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去卵巢致肾虚的腰椎间盘退变模型的实验研究

Hits: 2614 Download times: 1104 Received:May 13, 2013

作者	Author	单位	Unit	E-Mail
姚长风	YAO Chang-feng	上海中医药大学附属龙华医院, 上海 200032 安徽中医学院针灸骨伤临床学院, 安徽合肥 230038	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
赵永见	ZHAO Yong-jian	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
牛凯	NIU Kai	安徽理工大学医学院, 安徽淮南 232001
孙悦礼	SUN Yue-li	上海中医药大学附属龙华医院, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
李晨光	LI Chen-guang	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
唐德志	TANG De-zhi	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
舒冰	SHU Bing	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
卢盛	LU Sheng	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
周重建	ZHOU Chong-jian	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
梁倩倩	LIANG Qian-qian	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
施杞	SHI Qi	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China
王拥军	WANG Yong-jun	上海中医药大学附属龙华医院, 上海 200032 上海中医药大学脊柱病研究所, 上海 200032	Institute of Spine, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China Longhua Hospital, Shanghai University of Tranditional Chinese Medicine, Shanghai 200032, China	yjwang88@hotmail.com

期刊信息：《中国骨伤》2013年26卷，第12期，第1015-1022页

DOI：10.3969/j.issn.1003-0034.2013.12.011

基金项目:国家重点基础研究发展计划（“973”计划）基金资助项目（编号：2010CB530400）；国家自然科学基金重点项目（编号：30930111）；国家自然科学基金项目（编号：81102851，81102606，81102605，30973760，30901914）；上海市高校创新团队计划（沪教委科[2009]6号）；上海市教委“曙光”跟踪计划（编号：10GG20）

目的：观察去前肢卵巢对大鼠腰椎间盘和椎体骨密度的影响，建立大鼠肾虚型腰椎间盘退变模型，探讨椎间盘退变的内在机制、椎间盘退变和骨质疏松之间的关系.

方法：选择1月龄雌性SD大鼠30只，随机分为正常对照组、腰椎间盘退变组、肾虚型腰椎间盘退变组（复合模型组），每组10只.腰椎间盘退变组大鼠去除双前肢，肾虚型腰椎间盘退变组大鼠在去除双前肢3个月后，再去除双卵巢.8个月后，通过Micro-CT扫描观察椎体骨密度，藏红O-快绿染色法观测椎间盘组织形态学改变，免疫组织化学法观察Ⅱ、Ⅹ胶原在椎间盘中的蛋白表达，实时荧光定量聚合酶链式反应（real-time polymerase chain reaction，RT-PCR）法检测细胞外基质相关基因的表达，以评价去除双前肢和双卵巢对椎间盘退变和椎体骨密度的影响.

结果： Micro-CT扫描发现，肾虚型腰椎间盘退变组动物椎体骨质疏松明显；藏红O-快绿染色法显示椎间隙变窄，肾虚型腰椎间盘退变组椎间盘组织退变明显，软骨板发育不全；免疫组织化学染色显示肾虚型腰椎间盘退变组相对于正常对照组，椎间盘内Ⅹ型胶原表达增加，Ⅱ型胶原表达降低；RT-PCR分析发现，腰椎间盘退变组和肾虚型腰椎间盘退变组Ⅱ型胶原（typeⅡcollagen，Col2a1）基因的表达较正常对照组低，3组比较差异有统计学意义（P=0.000，P=0.000)；腰椎间盘退变组和肾虚型腰椎间盘退变组聚集蛋白聚糖（aggrecan-1，Agc1）基因的表达较正常对照组低，差异有统计学意义（P=0.000，P=0.000)；腰椎间盘退变组和肾虚型腰椎间盘退变组Ⅹ型胶原（type X collagen，Col10al）基因的表达较正常对照组高，差异无统计学意义；肾虚型腰椎间盘退变组基质金属蛋白酶-13（matrix metalloproteinase 13，MMP-13）基因的表达较正常对照组和腰椎间盘退变组高，3组比较差异有统计学意义（P=0.000，P=0.000）;腰椎间盘退变组和肾虚型腰椎间盘退变组聚集蛋白聚糖降解酶2（aggrecanase-2，ADAMTS-5）基因的表达较正常对照组高，差异有统计学意义（P=0.006，P=0.008）.

结论：采用去双前肢和去卵巢的方式建立的大鼠腰椎间盘退变模型，出现了肾虚“骨象”--骨质疏松，并在组织形态学、分子细胞生物学等方面的表现与临床肾虚型腰椎间盘退变更加相似.

[关键词]:腰椎椎间盘卵巢大鼠 Sprague-Dawley

Experimental study on lumbar intervetebral disc degeneration model with kidney deficiency by ovariectomizing

Abstract:

Objective: To observe effects of removing arms and ovarian on lumbar intervertebral disc and vertebral bone mineral density(BMD) by establishing rat model of lumbar intervetebral disc degeneration(IDD) with kidney deficiency,and to explore internal mechanism of disc degeneration,relationship between disc degeneration and osteoporosis.

Methods: Thirty Sprague-Dawley female rats aged one month were randomly divided into control group,lumbar IDD group and lumbar IDD with kidney deficiency group(combined group),10 rats in each group. Lumbar IDD group removed double arms,lumbar IDD with kidney deficiency group removed double arms after 3 months,both ovaries were removed. Vertebral bone mineral density were observed by Micro-CT scan;morphological changes were tested by safranine O-fast green staining;Ⅱ,Ⅹ collagen protein expression in the intervertebral disc were obsevered by immunohistochemistry;extracellular matrix gene expression were obsevered by real-time polymerase chain reaction(RT-PCR),in order to evaluate the effects of removed of forelimbs and double ovarian on degeneration and vertebral bone mineral density of intervertebral disc.

Results: Micro-CT scan showed osteoporosis in kidney deficiency group was obviously worse than other two groups;safranine O-fast green staining showed that intervertebral space became narrowed,intervertebral disc tissue degenerated obviously,chondral palte was underdeveloped in kidney deficiency group;immunohistochemistry showed that X collagen expression increased,typeⅡ collagen expression decreased in kidney deficiency group;RT-PCR showed that typeⅡcollagen expression in lumbar IDD group and kidney deficiency group was lower than control group,and had statistical meaning among three groups(P=0.000,P=0.000)； Agc 1 in lumbar IDD group and kidney deficiency group was lower than control group,and had statistical meaning among three groups(P=0.000,P=0.000)； while type X collagen expression was higher than control group,but no significant meaning;MMP-13 in lumbar IDD group and kidney deficiency group was higher than control group,with significant meaning compared among three groups(P=0.000,P=0.000)； aggrecanase-2 in lumbar IDD group and kidney deficiency group was higher than control group,with significant meaning compared among three groups(P=0.006,P=0.008).

Conclusion: Rats model of lumbar disc degeneration established by removed forelimbs and ovariectomized can occure "bone like"——osteoporosis,which is similar with clinical kidney lumbar disc degeneration in tissue morphology,molecular cell biology expression.

KEYWORDS:Lumbar vertebrae Intervertebral disk Ovary Rats Sprague-Dawley

引用本文，请按以下格式著录参考文献：

中文格式:	姚长风,赵永见,牛凯,孙悦礼,李晨光,唐德志,舒冰,卢盛,周重建,梁倩倩,施杞,王拥军.去卵巢致肾虚的腰椎间盘退变模型的实验研究[J].中国骨伤,2013,26(12):1015~1022
英文格式:	YAO Chang-feng,ZHAO Yong-jian,NIU Kai,SUN Yue-li,LI Chen-guang,TANG De-zhi,SHU Bing,LU Sheng,ZHOU Chong-jian,LIANG Qian-qian,SHI Qi,WANG Yong-jun.Experimental study on lumbar intervetebral disc degeneration model with kidney deficiency by ovariectomizing[J].zhongguo gu shang / China J Orthop Trauma ,2013,26(12):1015~1022

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