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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

小鼠胫骨三维合成图

Figure 1 [注]箭头所指为骨骺线最低端。
表 1

小鼠胫骨湿重及长度

Table 1
图 2

小鼠胫骨松质骨及皮质骨形态对比

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

小鼠腔骨松质骨骨密度和形态计量学指标

Table 2
表 3

小鼠腔骨皮质骨骨密度和形态计量学指标

Table 3

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我国各级医疗机构对儿童用基本药物剂型和规格的需求调查

随着经济的迅速发展和人们生活方式的改变,环境污染加剧,慢性病发病率升高;与此同时,职业人群也由以前单一职业危害因素暴露转变为多因素联合暴露[1-2]。由于制造业和充电电池中镉的广泛应用,镉污染和镉中毒事件屡见不鲜。肝、肾、呼吸道和骨骼是镉损伤的重要靶标,过量的镉染毒可以引起多种疾病[3-7]。有研究表明尿镉水平与血糖及糖尿病具有相关性且呈剂量依赖性[8]。据报道,糖尿病患者和镉暴露者均有肾脏及骨骼的损伤[9-13]。但是,镉暴露是否会加重糖尿病患者的骨损伤尚不清楚。胫骨位于小腿的内侧,是小腿骨中主要的承重骨骼,也是骨折的多发部位[14]。因此,本研究建立小鼠糖尿病模型,应用Micro-CT技术观察镉染毒对糖尿病小鼠胫骨的影响,为后续制定针对性的干预措施提供依据。

1   材料与方法

1.1   主要材料

1.1.1   主要试剂与动物

氯化镉(中国革新化工厂,中国),柠檬酸、多聚甲醛溶液(国药集团化学试剂有限公司,中国),柠檬酸三钠(天津市大茂化学试剂厂,中国),氯化钠注射液(湖南金健药业有限公司,中国),链脲佐菌素(Sigma-Aldrich,美国)。

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

小鼠胫骨三维合成图

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)。

表1

小鼠胫骨湿重及长度

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

小鼠胫骨松质骨及皮质骨形态对比

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)。

表2

小鼠腔骨松质骨骨密度和形态计量学指标

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)。

表3

小鼠腔骨皮质骨骨密度和形态计量学指标

3   讨论

小鼠胫骨由松质骨和皮质骨构成。松质骨存在于胫骨内部,其主要结构为骨小梁。正常情况下骨小梁排列紧密,呈无规则网状交织结构。如遇骨损伤或骨质疏松,骨小梁的数目、形态均可能改变,同时也会增加骨折的风险。作为研究骨骼的重要辅助工具,Micro-CT的分辨率极高,达微米级别[18],可在不破坏骨结构的情况下清晰地观察小鼠胫骨内部结构,很大程度上解决了骨组织形态计量学测量时的困难[19]。本研究应用Micro-CT技术,精确直观地反映出小鼠胫骨骨密度及形态。

本研究结果显示,各组小鼠胫骨湿重及骨长度均无差异。其原因可能是所研究小鼠已为成年小鼠,其骨长度已经达到成年水平;同时,糖尿病及氯化镉染毒对小鼠胫骨皮质骨影响较松质骨小,而皮质骨占骨重量的绝大部分。因此,小鼠胫骨湿重无差异的结果并不与小鼠胫骨松质骨微观形态改变的结果相违背。

流行病学研究表明,糖尿病患者中骨质疏松性骨折高发[20],镉染毒影响骨的发育[21-22]。结合本研究松质骨骨密度结果: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降低,说明镉染毒对松质骨已产生影响,此结果与秦波音等[23]针对镉染毒对大鼠胫骨骨微结构的活体Micro-CT的观察结果一致。与糖尿病模型组相比,联合组小鼠胫骨松质骨骨体积分数明显降低;同时,骨小梁数目减少,骨小梁分离度增大。此外,小鼠胫骨松质骨形态观察结果显示,与糖尿病组相比,联合组小鼠胫骨松质骨骨小梁数目减少、骨小梁厚度降低,排列疏松,提示镉染毒可导致糖尿病小鼠骨密度降低。针对皮质骨的研究同样显示,由于镉染毒,糖尿病小鼠皮质骨形态计量学部分指标受到影响。

影响骨代谢的因素众多,其中维生素D和钙结合蛋白的缺乏可导致机体钙稳态失调,出现低钙血症、骨质疏松等病变[24]。维生素D与Wnt信号通路作用关系明确,而Wnt信号通路与正常成骨过程关系紧密[25-26]。同时,一项meta分析研究表明,维生素D可有效调节糖代谢,改善2型糖尿病患者血糖水平[27]。镉染毒联合糖尿病对小鼠胫骨的影响可能与维生素D代谢密切相关。由于小鼠与人存在种属差异,了解镉染毒对糖尿病患者胫骨的影响,获得人群相关数据问题亟待解决;此外,其具体作用机制有待进一步研究。

综上所述,镉染毒可降低糖尿病小鼠胫骨松质骨骨体积分数、骨表面积体积比、骨小梁数目、骨密度,增加骨小梁分离度,说明镉染毒可导致糖尿病小鼠胫骨骨质疏松。

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