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2018, 35(6):531-535.doi:10.13213/j.cnki.jeom.2018.17626

Micro-CT study on tibia of diabetic mice post cadmium exposure

Department of Labor Hygiene and Environmental Health, School of Public Health, Soochow University, Suzhou, Jiangsu 215123, China

Accepted: 2017-11-17;  Published: 2018-07-06

Corresponding Author: LI Bing-yan, Email: bingyanli@suda.edu.cn;   ZHANG Zeng-li, Email: zhangzengli@suda.edu.cn  

[Objective] To investigate the effect of diabetic model mice combined with cadmium exposure on tibia by Micro-CT imaging.

[Methods] Forty SPF eight-week-old male C57BL6 mice were randomly divided into four groups:control group, cadmium chloride group, diabetes mellitus model group, and diabetes mellitus model combined with cadmium chloride group (combined exposure group). The cadmium chloride group was intraperitoneally injected with 3.5 mg/kg cadmium chloride 3 times per week for 4 weeks. The diabetes mellitus model was intraperitoneally injected with 40 mg/kg streptozotocin 5 days per week and detected for fasting plasma glucose after 1-2 weeks, and fasting plasma glucose ≥ 11.1 mmol/L indicated that the model was established. The diabetes mellitus model group was treated with cadmium chloride as previously described to establish a combined exposure group. The control group was intraperitoneally injected with normal saline at the same volume. After the treatment protocol, bone wet weight/body weight and bone length were measured after soft tissues were stripped from bone; right tibia of the mice was scanned by Micro-CT for three-dimensional reconstruction, and the measurements included bone mineral density, bone volume fraction, bone surface/bone volume, trabecular thickness, trabecular number, and trabecular separation.

[Results] No differences in bone wet weight per 100 g body weight and bone length were found among the four groups (P > 0.05). The morphological observation results showed that, compared with the control group, the other three groups had decreased trabecular number and trabecular thickness together with loose structure, and compared with the diabetes mellitus model group, the combined exposure group had more obvious such changes. Compared with the control group[(0.160±0.029) g/cm3, (14.781±4.754)%, (13.397±2.962) mm-1, (2.964±0.777) mm-1, and (0.118±0.013) mm], the bone mineral density, bone volume fraction, bone surface/bone volume, and trabecular number were decreased while trabecular separation of tibial cancellous bone were increased in the cadmium chloride group[(0.123±0.013) g/cm3, (7.972±2.404)%, (8.533±2.221) mm-1, (1.787±0.513) mm-1, and (0.147±0.015) mm] and the combined exposure group[(0.121±0.008) g/cm3, (8.571±1.710)%, (8.902±1.065) mm-1, (1.840±0.280) mm-1, and (0.154±0.008) mm] (Ps < 0.05); compared with the diabetes mellitus model group, all the above indicators were significanthy different in the combined exposure group (Ps < 0.05). However, there was no difference in the bone mineral density of tibial cortical bone among the four groups (P > 0.05). Compared with the control group[(5.973±0.418) mm-1], the diabetes mellitus model[(6.876±0.469) mm-1] and the combined exposure group[(7.568±0.595) mm-1] showed increased bone surface/bone volume of tibial cortical bone (P < 0.05); compared with the cadmium chloride group[(32.765±4.053)% and (6.038±0.579)mm-1], the combined exposure group[(39.901±3.386)% and (7.568±0.595)mm-1] showed increased bone volume fraction and bone surface/bone volume of tibial cortical bone (P < 0.05).

[Conclusion] Cadmium exposure could reduce bone volume fraction, bone surface/bone volume, trabecular number, and bone mineral density, and increase trabecular separation of tibial cancellous bone of diabetic mice, indicating osteoporosis of tibial cancellous bone.

Key Words: Micro-CT;  mouse;  cadmium;  diabetes;  tibia 

图 1


Figure 1 [注]箭头所指为骨骺线最低端。
表 1


Table 1
图 2


Figure 2 [注]A:胫骨松质骨;B:胫骨皮质骨。1:对照组;2:氯化镉染毒组;3:糖尿病模型组;4:联合组。箭头所指为松质骨骨小梁。
表 2


Table 2
表 3


Table 3



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1   材料与方法

1.1   主要材料

1.1.1   主要试剂与动物


40只SPF级雄性C57BL6小鼠,8周龄,体重20~ 25 g,由上海斯莱克实验动物有限责任公司提供,生产许可证号为SCXK(沪)2017-0005。

1.1.2   主要仪器

罗氏血糖仪(ACCU-CHEK Performa,德国),BSA124S分析天平(Sartorius,德国),YP1002N电子天平(上海菁海仪器有限公司,中国),BK-123002游标卡尺(Bunker,美国),Skyscan1174 Micro-CT断层扫描仪(香港梅林集团有限公司,中国),所需一次性注射器、烧杯、手术剪刀、镊子等器械均由苏州大学试验器材材料中心提供。

1.2   染毒方法

小鼠饲养于苏州大学独墅湖校区401-SPF级动物房[实验动物使用许可证编号:SCKK(苏)2017-0006]。饲养条件:昼夜周期为12 h,室温24℃,相对环境湿度55%,小鼠自由饮水进食。小鼠随机分为4组,每组10只,即对照组、氯化镉染毒组、糖尿病模型组、糖尿病模型联合氯化镉染毒组(以下简称为联合组)。糖尿病模型建立方法:以40 mg/kg链脲佐菌素腹腔注射,连续5 d,注射1~2周,期间监测血糖变化,以空腹血糖≥ 11.1 mmol/L为成模标准[15]。氯化镉染毒方法为每周3次,以3.5 mg/kg剂量腹腔注射氯化镉染毒,共染毒4周。对照组以同等体积生理盐水腹腔注射。镉染毒剂量选择依据及造模时间参考付静波等[16]关于亚慢性镉染毒小鼠的实验研究。待糖尿病模型建成后再行染镉处理;两组染毒方式与剂量为联合组同氯化镉组,糖尿病模型组同对照组。本研究通过苏州大学动物伦理委员会批准。

1.3   骨湿重及骨长度测量方法

分析天平称量小鼠剥离软组织后的胫骨湿重,以[骨湿重(mg)/体重(g)] × 100计算每100 g体重的胫骨湿重[17]。另用游标卡尺检测小鼠骨长度。

1.4   Micro-CT测量方法

应用Micro-CT技术,以分辨率为9.1 μm扫描小鼠右侧离体胫骨,应用CTVol、CTAn、CTVox软件进行三维重建分析。以图 1中箭头所指的骨骺线最低端为基线,向胫骨远端计数70层不作分析,继续向远端计数100层,分析其松质骨骨密度并获得形态计量学参数,再继续向远端计数300层不作分析,又计数100层分析皮质骨骨密度并获得形态计量学参数,构建小鼠胫骨松质骨及皮质骨三维重建结构图。

图 1


骨密度(bonemineral density)作为骨质量的重要指标之一,能够精确直观地反映骨质疏松程度;骨体积分数(bone volume/total volume)、骨表面积体积比(bone surface/total volume)均可间接反映骨量的多少;骨小梁厚度(trabecular thickness)可用于描述骨小梁结构、形态,同时也可反映骨量的变化,在骨小梁一定的情况下,其厚度越大,骨量越多。骨小梁数目(trabecular number)代表骨组织与非骨组织交点数量,骨小梁分离度(trabecular separation)代表骨小梁之间髓腔平均宽度。

1.4   统计学分析

运用SPSS 24.0对数据进行统计分析,数据用均数±标准差表示。采用方差分析进行组间比较,多组间两两比较采用Bonfferoni t检验,检验水准α=0.05。

2   结果

2.1   小鼠胫骨湿重及长度

表 1所示,与对照组相比,氯化镉染毒组、糖尿病模型组、联合组小鼠的每100 g胫骨湿重及骨长度差异均无统计学意义(P>0.05)。



2.2   小鼠胫骨松质骨及皮质骨形态

经Micro-CT扫描、三维构图后,对4组小鼠胫骨松质骨及皮质骨进行微观结构分析。如图 2所示,与对照组相比,氯化镉染毒组、糖尿病模型组、联合组小鼠胫骨松质骨骨小梁数目减少,骨小梁厚度明显降低,排列疏松。同时,与糖尿病模型组相比,联合组变化更加明显。然而,各组间胫骨皮质骨并无明显变化。

图 2


2.3   小鼠胫骨松质骨骨密度和形态计量学指标

表 2所示,与对照组相比,氯化镉染毒组、联合组小鼠胫骨松质骨骨密度下降(P < 0.05);与糖尿病模型组[(0.152± 0.017)g/cm3]相比,联合组胫骨松质骨骨密度下降(P < 0.05)。形态计量学指标检测结果显示,与对照组相比,氯化镉染毒组及联合组小鼠胫骨松质骨骨小梁分离度增加(P < 0.05),骨体积分数、骨表面积体积比、骨小梁数目降低(均P < 0.05)。同时,与糖尿病模型组相比,联合组小鼠胫骨松质骨骨小梁分离度增加(P < 0.05),骨体积分数、骨表面积体积比、骨小梁数目降低(均P < 0.05)。



2.4   小鼠胫骨皮质骨骨密度和形态计量学指标

表 3所示,与对照组相比,氯化镉染毒组、糖尿病模型组、联合组小鼠胫骨皮质骨骨密度差异无统计学差异(P>0.05)。形态计量学指标检测结果显示,与对照组相比,糖尿病模型组及联合组小鼠胫骨皮质骨骨表面积体积比增加(P< 0.05)。与氯化镉染毒组相比,联合组皮质骨体积分数及骨表面积体积比增加(P< 0.05)。



3   讨论







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