雌二醇对去卵巢大鼠基质GLA蛋白表达的影响

Effect of estrogen on the expression of matrix GLA protein in ovariectomized SD rats

目的 观察雌二醇对去卵巢SD大鼠基质GLA蛋白（MGP）表达的影响,探讨雌二醇在改善绝经后骨质疏松中的作用.方法 36只10月龄雌性SD大鼠,随机分为去卵巢组、雌二醇组、对照组,每组12只.去卵巢组、雌二醇组大鼠行双侧卵巢切除术,雌二醇组在去卵巢3周后给予苯甲酸雌二醇肌内注射;对照组仅切除卵巢附近的相当于卵巢大小的脂肪组织;各组大鼠每3周留取血清及尿液,至术后15周处死大鼠,取子宫、腰椎组织行HE染色观察病理学变化;双能X线骨密度仪（DEXA）测定大鼠腰椎骨密度;ELISA法检测血清MGP、雌二醇水平及尿液MGP水平;免疫组化法检测腰椎组织中未梭化MGP蛋白的表达,实时荧光定量PCR技术检测腰椎组织中MGP mRNA的表达.结果（1）术后15周,去卵巢组大鼠子宫内膜萎缩,腰椎骨小梁结构破坏最明显.术后15周血清雌二醇水平去卵巢组为 （454±66）pmol/L,对照组为（527±77）pmol/L,雌二醇组为（556±80）pmol/L,去卵巢组与其他两组分别比较,差异均有统计学意义（P＜0.05）.雌二醇组、去卵巢组、对照组骨密度分别为（0.279 ±0.024）、（0.263±0.030）、（0.295 ±0.024）g/cm2,去卵巢组与其他两组分别比较,差异均有统计学意义（P＜0.01）.（2）雌二醇干预后血清及尿液中MGP出现动态变化.去卵巢组及雌二醇组血清MGP水平均下降,术后9周去卵巢组下降至（104 ±6）ng/L,下降最明显,雌二醇组下降至（134±6）ng/L,两组比较,差异有统计学意义（P＜0.05）.对照组术前至术后15周尿液中MGP 水平无明显变化（P＞0.05）;雌二醇组从术后12周起尿液中MGP水平开始上升,至术后15周时为（110.0±3.4）ng/L,去卵巢组为 （86.5±2.5）ng/L,两组比较,差异有统计学意义（P＜0.05）.（3）免疫组化染色结果显示,去卵巢组腰椎组织中可见未梭化MGP蛋白呈棕色阳性表达,其他各组表达不明显.（4）腰椎组织中MGP mRNA相对表达水平在去卵巢组为1,雌二醇组为0.289±0.260,对照组为0.103 ±0.098,去卵巢组表达水平较其他各组明显增高,分别比较,差异均有统计学意义（P＜0.01）.结论 雌二醇可提高去卵巢大鼠MGP mRNA及蛋白的表达水平,可能在改善绝经后骨质疏松中发挥重要作用.

Objective To investigate the effect of estrogen on expression of matrix GLA protein （MGP）in ovariectomized Sprague-Dawley（SD）rats and the role of estrogen in improving postmenopausal osteoporosis.Methods Thirty-six SD female rats were allocated into 3 groups randomly,every 12 rats in ovariectomized group（OVX group）,estrogen group（E group）and control group（sham group）.Rats in OVX and E group all underwent bilateral ovariectomy,those rats in E group were given by estradiol benzoate intramuscularly after 3 weeks of ovariectomy.Rats in sham group underwent bilateral lipectomy near the ovary.All rats were kept the urine and the serum every three weeks and were sacrificed after 15 weeks.The pathology changes of uterus,lumbar vertebral bones were observed by immunohistochemistry.Bone mineral density（BMD）of lumbar vertebra of rats was determined by dual energy X ray absorptiometry（DEXA）.The content of MGP in serum and urine was determined by ELISA.Expression of underearboxylated matrix GLA Protein（MGP）was detected by immunohistochemistry.Relative quantification of MGP mRNA expression in lumbar vertebra bone was detected by Fluorescent real-time quantitative polymerase chain reaction.Results（1）After 15 weeks of ovariectomized,the endometrium of uterus and lumbar vertebra exhibit remarkable pathologic changes in OVX group.The serum estrogen of（454±66）pmol/L in OVX group were lower than in（527 ±77）pmol/L in sham group and（556 ±80）pmol/L in E group significantly （P 〈 0.05）.The BMD of lumbar vertebra of（0.263 ± 0.030）g/cm2 in OVX group were lower than （0.295 ±0.024）g/cm2 in sham group and（0.279 ±0.024）g/cm2 in E group significantly（P 〈0.01）.（2）The serum MPG protein in OVX group and E group showed decreased trends after ovariectomized,which were（104 ±64）ng/L in OVX group and（134 ±6）ng/L in E group at 9 weeks,which reached statistical difference（P 〈 0.05）.However,MGP in urine in sham group did not exhibit significant difference after 15 weeks of surgery（P 〉0.05）.The MGP in urine of E group showed increased trends after 12 weeks of surgery,which reached（110.0 ±3.4）ng/L at 15 weeks,in the mean time,it was found that（86.5 ±2.5）ng/L of MGP in urine in OVX group,which showed significant difference（P 〈 0.05）.（3）MGP could be observed in lumbar vertebra in OVX group by immunochemistry staining.In the other two groups,the expression of MGP was not dominant.（4）Relative quantification of MGP mRNA expression in lumbar vertebra was defined as 1 in OVX group,when compared with 0.289 ±0.260 in E group and 0.103 ±0.098 in sham group,the difference showed statistically significant（P 〈 0.01）.Conclusion Estrogen could increase the expression of MGP mRNA and protein in ovariectomized rats and might play an important role in improving postmenopausal osteoporosis.