生理学报 Acta Physiologica Sinica, December 25, 2006, 58 (6): 573-576
研究论文
雌激素改善维生素D受体基因敲除雌性小鼠的骨、钙代谢
李冰燕，童 建，张增利*
苏州大学放射医学与公共卫生学院，苏州 215123
摘 要：以维生素D受体基因敲除雌性小鼠为模型，研究雌激素对骨、钙代谢的调节作用。外源性给予雌二醇一个月后， 观察小鼠血钙水平的变化，同时测定小鼠骨密度，并利用胫骨非脱钙 von Kossa染色观察钙化的骨小梁和未钙化的类骨质面积的变化。结果显示，外源性给予雌二醇一个月后，维生素 D受体基因敲除小鼠的血钙水平，从(2.10±0.37) mmol/L上升到(2.80±0.41) mmol/L (P<0.05); 骨密度从(0.037±0.006) g/cm2增高到(0.048±0.007) g/cm2，显著改善(P<0.05)；钙化骨小梁面积显著增加，未钙化的类骨质面积显著缩小。结果提示，外源性雌二醇对骨、钙代谢具有非依赖于维生素 D的正向调节作用。
关键词：维生素D受体；雌激素；钙平衡；骨骼矿化
中图分类号：R335.5
Exogenous estrogen improved calcium homeostasis and skeletal mineralization in vitamin D receptor gene knockout female mice
LI Bing-Yan, TONG Jian, ZHANG Zeng-Li*
Radiation Medical and Public School of Soochow University, Suzhou 215123, China
Abstract: It is well known that estrogen can inhibit bone absorption, decrease bone turnover and preserve bone mass. Some studies indicated that the effect of estrogen on calcium and bone is relative to vitamin D system, while others also reported that this effect of estrogen is independent of vitamin D. The genomic effect of 1α, 25(OH)2D3 is mediated by the nuclear vitamin D receptor (VDR) in a ligand-dependent manner. Hypocalcemia, hyperparathyroidism and osteomalacia are developed in VDR gene knockout mice. To determine whether the effect of estrogen on calcium and bone is dependent on VDR, this study examined the effect of exogenous estrogen on calcium and bone homeostasis in VDR gene knockout mice. Male and female wild type (WT) and VDR gene knockout heterozygous mice were mated each other and the genotyping of their offsprings were determined by PCR. At age of 21-day, WT and knockout mice were weaned and treated by one of three different regimens: (1) WT-vehicle group: the WT mice were injected with normal saline; (2) VDR KO-vehicle group: the VDR gene knockout mice were injected with normal saline; (3) VDR KO-E group: the VDR gene knockout mice were subcutaneously injected with estradiol, 0.2 μg per mouse, once daily for 1 month. The bone mineral density (BMD) of mice was measured using dual-energy X-ray absorptiometry. All mice were sacrificed at age of 50-day. Blood was taken by heart puncture under anesthesia and serum calcium was measured by autoanalyser.Tibiae were removed, fixed and embedded with the methylmethacrylate (MMA), and undecalcified sections were cut. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue. Static histomorphometric analyses were performed on those stained sections. The results showed that the serum calcium level was (2.10?.37) mmol/L in the VDR KO-vehicle mice and rose to (2.80?.41) mmol/L in the VDR KO-E mice although it was still lower than WT-vehicle mice [(3.10?.48) mmol/L]. BMD and mineralized trabeculer volume were increased significantly in VDR KO-E group compared with that in VDR KO-vehicle group. These results suggest that exogenous estrogen can improve calcium absorption and skeletal mineralization in a VDR-independent manner.
Key words: vitamin D receptor; estrogen; calcium homeostasis; skeleton mineralization
雌激素和维生素D都是重要的骨、钙代谢调节激素。它们调节骨、钙代谢的机理有相似的方面，也有不同的方面。同时，两者之间存在相互作用。目前，对雌激素和维生素 D在调节骨、钙代谢过程中的相互作用机理并不完全清楚。许多研究者认 为雌激素是通过调节肠道维生素D受体(vitamin D receptor,VDR)的表达来影响钙的吸收[1-3]，此外，雌激素还可以影响维生素D对成骨细胞的作用[4]。近年来，也有报道认为雌激素在调节钙吸收与骨代 谢方面还有完全不依赖于维生素D系统的途径[5,6]。因为VDR基因敲除(vitamin D receptor gene knockout mice, VDR-/-)小鼠不存在维生素D的基因组作用，所 以它是研究雌激素与维生素D系统相互作用的理想动物模型。本研究利用 VDR-/-小鼠探讨雌激素调节钙和骨代谢的机理。
1 材料与方法
1.1 动物及分组 VDR-/-小鼠由哈佛大学Dr. Marie教授赠送。以配制饲料喂养，饲料的主要成分是：维生素 D 2.2 IU/g、钙2%、磷1.25%。小鼠3周龄时断奶、鉴定基因型。12只3周龄的雌性VDR -/-小鼠分成两组，雌激素组(VDR KO-E)每日皮下注射雌二醇0.2 mg；溶剂对照组(VDR KO-vehicle)每日皮下注射生理盐水；用同窝野生型(wild type, WT)小鼠作WT对照组(WT-vehicle)，每日皮下注射生理盐水。给药后一个月取样分析。
1.2 小鼠基因型鉴定 在小鼠断奶分窝时，剪取约0.5 cm的鼠尾。用酚-氯仿法抽取基因组DNA。用PCR法鉴定小鼠基因型。WT小鼠VDR基因位点引物序列：正义链 5'-AGCCGGCAGGCCTGGCAGTGTC-3'；反义链：5'-CCACCTGCCTCTGCCTCCCAAG-3'；掺入的neomycin基因位点引物序列：正义链：5'-GGAGAGGCTATTCGGCTATGAC-3'；反义链：5'-CGCATTGCATCAGC CATGATGG-3'。退火温度均为63℃，均为30个循环。
1.3 血钙水平测定 麻醉小鼠，心脏穿刺取血，分离血清。使用全自动生化分析仪检测血清钙。
1.4 骨密度测定 使用XR-36型双能X线骨密度仪，将小鼠置于有机玻璃板上，用双能 X线骨密度仪测量。扫描速度为60.0 mm/s，步距为1.0 mm×1.0 mm，精确度和准确度均为1%。
1.5 von Kossa染色 固定过的胫骨脱水塑料包埋，制作非脱钙骨切片。von Kossa染色，甲苯胺蓝复染。采用HPIAS-1000高清晰度彩色病理图文分析系统分析钙化骨小梁面积(trabecular area, Tb.Ar, %)和未钙化的类骨质面积(osteoid volume/bone volume, OV/BV, %)。
1.6 统计学处理 实验数据均采用mean±SD，用Windows SPSS 10.0软件进行方差分析。
2 结果
2.1 小鼠基因型鉴定结果
小鼠基因组DNA的PCR结果见图1。WT小鼠(+/+)只出现一条带(382bp)。VDR-/-小鼠(-/-)也只出现一条带(290 bp)。这是因为在VDR-/-小鼠的基因组中，neomycin 基因取代了VDR基因的第三个外显子。杂合子(+/-)出现两条带。
2.2 血钙水平
外源性给予雌二醇一个月后，与溶剂对照组相比，雌激素组小鼠血钙水平显著增高[(2.80±0.41) vs (2.10±0.37) mmol/L，P<0.05，图2]，但仍然低于WT对照组小鼠[(3.10±0.48) mmol/L]。
2.3 小鼠骨密度
外源性给予雌二醇一个月后，与溶剂对照组小鼠相比，雌激素组小鼠骨密度显著增高[(0.037±0.06)vs(0.048±0.007)g/cm2，P<0.05，图3]，但是仍然低于WT对照组。
2.4 von Kossa 染色
VDR-/-小鼠雌二醇处理一个月后，与溶剂对照 组小鼠相比，钙化骨小梁面积显著增加，未钙化的类骨质面积显著缩小(图4)。
3 讨论
维生素D在机体骨和钙代谢过程中起着重要的作用。从皮肤和肠道吸收的维生素D经过肝脏和肾脏两次羟化后形成其活性形式——1,25二羟基维生素D3。后者通过位于细胞核的VDR发挥生理作用。VDR -/-小鼠表现出低血钙、骨质软化等佝偻病样表型。
雌激素是另一个对骨和钙代谢具有重要调节作用的激素。它可以增加碱性磷酸酶和I型胶原蛋白的表达，调节甲状旁腺激素对成骨细胞的反应性[7]。而雌激素缺乏可以导致破骨细胞活性增加、骨转换加快、肾脏钙排泄增加[8]和肠道钙吸收减少[9]。因此，雌激素缺乏是引起绝经后女性骨质疏松的主要 原因。目前，用雌激素替代治疗可以明显改善女性绝经后骨质疏松症[10]。但这些结论大多数来自于绝经后的妇女或卵巢切除后的动物模型。雌激素是否 可以改善VDR-/-小鼠的骨软化表型尚未见报道。曾 有文献报道雌激素可以增加VDR-/-小鼠肠道钙相关 基因的表达[5]。本实验发现，雌激素可以提高VDR-/-小鼠的血钙水平可能是与钙相关基因表达增加的结果。此外，雌激素治疗后，小鼠骨密度、钙化骨小梁面积明显增加，未钙化的类骨质面积显著缩小。这 说明雌激素改善了VDR-/-小鼠的骨软化表型。这种 现象可以从两个方面解释，一方面可能是雌二醇引起血钙水平升高后的间接作用；另一方面是雌二醇 的直接作用。
在研究了雌激素和维生素D各自对骨、钙代谢调节的基础上，雌激素和维生素D在调节骨、钙代谢方面的联合作用逐渐受到国内外学者的重视[11,12]。VDR-/-小鼠因为没有维生素D的基因组作用，所以 它是研究雌激素与维生素D相互作用的理想动物模型。关于维生素D是否参与了雌激素调节的骨、钙 代谢过程有两种观点。第一种观点认为雌激素对骨、钙代谢的调节是通过调节VDR表达而实现的 [13,14]。卵巢切除的病人肠道对活性维生素D的反应性下降，钙吸收障碍；补充雌激素后，指标恢复正常[15]。这说明雌激素缺乏造成的钙吸收障碍与肠道VDR减少有关。第二种观点认为雌激素对骨、钙的调节作用 与维生素D系统没有直接关系。Colin等发现卵巢切除后补充雌激素的大鼠肠道VDR表达没有显著改变[6]。本实验的结果支持第二种观点，因为雌激素明显 改善了VDR-/-小鼠的骨、钙代谢。这说明雌激素可以 不依赖于VDR而影响骨、钙代谢。
本实验结果证明，雌激素可以以维生素D非依赖的方式正向调节骨、钙代谢，但这并不排除维生素D介导的雌激素作用。在正常的机体内这两种作用方式可能同时存在，但是其详细机制尚不清楚。
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