维生素D的受体基因多态性及其血清浓度对骨关节结核易感性的影响

发布时间：2018-05-05 20:42

本文选题：骨关节结核 + 维生素D　；参考：《南方医科大学》2017年硕士论文

【摘要】：一、研究背景近年来有研究发现,除环境与生活方式等外界因素外,个体的遗传背景是结核病发生的另一种影响因素。目前已经发现多个基因位点与结核病发病密切相关,其中维生素D受体(vitaminDreceptor,VDR)是其中主要的候选基因之一。迄今为止,已发现不少于25个VDR基因多态性位点,其中研究较多的包括ApaⅠ、Bsm Ⅰ、FokⅠ、TaqⅠ四个位点,是调节骨代谢的主要位点。此外,许多VDR目的基因是通过一个保守的内分泌途径来调节维生素D的代谢,所以VDR蛋白分子或其功能上的任何变化都可能影响维生素D的代谢,从而影响骨关节结核的易感性。目前有关VDR基因多态性对于肺结核易感性的研究在国内外已有较多报道,但VDR基因多态性对骨关节结核易感性是否有影响,国内外相关性研究较少。至今尚无针对广东地区汉族人群维生素D受体基因多态性与骨关节结核易感性的研究。同时,作为VDR的配体,维生素D的血清浓度是否影响骨关节结核的易感性,目前尚不明确。二、研究目的1、通过检测骨关节结核病人与非骨关节结核者的维生素D受体基因多态性,探讨维生素D受体基因多态性与中国广东地区汉族人群骨关节结核易感性的关系。2、通过检测骨关节结核病人与非骨关节结核者的维生素D血清浓度,探讨维生素D血清浓度与中国广东地区汉族人群骨关节结核易感性的相关性。三、材料与方法1、研究对象与分组1.1结核组收集2015年5月至2016年12月南方医院、广州市胸科医院、广州华侨医院、广州军区总医院、广州中医药大学第一附属医院、河源市中医院收治的广东地区汉族罹患骨关节结核患者133例,男85例,女48例,年龄8-84岁,平均45.5岁。术前血液化验指标及放射影像学符合骨关节结核的诊断,术后病灶病理切片证实结核病感染征象,病灶组织分离培养结核分枝杆菌阳性或抗酸染色阳性,纳入结核组。1.2对照组2015年5月至2016年12月,选取与病例组无血缘关系的130例广东地区汉族志愿者作为对照。入选者均来自河源市中医院健康查体者;年龄20-76岁,平均43.4岁;其中男性68例,女性62例。均经问病史查体及胸片检查均排除肺结核及骨关节结核病史。2、样本采集两组研究对象在次日凌晨空腹抽取外周静脉血2ml,取1ml置于EDTA抗凝管中,静置在室温(20-25℃C)1小时后,2000转/分离心10分钟,得到的血清置于-80℃C冰箱冻存待检。剩余1ml,提取VDR全基因组DNA,置于-80℃C冰箱待检。3、维生素 D 多态位点 rs7975232(aⅠ)、rs1544410(Bsm Ⅰ)、rs2228570(FokⅠ)、rs731236(TaqⅠ)的检测应用SNaPshot技术检测两组对象维生素D受体基因各位点多态性,并进行VDR基因分型。4、血清维生素D的测定检测血清25-羟维生素D(250HD)是目前评价血清维生素D状态的最佳方法。应用双抗体夹心法(Elisa)测定标本中人25-羟维生素D水平,评价人体血清维生素D的浓度。5、数据分析采用SPSS软件(version 20.0;SPSS,Chicago,IL)进行统计学分析。计量资料以均数±标准差(x±s)表示(本研究的计量资料符合正态分布),计数资料用频数表示。采用卡方检验比较分类变量及计数资料;采用独立样本t检验比较连续变量。双侧P0.05表示差异有统计学意义。四、结果1、Hardy-Weinberg遗传平衡检验根据Hardy-Weinberg遗传平衡定律计算对照组各基因型的预期值。经卡方检验各观察值与期望值之间分布近似,P值均0.05,提示对照组样本来自于遗传平衡群体,有较好代表性。2、一般情况比较两组研究对象在年龄、性别构成方面的差异无统计学意义(P0.05)。3、两组VDR基因型分布频率比较维生素D受体基因Apa Ⅰ位点的基因型分别为野生型AA和突变型Aa、aa,三种基因型在结核组和对照组中的分布差异有统计学意义(P0.05);其中结核组中AA基因型的分布频率为49.9%,显著高于对照组(33.1%),与Aa基因型相比,罹患骨关节结核风险更大(OR=1.924,P＜0.05,95%CI为1.141～3.245)。维生素D受体基因Bsm Ⅰ位点的基因型分别为野生型BB和突变型Bb、bb,但在结核组和对照组内未能测到突变型bb。两个基因型在两组内的分布差异无统计学意义(P0.05)。维生素D受体基因Fok Ⅰ位点的基因型分别为野生型FF和突变型Ff、f、,三种基因型在两组中的分布差异有统计学意义(P0.05);其中结核组中ff基因型的分布频率为22.6%,显著低于对照组36.2%,与FF、Ff基因型相比,罹患骨关节结核风险更小(OR=0.458,P0.05,95%CI 为 0.235～0.893;OR=0.549,P0.05,95%CI 为 0.306～0.982)。维生素D受体基因Taq Ⅰ位点的基因型分别为野生型TT和突变型Tt、tt,但在结核组和对照组内未能测到突变型tt。两个基因型在两组内的分布差异无统计学意义(P0.05)。4、两组血清维生素D浓度比较结核组与正常对照组的250HD浓度均数分别为25.72±6.21、27.27±5.53ng/mL,两组浓度的差异有统计学意义(P0.05)。五、结论1、维生素D受体基因多态性位点rs7975232(ApaⅠ)、rs2228570(FokⅠ)的基因型在两组间分布频率存在差异,提示其多态性可能与广东地区汉族人群骨关节结核相关;其中ApaⅠ-AA基因型可能是骨关节结核的易感基因型,而Fok Ⅰ-ff基因型可能是骨关节结核的保护基因型。rs1544410(Bsm Ⅰ)、rs731236(TaqⅠ)基因型分布在两组间不存在差异,提示其多态性可能与广东地区汉族人群骨关节结核无相关性。2、结核组患者血清25-羟维生素D浓度较正常组低,提示血清25-羟维生素D浓度可能与广东地区汉族人群骨关节结核发病相关。
[Abstract]:1. In recent years, studies have found that the genetic background of individuals is another factor affecting the occurrence of tuberculosis in addition to external factors such as environment and lifestyle. At present, multiple gene loci have been found to be closely related to tuberculosis, and vitamin D receptor (vitaminDreceptor, VDR) is one of the major candidate genes. Up to now, no less than 25 VDR gene polymorphisms have been found, and many of them include Apa I, Bsm I, Fok I, and Taq I, which are the main sites for regulating bone metabolism. In addition, many VDR target genes regulate the metabolism of vitamin D through a conservative endocrine pathway, so VDR protein molecules or their functional functions. What changes may affect the metabolism of vitamin D and thus affect the susceptibility of bone and joint tuberculosis. The research on the susceptibility of VDR gene polymorphism to tuberculosis has been reported at home and abroad, but the polymorphism of VDR gene has an impact on the susceptibility to tuberculosis of bone and joint, and there are few studies at home and abroad. So far, there is no reference to Guangdong. Study on the polymorphism of vitamin D receptor gene and susceptibility to joint tuberculosis in the Han population. At the same time, as the ligand of VDR, whether the serum concentration of vitamin D affects the susceptibility to bone and joint tuberculosis is not yet clear. Two. Objective 1 to detect the multiple vitamin D receptor genes in people with osteoarticular tuberculosis and non osteoarticular tuberculosis. The relationship between vitamin D receptor gene polymorphism and the susceptibility of bone and joint tuberculosis in Han population in Guangdong, China,.2. The correlation between the serum concentration of vitamin D and the susceptibility of human bone and joint tuberculosis in Guangdong area of China was investigated by detecting the serum concentration of vitamin D in patients with tuberculosis and non osteoarticular tuberculosis. Three, three, Materials and methods 1. The subjects and group 1.1 tuberculosis group were collected from May 2015 to December 2016 to collect 133 cases of bone joint tuberculosis in the Han nationality in Guangdong, Guangzhou Chest Hospital, Guangzhou Overseas Chinese Hos, Guangzhou Overseas Chinese Hos, the First Affiliated Hospital of Guangzhou University of Chinese Medicine, 85 men and 48 women. The age of 8-84 years old, average 45.5 years old. Preoperative blood test indexes and radiological imaging accords with the diagnosis of tuberculosis of bone and joint. The pathological sections of the lesion confirm the signs of tuberculosis infection after operation. The tissue isolation and culture of Mycobacterium tuberculosis positive or positive acid stain positive, included in the tuberculosis group.1.2 control group from May 2015 to December 2016, and there is no blood in the case group. 130 cases of Han volunteers in Guangdong area were selected as control. All the participants were selected from the health checkup in Heyuan Hospital of Traditional Chinese Medicine; the age 20-76 years old, with an average of 43.4 years old, including 68 males and 62 women, all excluded tuberculosis and the history of tuberculosis of bone and joint.2 through the examination of medical history and chest examination. The samples were collected in the morning empty in the morning of the next day. Abdominal vein blood 2ml was extracted and 1ml was placed in EDTA anticoagulant tube. After 1 hours at room temperature (20-25 C C), 2000 turns / separated heart 10 minutes. The obtained serum was placed at -80 C C refrigerator to be frozen to be checked. The remaining 1ml was extracted from VDR whole genome DNA, at -80 C C refrigerator to be tested.3, vitamin I polymorphic loci (Fok I), rs731236 (Taq I) detection and application of SNaPshot technology to detect the polymorphism of two groups of vitamin D receptor genes and VDR gene typing.4. Serum vitamin D determination and detection of serum 25- hydroxyvitamin D (250HD) is the best method to evaluate the status of vitamin D in serum. The level of human 25- hydroxyvitamin D was used to evaluate the concentration of vitamin D in human serum. The data analysis was analyzed by SPSS software (version 20; SPSS, Chicago, IL). The measurement data were expressed in mean + standard deviation (x +) (x +.), and the count data were expressed in frequency. The classification variables were compared by chi square test. And counting data, using independent sample t test to compare continuous variables. Bilateral P0.05 showed statistical significance. Four, results 1, Hardy-Weinberg genetic balance test calculated the expected values of each genotype in the control group according to the Hardy-Weinberg genetic balance law. The distribution of each observation value and the expected value was approximately 0.05, and the value of P was 0.05, The sample of the control group came from the genetic balance group and had a better representative.2. In general, there was no significant difference between the two groups of subjects in age and the gender composition (P0.05).3. The two groups of VDR genotype frequencies compared the genotype of the vitamin D receptor gene Apa I loci to the wild type AA and the mutant Aa, AA, and three bases, respectively. The distribution of the type in the tuberculosis group and the control group was statistically significant (P0.05); the frequency of AA genotype in the tuberculosis group was 49.9%, significantly higher than that of the control group (33.1%). Compared with the Aa genotype, the risk of osteoarticular tuberculosis was greater (OR=1.924, P < 0.05,95%CI 1.141 to 3.245). The genotype of the vitamin D receptor gene Bsm I loci Wild type BB and mutant Bb, BB, but there was no significant difference in the distribution of two genotypes of mutant bb. in the two groups (P0.05) in the tuberculosis group and the control group. The genotype of the vitamin D receptor gene Fok I loci were the wild type FF and the mutant Ff, F, and the differences in the distribution of the three genotypes in the two groups were statistically significant Significance (P0.05); the distribution frequency of FF genotypes in tuberculosis group was 22.6%, significantly lower than that of control group 36.2%. Compared with FF and Ff genotype, the risk of osteoarticular tuberculosis was less (OR=0.458, P0.05,95%CI was 0.235 ~ 0.893; OR=0.549, P0.05,95%CI was 0.306 to 0.982). The genotype of the Taq I loci of the vitamin D receptor gene was wild-type TT. And mutant Tt, TT, but in the tuberculosis group and the control group, the distribution difference of the mutant tt. two genotypes in the two groups was not statistically significant (P0.05).4. The concentration of serum vitamin D in the two groups compared with the normal control group was 25.72 + 6.21,27.27 + 5.53ng/mL, respectively, and the difference between the two groups was statistically significant P0.05. (P0.05). Five, conclusion 1, vitamin D receptor gene polymorphism site rs7975232 (Apa I), rs2228570 (Fok I) genotype distribution frequency differences between the two groups, suggesting that the polymorphism may be associated with bone joint tuberculosis in Guangdong Han population, and Apa I -AA genotypes may be the susceptible genotype of bone joint tuberculosis, and Fok I -ff. The genotype may be the protective genotype.Rs1544410 (Bsm I) of bone joint tuberculosis. The genotype distribution of rs731236 (Taq I) is not different between the two groups, suggesting that the polymorphism may be not related to the.2 of the bone and joint tuberculosis in the Han population of Guangdong, and the serum 25- hydroxyvitamine D concentration is lower than that of the normal group, suggesting the serum 25- hydroxyvitamin D. The concentration may be related to the incidence of osteoarticular tuberculosis in Han nationality in Guangdong area.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R529.2

【参考文献】