慢性肾功能不全幼鼠脊柱椎体生长板甲状旁腺激素相关蛋白受体低表达对椎体发育的影响

Effect of low expression of PTHrp receptor in spinal vertebral growth plate on vertebral body development in chronic renal insufficiency rats

目的探讨慢性肾功能不全幼鼠脊柱椎体生长板区甲状旁腺激素相关蛋白(Parathyroid hormone-related protein,PTHrp)受体表达对椎体发育的影响。方法将60只2周龄的雄性Sprague-Dawley大鼠按每组20只,平均分为假手术组、模型组和治疗组。假手术组大鼠切开暴露左侧输尿管后缝合,模型组大鼠行左侧输尿管结扎,治疗组大鼠行左侧输尿管结扎并用依那普利治疗。术后4周,3组各处死10只大鼠并检测其血PTHrp浓度。拍摄脊柱正位X线片测量腰椎1~腰椎6长度,组织学切片观测脊柱椎体生长板增殖区组成细胞柱的细胞数量,免疫组织化学检测椎体生长板区PTHrp受体表达。将每组剩余大鼠处死后提取椎体生长板区软骨细胞进行体外培养至P3代,原位杂交检测PTHrp受体mRNA表达,并应用PCR行定量分析;免疫荧光检测PTHrp受体蛋白表达,并用Western-blot行定量分析。应用EDU技术检测细胞24 h增殖率。最后应用单因素方差分析对各组间差异进行统计学分析。结果假手术组、模型组和治疗组大鼠血PTHrp浓度分别为(0.48±0.14)ng/L、(1.05±0.21)ng/L和(0.70±0.19)ng/L,另两组与假手术组比较,差异均有统计学意义(P均<0.05)。模型组和治疗组腰椎1~腰椎6长度分别为(44.6±11.5)mm和(46.8±13.5)mm,均较假手术组(58.1±9.6)mm短,且差异有统计学意义(P均<0.05);模型组和治疗组椎体生长板增殖区细胞柱细胞数分别为(2.3±1.1)个和(2.1±1.4)个,均较假手术组(4.1±1.2)个减少,且差异有统计学意义(P均<0.05)。免疫组织化学结果显示,假手术组PTHrp受体高表达,定量分析评分为(7.1±1.8)分;治疗组次之,为(5.4±2.1)分,模型组表达最少,为(3.6±1.9)分,组间比较,差异均有统计学意义(P均<0.05)。模型组和治疗组体外培养软骨细胞内PTHrp受体mRNA相对表达量分别为0.24±0.14和0.36±0.13,均低于假手术组1.01±0.32,且差异有统计学意义(P均<0.05)。模型组和治疗组细胞24 h增殖率分别为(8.7±2.6)%和(14.3±7.1)%,均低于假手术组(21.4±7.3)%,且差异有统计学意义(P均<0.05)。结论慢性肾功能不全幼鼠血液PTHrp浓度增高,椎体生长板区软骨细胞PTHrp受体由于负反馈原因表达减少,PTHrp无法发挥促进增殖作用,最终导致椎体发育障碍。

Objective To explore the effect of low expression of PTHrp receptor in spinal vertebral growth plate on vertebral body development in chronic renal insufficiency rats.Methods A total of 60 male 2-week-old Sprague-Dawley rats were randomly divided into three groups of sham+saline,surgery+saline and surgery+enalapril(n=20 each).Left ureteral obstruction was surgically created for inducing chronic renal insufficiency.The rats were sacrificed at 4 weeks postoperatively and the length of lumbar vertebra was measured from the first to the sixth on spinal radiography.The concentration of PTHrp was detected by intracardiac blood sampling.The chondrocyte number of columnar structure in growth plate proliferative zone of spinal vertebra was measured and the expression of PTHrp receptor in growth plate detected by immunohistochemistry.Chondrocytes from growth plate in three groups were isolated and cultured in vitro into P3 generation.The expression of PTHrp receptor mRNA was detected by in situ hybridization and quantitative polymerase chain reaction(PCR)performed.The expression of PTHrp receptor protein was detected by immunofluorescence and quantitative analysis completed by Western blot.The chondrocyte proliferation rate at 24 h was detected by EDU.Univariate analysis of variance was performed for comparing among three groups.Results Blood concentrations of PTHrp in sham+saline group were lower than those in surgery+saline and surgery+enalapril groups and surgery+enalapril group was lower than surgery+saline group[sham+saline(0.48±0.14)ng/L vs.surgery+saline(1.05±0.21)ng/L surgery+enalapril(0.70±0.19)ng/L](all P<0.05).The length of lumbar vertebra from the first to the sixth[sham+saline(58.1±9.6)vs.surgery+saline(44.6±11.5)vs.surgery+enalapril(46.8±13.5)mm]and chondrocyte number of column structure[sham+saline(4.1±1.2)vs.surgery+saline(2.3±1.1)vs.surgery+enalapril(2.1±1.4)]in growth plate proliferative area were higher in sham+saline group than those both in surgery+enalapril and surgery+saline groups(all P<0.05).Immunohistochemistry of PTHrp receptor showed high expression in sham+saline group,followed by surgery+enalapril group and the least expression in surgery+saline group[sham+saline(7.1±1.8)points vs.surgery+saline(3.6±1.9)points surgery+enalapril(5.4±2.1)score].The levels of PTHrp receptor mRNA of sham+saline group was higher than those in surgery+saline and surgery+enalapril groups[sham+saline(1.01±0.32)fold vs.surgery+saline(0.24±0.14)fold vs.surgery+enalapril(0.36±0.13)fold].The levels of PTHrp receptor protein of sham+saline group was higher than those both in surgery+saline and surgery+enalapril groups[sham+saline(0.51±0.18)vs.surgery+saline(0.16±0.07)vs.surgery+enalapril(0.37±0.14)].The 24 h chondrocyte proliferation rate of sham+saline group was higher than those both in surgery+saline and surgery+enalapril groups[sham+saline(21.4±7.3)%vs.surgery+saline(8.7±2.6)%vs.surgery+enalapril(14.3±7.1)%](all P<0.05).Conclusions An increase of PTHrp concentration in blood and a decrease of PTHrp receptor in chondrocytes from spinal vertebra growth plate may lead to lowered chondrocyte proliferation rate and growth retardation of spinal vertebra in chronic renal insufficiency rats.