遗传高钙尿结石大鼠肾组织钙敏感受体mRNA异常表达及其意义

目的 观察肾钙敏感受体（CaSR）在特发性高钙尿症（IH）发病中的作用。方法 雄性遗传性高钙尿结石（GHS）大鼠和正常野生型SD大鼠各6只，相同的饮食和饲养条件下，检测各组大鼠血Ca^2＋、P5^＋、1，25（OH）2D3和连续2个24h尿Ca^2＋的水平，使用半定量逆转录-聚合酶链反应（RT-PCR）检测肾组织钙敏感受体mRNA表达，PCR产物纯化后进行DNA直接测序。结果两组大鼠血Ca^2＋、P5^＋、1，25．（OH）2D3水平相似，均在正常范围内，而GHS组平均24h尿Ca^2＋排泄量为正常对照（NC）组的3．36倍[分别为（2．66±0．64）、（0．79±0．06）mg／d]，两组比较差异有统计学意义（t=5．82，P〈0．01）；GHS组CaSR mRNA平均相对吸光度（A）值为1．354±0．108，NC组为0．602±0．218，差异有统计学意义（t=6．19，P〈0．01）；CaSR基因第7外显子区域cDNA测序结果与Genebank（Accession：NM-016996）公布的碱基序列比较，GHS组有4只大鼠存在第3072位碱基G／A替换，使第1024位谷氨酸（Glu）变为赖氨酸（Lys），其余2只和NC组均与Genebank公布的碱基序列一致。结论 肾组织CaSRmRNA高表达及其第7外显子Glu1024Lys替换可能是特发性高钙尿症发病的分子机制之一。     

大鼠葡萄糖转运体3真核表达载体的构建及其在293细胞中的表达

目的构建大鼠葡萄糖转运体3（GLUT3）的真核表达载体，为进一步研究葡萄糖转运体3对缺血缺氧脑细胞的保护作用奠定基础。方法以逆转录一聚合酶链反应（RT-PCR）的方法从大鼠脑组织中获取GLUT3全长cDNA片段，将其克隆至真核表达质粒pcDNA3．1（＋）中，构建重组真核表达质粒pcDNA3．1（＋）-Glut3，随后用Lipofectamine^TM2000介导转染HEK293细胞，以RT-PCR的方法检测重组质粒在mRNA水平的表达，以免疫组织化学的方法检测重组质粒在蛋白水平的表达。结果克隆的大鼠GLUT3全长cDNA大小约1．5kb且无碱基突变，以构建的重组真核表达质粒pcDNA3．1（＋）-Glut3转染293细胞后，在基因及蛋白表达水平均检测到了葡萄糖转运体3的表达。结论成功构建了携带大鼠葡萄糖转运体3的真核表达载体pcDNA3．1（＋）-Glut3，且证实其可在293细胞中成功表达目的基因。     

PCR-RFLP检测维生素D受体基因启动子区多态性

目的建立一种简便、实用、重复性好的人维生素D受体（vitamin D receptor，VDR）基因5’端启动子区Fokl酶切位点多态性的检测方法，以利于VDR基因起始密码多态性与骨质疏松等疾病相关性的研究。方法应用聚合酶链反应（PCR）特异性扩增VDR基因5’端启动子区，扩增产物用限制性内切酶Fokl酶切，10％聚丙酰胺凝胶电泳观察酶切结果。结果应用PCR—RFLP法检测了98例健康汉族中老年人VDR基因型，FF、Ff、ff基因型分布频率分别为47％、43％、10％。结论该方法简便、准确、实用，适用于院校及医院实验室的研究和分子流行病学调查。     

健脾方对去势大鼠维生素D代谢的影响

目的采用放射免疫方法与分子生物学方法从VitD代谢的角度探讨健脾方防治原发性骨质疏松症的机制。方法建立卵巢切除致骨质疏松大鼠模型,运用骨组织病理学、血液生物化学等方法综合观察健脾方对模型大鼠骨密度、尿吡啶酚（PYD）和血清骨钙素（BGP）的影响;同时应用放射免疫方法与逆转录聚合酶链式反应观察健脾方对模型大鼠血清1,25（OH）2D3和小肠、肾脏组织维生素D受体基因（VDR mRNA）表达的影响。结果健脾方能升高模型大鼠的骨密度,降低模型大鼠血清1,25（OH）2D3的含量（P〈0.05）,同时能上调模型大鼠小肠、肾脏VDR mRNA的表达。结论健脾方能增强机体对血清1,25（OH）2D3反应敏感性,上调小肠与肾脏VDR mRNA的表达,调节VitD代谢。     

遗传高钙尿结石大鼠肾小管上皮细胞基底膜钙转运蛋白的异常表达及意义

目的 观察肾小管上皮细胞基底膜钙转运蛋白细胞膜钙泵1b(PMCA1b)和钠钙交换体.(NCX1)在遗传性高钙尿结石(GHS)大鼠肾组织的表达及其在特发性高钙尿症(IH)发病中的作用.方法 雄性遗传高钙尿结石大鼠和正常野生型SD大鼠各6只,采用实时荧光定量PCR检测PMCA1b和NCX1 mRNA表达,westem blot方法 检测其蛋白表达.结果 GHS大鼠与正常对照(NC)组大鼠的NCX1在mRNA水平和蛋白水平比较差异均无统计学意义(P＞0.05);GHS大鼠与NC大鼠的PMCA1b在mRNA水平比较差异无统计学意义(P＞0.05),GHS大鼠的PMCA1b蛋白平均相对吸光度值(A)为(0.18±0.05),NC组为(0.43±0.07),差异有统计学意义(P＜0.01).结论 PMCA1b蛋白表达下降可能是特发性高钙尿症形成的发病机制之一.     

MGP基因的重组及在大肠杆菌中的表达

目的重组骨质疏松候选基因基质Gla蛋白（MGP）基因使其蛋白在大肠杆菌中高效表达。方法利用反转录聚合酶链反应（RT—PCR）从正常人肺组织总RNA中扩增出MGP基因cDNA序列，与克隆pGEM—Teasy载体相连，测序为完整的编码序列后与表达载体pTrcHisB构建重组体，转化入大肠杆菌Top10后用IPTG诱导，Western bloting证实蛋白表达。结果克隆至pGEM-Teasy载体及pTreHisB载体中的MGP基因cDNA序列与基因库完全一致。转入大肠杆菌后经IPTG诱导有蛋白的表达，Western bloting证实诱导后2、3、4h蛋白的表达量显著增加。结论成功重组的人MGP基因，重组体在大肠杆菌内能成功高效地表达。IPTG诱导后蛋白的表达为时间依赖性。     

维生素D受体基因多态性对脊柱结核易感性的影响

目的：探讨维生素D受体（vitamin D receptor，VDR）基因多态性与中国湖南省汉族人群脊柱结核易感性的关系。方法：选取2004年10月至2006年2月我院收治的湖南省汉族新发脊柱结核患者42例（病例组）及志愿者64例（对照组），应用聚合酶链反应-限制性片断长度多态性分析技术检测两组对象VDR基因FokⅠ酶切位点多态性，并进行VDR基因分型。结果：VDR—FF、VDR—Ff与VDR—ff三种基因型在病例组和对照组中的分布频率分别为14．29％、35．71％、50％和23．45％、54．69％、21．86％；两组组间比较有显著性差异（P〈0．05）．其中VDR—ff基因型在病例组中的分布频率明显高于对照组，比值比（odds ratio，OR）为3．571（P〈0．05）．其95％可信区间（confidence interval，CI）为1．561～8.167。结论：VDR基因FokⅠ酶切值点多念性与湖南省汉族人群脊柱结核的易感性相关，VDR—ff基因型可能是其易感基因型。     

维生素D受体基因TaqⅠ和ApaⅠ位点单核苷酸多态性与特发性低枸橼酸尿症关系的研究

目的：探讨维生素D受体（VDR）基因TaqⅠ和ApaⅠ位点单核苷酸多态性与特发性低枸橼酸尿症的关系及其临床意义。方法：实验筛选出无特发性低枸橼酸尿症者50名及特发性低枸橼酸尿症患者21名．通过PCR—RFLP技术检测VDR基因TaqⅠ及ApaⅠ位点单核苷酸多态性，并分析其与特发性低枸橼酸尿症之间的相关性。结果：两组间VDR基因ApaⅠ位点各基因型频率差异无统计学意义（P〉0．05）；而TaqⅠ各基因型频牢差异有统计学意义（P〈0．05）．在特发性低枸橼酸尿症患者组中TT型较为多见。且两组人群中基因型为TT者21h尿枸橼酸含量明显低于同组的其他基因型（P〈0．05）。结论1特发性低枸橼酸尿症与VDR—TaqⅠ单核降酸多态性间存在遗传相关性，而与VDR ApaⅠ单核苷酸多态性关系不明显。VDR基因TaqⅠ位点的TT型基因有望成为特发性低枸橼酸尿症的遗传标志基因。     

肾癌G250抗原基因真核表达载体的构建和序列测定

目的构建肾癌G250抗原基因编码区序列的直核表达载体并进行序列测定。方法从肾癌组织中提取总RNA，采用RT-PCR技术扩增肾癌G250抗原的基因编码区序列，将PCR片段定向克隆至真核表达载体pcDNA3．0，并进行序列测定。结果肾癌组织RT-PCR扩增后，均产生特异性条带，位置在1000bp和1600bp之间。与预定相符。pcDNA3．0-G250分别用Hind Ⅲ、Xho Ⅰ双酶切后产生2条带。均与预定位置相符；抗原基因编码区序列与Genbank登录号BC014950文献报道的序列同源性为100％，目的基因与载体正确连接。结论成功克隆了肾癌G250抗原的基因编码区序列，并构建了其真核表达载体pcDNA3．0-G250，为核素标记的G250单克隆抗体在诊断和治疗的心用及G250基因修饰的树突状细胞疫苗的肾癌生物治疗提供了依据。     

人瘦素蛋白在大肠杆菌中的融合表达和纯化

目的构建人瘦素因子（Leptin）的原核表达质粒pGEX-4T-3-LEP，并诱导该基因在原核细胞中大量表达。方法采用逆转录-聚合酶链反应（RT—PCR）和DNA重组技术，从人皮肤成纤维细胞内将编码人Leptin的cDNA序列克隆至原核表达载体pGEX-4T-3上，选取阳性克隆扩增后，提取DNA进行酶切鉴定和测序。同时应用SDS-PAGE和蛋白质印迹方法对其表达产物进行特异性鉴定。结果克隆出的Leptin基因的cDNA由469bp组成，包括翻译起始密码子、编码序列和终止密码子等部分，含有Letinp基因的cDNA的表达质粒pGEX-4T-3在DH5a细菌体内能够表达，并且随着诱导时间的延长，Leptin基因的表达量增加，重组Leptin蛋白主要存在于包涵体中。结论Leptin基因可以在原核细胞中获得表达，为深入研究该蛋白在创伤愈合中的具体作用奠定了基础。     

Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status,insulin secretory capacity,and VDR genotype in Bangladeshi Asians

Associations have been reported between vitamin D receptor (VDR) gene polymorphisms, type 1 diabetes, insulin secretion, and the insulin resistance syndrome. As VDR polymorphisms have no known functional significance, these findings may implicate a variant of the VDR gene or a locus in linkage disequilibrium with the VDR. We have examined VDR mRNA and VDR protein levels in relation to VDR polymorphisms (41 Bangladeshi subjects) and analyzed insulin secretory capacity (143 Bangladeshi subjects), allowing for other known determinants. Peripheral blood mononuclear cells (PBMCs) from subjects who had been genotyped for BsmI, ApaI, TaqI, and FokI VDR restriction fragment length polymorphisms were used for both total VDR mRNA quantitation (using TaqMan) and measurement of VDR protein levels (using a specific micro-immunoassay). Stepwise multiple regression analyses were used (to P < 0.05) to analyze the data. For the insulin secretion index, the best-fit model (n = 143, P < 0.0001) gave age (P = 0.002), TaqI (P < 0.0001), and BMI (P = 0.001) as independent determinants; with the inclusion of VDR mRNA and VDR protein levels, VDR mRNA was the sole independent determinant (n = 41, P = 0.024). However, the best-fit model for VDR mRNA (P = 0.004) gave FokI (P = 0.044) and TaqI (P = 0.04) genotypes and insulin secretory capacity (P = 0.042) as independent determinants. For VDR protein levels, the best-fit model (P = 0.006) gave TaqI genotype (P = 0.005) and circulating 1,25-dihydroxyvitamin-D levels (P = 0.03) as independent determinants. In conclusion, these studies confirm an association between VDR polymorphisms and insulin secretory capacity and demonstrate the VDR genotype to be a significant determinant of VDR mRNA and VDR protein levels in PBMCs, providing functional support to previously described genetic associations with the VDR gene. Furthermore, VDR expression has been shown to be a determinant of insulin secretory capacity.     

Haplotype analysis of VDR gene polymorphisms: a meta-analysis

Introduction: Although many studies have addressed the relationship between multiple individual polymorphisms in the vitamin D receptor (VDR) gene and bone health, few have analyzed this data in terms of haplotypes. We performed a meta-analysis of studies with data on the BsmI, ApaI, and TaqI polymorphisms in order to (a) estimate haplotype frequencies, (b) determine linkage disequilibrium (LD), and (c) estimate the magnitude of the association between haplotypes and osteoporosis/bone mineral density (BMD). Methods: Haplotypes were inferred using the expectation-maximization algorithm (EM); log-linear models were used to determine association with osteoporosis; and regression analysis with variance components was used to determine association with BMD. Results: Our results indicate that the most common haplotype for the VDR gene, regardless of ethnicity, is baT, followed by BAt and bAT in Caucasians, and bAT and BaT in Asians. This indicates strong LD between the BsmI and TaqI polymorphisms. We demonstrate a gain in power when considering the haplotypes rather than the individual polymorphisms separately, i.e., although BsmI, ApaI, and TaqI were not significantly associated with osteoporosis on their own, the haplotypes Bat and BAt were significantly associated, with an OR of approximately 4. Conclusion: We have applied haplotype analysis to the VDR polymorphisms and bone measures. We also highlight a number of methodologic issues, including linkage disequilibrium, the robustness of the EM algorithm in this context, and the potential for exploring effect modification.     

维生素D受体基因多态性与白癜风的相关性研究

目的：探讨维生素D受体基因多态性与白癜风的相关性。方法：采用聚合酶链反应和限制性片段长度多态性方法，对749例白癜风患者和763例健康人的维生素D受体基因型进行分析。结果：白癜风患者维生素D受体BsmI、ApaI、TaqI位点基因型的分布与正常对照组相比有显著性差异，bb、aa、tt基因型在白癜风患者中频率较高，FokI位点基因型的分布与对照组无明显差异。单倍体型分析表明FokI位点和BsmI位点，BsmI位点和ApaI位点，BsmI和TaqI位点，ApaI位点和TaqI位点之间存在较强的连锁不平衡，白癜风患者中fbAT、FbAT和FbaT单倍体型的频率显著高于对照组。结论：维生素D受体基因多态性与白癜风有明显的相关性。携带维生素D受体基因纯合子bb、aa或tt基因型可能会增加对白癜风的易感性。     

维生素D受体激活剂在慢性肾病及其并发症中的作用

慢性肾脏疾病是常见的严承影响公众健康的疾病,可导致血清中活性维生素D下降,并发甲旁亢、肾性骨病以及心血管疾病等.最近的研究表明,维生素D受体激活剂对于慢性肾病及其并发症有显著的疗效,可减少此类患者的死亡率并提高其生存质量.本综述将探讨维生素D受体激活剂在慢性肾病及其并发症中的作用.     

维生素D受体激活剂在慢性肾病及其并发症中的作用

慢性肾脏疾病是常见的严承影响公众健康的疾病,可导致血清中活性维生素D下降,并发甲旁亢、肾性骨病以及心血管疾病等.最近的研究表明,维生素D受体激活剂对于慢性肾病及其并发症有显著的疗效,可减少此类患者的死亡率并提高其生存质量.本综述将探讨维生素D受体激活剂在慢性肾病及其并发症中的作用.     

VDR Genotype and Response to Etidronate Therapy in Late Postmenopausal Women

Twenty-four late postmenopausal women with osteoporosis were studied. The patients were separated in three subgroups according to the BsmI polymorphism of the vitamin D receptor (VDR) gene: BB (n= 8), Bb (n = 10) and bb (n = 6). They did not differ in age (mean ages were 66.0 years, 65.9 years and 63.9 years, respectively), years after menopause (18.7 years, 18.1 years and 18.4 years) or body weight (64.9 kg, 65.3 kg and 63.8 kg), the variables known to be associated with bone mineral density (BMD). The results show that the response to antiresorptive bisphosphonate therapy in combination with calcium supplementation is modified by VDR genotype. The lumbar spine BMD increased significantly faster in the BB and Bb groups (7.3% and 7.0%, respectively) compared with the bb group (2.5%) during 1 year of cyclic etidronate therapy (400 mg/day) and calcium supplementation (1000 mg/day). The biochemical marker of bone resorption (urinary hydroxyproline excretion) as well as the bone formation marker (serum levels of osteocalcin) decreased during the treatment. With respect to VDR genotype, a significantly higher decrease in osteocalcin level was observed in bb as compared with BB subjects. We conclude that the VDR genotype is involved in an individual’s response to cyclic etidronate therapy with calcium supplementation. Received: 12 December 1998 / Accepted: 18 March 1999     

Effect of VDR gene polymorphisms on osteocalcin secretion in calcitriol-stimulated human osteoblasts

BACKGROUND: The impact of vitamin D receptor (VDR) gene polymorphisms in bone metabolism remains controversial. Some authors have found a beneficial effect of some VDR gene polymorphisms, while others found no differences, or even a lower bone mass in subjects with the same type of polymorphisms. The aim of this study was to assess if the VDR gene polymorphisms could have an effect on the calcitriol-stimulated osteocalcin in human osteoblasts. METHODS: Osteoblasts were obtained from human femoral necks replaced because of osteoarthritis. Bones were cut into pieces of 1 to 2 mm and placed in a nylon mesh. After the migration of osteoblasts, the pieces were collected and cultured with different concentrations of calcitriol (10(-8), 10(-9), and 10(-1)0 mol/L). After 48 hours of incubation with calcitriol, the osteocalcin secreted into the medium (corrected by either total proteins or total DNA content) was measured. The DNA was extracted from the osteoblasts, amplified by polymerase chain reaction (PCR), and analyzed for target sequences sites of the BsmI, ApaI, TaqI, and FokI restriction enzymes. RESULTS: The response observed in osteocalcin secretion in the bb or TT genotypes doubled the response observed in the BB or tt genotypes (calcitriol 10(-8) and 10(-9) mol/L). A slight trend was also observed with the aa genotype. Men showed higher levels of osteocalcin secretion than women. Age did not show any influence in osteocalcin secretion. CONCLUSION: VDR alleles and gender demonstrated an effect on the osteocalcin secretion. BB or tt genotypes, and also the "A" allele, showed the lowest calcitriol-stimulated osteocalcin secretion.