Calbindin-D28k对小鼠骨质生长的影响

目的 探讨小鼠肾脏Calbindin-D28k(CaBP-D28k)在钙代谢中的作用.方法 构建维生素D受体(Vitamin D Recep-tor,VDR)/Calbindin-D28k双基因剔除小鼠模型,在普通及高钙饮食下,检测小鼠体重、摄食量、血尿参数值、下肢骨的长度、密度以及胫骨切片组织学染色等.结果 普通饮食下,VDR(-/-)/CaBP-D28k(-/-)小鼠发育更迟缓,体重比VDR(-/-)小鼠轻42%,尿钙的分泌更高,发展为更严重的佝偻病骨表型,表现为骨密度较低和骨小梁区生长板变形更多.高钙饮食下,VDR(-/-)及VDR(-/-)/CaBP-D28k(-/-)小鼠的血钙离子正常,VDR(-/-)/CaBP-D28k(-/-)小鼠的骨骼异常未得到完全纠正.结论 CaBP-D28k对骨质的生长发育起了重要作用,它对钙代谢的作用大都可被CaBP-D9k补偿.     

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。     

钙对染铅大鼠骨骼TRPV通路中相关基因mRNA表达的影响北大核心CSCD

目的研究不同剂量钙对染铅大鼠骨骼TRPV通路中相关基因mRNA表达的影响,从分子水平探讨其干预机制,为适量补钙预防铅中毒提供理论依据。方法选用(50±5)g的健康雄性SD大鼠60只,适应性喂养7d后,随机分为五组:对照组(基础饲料,去离子水)、染铅组(基础饲料,0.2%醋酸铅水)、试验Ⅰ组(基础饲料添加0.5%碳酸钙,0.2%醋酸铅水)、试验Ⅱ组(基础饲料添加1.0%碳酸钙,0.2%醋酸铅水)、试验Ⅲ组(基础饲料添加2.0%碳酸钙,0.2%醋酸铅水)。饲养60d后,采集大鼠后肢股骨,采用荧光定量RT-PCR法测定TRPV5、TRPV6、NCX1和PMCA1b基因的mRNA表达量。结果与对照组比较,染铅组TRPV5和TRPV6 m RNΑ表达量极其显著下降(P<0.01),而NCX1和PMCA1b m RNΑ表达量极显著上升(P<0.01);与染铅组比较,试验Ⅰ组TRPV5和TRPV6 m RNΑ表达量显著上升(P<0.05),而NCX1 m RNΑ表达量显著下降(P<0.05),试验Ⅱ组TRPV5和TRPV6 m RNΑ表达量极显著上升(P<0.01),而NCX1和PMCA1b m RNΑ表达量显著下降(P<0.01或P<0.05),试验Ⅲ组TRPV6 m RNΑ表达量显著上升(P<0.05),而NCX1 m RNΑ表达量显著下降(P<0.05)。结论适量钙的补充可减轻铅暴露对骨骼TRPV通路的干扰。     

维生素D受体敲除鼠肾脏、心、脑、结肠组织及血浆中肾素表达状况的研究

目的探讨维生素D(VD)受体敲除鼠肾脏、心、脑、结肠组织及血浆肾素表达情况。方法将C57BL/6源性小鼠分为野生组和维生素D受体敲除(VDR)组,脱颈处死后取相应的肾脏组织、心脏组织、脑组织、结肠组织及血液。应用免疫荧光、实时定量PCR等方法测定肾脏、心脏、脑、结肠及血浆中肾素水平。结果 VDR敲除鼠的肾脏、心脏、脑、结肠及血液中的肾素均强烈表达。结论 VD抑制肾素在肾脏、心脏、脑、结肠中的表达。系统血浆中的肾素可能由肾脏、心脏、脑、结肠等器官共同贡献,而不是单一的由肾脏产生。     

MARCO基因敲除小鼠模型鉴定

目的通过基因分型、基因测序与蛋白免疫印迹法鉴定MARCO基因敲除小鼠模型。方法将经成簇的规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)技术获得16种碱基敲除的MARCO~(-/-)亲代C57BL/6小鼠(雌、雄鼠各8只)与MARCO~(+/+)的亲代C57BL/6雌性小鼠(8只)合笼繁殖,获得子一代MARCO~(+/-)小鼠;再以MARCO~(+/-)小鼠自交得到MARCO~(-/-)足够数量的纯合小鼠。采用基因测序对小鼠基因型进行鉴定,采用蛋白免疫印迹法检测MARCO蛋白相对表达水平。结果经过1年的合笼繁殖,共繁殖5代,有5种敲除不同碱基数(-11、-25、-36、-46、-61 bp)的MARCO~(-/-)基因型稳定遗传;MARCO~(-/-)、MARCO~(+/-)、MARCO~(+/+)基因型比例约为1∶2∶1,符合孟德尔遗传定律。以MARCO(-11 bp)为例,共获得子五代(F5代)MARCO~(-/-)小鼠42只,MARCO~(+/-)小鼠92只,MARCO~(+/+)小鼠48只;上述3种基因型F5代小鼠出生后第4、6和8周的体质量分别比较,差异无统计学意义(P0.05)。与MARCO~(+/+)小鼠和MARCO~(-/-)(-36 bp)、MARCO~(-/-)(-61 bp)小鼠比较,MARCO~(-/-)(-11 bp)和MARCO~(-/-)(-46 bp)小鼠的MARCO蛋白相对表达水平均下调(P0.05),选择该2种小鼠模型作为MARCO基因敲除小鼠模型。结论成功鉴定MARCO基因敲除小鼠模型,为深入研究MARCO基因在小鼠矽肺纤维化中的作用及调控机制奠定基础。     

载脂蛋白E基因敲除小鼠血脂及病理组织学观察

目的观察载脂蛋白E（ApoE-/-）基因敲除小鼠血脂及病理组织学变化。方法选择24只ApoE-/-基因敲除小鼠,雌雄各半,另选同龄的雌雄各半C57BL/6J小鼠24只为对照,观察小鼠血脂及心脏病理形态学变化。结果 ApoE-/-基因敲除小鼠组总胆固醇（TC）、低密度脂蛋白胆固醇（LDL）水平较对照组明显增高（P〈0.05）;ApoE-/-基因敲除小鼠组高密度脂蛋白胆固醇（HDL-C）与对照组相比无差异（P〉0.05）;ApoE-/-基因敲除小鼠组动脉粥样硬化斑块大小较对照组明显增高（P〈0.05）;ApoE-/-基因敲除小鼠心肌发生粥样硬化病变改变。结论 ApoE-/-基因敲除小鼠更容易引起动脉粥样硬化病灶形成。     

α-细辛醚对Fmr1基因敲除小鼠的自主活动的干预作用

目的 探讨α-细辛醚对Fmr1基因敲除小鼠的自主活动的干预作用.方法 选取30日龄Fmr1基因敲除小鼠（KO小鼠）和FVB野生型小鼠（WT小鼠）为研究对象,将KO和WT两种类型的小鼠分别分为7小组,每组15只.其中1组作为对照组给予生理盐水,另外6小组连续腹腔注射不同剂量α-细辛醚（3 mg/kg、6 mg/kg、9 mg/kg、12 mg/kg、24 mg/kg、36mg/kg）5天,用药第5天进行自主活动行为学实验,观察α-细辛醚能否改善KO鼠的过度活动的表型.结果 在行为学自主活动实验中,KO鼠的活动次数比WT鼠的活动次数多,站立次数比WT鼠的站立次数少,差异具有统计学意义（P＜0.05）;使用α-细辛醚后,KO鼠的活动次数明显减少,站立次数明显增多,差异均具有统计学意义（P均＜0.05）.结论 α-细辛醚能改善KO鼠的的活动过度的表型,可能对Fmr1基因敲除小鼠有治疗作用.     

Fmr1基因敲除小鼠的被动回避行为及海马GAD的表达变化

目的 探讨Fmr1基因敲除小鼠的学习记忆功能和海马的谷氨酸脱羧酶(GAD)表达变化的关系.方法 对4周龄和6周龄的基因敲除型(KO)鼠和野生型(WT)鼠分别连续进行2天的被动回避行为的避暗和跳台实验观察后使用免疫印迹技术检测海马GAD的表达变化,根据所获得的数据进行多因素方差分析处理.结果 避暗实验中,4周龄和6周龄KO鼠潜伏期比WT鼠明显少(P＜0.05);而KO鼠的错误次数比WT鼠明显多(P ＜0.05);4周龄和6周龄KO鼠或WT鼠的潜伏期、错误次数无差异(P＞0.05),同周龄WT鼠第一天的潜伏期和错误与第二天相比无差异(P＜0.05).跳台实验中,4周龄和6周龄KO鼠的潜伏期比WT鼠明显少(P＜0.05);而KO鼠的错误次数比WT鼠明显多(P＜0.05);4周龄和6周龄KO鼠或WT鼠的潜伏期、错误次数无差异(P＞0.05);同周龄第一天KO鼠的潜伏期和错误次数与第二天相比无差异(P ＞0.05);同周龄第一天WT鼠的潜伏期和错误次数与第二天相比有显著差异(P＜0.05).GAD65/67蛋白在KO鼠海马表达比WT鼠增多(P＜0.05);随着周龄的增加,6周龄KO鼠或WT鼠的GAD 65/67蛋白表达比4周龄表达增多(P＜0.05).结论 4周龄和6周龄Fmr1基因敲除小鼠存在认知功能障碍,海马的GAD的表达异常变化可能介导Fmr1基因敲除小鼠学习记忆障碍.     

脂联素基因敲除小鼠血管钙化的研究

目的 观察脂联素基因敲除小鼠主动脉组织病理学和组织化学的特性. 方法 SPF级6周龄雄性脂联素基因敲除小鼠纯合子(Adiponectin-/-)30只随机分为5组,第1、2、3组分别给予普通膳食喂养10、20、30周,第4组每2周经颈静脉注射空白腺病毒载体(即β-半乳糖甘酶腺病毒载体)并予普通膳食喂养30周,第5组每2周经颈静脉注射重组脂联素腺病毒载体并予普通膳食喂养30周;另随机选取SPF、级6周龄雄性野生型小鼠(WT)6只为正常对照组,给予普通膳食喂养30周.采用酶法测定血糖浓度,放射免疫法测定血浆胰岛素和脂联素水平.分离小鼠胸主动脉置4%多聚甲醛固定,石蜡包埋,连续切片,行茜素红钙化染色.分离主动脉弓到髂骨分支的动脉,用比色法测定10%甲酸抽提的钙含量.超声破碎胸主动脉,用Bradford法测总蛋白含量,离心后取上清液采用对硝基苯酚法测定ALP活性. 结果经普通膳食喂养的各组脂联素基因敲除小鼠与野生型小鼠在体重、血糖、血胰岛素水平方面无明显区别.与野生型小鼠、喂养10及20周的脂联素基因敲除小鼠相比,喂养30周的脂联素基因敲除小鼠出现了轻度的动脉钙化,其动脉的钙含量及ALP活性升高.通过对脂联素基因敲除小鼠进行外源性脂联素的补充,抑制了动脉钙化的出现及ALP活性升高. 结论在普通膳食喂养30周后,脂联素基因敲除小鼠出现轻度的动脉钙化,其机制可能与动脉中升高的ALP活性有关;外源性脂联素的补充可抑制脂联素基因敲除小鼠动脉钙化的发生,提示脂联素为动脉钙化的保护因子.     

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。     

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.     

1,25 dihydroxycholecalciferol-mediated calcium absorption and gene expression are higher in female than in male mice

Recent advances in bone and calcium (Ca) metabolism have relied upon genetically modified mice. However, although human studies have identified gender as an important modulator of Ca metabolism, its effect on Ca metabolism has not been examined in mice. Here we examined basal and vitamin D-regulated Ca absorption (in situ ligated loops) and mRNA levels for the apical membrane calcium channel, TRPV6, and the calcium binding protein, calbindin D(9k) (CaBP) mRNA levels (real-time PCR) in duodenum of female and male mice. At 2 mo of age, females fed a 5 g Ca/kg diet had higher Ca absorption (62.3 +/- 4.8 vs. 47 +/- 3.6%) and TRPV6 mRNA levels than males even though plasma 1,25 dihydroxyvitamin D [1,25(OH)(2) D] was not different. In mice fed high (20 g/kg), normal (5 g/kg), or low (0.2 g/kg) Ca diets for 7 d to alter plasma 1,25(OH)(2) D (91 +/- 12, 322 +/- 25, and 587 +/- 43 pmol/L, respectively), the relation between Ca absorption (slope = 0.116 vs. 0.084, P = 0.021) or duodenum TRPV6 mRNA (slope = 0.042 vs. 0.025, P = 0.034) and circulating 1,25(OH)(2) D was steeper in females. After a single 1,25(OH)(2) D injection (200 ng/100 g body weight), peak induction of TRPV6 mRNA was 2-fold greater (at 6 h) and CaBP mRNA was 20% higher in females (at 16 h). Duodenal vitamin D receptor mRNA levels did not differ between genders. Our data indicate that female mice are more sensitive to changes in serum 1,25(OH)(2) D levels than males and that this must be considered when using mice to study calcium and bone biology.     

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.     

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。     

Long-Term Vitamin D3 Supplementation Does Not Prevent Colonic Inflammation or Modulate Bone Health in IL-10 Knockout Mice at Young Adulthood

Inflammatory bowel disease (IBD) is an idiopathic disease that can impair bone metabolism. Low vitamin D status has been implicated in its progress. This study used interleukin (IL)-10 knockout (KO) mice, that develop an intestinal inflammation when housed in a non-sterile environment, to determine if supplementation with vitamin D₃ throughout life could mitigate inflammation and attenuate the lower bone mineral content (BMC) and density (BMD), and bone strength. Female IL-10 KO mice were randomized 25 or 5000 IU vitamin D₃/kg diet throughout pregnancy and lactation. At weaning, offspring received the same or opposite diet as their mother until age three months. Body weight growth was similar among groups within a sex. At three months of age, there were no differences in inflammation and gene expression in the colon of offspring. Male offspring exposed to continuous 25 IU vitamin D₃/kg diet had lower (p < 0.001) colonic VDR expression and those exposed only to low vitamin D₃ until weaning had higher serum IL-6. There were no differences in femur or vertebral BMC, BMD or bone strength. In summary, long-term exposure to vitamin D₃ did not attenuate intestinal inflammation or preserve bone mineral or bone strength. Thus, supplementation with vitamin D₃ does not exert anti-inflammatory effects in this mouse model that mimics human inflammatory bowel disease.