【作者】 陶春香；

【导师】 苗登顺；

【作者基本信息】 南京医科大学 ， 人体解剖学， 2008， 硕士

【摘要】 为了研究钙敏感性受体（CaSR）和活性维生素D[1,25（OH）₂D₃]在钙磷平衡和骨骼代谢中的相互作用及机制,我们建立了CaSR和25羟维生素D-1-α羟化酶[1α（OH）ase]双基因敲除[CaSR^-/-1α（OH）ase^-/-]小鼠模型,比较分析了2周龄CaSR^-/-1α（OH）ase^-/-小鼠与相应单基因敲除及野生型（WT）同窝小鼠的表型差异。与先前的研究结果一致,约85%的CaSR^-/-小鼠在出生后两周内死亡,幸存至两周者体格小,体重轻,血钙明显升高,血磷降低,血甲状旁腺素（PTH）水平明显升高,甲状旁腺肥大。两周龄1α（OH）ase^-/-小鼠表现为轻度低钙低磷血症,血PTH升高50%,甲状旁腺中度肥大,但能存活到成年。CaSR^-/-1α（OH）ase^-/-小鼠生存期较CaSR^-/-小鼠延长,2周和4周龄CaSR^-/-1α(（OH）ase^-/-小鼠生存率分别为91%和42%。2周龄CaSR^-/-1α（OH）ase^-/-小鼠体重较CaSR^-/-小鼠明显增加,血钙正常,血磷较CaSR^-/-和1α（OH）ase^-/-小鼠低,血PTH水平较CaSR^-/-小鼠更高。这些结果说明,1α（OH）ase基因敲除能通过纠正CaSR^-/-小鼠的高钙血症而延长其存活期,但由于更严重的高PTH血症致使CaSR^-/-1α（OH）ase^-/-小鼠不能存活至成年。为了明确CaSR和1,25（OH）₂D₃在骨骼生长发育过程中的相互作用,我们通过影像学和组织学比较分析了2周龄不同基因型小鼠骨骼的表型差异。CaSR^-/-小鼠表现为严重的骨骼生长障碍,包括长骨短小,钙化的次级骨化中心缺如,骨密度降低,但干骺端小梁骨密度增加。1α（OH）ase^-/-小鼠长骨略短,钙化的骺端较小,骨密度轻度降低。CaSR^-/-1α（OH）ase^-/-小鼠长骨长度、皮质骨和小梁骨密度均较CaSR^-/-小鼠明显增加。这些结果说明1α（OH）ase基因敲除能够部分改变CaSR缺失引起的严重的骨骼生长发育。为了明确CaSR和1,25（OH）₂D₃在软骨内成骨中的相互作用,我们通过组织学和免疫组织化学的方法分析比较了2周龄不同基因型小鼠生长板的表型差异。CaSR^-/-小鼠表现为次级骨化中心形成延迟,增殖细胞核抗原（PCNA）阳性软骨细胞减少,软骨细胞肥大带明显增宽。1α（OH）ase^-/-小鼠生长板和肥大带宽度较WT小鼠稍有增宽。在2周龄CaSR^-/-1α（OH）ase^-/-小鼠,次级骨化中心已开始形成,肥大软骨细胞中已开始出现血管和成骨细胞。生长板和肥大带宽度较CaSR^-/-小鼠明显变窄,而PCNA阳性软骨细胞则较CaSR^-/-小鼠明显增加。鉴于甲状旁腺素相关蛋白（PTHrP）在软骨细胞增殖和分化中起有重要作用,我们通过PTHrP免疫组织化学染色发现PTHrP阳性细胞在CaSR^-/-小鼠明显减少,而在CaSR^-/-1α（OH）ase^-/-小鼠则较CaSR^-/-小鼠有所增加,但没有达到WT小鼠的水平。这些结果说明,1α（OH）ase基因敲除能够通过上调PTHrP,增加软骨细胞的增殖而起到部分纠正CaSR缺失引起的软骨内成骨的严重障碍。为了明确CaSR和1,25（OH）₂D₃在骨形成和骨吸收中的相互作用,我们通过组织学,组织化学和免疫组织化学及图像分析检测了两周龄不同基因型小鼠骨组织的表型差异。与WT小鼠相比,成骨细胞数、小梁骨容量和骨钙素（OCN）阳性面积在CaSR^-/-小鼠均明显增加;在1α（OH）ase^-/-小鼠则有所减少,而在CaSR^-/-1α（OH）ase^-/-小鼠增加更为明显。抗酒石酸酸性磷酸酶（TRAP）阳性破骨细胞数在CaSR^-/-小鼠增加,在1α（OH）ase^-/-小鼠减少;而在CaSR^-/-1α（OH）ase^-/-小鼠与WT小鼠相似。NF-κB受体活化素配体（receptoractivatior of NF-κB ligand,RANKL）阳性细胞和面积变化与成骨细胞的变化一致。这些结果说明,CaSR缺失引起的成骨细胞骨形成增加是由于PTH过代偿作用的结果。本研究进一步阐述了CaSR和1,25（OH）₂D₃在调节钙磷平衡,甲状旁腺细胞生长和PTH产生以及软骨内成骨和成骨细胞骨形成中的相互作用的机制,为后续研究和临床相关疾病的诊治提供了实验和理论依据。更多还原

【Abstract】 To assess mechanism of interaction between calcium sensing receptor（CaSR）and active vitamin D[1,25（OH）₂D₃]on calcium and phosphate homeostasis and on skeletal metabolism,we created a CaSR and 25-hydroxyvitamin D-1-hydroxylase[1（OH）ase]double gene knockout[CaSR^-/-1α（OH）ase^-/-]animal model and compared their phenotypes with those of each single gene knockout and wild-type animals at 2-weeks of ages.Our results confirmed that around 85%CaSR^-/-mice died at within 2 weeks after birth.Size of body and body weight were reduced markedly,serum calcium levels were raised,serum phosphorus levels were decreased and serum parathyroid hormone（PTH）levels were raised significantly,the size of parathyroid glands was enlarged in 2-week-old CaSR^-/-mice compared to their WT littermates.1α（OH）ase^-/-mice are viable until adult.Serum calcium and phosphorus levels were reduced,serum PTH levels were raised to 150%of WT mice and the size of parathyroid glands was enlarged moderately in 2-week-old 1α（OH）ase^-/-mice compared to their WT littermates. CaSR^-/-1α（OH）ase^-/-mice were survival longer than CaSR^-/-mice,their survival rate reached 91%and 42%at 2 and 4 weeks of ages, respectively,and their serum calcium levels were normalized.Size of body and body weight were increased,serum phosphorus levels were reduced,and PTH levels were raised and the size of parathyroid glands was enlarged in 2-week-old CaSR^-/-1α（OH）ase^-/-mice compared to age-matched CaSR^-/-littermates.These results indicate that deletion of 1α（OH）ase gene in CaSR^-/-mice makes animal survival longer due to rescued hypercalcemia,however,CaSR^-/-1α（OH）ase^-/-mice cannot survival until adult due to more severe hyperparathyroidism.To assess the interaction between CaSR and 1,25（OH）₂D₃ on skeletal growth and development,the skeletal phenotypes of mice with different genotypes were analyzed at 2-weeks of ages by radiography,micro-CT and histology.CaSR^-/-mice displayed severe skeletal growth retardation including shorter long bones without calcified secondary ossification and reduced bone mineral density （BMD）,however,BMD was increased in trabecular bone at the metaphyseal region.The length of long bones was slight shorter, calcified epiphysis was smaller and BMD was reduced in 1α（OH）ase^-/- mice compared to their WT littermates.The length of long bones and BMD in cortical and trabecular bone were increased significantly in CaSR^-/-1α（OH）ase^-/-mice compared to CaSR^-/-littermates.These results indicate that the deletion of 1α（OH）ase gene in CaSR^-/-mice can rescue partially severe skeletal growth retardation. To assess the interaction between CaSR and 1,25（OH）₂D₃ on endochondral bone formation,the phenotypes of growth plates in mice with different genotypes were analyzed at 2-weeks of ages by histology and immunohistochemical staining for proliferating cell nuclear antigen(PCNA and parathyroid hormone related peptide （PTHrP）.The formation of the secondary ossification was delayed, PCNA positive chodrocytes were reduced and the hypertrophic zone of growth plates was enlarged in CaSR^-/-mice.The thickness of growth plates and hypertrophic zone were slight enlarged in 1α（OH）ase^-/-mice. The secondary ossification appeared at 2 weeks of ages with vessels and osteoblasts among hypertrophic chondrocytes in CaSR^-/-1α（OH）ase^-/-mice.The thickness of growth plates and hypertrophic zone were reduced and PCNA positive chondrocytes were increased significantly in CaSR^-/-1α（OH）ase^-/-mice compared to CaSR^-/- littermates.In view of the fact,PTHrP plays an important role in the proliferation and differentiation of chondrocytes,we examined the expression of PTHrP in chondrocytes by immunostaining.Results showed that PTHrP positive chondrocytes were reduced markedly in CaSR^-/-mice compared to their WT littermates and were increased in CaSR^-/-1α（OH）ase^/-mice compared to CaSR^-/-littermates although they did not reached to WT levels.Consequently,the deletion of 1α（OH）ase gene in CaSR^-/-mice rescued partially severe skeletal growth retardation may mediated through the up-regulation of PTHrP which stimulates the proliferation of chodrocytes.To assess the interaction between CaSR and 1,25（OH）₂D₃ on osteoblastic bone formation and osteoclastic bone resorption,the phenotypes of bone tissues in mice with different genotypes were analyzed at 2-weeks of ages by histology,histochemical staining, immunostaining and image analysis.Osteoblast number,trabecular bone volume and osteocalcin positive areas were increased significantly in CaSR^-/-mice and were decreased in 1α（OH）ase^-/-mice compared to their WT littermates,whereas these parameters were further increased in CaSR^-/-1α（OH）ase^-/-mice even compared to CaSR^-/-littermates.The number of TRAP positive osteoclasts were increased significantly in CaSR^-/-mice and were decreased in 1α（OH）ase^-/-mice,was normalized in CaSR^-/-1α（OH）ase^-/-mice.Alterations of RANKL positive osteoblasts and positive areas were consistent with those of osteoblasts. Our findings imply that increased osteoblastic bone formation in CaSR deficient mice results from over compensation of PTH.This study further expound the mechanism of interaction between CaSR andl,25（OH）₂D₃ on calcium and phosphate homeostasis,the cell growth of parathyroid glands and PTH production,endochodral and osteoblastic bone formation.It provides experimental and theoretical evidence for subsequent research and the diagnosis and treatment of related diseases.更多还原