小鼠Calbindin-D28k基因对钙代谢的影响

目的：探讨Calbindin-D28k对钙代谢的影响及作用。方法：制备维生素D受体（VDR）／Calbindin-D28k双基因剔除小鼠模型，常规及高钙饮食下，检测小鼠体重、摄食量、血尿参数值及甲状旁腺大小等。结果：常规饮食下，双基因剔除小鼠发育更迟缓，体重比VDR单基因剔除小鼠轻42％。尿钙的分泌更高，并发展为严重的继发性甲状旁腺功能亢进。高钙饮食下，VDR及双基因剔除小鼠的血钙离子水平恢复正常。结论：CaBP-D28k对钙代谢平衡起了重要的作用，它的作用大都被CaBP-D9k代偿。     

上皮钙通道基因在维生素D受体和Calbindin-D28k双基因敲除鼠中的变化

目的研究上皮钙通道TRPV5和TRPV6基因与维生素D受体(VDR)和钙结合基因Calbindin-D28k的关系,以及肾钙的重吸收减少是否与TRPV5和TRPV6基因表达减少有关。方法制备VDR和CaBP-D28k双基因敲除小鼠,普通和高钙食物喂养下,检测野生型鼠(WT),CaBP-D28(-/-),VDR(-/-),和VDR(-/-)/CaBP-D28k(-/-)鼠的体重、摄食量及血尿参数值,用实时RT-PCR的方法检测各种鼠肾脏TRPV5和TRPV6的mRNA水平。结果普通饮食下,双基因敲除小鼠尿钙的分泌和血清甲状旁腺激素水平更高。TRPV5和TRPV6两者的表达在CaBP-D28k敲除鼠中均无变化,而在VDR敲除鼠和双基因敲除鼠中下调的幅度相当。高钙饮食下,VDR及双基因敲除小鼠的血离子钙水平正常。所有小鼠这两个基因的表达普遍降低,而在VDR和双基因敲除鼠中的表达更低。结论这些结果证实了上皮钙通道基因受钙和维生素D的调节,CaBP-D28k的缺失对两者无影响。肾钙的重吸收减少与TRPV5和TRPV6基因表达无明显关系。     

甲状旁腺素、维生素D_3对Caco-2细胞钙结合蛋白D9k mRNA表达的影响

目的探讨甲状旁腺素(PTH)对小肠细胞钙结合蛋白(CaBP)D9k mRNA表达的影响以及探讨PTH是否影响1,25-(OH)2-D3对Caco-2细胞钙结合蛋白D9k mRNA表达的促进作用。方法以人结肠癌上皮细胞株Caco-2细胞作为小肠细胞体外模型,PTH分三个剂量10-8、10-9和10-12mol/L分别干预Caco-2细胞5、10、20、40和80min;10-8mol/L1,25-(OH)2-D3干预Caco-2细胞2、4、8、16和24h,溶剂对照为0.1%乙醇;10-8mol/L1,25-(OH)2-D3联合以上三剂量PTH干预Caco-2细胞2、4、8、16和24h,溶剂对照亦为0.1%乙醇。运用RT-PCR方法进行CaBP-D9k mRNA测定,以GAPDH作为内对照。结果10-8mol/LPTH作用20min,10-12mol/L作用10min,CaBP-D9k mRNA表达量高于空白组,其余时间点低于空白组;10-8mol/L1,25-(OH)2-D3干预Caco-2细胞2、4、8、16和24h,CaBP-D9k mRNA的表达均高于溶剂对照(0.1%乙醇);与10-8mol/L1,25-(OH)2-D3单独作用比较,10-8mol/L1,25-(OH)2-D3联合以上三剂量PTH作用,CaBP-D9k mRNA相对表达量均低于单独作用的表达量。结论10-8mol/L、10-12mol/LPTH可能促进Caco-2细胞CaBP-D9k mRNA瞬时(1~20min)合成增加;10-8mol/L1,25-(OH)2-D3可明显诱导Caco-2细胞CaBP-D9k mRNA的表达;PTH可能抵消或者抑制1,25-(OH)2-D3促进Caco-2细胞CaBP-D9k mRNA表达的作用。     

高脂饮食对载脂蛋白E缺乏鼠维生素D受体表达及一氧化氮合酶活性影响研究

目的了解高脂饮食对载脂蛋白（apoE）缺乏鼠（apoE^-/-）维生素D受体（VDR）表达及内皮型一氧化氮合酶（eNOS）活性影响,探讨VDR在动脉粥样硬化（AS）形成的意义及可能机制。方法apoE^-／-小鼠与作为对照的C57BLP6J小鼠分别按数字表法分为正常食物组与高脂食物组,采用竞争蛋白结合放射免疫法检测小鼠血25-（OH）D水平,采用免疫荧光化学法及RT-PCR法检测小鼠主动脉VDR表达,应用硝酸还原酶法检测小鼠血一氧化氮（NO）含量和eNOS酶活力。结果高脂饮食进一步加重apoE-/-小鼠As病变、降低血25-（OH）D水平[血25-（OH）D：正常饲料C57BLP6J小鼠、高脂饲料C57BLP6J小鼠、正常饲料apoE。一小鼠、高脂饲料apoE-/-小鼠分别为[（26．44±1．28）ng／mL、（22．68±2．07）ng／mL、（1-7.46±4.2．22）ng／mL、（15．88±0．97）ng／mL,P〈0．01]。高脂饮食进一步上调apoE-/-小鼠主动脉VDR蛋白与mRNA表达水平[VDR蛋白分别为0．244±0．088、0．346±0．132、0．547±0．128、0．768±0．162,VDtlmRNA分另U为、0．228±O．08．3、0．375±0．103、0．451±0．117、0．597±0．131,P均〈0．01]。高脂饮食致apoE一小鼠血NO水平、eNOS酶活力明显增高[NO：（39．74±4．81）μmol／L、（48．1±5．24）ixmol／L、（67．34±6．14）tzmol／L、（86．74±8．05）txmol／L;eNOS：（8．6-t-O．77）u／L、（12．28±1．42）U／L、（15．96-t-O．92）U／L、（18．68±1．15）U／L,P均〈0．01]。血25-（OH）D水平与血浆中NO含量、eNOS酶活力及VDR表达呈负相关（P〈0．01）。结论apoE-/-小鼠血25-（OH）D水平降低,VDR表达量明显上调,血NO含量、eNOS酶活力增高,高脂饮食可进一步降低血25-（OH）13、NO水平,上调主动脉VDR表达,增加eNOS酶活力。高脂饮食、维生素D、apoE相互作用,影响As病变可能与NO、eNOS有关。     

活性维生素D_3及受体在肾脏病中作用

维生素D是人体内不可缺少的生物活性物质,也是必需维生素之一.有学者认为[1],维生素D是一种激素,因为它具有激素样生理特性.多数植物和动物都能在日光下合成维生素D.随着对维生素D体内代谢研究发现,其活性代谢产物-活性维生素D3,即1,25-二羟维生素D3[1,25-(OH)2D3],不仅在调节机体钙磷代谢平衡方面有重要作用,而且还对许多组织细胞增殖、分化、造血系统、免疫系统以及内分泌源性激素分泌也发挥着重要调节作用,同时它的生物效应由细胞内特异维生素D受体(VDR)介导[2].本文对1,25-二羟维生素D3与VDR在肾脏病方面作用研究进展综述如下.     

膳食钙降低饮食诱导肥胖大鼠体重的机制

目的 研究膳食钙对饮食诱导肥胖大鼠的治疗作用及其机制。方法 以雄性Wistar大鼠为实验对象，用高脂饮食诱导大鼠肥胖模型。将肥胖大鼠按体重随机分为4组：A组为基础饲料组，B组为高脂低钙组(含0．25％钙)，C组为高脂正常钙组(含0．5％钙)，D组为高脂高钙组(含1％钙)。于第10周末处死动物，计算脂／体比，测定血糖、血脂和激素水平。结果 高钙膳食降低饮食诱导肥胖大鼠的体重和体脂含量，改善血脂代谢紊乱状态和胰岛素抵抗；膳食高钙通过促进瘦素分泌，降低神经肽Y分泌，升高血中游离T3和生长激素水平，起到降低饮食诱导肥胖大鼠体重和体脂含量的作用。结论 高钙膳食可以影响血中瘦素、神经肽Y、T3和生长激的水平，从而降低饮食诱导肥胖大鼠的体重。     

γ-氨基丁酸拮抗2型糖尿病的作用及机制探讨

目的 探讨 γ-氨基丁酸(GABA)拮抗2型糖尿病(T2DM)作用及机制,为T2DM防治提供依据.方法 将高脂饮食联合链脲佐菌素建立的T2DM模型小鼠分为T2DM组和T2DM+GABA组,普通饲料喂饲的小鼠分为对照组和GABA组,每组8只.T2DM+GABA与GABA组小鼠饮用含2g/L GABA蒸馏水,对照组和T2D...     

饮食限制对高脂小鼠体重、血脂和学习记忆的影响研究

目的研究饮食限制对高脂小鼠体重、降血脂、学习记忆和抗氧化能力的影响。方法建立高脂小鼠模型,随机分高脂饲料对照组、普通饲料对照组、DR20%高脂饲料组、DR40%高脂饲料组、DR20%普通饲料组和DR40%普通饲料组,每组11只;饮食限制21 d后,测体重、脏器系数和血清中总胆固醇(TC)和甘油三酯(TG)含量。另建立高脂小鼠模型,随机分模型对照组、DR20%组、DR 40%组和DR60%组,每组10只,饮食限制21 d后,测定小鼠学习记忆能力及心、肝中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果各饮食限制组小鼠TC、TG、体重、脂肪系数和Lee`s指数均明显低于高脂饲料对照组(P<0.01);水迷宫结果显示,各饮食限制组小鼠潜伏均明显小于对照组(P<0.05),DR40%组小鼠学习记忆增强最明显;DR20%组小鼠心、肝中SOD活性均明显提高(P<0.05)。结论饮食限制具有减肥、降血脂作用,增强高脂小鼠的学习记忆和抗氧化能力,限食20%～40%最为有益。     

钙和维生素D抑制高脂饮食诱发的前列腺上皮细胞增生

本研究目的是观察饮食中添加钙和维生素D对小鼠前列腺上皮细胞增生的影响。将四周龄小鼠随机分成三组，分别给予对照AIN—76A饲料、高脂低钙低维生素D饲料（试验饲料Ⅰ）和高脂加钙和维生素D饲料（试验饲料Ⅱ）。饲养9周后终止试验，小鼠植入渗透泵，灌注溴脱氧尿苷（BrdU）72小时。结果显示，试验饲料Ⅰ组小鼠前列腺背叶上皮细胞BrdU标记指数与对照组比较明显升高（P＜0．05），而试验饲料Ⅱ组的BrdU标记指数与对照组相似（P＞0．05）。本实验研究发现证明，高脂低钙低维生素D饮食能诱发小鼠前列腺背叶上皮细胞增生，同时提示饮食中添加钙和维生素D有助于预防高脂低钙低维生素D饮食的这种不良作用。     

地中海贫血患儿维生素D水平及影响因素分析

目的了解我国地中海贫血儿童维生素D(vitamin D,Vit D)水平状况。方法 81例各型地中海贫血患儿纳入研究,收集临床资料,计算体质指数Z分值(body mass index Z-score,BMI-Z分值);测定血清25-(OH)D、钙和磷水平;采用PCR-RFLP方法分析Vit D受体(vitamin D receptor,VDR)基因Fok I、Bsm I、Apa I位点多态性。结果地中海贫血患儿25-(OH)D水平为(23.7±9.2)ng/mL,显著低于对照组的(28.3±10.5)ng/mL(P0.05);地中海贫血合并Vit D缺乏或不足的发生率为66.7%,比对照组高;重型地中海贫血Vit D缺乏或不足者检出率88.9%,中间型64.0%,轻型48.3%。所有患儿血清钙、磷浓度均正常。BMI-Z分值≤-1与Vit D缺乏或不足相关,而VDR基因多态性、性别及居住地并非危险因素。结论地中海贫血患儿Vit D缺乏或不足发生率高,且与贫血严重程度以及营养状态相关。     

Vitamin D receptor null mutant mice fed high levels of calcium are fertile

Vitamin D receptor (VDR) null mutant mice provide a model to investigate the possible effect of vitamin D on female reproduction. Infertility in these mice has been reported but it is uncertain whether the infertility results from a lack of VDR or from the hypocalcemia that results from a lack of VDR. VDR null mutant mice and wild-type controls were fed a nonpurified, high calcium or medium calcium diet, plus a diet containing lactose and their reproductive efficiency was examined. VDR null mutant mice fed a nonpurified diet were hypocalcemic and were found to be largely infertile with 14% fertility, while the fertility percentage of normocalcemic VDR null mutant mice and wild-type mice was between 86% and 100%. A high calcium or medium calcium diet maintained 100% fertility in the VDR knockout mice; removal of the lactose from this diet did not diminish reproductive capability. Reproductive capacity of VDR null mutant mice was analyzed when they were fed purified diets containing 0.02-2% calcium. Mutant mice fed a low calcium diet (0.47%) had a lower reproductive efficiency than VDR null mutant mice fed a diet that resulted in normal serum calcium concentrations. Thus, high dietary calcium levels are required for normal reproduction in VDR null mutant female mice. It seems that the defect in reproduction reported previously for VDR null mutant mice is not the lack of a direct effect of 1,25-dihydroxycholecalciferol on reproductive function but is the result of hypocalcemia.     

Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA

To study the role of calbindin D(9k) (CaBP) and epithelial calcium channel ECaC2 in intestinal calcium (Ca) absorption, vitamin D receptor knockout (KO) and wild-type (WT) mice were fed either 0.5% Ca or a 2.0% Ca rescue diet starting at 21 d of age. Ca absorption and parameters involved in this process were measured at 60 or 90 d of age. Compared with WT, KO mice fed the 0.5% Ca diet had higher plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], and lower plasma Ca and insulin-like growth factor-I (IGF-I). Duodenal Ca absorption (% Ca absorbed) in KO mice was reduced 71% relative to WT mice and was associated with 55% lower CaBP mRNA, 47% lower CaBP protein and 95% lower ECaC2 mRNA levels. Compared with WT mice, the percentage of Ca absorbed in KO mice fed the 0.5% Ca diet was inappropriately low for the level of duodenal CaBP. The 2% Ca rescue diet normalized plasma Ca, prevented osteomalacia, increased growth and plasma IGF-I levels, but did not normalize plasma PTH or 1,25(OH)(2)D(3) in KO mice. In addition, the relationship between CaBP protein and the percentage of Ca absorbed was normalized, whereas ECaC2 mRNA fell to near zero. Our data demonstrate that higher CaBP levels do not ensure high rates of duodenal Ca absorption and that transcellular Ca absorption can occur even when ECaC2 gene expression is very low. In addition, our data suggest that the 2% Ca diet promotes a vitamin D receptor-independent anabolic effect on bone formation and calcium absorption, leading to improved calcium balance even in the presence of high PTH levels.     

High dietary vitamin D prevents hypocalcemia and osteomalacia in CYP27B1 knockout mice

Mice lacking 25-hydroxycholecalciferol [25(OH)D]-1alpha-hydroxylase (CYP27B1) are growth retarded, hypocalcemic, and have poor bone mineralization. We tested whether high dietary cholecalciferol (VD3) could exert effects in the absence of CYP27B1 in vivo. Weanling male wild-type (WT) and CYP27B1 knockout (KO) mice were fed either a 2% calcium (Ca), 20% lactose rescue diet or an AIN93G diet (0.5% Ca, 0.4% phosphorus) containing 1000 (1K, the rodent requirement, 25 microg), 10,000 (10K, 250 microg), or 20,000 (20K, 500 microg) IU VD3/kg diet until 12 wk when blood and tissues were taken. Serum 25(OH)D was >90 nmol/L in the 1K diet group and increased >4-fold in mice fed 10K and 20K diets. The 1K diet impaired growth and caused hypocalcemia in KO mice; the 10K and 20K diets were as effective as the high Ca rescue diet in preventing these outcomes. High VD3 restored expression of vitamin D-regulated genes in intestine (calbindin D(9K)) and kidney (CYP27B1, 24-hydroxylase, calbindin D(9K)) of KO mice. Micro-computed tomography of femora revealed complete recovery of cortical bone in KO mice fed either the rescue or 10K diets but only partial recovery of trabecular bone measures (e.g. 40% lower bone volume, 20% lower trabecular thickness, and 23% increase in trabecular separation). These data show that very high serum 25(OH)D can influence Ca and bone metabolism independent of its conversion to 1,25 dihydroxycholecalciferol. However, neither high dietary Ca nor high dietary VD3 is sufficient to fully recover the phenotype of CYP27B1 KO mice.     

Effect of high vegetable protein diets on urinary calcium loss in middle-aged men and women

OBJECTIVE: To determine the effect of high-protein diets, which have recently been promoted for their health benefits, on urinary calcium losses and bone turnover in older subjects. DESIGN: Randomized controlled cross-over study. SETTING: Teaching hospital and university. SUBJECTS: Twenty hyperlipidemic men and postmenopausal women (age 56+/-2 y) completed the study. INTERVENTION: One-month test and control phases during which subjects consumed equi-energy metabolic diets high in calcium (1578 and 1593 mg/day, respectively). On the test diet 11% of total dietary energy from starch in the control bread was replaced by protein (wheat gluten), resulting in 27% of energy from protein on the test diet vs 16% on the control diet. MAIN OUTCOME MEASURE: Urinary calcium excretion. RESULTS: Compared with the control diet, at week 4, the test diet increased mean (+/-s.e.m.) 24 h urinary output of calcium (139+/-15 vs 227+/-21 mg, P=0.004). The treatment difference in urinary calcium loss correlated with the serum anion gap as a marker of metabolic acid production (r=0.57, P=0.011). Serum calcium levels were marginally lower 2.41+/-0.02 vs 2.38+/-0.02 mmol/l (P=0.075), but there was no significant treatment difference in calcium balance, possibly related to the high background calcium intake on both diets. CONCLUSION: In the presence of high dietary calcium intakes the vegetable protein gluten does not appear to have a negative effect on calcium balance despite increased urinary calcium loss.     

The effect of a high-protein, high-sodium diet on calcium and bone metabolism in postmenopausal women and its interaction with vitamin D receptor genotype

The influence of a high-Na, high-protein (calciuric) diet on Ca and bone metabolism was investigated in postmenopausal women (aged 50-67 years) who were stratified by vitamin D receptor (VDR) genotype. In a crossover trial, twenty-four women were randomly assigned to a diet high in protein (90 g/d) and Na (180 mmol/d) or a diet adequate in protein (70 g/d) and low in Na (65 mmol/d) for 4 weeks, followed by crossover to the alternative dietary regimen for a further 4 weeks. Dietary Ca intake was maintained at usual intakes (about 20 mmol (800 mg)/d). Urinary Na, K, Ca, N and type I collagen cross-linked N-telopeptide (NTx; a marker of bone resorption), plasma parathyroid hormone (PTH), serum 25-hydroxycholecalciferol (25(OH)D3), 1,25-dihydroxycholecalciferol (1,25(OH)2D3), osteocalcin and bone-specific alkaline phosphatase (B-Alkphase) were measured in 24 h urine samples and fasting blood samples collected at the end of each dietary period. The calciuric diet significantly (P<0.05) increased mean urinary Na, N, K, Ca and NTx (by 19 %) compared with the basal diet, but had no effect on circulating 25(OH)D3, 1,25(OH)2D3, PTH, osteocalcin or B-Alkphase in the total group (n 24). There were no differences in serum markers or urinary minerals between the basal and calciuric diet in either VDR genotype groups. While the calciuric diet significantly increased urinary NTx (by 25.6 %, P<0.01) in the f+ VDR group (n 10; carrying one or more (f) Fok I alleles), it had no effect in the f- VDR group (n 14; not carrying any Fok I alleles). It is concluded that the Na- and protein-induced urinary Ca loss is compensated for by increased bone resorption and that this response may be influenced by VDR genotype.