慢性肾衰竭大鼠股骨皮质骨及松质骨Cbfa1表达与骨密度间的关系

发布时间：2018-07-03 02:26

本文选题：慢性肾衰竭 + 核心结核因子-a1　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：慢性肾脏病矿物质和骨代谢紊乱（CKD-MBD）是慢性肾脏病（ChronicRenal Failure，CKD）患者重要并发症。肾性骨营养不良是CKD-MBD中骨的异常表现。肾性骨营养不良诊断的“金标准”为骨活检，但由于其临床操作复杂等问题，未被广泛应用。骨的评价存在较大困难。核心结合因子-a1（cbfa1）在成骨细胞发育、分化和骨形成过程中起着至关重要的作用，且其表达的改变可反映肾性骨营养不良的严重程度。本研究通过检测慢性肾衰竭大鼠模型股骨cbfa1的表达情况及相应部位的骨密度，探讨两者间的关系，为临床诊断CKD-MBD中肾性骨营养不良找到更好的评价指标。方法：本研究采用实验-对照研究方法。 1实验对象：清洁级、健康、雄性SD大鼠；体质量为180~200g，共14只；于河北医科大学第四医院动物中心屏障环境适应性饲养1周后，随机分为正常对照组、慢性肾衰竭模型组2组。 2实验方法：动物模型构建：应用硫酸腺嘌呤悬浊液灌胃法构建慢性肾衰竭大鼠模型，1-3周给予硫酸腺嘌呤悬浊液（硫酸腺嘌呤溶于1.5%甲基纤维素中制成100g/l的悬浊液）6ul/g每天灌胃给药，4-8周给予1.5%甲基纤维素6ul/g隔日灌胃。正常对照组给予甲基纤维素灌胃给药，给予1.5%甲基纤维素6ul/g在1-3周每天灌胃，在4-8周隔日灌胃给药。 3标本留取及检测：8周后采用心脏取血法收集大鼠血标本检测其血肌酐、尿素氮、钙、磷等相关生化指标血清浓度。取左侧股骨，立即将股骨用生理盐水浸湿的纱布包裹，存入-80℃以下的冰箱中做骨密度试验。测量各组大鼠股骨的整体骨密度（Whole-Bone Mineral Density,W-BMD）、松质骨骨密度（Trabecular-Bone Mineral Density,T-BMD）、皮质骨骨密度（Cortical-Bone Mineral Density,C-BMD）。取右侧股骨。置入EDTANa2脱钙液脱钙，用于免疫组化染色及HE染色，检测大鼠股骨中Cbfa1蛋白的表达及松质骨和皮质骨的形态学改变。 4统计学处理：应用SPSS13.0统计学软件进行统计学处理，符合正态分布的计量资料用均数±标准差（x s）表示，两组间均数比较用t检验，相关分析符合双变量正态分布作Pearson积差相关分析，，余者采用秩和检验和Spearman相关分析。P0.05认为差异有统计学意义。结果： 1慢性肾衰竭大鼠模型的构建慢性肾衰竭组大鼠血肌酐、尿素氮明显高于正常对照组组大鼠血清水平（P0.05）；慢性肾衰竭模型组血清钙浓度（2.10±0.01mmol/L）低于正常对照组血清钙浓度（2.34±0.03mmol/L），慢性肾衰竭模型组血清磷浓度（2.21±0.03mmol/L）高于正常对照组血清磷浓度（1.75±0.07mmol/L），差异均有统计学意义（P0.001）。慢性肾衰竭组大鼠肾脏HE染色亦符合慢性肾衰竭改变；慢性肾衰竭大鼠股骨HE染色：慢性肾衰竭模型组与正常对照组相比：小梁数增多，而脂肪空泡数减少，且皮质厚度变薄 2慢性肾衰竭大鼠股骨骨皮质骨及松质骨Cbfa1表达的改变慢性肾衰竭模型组股骨松质骨Cbfa1表达（0.01539±0.00016meD）多于正常对照组股骨松质骨Cbfa1表达（0.01075±0.00026meD），慢性肾衰竭模型组股骨皮质骨Cbfa1表达（0.00367±0.00022meD）少于正常对照组股骨皮质骨Cbfa1表达（0.01143±0.00078meD）。差异均有统计学意义（P0.001）。 3慢性肾衰竭大鼠股骨皮质骨及松质骨骨密度的改变慢性肾衰竭模型组W-BMD（0.1405±0.0016g/cm2）低于正常对照组W-BMD（0.1571±0.0019g/cm2），慢性肾衰竭模型组C-BMD（0.1314±.0028g/cm2）低于正常对照组C-BMD（0.1550±.0057g/cm2），慢性肾衰竭模型组T-BMD（0.1662±0.0013g/cm2）高于正常对照组T-BMD（0.1583±0.0024g/cm2），差异均有统计学意义（P0.001）。 4慢性肾衰竭大鼠股骨骨皮质骨及松质骨Cbfa1表达与骨密度及生化指标的关系慢性肾衰竭大鼠股骨皮质骨骨密度与皮质骨Cbfa1蛋白表达做相关分析呈正相关（r=0.805，P0.001），慢性肾衰竭大鼠股骨松质骨骨密度与松质骨Cbfa1蛋白表达做相关分析呈正相关（r=0.844，P0.001）。慢性肾衰竭大鼠股骨整段骨密度与血清钙浓度做相关分析呈正相关（r=0.730，P0.001），慢性肾衰竭大鼠股骨整段骨密度与血清磷浓度做相关分析呈负相关（r=-0.652，P0.001）。结论： 1慢性肾衰竭大鼠股骨发生骨重建，皮质骨骨密度降低，松质骨骨密度升高。 2慢性肾衰竭大鼠股骨皮质骨Cbfa1表达减少，松质骨Cbfa1表达增多，可能与同部位骨密度改变相关。
[Abstract]:Objective:
Mineral and bone metabolic disorders (CKD-MBD) of chronic kidney disease (ChronicRenal Failure, CKD) is an important complication in patients with chronic renal disease (CKD). Renal osteonystrophy is an abnormal manifestation of bone in CKD-MBD. The "gold standard" for the diagnosis of renal osteonystrophy is a bone biopsy, but it is not widely used because of its complicated bed operation. Bone evaluation The core binding factor -a1 (Cbfa1) plays a vital role in the development, differentiation and bone formation of osteoblasts, and its expression changes can reflect the severity of renal osteogenesis. This study examined the expression of Cbfa1 in the femur and the bone mineral density in the corresponding part of the rat model of chronic renal failure. The relationship between them can provide better indicators for clinical diagnosis of renal osteo malnutrition in CKD-MBD.
Method:
The experimental control method was used in this study.
1 subjects: clean grade, healthy, male SD rats; body mass of 180~200g, a total of 14. After 1 weeks of environmental adaptation in the animal center barrier of the fourth hospital of Hebei Medical University, they were randomly divided into normal control group and 2 group of chronic renal failure model group.
2 experimental method: Animal Model Construction: the rat model of chronic renal failure was constructed with adenine sulphate suspense solution, and the adenine sulfate suspension was given for 1-3 weeks (100g/l suspension of adenine sulphate dissolved in 1.5% methylcellulose) 6ul/g every day, and 1.5% methylcellulose was given to the stomach for 4-8 weeks. The rats in the treatment group were given methylcellulose, and 1.5% methylcellulose 6ul/g was administered every day for 1-3 weeks. The rats were administrated on the next 4-8 weeks.
3 specimen retention and detection: after 8 weeks, the blood samples of rats were collected by cardiac blood sampling to detect the serum concentration of serum creatinine, urea nitrogen, calcium, phosphorus and other biochemical indexes. The left femur was taken and the femur was soaked with saline, and the bone density test was done in the refrigerator below -80 C. The whole bone of the femur was measured in each group. Whole-Bone Mineral Density (W-BMD), cancellous bone mineral density (Trabecular-Bone Mineral Density, T-BMD), cortical bone mineral density (Cortical-Bone Mineral Density, C-BMD). The right femur was taken. The decalcification of the decalcification solution was used to detect the expression of the protein in the femur and the cancellous bone and cortex of the femur. The morphological changes of the bone.
4 statistical processing: statistical processing with SPSS13.0 statistics software, the measurement data conforming to normal distribution are expressed in mean number + standard deviation (x s). The average number of the two groups is compared with t test. The correlation analysis conforms to the bivariate normal distribution as the Pearson product difference correlation analysis. The surplus uses the rank sum test and the Spearman correlation analysis.P0.05 to think the difference.P0.05 is poor. The difference is statistically significant.
Result:
The construction of 1 rat model of chronic renal failure
The serum creatinine and urea nitrogen in the chronic renal failure group were significantly higher than that of the normal control group (P0.05). The serum calcium concentration (2.10 + 0.01mmol/L) in the chronic renal failure model group was lower than that of the normal control group (2.34 + 0.03mmol/L), and the serum phosphorus concentration (2.21 + 0.03mmol/L) in the chronic renal failure model group was higher than that of the normal control group. The difference of phosphorus concentration (1.75 + 0.07mmol/L) was statistically significant (P0.001). HE staining in the kidney of chronic renal failure rats also conformed to the change of chronic renal failure; HE staining in the femur of chronic renal failure rats: the number of trabeculae increased, the number of fat vacuoles decreased and the thickness of the cortex was thinner in the chronic renal failure model group than in the normal control group.
2 expression of Cbfa1 in femoral cortical bone and cancellous bone in rats with chronic renal failure
The expression of Cbfa1 (0.01539 + 0.00016meD) of the femoral cancellous bone in the model group of chronic renal failure (0.01539 + 0.00016meD) was more than that of the femoral cancellous bone in the normal control group (0.01075 + 0.00026meD). The expression of Cbfa1 in the femoral cortical bone (0.00367 + 0.00022meD) in the chronic renal failure model group was less than that of the femoral cortical bone Cbfa1 (0.01143 + 0.00078meD) in the normal control group. The differences were all statistically significant. Learning meaning (P0.001).
3 changes in bone mineral density of femoral cortical bone and cancellous bone in chronic renal failure rats
The W-BMD (0.1405 + 0.0016g/cm2) in the model group of chronic renal failure was lower than that of the normal control group (0.1571 + 0.0019g/cm2). The C-BMD (0.1314 +.0028g/cm2) in the chronic renal failure model group was lower than that of the normal control group (0.1550 +.0057g/cm2). The T-BMD (0.1662 + 0.0013g/cm2) in the chronic renal failure model group was higher than that of the normal control group (0.1583 + 0.0024g/cm2). The difference was higher than that of the normal control group (0.1583 + 0.0024g/cm2). There were statistical significance (P0.001).
4 the relationship between Cbfa1 expression in bone cortex and cancellous bone of femur and bone mineral density and biochemical indexes in rats with chronic renal failure
The bone mineral density of the femoral cortical bone of the rats with chronic renal failure was positively correlated with the correlation analysis of the cortical bone Cbfa1 protein expression (r=0.805, P0.001). The bone mineral density of the femur cancellous bone in the chronic renal failure rats was positively correlated with the correlation analysis of the Cbfa1 protein expression of the cancellous bone (r=0.844, P0.001). The whole bone mineral density and serum calcium concentration in the slow renal failure rats were made. Correlation analysis was positively correlated (r=0.730, P0.001), and there was a negative correlation between whole bone mineral density and serum phosphorus concentration in chronic renal failure rats (r=-0.652, P0.001).
Conclusion:
1 the bone remodeling of femur in chronic renal failure rats, the decrease of cortical bone density and the increase of bone density of cancellous bone.
2 the expression of Cbfa1 in the cortical bone of femur in chronic renal failure rats was decreased, and the expression of Cbfa1 in cancellous bone increased, which may be related to the change of bone mineral density in the same position.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5;R580

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