［摘要］目的：研究雌激素受体（estrogen receptor,ER）基因多态性在中国北京地区绝经后的汉族妇女中的分布及其与骨密度的相关性。方法：采用聚合酶链式反应-限制性片段长度多态性（PCR-restriction fragment length polymorphisms,PCR-RFLP）方法研究ER基因的Xba Ⅰ和Pvu Ⅱ酶切多态性，检测骨密度，通过方差分析探讨ER基因的多态性分布与骨密度的关系。结果：ER基因Pvu Ⅱ酶切多态性分布与桡骨松质骨以及桡骨密质骨的骨密度之间不存在相关性，而ER基因Xba Ⅰ酶切多态性分布与桡骨松质骨以及桡骨密质骨的骨密度之间存在相关性，XX基因型骨密度值最低，xx基因型骨密度值最高。结论：ER基因Xba Ⅰ酶切多态性分布与桡骨松质骨以及桡骨密质骨的骨密度之间显著相关(P＜0.05)。本研究为探讨骨质疏松症在分子生物学范畴的发病机制及预防与预后提供了有益的依据。

［中图分类号］R322.65［文献标识码］A

［文章编号］1000-1530（2000）06-0508-04

Study on the association of estrogen receptor genotypes with bone mineral density in Chinese postmenopausal Han women in Beijing

GUAN Jing，DAI Zhao-Heng，SHEN Huan，TIAN Li，GAO Bo-Shan，YUE Ming-Gang

(Department of Gynecology and Obstetrics,Peking University People's Hospital,Beijing100044,China)

ABSTRACTObjective：To investigate the distribution of polymorphism of estrogen receptor (ER) gene in postmenopausal Han women in Beijing as well as its relationship with bone mineral density (BMD).Methods：Xba Ⅰ,and PvuⅡ polymorphisms of ER gene were studied by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) and BMD were determined by DEXA (dual-energy X-ray absorptiometry).The relationship between BMD and polymorphism of ER gene was studied by variance analysis.Results：Pvu Ⅱ polymorphism of ER gene was not associated with BMD of spongy and compact bone of radius;while Xba Ⅰ polymorphism of ER gene was associated with BMD of spongy and compact bone of radius.The lowest BMD was found with XX genotype while the highest BMD was found with xx genotype.Conclusion:There is high correlation between of Xba Ⅰ polymorphism and BMD of spongy and compact bone of radius.Our study suggested some bases to explore the pathogenesis of osteoporosis and to prevent the development of osteoporosis.

KEY WORDSReceptors,estrogen;Bone density;Postmenopause;Restriction fragment length polymorphisms

Bone mineral density (BMD) is influenced by genetic and environmental factors.Studies showed that the vitamin D receptor (VDR) accounted for 75% of genetic factors of BMD^［1］,besides,estrogen receptor (ER) also influenced BMD.It was found that Xba Ⅰ and Pvu Ⅱ polymorphisms of ER had significant influences on BMD of Japanese,and that PPxx genotype was associated with low BMD^［2］.

The change of the single basic group of ER gene intron Ⅰ leads to the recognition of genes by restriction endonuclease Xba Ⅰ and Pvu Ⅱ respectively.Through the recognition of genes by endonuclease,different alleles of different individual ER's are differentiated.

In this research,we studied the relationship between BMD and polymorphism of ER gene among 99 healthy postmenopausal Chinese Han women by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

1CLINICAL MATERIALS AND METHODS

1.1Subjects

99 healthy Beijing postmenopausal Han women between 49 and 55 years of age with no history of hormone replacement therapy (HRT) were studied.Women with diseases that affect BMD were excluded.

1.2Methods

1.2.1Bone DensitometryBMD was measured at spongy and compact bone of radius by dual-energy X-ray absorptiometry (Israel Dexa DX-10).

1.2.2Isolation of Genomic DNA 2 ml peripheral blood was used to isolate DNA with Genomic DNA Purification kit (Promega) as recommended.The purified DNA was resolved in Tris EDTA (TE) buffer.

1.2.3PCRGenomic DNA was amplified using polymerase chain reaction (PCR) with 10 mmol*L^－1 Tris-HCl,50 mmol*L^－1 KCl,2 mmol*L^－1 MgCl₂,200 μmol*L^－1 dNTP (Takara) and 0.2μmol*L^－1 of each primer.The oligonucleotide primers used to determine the Xba Ⅰ and Pvu Ⅱ polymorphisms in the ER gene were:forward,5′- CTGCCACCCTATCTGTATCTTTTCCTATTCTCC- 3′;reverse,5′-TCTTTCTCTGCC-

ACCCTGGCGTCGATTATCTGA-3′^［2］.The conditions of PCR cycle were:denaturation at 94℃ for 5 min followed 30 circles of (1) denaturation at 94℃ for 30 s;(2) annealing at 62℃ for 40 s;(3) extension at 72℃ for 60 s;extension at 72℃ for 5 min was performed after PCR cycle^［2］.

1.2.4GenotypingThe PCR products of 1.3 kb fragments were digested with the Xba Ⅰ and Pvu Ⅱ restriction endonucleases (Takara) and separated on agarose gel.PP and XX,signifying the absence of restriction sites on both alleles,showed 1.3kb fragment,and pp,signifying the presence of restriction sites on both alleles were digested into two fragments (0.85 kb and 0.45 kb).The cutting fragment of Xba Ⅰ polymorphism near the Pvu Ⅱ RFLP site revealed two fragments (0.9kb and 0.4 kb) labeled as xx.

1.3Statistical Analysis

To determine whether the proportions observed in our data were those to be expected in a random mating equilibrium population,they were explored using the χ² method under the Hardy-Weinberg law^［3］.Distribution of characteristics in each genotype and the effect of the polymorphism of the ER gene on BMD of each skeletal site was evaluated with one-way ANOVA test^［3］.A Р value less than 0.05 was considered statistically significant.

2RESULTS

2.1The ERG polymorphism in northera Chinese postmenopausal Han women in Beijing

The ditribution of Pvu Ⅱ RFLP was:PP 13,Pp 61,pp 25.The ditribution of Xba Ⅰ RFLP was:XX 7,Xx 48,xx 44.In the combination of two RFLPs,all nine genotypes were detected in our data (Figure 1 and Table 1).

2.2The ERG Pvu Ⅱ and Xba Ⅰ genotype of postmenopausal women in Beijing and the background material

The postmenopausal women's genotype and background materials were given in Table 2.There was no significant difference in age,age of menolipsis,height,and body weight among different groups.

2.3The relationships between ERG Pvu Ⅱ polymorphism and BMD in postmenopausal women in Beijing

Through this study the distribution of Pvu Ⅱ polymorphism was not found to be related to radii spongy bone and compact bone (P＞0.05,Table 3).

Figure 1The results of electrophoresis for ER gene Pvu Ⅱ and Xba Ⅰ endonuclease digestion

Table 1Genotypes at Pvu Ⅱ and Xba Ⅰ polymorphic sites

Pvu Ⅱ genotypes Xba Ⅰ genotypes XX Xx xx PP 4 7 2 Pp 2 35 24 pp 1 6 18

2.4The relationships between the distribution of ER Xba Ⅰ polymorphism and BMD in postmenopausal women

It was found that the distribution of Xba Ⅰ polymorphism w