骨内H型血管在去势骨质疏松症模型中的变化

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目的:通过建立并观察骨质疏松小鼠模型中骨内H型血管(CD31/EMCN染色双阳性)的表现,探讨骨密度与骨内H型血管的变化关系。方法:选取雌性8周龄野生型C57BL/6J小鼠30只,分为假手术组、切除卵巢组(ovariectomy,OVX组),每组15只。假手术组手术中仅暴露双侧卵巢,并切除其周围少许脂肪组织;OVX组手术中完整切除双侧卵巢。4周后取材股骨标本以无菌纱布包裹暂存于生理盐水中,以备Micro CT扫描观察骨密度及骨小梁显微结构的变化。取材胫骨标本并剔除其表面软组织,经固定、脱钙、脱水及包埋等处理,进行免疫荧光染色观察两组小鼠骨标本中H型血管的变化,检测两组数据并行统计学分析。结果:经Micro CT扫描,OVX组骨密度为(0.11±0.01)g/cmn 3,假手术组为(0.21±0.01)g/cmn 3,差异有统计学意义(n P=0.001)。骨小梁显微结构发生变化:OVX组骨小梁体积分数为11.52%±1.77%,假手术组为25.87%±1.31%,差异有统计学意义(n P<0.05);OVX组骨小梁数目为(1.67±0.33)个/mm,假手术组为(2.95±0.82)/mm,差异无统计学意义(n P=0.07);OVX组骨小梁厚度为(0.06±0.01)mm,假手术组为(0.07±0.01)mm,差异有统计学意义(n P=0.021)。OVX组骨小梁间隙为(0.29±0.15)mm,假手术组为(0.19±0.01)mm,差异有统计学意义(n P<0.05)。除两组间骨小梁数目无统计学意义以外,其余两组骨小梁参数间的差异存在统计学意义(n P<0.05)。两组小鼠的骨标本经免疫荧光染色的标准流程处理后,分别计算两组骨标本同一部位H型血管的显像面积,OVX组H型血管的面积占比为9.14%±0.99%,而假手术组H型血管面积占比为29.33%±1.22%,OVX小鼠胫骨干骺端H型血管较假手术组显著减少,两组间差异存在显著的统计学意义(n P<0.05)。n 结论:在切除小鼠双侧卵巢模拟绝经后骨质疏松症的模型中,骨密度及骨小梁显微结构发生变化的同时也存在骨内H型血管的改变,这提示H型血管可能参与了绝经后骨质疏松症的发生。“,”Objective:To explore the changes of bone mineral density (BMD) and type H vessel, which was recently identified as strongly positive for CD31 and Endomucin (CD31n hiEmcnn hi) in long bone from ovariectomized (OVX) mice compared withSham group.n Methods:C57BL/6Jwild-type mice were used for experiments and bone tissuewas collected. Eight-week-old female mice were randomly divided into bilateral ovariectomy (OVX) and a sham operation (Sham). The bilateral ovaries were exposed and removed in the OVX group. In the sham group, the ovaries were only exposed but left intact. After 4weeks, these mice were killed for experiment and the femurs were collected for Micro CT scanning in order to observe the changes of bone mineral density (BMD) and trabecular indexes, including bone volume (BV), total volume of interest (TV), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N). The fresh tibia of each mouse was fixed, decalcified, dehydrated and embedded for immunostaining. All experimental data were analyzed with n t-test.n Results:Mouse femora from two groups were dissected at 4 week time points, and the attached soft tissue was completely removed for Micro CT scanning. BMD in OVX is 0.11±0.01 g/cmn 3 and 0.21±0.01 g/cmn 3 in Sham, which indicated the BMD in OVX significantly decreased. The results showed significant difference between the groups (n P=0.001). The microarchitecture in trabecular bone changed. BV/TV in OVX is 11.52%±1.77% and 25.87%±1.31% in Sham, which indicated the BV/TV in OVX significantly decreased. The results showed significant difference between the groups (n P<0.05). Tb.N in OVX is 1.67±0.33/mm and 2.95±0.82/mm in Sham, which indicated the Tb.N in OVX slightly decreased. The results showed no significant difference between the groups (n P=0.066). Tb.Th in OVX is 0.06±0.01 mm and 0.07±0.01 mm in Sham, which indicated the Tb.Th in OVX significantly thinned. The results showed significant difference between the groups (n P=0.021). Tb.Sp in OVX is 0.29±0.15 mm and 0.19±0.01 mm in Sham, which indicated the Tb.Sp in OVX significantly increased. The results showed significant difference between the groups (n P<0.05). In the groups BMD decreased and trabecular microstructure was broken. Both BMD and trabecular indexes (BV/TV, Tb. Th, Tb. Sp) showed significant changes in OVX group compared with Sham (n P<0.05) except Tb.N. We next examined the expression of CD31 and EMCN via immunostaining in order to observe the changes of type H vessel.By immunostaining, the percentage of HV/TV in OVX group was 9.14%±0.99% and 29.33%±1.22% in the sham-operated mice. Dramatically decreased type H vessels in the metaphysis of OVX mice were observed compared with that of Sham control mice. The results showed significant difference between the groups (n P<0.05).n Conclusion:In this study, ovariectomized mice, a widely used model for postmenopausal osteoporosis, exhibited significantly reduced type H vessels accompanied by reduced BMD, which indicatedtype H vessel involved in the occurrence of postmenopausal osteoporosis.
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