骨保护素基因修饰的骨髓间充质干细胞治疗大鼠骨质疏松
摘要
目的探究骨保护素(OPG)基因修饰的骨髓间充质干细胞(BMSCs)治疗SD大鼠骨质疏松的可行性及疗效。方法提取并培养SD大鼠BMSCs,用携带OPG基因的慢病毒(LV-OPG-GFP)感染BMSCs,构建成OPG基因修饰的BMSCs,并将其通过尾静脉注射至去卵巢SD大鼠骨质疏松模型体内,其中A组,注射等量生理盐水;B组,BMSCs,1×10~6/kg;C组,注射LV-BMSCs,1×10~6/kg;D组,注射LV-OPG-GFP-BMSCs,1×10~6/kg。通过对SD大鼠的骨密度、血钙、血磷及血清中骨碱性磷酸酶(B-ALP)和Ⅰ型胶原C端肽(CTX-1)水平进行检测,统计并分析治疗效果及可行性。结果成功提取出SD大鼠BMSCs,细胞呈梭形,呈典型的鱼群样或漩涡样生长。LV-OPG-GFP感染BMSCs 72 h后,用倒置荧光显微镜观察发现,实验组OPG已成功转入BMSCs中并能稳定表达,细胞转染率可达80%以上,PCR后电泳显示,实验组OPG mRNA在(500～700)bp处出现明显的条带。模型构建术后3个月,模型组SD大鼠骨密度下降明显,与对照组相比差异有统计学意义(P<0.05)。LV-OPG-GFP-BMSCs尾静脉注射治疗后3个月,骨密度检测结果显示D组骨密度显著高于A、B、C组(P<0.05),但与正常组相比,仍略显不足(P<0.05);血清中钙、磷检测结果显示,D组及正常组血钙含量均显著低于A、B、C三组(P<0.05),而磷含量均显著高于A、B、C三组(P<0.05),而D组与正常组相比无明显差异(P>0.05);血清中B-ALP、CTX-1检测结果显示,D组及正常组B-ALP、CTX-1水平均显著低于A、B、C三组(P<0.05),D组B-ALP与正常组相比无明显差异(P>0.05),而CTX-1水平比正常组稍高(P<0.05)。结论通过尾静脉注射LV-OPG-GFP-BMSCs,以"全身给药"的方式,治疗SD大鼠骨质疏松,未出现明显的毒副作用,治疗效果良好,方法可行,可为临床骨质疏松的治疗提供一定实验依据。
引文
1 Kanis JA,Rd ML,Christiansen C,et al.The diagnosis of osteoporosis[J].J Bone Miner Res,1994;9(8):1137-41.
2 Cano A,Chedraui P,Goulis DG,et al.Calcium in the prevention of postmenopausal osteoporosis:EMAS clinical guide[J].Maturitas,2018;107:7-12.
3 李宏超,宋慧.抗骨吸收和促骨形成药物在骨质疏松症治疗中的联合应用[J].中国骨质疏松杂志,2018;24(3):399-405.
4 Lin X,Xiong D,Peng YQ,et al.Epidemiology and management of osteoporosis in the people's republic of China:current perspectives[J].Clin Int Aging,2015;25(10):1017-33.
5 白璧辉,谢兴文,李鼎鹏,等.我国近5年来骨质疏松症流行病学研究现状[J].中国骨质疏松杂志,2018;24(2):253-8.
6 Tu KN,Lie JD,Wan C,et al.Osteoporosis:a review of treatment options[J].PT,2018;43(2):92-104.
7 张丽卿,雷波,王建宝,等.骨保护素在类风湿关节炎的临床意义[J].中国药物与临床,2018;18(4):515-7.
8 乔松,许琦,叶子青,等.骨保护素对高龄骨质疏松症患者骨代谢的影响[J].解放军医学杂志,2015;40(6):472-4.
9 Karatay CC,Balkarli A,Elmas L,et al.Osteoporosis frequency and the association of rank,rankl,osteoprotegerin gene polymorphisms with osteoporosis in gout patients[J].Ann Rheumatic Dis,2016;75(2):1177-8.
10 Khassawna TE,Merboth F,Malhan D,et al.Osteocyte regulation of receptor activator of NF-κB ligand/osteoprotegerin in a sheep model of osteoporosis[J].Am J Pathol,2017;187(8):1686-99.
11 Pobeha P,Petrasova D,Tkacova R,et al.Circulatory osteoprotegerin is related to osteoporosis of the hip in patients with COPD[J].Respir Med,2014;108(4):621-7.
12 付应霄,顾建红,赵鸿雁,等.骨保护素对破骨细胞活性的影响[J].中国兽医学报,2013;33(5):738-41.
13 廖锋,刘士博,刘瑶,等.重组人骨保护素抑制破骨细胞及促进羟磷灰石修复去势大鼠下颌骨缺损的研究[J].华西口腔医学杂志,2018;36(4):367-71.
14 刘庆羊,孙自强,赵鸿雁,等.自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制[J].畜牧兽医学报,2018;49(8):1761-9.
15 Girolamo LD,Lucarelli E,Alessandri G,et al.Mesenchymal stem/stromal cells:a new "cells as drugs" paradigm.Efficacy and critical aspects in cell therapy[J].Curr Pharm Des,2013;19(13):2459-73.
16 Yang Y,Rossi FM,Putnins EE.Periodontal regeneration using engineered bone marrow mesenchymal stromal cells[J].Biomaterials,2010;31(33):8574-82.
17 张阔,洪寿海,何芹芹,等.大鼠清醒状态下尾静脉注射的快速操作技巧与体会[J].中国比较医学杂志,2016;26(12):81-4.
18 Minguell JJ,Allers C,Lasala GP.Mesenchymal stem cells and the treatment of conditions and diseases:the less glittering side of a conspicuous stem cell for basic research[J].Stem Cells Dev,2013;22(2):193-203.
19 Anonymous.Management of osteoporosis in postmenopausal women:2010 position statement of the North American Menopause society[J].Menopause,2010;17(1):25-54.
20 Simonet WS,Lacey DL,Dunstan CR,et al.Osteoprotegerin:a novel secreted protein involved in the regulation of bone density[J].Cell,1997;89(2):309-19.
21 刘艺,张旗.骨保护素在牙根吸收中保护作用的研究进展[J].口腔医学,2015;35(11):979-82.
22 邓红霞,闫云飞,于钧.骨保护素、胰岛素样生长因子-1对骨关节炎骨重建作用的研究进展[J].中华地方病学杂志,2016;35(7):534-9.
23 李锋,陶天遵,陶树清.移植治疗骨质疏松研究进展[J].中国骨质疏松杂志,2010;16(9):683-6.
24 李春雯,汪煌,史晓林.去卵巢骨质疏松大鼠骨组织OPG/RANKLmRNA动态表达研究[J].中国中医骨伤科杂志,2012;20(11):3-5,8.
25 Hamdy NA.Targeting the RANK/RANKL/OPG signaling pathway:a novel approach in the management of osteoporosis[J].Curr Opin Investig Drugs,2007;8(4):299-303.
26 Liu W,Zhang X.Receptor activator of nuclear factor-κB ligand (RANKL)/RANK/ osteoprotegerin system in bone and other tissues (Review)[J].Mol Med Rep,2015;11(5):3212-8.
27 Kim J,Ko J.A novel PPARγ2 modulator sLZIP controls the balance between adipogenesis and osteogenesis during mesenchymal stem cell differ[J].Cell Death Differ,2014;21(10):1642-55.
28 Liao L,Yang X,Su X,et al.Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow[J].Cell Death Dis,2013;4(4):e600.
29 李延红,党晓谦,龚福太,等.骨质疏松动物模型的研究进展及文献回顾[J].中国组织工程研究,2018;22(12):1956-61.
30 张春丽,李忠海,周颖,等.构建骨质疏松动物模型建模方法的改进及评价[J].中国组织工程研究,2016;20(5):754-9.
31 王振恒,赵建宁,王瑞.骨质疏松动物模型研究进展[J].中国骨质疏松杂志,2012;18(7):656-62.
2 Cano A,Chedraui P,Goulis DG,et al.Calcium in the prevention of postmenopausal osteoporosis:EMAS clinical guide[J].Maturitas,2018;107:7-12.
3 李宏超,宋慧.抗骨吸收和促骨形成药物在骨质疏松症治疗中的联合应用[J].中国骨质疏松杂志,2018;24(3):399-405.
4 Lin X,Xiong D,Peng YQ,et al.Epidemiology and management of osteoporosis in the people's republic of China:current perspectives[J].Clin Int Aging,2015;25(10):1017-33.
5 白璧辉,谢兴文,李鼎鹏,等.我国近5年来骨质疏松症流行病学研究现状[J].中国骨质疏松杂志,2018;24(2):253-8.
6 Tu KN,Lie JD,Wan C,et al.Osteoporosis:a review of treatment options[J].PT,2018;43(2):92-104.
7 张丽卿,雷波,王建宝,等.骨保护素在类风湿关节炎的临床意义[J].中国药物与临床,2018;18(4):515-7.
8 乔松,许琦,叶子青,等.骨保护素对高龄骨质疏松症患者骨代谢的影响[J].解放军医学杂志,2015;40(6):472-4.
9 Karatay CC,Balkarli A,Elmas L,et al.Osteoporosis frequency and the association of rank,rankl,osteoprotegerin gene polymorphisms with osteoporosis in gout patients[J].Ann Rheumatic Dis,2016;75(2):1177-8.
10 Khassawna TE,Merboth F,Malhan D,et al.Osteocyte regulation of receptor activator of NF-κB ligand/osteoprotegerin in a sheep model of osteoporosis[J].Am J Pathol,2017;187(8):1686-99.
11 Pobeha P,Petrasova D,Tkacova R,et al.Circulatory osteoprotegerin is related to osteoporosis of the hip in patients with COPD[J].Respir Med,2014;108(4):621-7.
12 付应霄,顾建红,赵鸿雁,等.骨保护素对破骨细胞活性的影响[J].中国兽医学报,2013;33(5):738-41.
13 廖锋,刘士博,刘瑶,等.重组人骨保护素抑制破骨细胞及促进羟磷灰石修复去势大鼠下颌骨缺损的研究[J].华西口腔医学杂志,2018;36(4):367-71.
14 刘庆羊,孙自强,赵鸿雁,等.自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制[J].畜牧兽医学报,2018;49(8):1761-9.
15 Girolamo LD,Lucarelli E,Alessandri G,et al.Mesenchymal stem/stromal cells:a new "cells as drugs" paradigm.Efficacy and critical aspects in cell therapy[J].Curr Pharm Des,2013;19(13):2459-73.
16 Yang Y,Rossi FM,Putnins EE.Periodontal regeneration using engineered bone marrow mesenchymal stromal cells[J].Biomaterials,2010;31(33):8574-82.
17 张阔,洪寿海,何芹芹,等.大鼠清醒状态下尾静脉注射的快速操作技巧与体会[J].中国比较医学杂志,2016;26(12):81-4.
18 Minguell JJ,Allers C,Lasala GP.Mesenchymal stem cells and the treatment of conditions and diseases:the less glittering side of a conspicuous stem cell for basic research[J].Stem Cells Dev,2013;22(2):193-203.
19 Anonymous.Management of osteoporosis in postmenopausal women:2010 position statement of the North American Menopause society[J].Menopause,2010;17(1):25-54.
20 Simonet WS,Lacey DL,Dunstan CR,et al.Osteoprotegerin:a novel secreted protein involved in the regulation of bone density[J].Cell,1997;89(2):309-19.
21 刘艺,张旗.骨保护素在牙根吸收中保护作用的研究进展[J].口腔医学,2015;35(11):979-82.
22 邓红霞,闫云飞,于钧.骨保护素、胰岛素样生长因子-1对骨关节炎骨重建作用的研究进展[J].中华地方病学杂志,2016;35(7):534-9.
23 李锋,陶天遵,陶树清.移植治疗骨质疏松研究进展[J].中国骨质疏松杂志,2010;16(9):683-6.
24 李春雯,汪煌,史晓林.去卵巢骨质疏松大鼠骨组织OPG/RANKLmRNA动态表达研究[J].中国中医骨伤科杂志,2012;20(11):3-5,8.
25 Hamdy NA.Targeting the RANK/RANKL/OPG signaling pathway:a novel approach in the management of osteoporosis[J].Curr Opin Investig Drugs,2007;8(4):299-303.
26 Liu W,Zhang X.Receptor activator of nuclear factor-κB ligand (RANKL)/RANK/ osteoprotegerin system in bone and other tissues (Review)[J].Mol Med Rep,2015;11(5):3212-8.
27 Kim J,Ko J.A novel PPARγ2 modulator sLZIP controls the balance between adipogenesis and osteogenesis during mesenchymal stem cell differ[J].Cell Death Differ,2014;21(10):1642-55.
28 Liao L,Yang X,Su X,et al.Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow[J].Cell Death Dis,2013;4(4):e600.
29 李延红,党晓谦,龚福太,等.骨质疏松动物模型的研究进展及文献回顾[J].中国组织工程研究,2018;22(12):1956-61.
30 张春丽,李忠海,周颖,等.构建骨质疏松动物模型建模方法的改进及评价[J].中国组织工程研究,2016;20(5):754-9.
31 王振恒,赵建宁,王瑞.骨质疏松动物模型研究进展[J].中国骨质疏松杂志,2012;18(7):656-62.