摘要
目的针对DKK1基因构建两段小干扰RNA(siRNA)真核表达载体,观察其对骨质疏松(OP)大鼠的治疗作用。方法12周龄Wistar雌性大鼠随机分为5组,对照组、模型组、空载体组、DKK1-siRNA1组及DKK1-siRNA2组,每组10只。对照组肌注生理盐水,其他组肌注地塞米松造模,连续12 w,鉴定骨质疏松模型是否构建成功。构建两段DKK1-siRNA真核表达载体,尾静脉注射治疗组大鼠,对照组和模型组注射等体积PBS,空载体组注射等体积空载体。骨切片观察载体是否靶向到骨,RTPCR检测股骨DKK1 mRNA的表达,免疫组化法检测股骨DKK1蛋白的表达水平。ELISA检测血清中骨碱性磷酸酶(BALP)、Ⅰ型胶原蛋白(Col-Ⅰ)和抗酒石酸酸性磷酸酶(TRAP)的水平;测定股骨骨密度及病理结构变化。结果 (1)双酶切和基因测序鉴定结果表明siRNA真核表达载体构建成功;(2)模型组、空载体组及治疗组大鼠造模后,近端股骨骨密度较对照组明显降低(P<0.05);病理改变为骨小梁数目明显减少、断裂,而对照组骨小梁结构较完整;(3)DKK1-siRNA质粒载体靶向到骨;(4)治疗组大鼠血DKK1-mRNA及股骨DKK1蛋白的表达明显下降;(5)与模型组和空载体组相比,治疗组血清中BALP与Col-Ⅰ水平明显升高,TRAP水平明显下降(P<0.05);(6)治疗后各组大鼠近端股骨骨密度的差异无统计学意义(P>0.05);(7)治疗组大鼠股骨有新生骨小梁形成,骨小梁破坏减轻。结论 DKK1-siRNA促进骨形成,抑制骨吸收,减轻骨结构的破坏。
Objective To construct two vectors expressing siRNA targeting DKK1 gene and to investigate the treatment effect on rat osteoporosis（OP）.Methods Twelve-week-old OP rats were randomly divided into 5 groups,including control group,model group,empty vector group,DKKl-siRNAl group,and DKKl-siRNA2 group,with 10 rats in each group.Rats in latter 4 groups were given dexamethasone for 12 weeks continuously,but rats in control group were injected normal saline.The model of osteoporosis was identified.Two vectors containing DKK1-silencing siRNA were constructed successfully.They were injected through tail vein of OP rats in therapy group,while rats in the control group and model group were injected with the same volume of PBS,and rats in empty vector group were injected with the same volume of empty vector.Bone tissue was sliced to observe if siRNA can target to bone.The expression of DKK1 mRNA in the femur was detected using RT-PCR and DKK1 protein was detected using immunohistochemistry.Serum levels of BALP,Col-I,and TRAP in each group were detected using ELISA method.Bone mineral density and the pathological changes of the femur were observed.Results（1） siRNA eukaryotic expression vectors were successfully constructed.（2） Comparing to control group,bone mineral density of the proximal femurs in model group,empty vector group,and DKK1-siRNA groups decreased significantly（P〈0.05）.The number of trabecular reduced and the trabecular broke in these groups,but the trabecular remained intact in control group.（3） DKK1-siRNA can target to the bone.（4）Expression of DKKl-mRNA in blood and DKK1 protein in bone were significantly decreased in therapy groups.（5） Comparing to model group and empty vector group,serum BALP and Col-I levels in therapy groups significantly decreased（P〈0.05）,but TRAP level increased significantly（P〈0.05）.（6） Bone mineral density of the proximal femur of rats between each group had no significant difference（P〈0.05）.（7） Formation of new trabeculae and re
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2015年第2期125-131,151,共8页
Chinese Journal of Osteoporosis
基金
内蒙古自治区自然科学基金项目(2012MS1106)
内蒙古自治区高等学校科学研究项目(NJZZ122163)
