| Osteoporosis is a systemic metabolic bone disease, characterized by reduced bone mass and deterioration of bone structure, increased bone fragility and increased fracture risk.It is increasing to occur with the aging of the world population, which is called"soundless epidemic disease". It has been become as an important health and social problem in the world. However, it is not clear about its pathogenesis at present. It is considered that osteoporosis is affected by many factors and bone density is the golden diagnosed index to it. More and more researches showed that low bone density is the main risk factor of osteoporosis. Vitamin D is necessary in normal skeletal metabolism which mediates many biological effects. Belonging to the member of steroid and thyroid hormones receptor's superfamily, Vitamin D receptor (VDR) is a biomacromolecule in the nucleus which mediate 1,25(OH)2D3 to play its biological effects.In 1994 Morrison et al first reported that VDR gene polymorphisms was associated with bone density. Now VDR has been considered as one of the most general studied genes which associated to the bone metabolism. There are not conclusion about whether it is the main candidated gene to regulate bone mass,and whether its gene polymorphism is associated with bone density and might be the index to predict early osteoporosis.At present, there are not any saft and effective cure ways to help the pultaceous bone to recover from its previous structure.Especially, the pathogenesis of osteoporosis is very complicated which was affected by race, locality, environment et al. Osteoporosis can't be effectively resolved until it is distinguished by different race, locality and groups.It has been showed that VDR gene polymorphism varies in different country, race, even in different locality in the same country such as China. There are different results about the relationship between VDR polymorphisms and bone metabolism to offer the reference how to prevent osteoporosis locally. It is more important to how to prevent it than to treat it because it is impossible to irreverse osteoporosis in clinic. The purpose of this study is to research the effect VDR gene on bone metabolism with molecular biological method in order to provide the theoretical basis on preventing the local female osteoporosis and its complications.1. The history of past illness of the participants was inquired in detail and the number of their childbirth's was counted statistically. The participants'height and weight were measured regularly.The accuracy were 0.1cm and 0.1kg repectively. 200 participants were collected, between 24-51 years old, and the average age was 34.3±8.5 years old. The selected criteria was:people who have worked in the first hospital of Jilin university and have lived in Changchun at least 10 years with normal body condition, regular menstruation, healthy pre-menopausal women of Chinese Han population,no blood relationship between participants, no production and lactation within one year, no smoking, no bone surgical trauma, no ovary excision, no any kinds of disease affecting bone metabolism such as congenital bone aberration, osteomalacia, poliomyelitis, diabetes mellitus, bone tumor, severe liver and kidney disease, collagen disease, hyper- or hypoparathyroidism and hyper- or hypothyroid, other bone joint disease, not taking those medicine affecting bone metabolism within one year such as estrogen and active Vitamin D et al and not taking cortin medicine before.2. Twenty milliliter fasting venous blood samples in participants were taken and of 10 ml was dealt with anticoagulant EDTA Na2, serum was separated from it after centrifuged at 3000 rpm/min for 5 min, then stored in -20℃refrigerator. 186 effective samples.The polymorphism of their VDR gene restriction fragment length's were measured by molecular biology technique. There were many kinds of polymorphism with VDR gene, and this research studyed the polymorphism of VDR gene Bsm I. The genomic DNA was extracted from blood lymphocytes by a traditional hydroxybenzene-chloroform-ethanol procedure. Then DNA of them, was amplified by PCR method.Finally PCR amplifying products were identified with Bsm I restriction endonuclease. The results were recorded with EYE-Ⅱgel photograph system.3. Bone gla protein (BGP) level was measured by radio-immunity assay method in frigorific serum. All samples to measur were dealt in a time following to the kit instruction. The error within one batch was less than 2.61% and error between different batches was less than 5.74%.γradio-immunity assay instrument called DPC-γ-C12 model was produced by German.4. Bone mineral density was detected with DEXA method (Lunnar company, the United States) by a specialist at the department of nuclear medicine. Lumbar spine 2-4 normotopia bone density was detected and the results were analyzed by microcomputer.5. Data was done using SAS statistical package. The distribution of genotype and allele gene was statisticed by gene counting method. Chi-square test, Spearman ranking correlation and t test were used in this study. The results were recorded as x±s and there was statistic significance when P<0.05.6. Analysis correlation between VDR gene polymorphism, BGP and BND; between weight, age, height, and BGP,BMD; between delivery frequency and BGP, BMD.7. Results(1) PCR products were distributed with restriction endonuclease Bsm I.Genotype Bb of them was 23/186 examples, about 12.4%, bb was 163/186 examples, about 87.6%, and BB type was not observed.Compare them to those health women in U.S.A, Australia, France and Japan, Kazak in Xinjiang, Han in Xinjiang and in Shanghai. This research showed there was significant difference in the genotype VDR distribution frequency between those in Changchun district to those in the United States, Australia, France and Kazak in Xinjiang (P<0.05). There was no significant difference between those in Japan, Han population in Xinjiang and Shanghai district (P>0.05).(2) Two types of VDR genotype were found in this research: Bb and bb. The BGP average of genotype Bb was 4.60±1.14μg.L-1 (n=23), and that of genotype bb was 4.53±1.27μg.L-1 (n=163). There is no significant difference between them by t test (P>0.05).(3) The BMD average of genotype Bb was 1.19±0.09 g.cm-2 (n=11), and that of genotype bb was 1.15±0.11g.cm-2 (n=41). There is no significant difference between them by t test (P>0.05).(4) There is no significant difference between weight, age, height and BGP by the linear regression analysis (P>0.05).(5) There is no significant difference between weight, age, height and BMD by the linear regression analysis (P>0.05).(6) The correlation between childbirth frequency and BGP was analysized and showed that in 186 participants, BGP average was 4.76±1.29μg.L-(1n=67), 4.46±1.13μg.L-1 (n=105), 4.29±1.19μg.L-1 (n=105) and 4.74±2.23μg.L-1 (n=2) when their childbirth frequency was 0, 1, 2 and 3 respectively. There is no significant correlation between childbirth frequency and BGP (r=-0.09, P>0.05).(7) The correlation between childbirth frequency and BMD was analysized and showed that BMD average was 1.15±0.08 g.cm-2 (n=14), 1.16±0.12 g.cm-2 (n=33), 1.17±0.06 g.cm-2 (n=4)and 1.14 g.cm-2 (n=1) when their childbirth number was 0, 1, 2 and 3 respectively. There is no significant correlation between childbirth frequency and BMD (r=0.06, P>0.05).(8) The linear correlation was analysized between BGP and BMD and showed that there is no significant correlation between them (r=-0.01, P>0.05).8. Conclusion(1) This study showed the gene polymorphism of VDR in pre-menopausal women of Han population in Changchun district. Most of them were bb type and few of them were BB type.This result was inconsistent with those of other district in China.(2) There was no significant correlation between weight, age, height, childbirth frequency,BGP, and BMD. (3) There was no significant correlation between Bsm I polymorphism of VDR gene and BGP, BMD in pre-menopausal women of Han population in Changchun district.This research indicated that:1 the distribution of VDR gene in different race and locality was different; 2 The expression of VDR genotype was affected by intensively environmental stimulation such as psychic and psychology factors, living habits, reasonable diet, quantificational exercises et al. VDR plays different roles in different groups, environment and physiological conditions. In summarize, bone mineral density and osteoporosis were not predicted only by VDR allelic genotype.Molecular biology technique was used in this study in order to keep up with the level of study about bone metabolism in the world. The previous researches about relationship between VDR gene and bone metabolism only were limited to the post-menopause women.This is the first research to study the mechanism of osteoporosis in women from Changchun district by taking the pre-menopause women as the subjects. This research demonstrated that osteoporosis was affected mainly by non-genetic factors, which further confirmed that osteoporosis could be prevented by adjusting bad living habits, incorrect diet and some disease and it could offer the theorial basis to prevent osteoporosis and its complications effectively in the local women. This study also asked the doctors to understand the pathogenesis of osteoporosis in special locality before treat them so that they could scientifically and effectively prevent osteoporosis. This research may have the far-reaching significance in improving public health, living level and saving enormous medical expenditures. |
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