was 101.500μg/kg, and in selenium-deficient diet was 1.118μg/kg. Rats were fed for 4 weeks with selenium-deficient or normal diet, respectively. After successful build up of the low selenium rat model, normal diet group was then subdivided into 2 sub-groups: normal group(n = 8) and normal diet plus low T-2 toxin group(n = 8);and selenium-deficient diet group was also subdivided into 2 sub-groups: selenium-deficient group ( n = 8 ) and selenium-deficient diet plus T-2 toxin group ( n = 8 ) . T-2 toxin of 100 μg·kg-1·d-1 was administered by intragastric administration for 30 days. Then the rats were sacrificed, and their knee joints were processed for histopathological evaluation. MMP-1 and MMP-13 locations in cartilages were performed by inmmunohistochemistry. Human chondrocytes C28/I2 were cultured in vitro. The experiment was divided into 4 groups: empty vector plasmid group, MMP-13 promoter plasmid group, MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40 μg/L T-2 toxin group. MMP-13-luciferase reporter plasmid and vector plasmid were transiently transfected into C28/I2 cells for 24 hours, and then treated with 20 - 40 μg/L T-2 toxin for 24 hours. Transactivation of human MMP-13 promoter was analyzed using luciferase reporter constructs containing sequences spanning-1602 to+20 bp in C28/I2 chondrocytes. Results The percentages of chondrocytes staining for MMP-1 in the superficial and middle zones of KBD samples [(29.73 ± 10.12)%, (28.27 ± 0.91)%] were significantly higher than those of controls[(2.47 ± 0.11)%, (0.00 ± 0.00)%, all P < 0.05]. The percentages of chondrocytes staining for MMP-13 in the superficial and middle zones of KBD samples [(13.21 ± 4.32)%, (41.85 ± 6.32)%] were significantly higher than those of controls[(5.72 ± 0.31)%, (0.00 ± 0.00)%, all P<0.05]. The percentages of chondrocytes staining for MMP-13 in the superficial and middle zones of rats fed with selenium-deficient diet plus T-2 toxin group[(13.21 ± 4.32)%, (61.85 ± 8.68)%] were significantly higher than those of the normal and selenium-deficient groups[(2.43 ± 0.22)%, (5.89 ± 0.69)%, (3.03 ± 0.29)%, (25.99 ± 0.57)%, all P < 0.05]. Moreover, T-2 toxin activated the MMP-13 promoter detected with luciferase reporter assays in C28/I2 cells. The luciferase activities in MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40μg/L T-2 toxin group(0.082 78 ± 0.008 40, 0.103 35 ± 0.013 19) were significantly higher than those in empty vector plasmid group and MMP-13 promoter plasmid group(0.024 19 ± 0.000 96, 0.040 32 ± 0.003 56, all P < 0.05). Conclusions These data suggest that T-2 toxin induces cartilage matrix degradation through up-regulation of MMP-13 promoter expression. Increased MMPs staining intensity in KBD cartilage and the rat KBD model of T-2 toxin poisoning under selenium deficient conditions suggest that matrix degradation appear to be driven by MMPs activity.
