摘要
目的观察慢性氟中毒对大鼠骨组织破骨细胞中基质金属蛋白酶9（MMP-9）蛋白、mRNA表达及骨细胞分化成熟过程的影响。方法36只健康SD大鼠，体质量100～120g，雌雄各半，按体质量采用随机数字表法分为3组（每组12只，雌雄各半）。对照组饮用自来水（含氟量〈1mg／L）：低氟组、高氟组分别饮用含5、50mg／L氟化钠的自来水。饲养8个月后，收集24h尿液，股动脉放血处死大鼠并取股骨。氟离子选择电极法检测各组大鼠尿氟、骨氟含量；酶联免疫吸附法（ELISA）检测大鼠血清抗酒石酸酸性磷酸酶5b（TRACP5b）含量；苏木精-伊红（HE）染色法观察大鼠股骨下段骨组织的病理学改变，应用图像分析系统进行形态计量学测量；免疫组织化学方法和原位杂交技术分别检测大鼠骨组织破骨细胞中MMP．9蛋白、mRNA的表达。结果大鼠尿氟、骨氟含量组间比较差异有统计学意义（F值分别为400．612、48．229，P均〈0．05）。其中低氟组、高氟组大鼠尿氟[（6．09±0．56）、（7．69±0．64）mg／L]和骨氟[（12．65±3．07）、（26．53±5．88）mg／kg]均高于对照组[（1．36±0．51）mg／L，（0．67±0．16）mg／kg，P均〈0．05]，且随染氟剂量的增加而逐渐升高（P均〈0．05）。大鼠血清TRACP5b含量组间比较差异有统计学意义（F=9．607，P〈0．05）。其中低氟组、高氟组大鼠血清TRACP5b含量[（1．86±0．13）、（1．92±0．22）U／L]均高于对照组[（1．57±0．20）U／L，P均〈0．05]。HE染色光镜下观察，染氟组大鼠表现为骨质硬化。大鼠破骨细胞中MMP-9蛋白、mRNA表达组间比较差异有统计学意义（F值分别为365．727、331．382，P均〈0．05）。其中低氟组、高氟组大鼠MMP-9蛋白（108．18±1．97、119．28±1．76）和mRNA（89．44±2．86、102．14±2．39）表达均高于对照组（97．22±2．24，78．51±1．16，P均〈0．05），且均随染氟剂量的增加而逐渐升高（P均〈0．05）。结论慢性氟中毒影响大鼠骨组织破骨细胞中MMP-9蛋白、mRNA的表达．并通过调节二者的表达水平影响破骨细胞的分化、成熟过程。
Objective To investigate the effects of chronic fluorosis on the expressions of matrix metalloproteinase-9 （MMP-9） mRNA and protein and the differentiation and maturation process of bone cell in the osteoclast of bone tissue of rats. Methods According to body weight, thirty-six healthy SD rats（body mass 100 - 120 g） were divided into three groups by random number table, twelve in each group, half male and half female. The rats of control group were given tap water（NaF 〈 1 mg/L）, and rats of low-fluorine and high-fluorine groups were fed with tap water containing 5 and 50 mg/L NaF to establish chronic fluorosis model. Rats were sacrificed after eight months; the contents of urinary fluoride in 24 hours and bone fluoride were analyzed by fluoride selective electrode. Serum content of tartrate resistant acid phosphatase 5b（TRACP5b）was detected by enzyme-linked immunosorbent assay（ELISA）. The paraffin section of bone tissue was stained by hematoxylin-eosin （HE） and pathological morphometry was observed under optical microscope. The protein and mRNA levels of MMP- 9 in the osteoclast of bones were detected by immunohistochemistry （IHC） and in situ hybridization （ISH）, respectively. Results The differences of fluoride contents of urine and bone in rats were statistically significant between groups（F = 400.612, 48.229, all P 〈 0.05）. Fluoride contents of urine and bone were increased in low- fluorine and high-fluorine groups[（6.09 ± 0.56）, （7.69 ± 0.64 ）mg/L, （12.65 ± 3.07）, （26.53 ± 5.88）mg/kg] compared to the control groups[ （1.36 ± 0.51）mg/L, （0.67 ± 0.16）mg/kg, all P 〈 0.05], and the fluoride contents of urine and bone were gradually increased with increasing fluoride doses（all P 〈 0.05）. The difference of TRACPSb content in serum was statistically significant between groups （F = 9.607, P 〈 0.05）, in low-fluorine and hlgh-fluorine groups, the TRACP5b contents[ （1.86 ±0.13）, （1.92 ± 0.22）U/L] were higher than that of control group [ （1.57 ± 0.20）U/L, all P 〈 0.05 ]. The pathological examination showed osteosclerosis in fluoride exposed groups. The differences of MMP-9 mRNA and protein expressions were Statistically significant between groups （F = 365.727, 331.382, all P 〈 0.05）. Compared to the control groups（97.22± 2.24, 78.51±1.16）, the expressions of MMP-9 protein（108.18 ± 1.97, 119.28± 1.76） and mRNA（89.44± 2.86, 102.14± 2.39） were increased（all P 〈 0.05）, and the expressions of MMP-9 mRNA and protein were gradually increased with increasing fluoride doses （all P 〈 0.05）. Conclusions Chronic fluorosis might influence osteoclast differentiation and maturation process through regulating the expression levels of MMP-9 protein and mRNA.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2014年第2期133-137,共5页
Chinese Journal of Endemiology
基金
国家自然科学基金（81260419）
科技部国际合作重点项目（2010DFB30530）
贵州省科技厅社会发展攻关项目（黔科合SY[2011]3006号）
关键词
氟中毒
牙
基质金属蛋白酶9
免疫组织化学
原位杂交
Fluorosis, Dental
Matrix metalloproteinase-9
Immunohistochemistry
In situ hybridization
