论文部分内容阅读
[摘要]目的探讨多聚蛋白多糖（Aggrecan）过表达载体修饰骨髓间充质干细胞（BMSCs）来源外泌体联合丝素蛋白支架对新西兰大白兔软骨缺损的修复作用。方法培养兔源性BMSCs，流式细胞仪检测第2代BMSCs表面蛋白CD44、CD29、CD34和CD45表达。慢病毒Aggrecan过表达质粒转染BMSCs，超速离心法提取外泌体，Western Blot方法检测外泌体分子标志物CD81和CD63的表达。构建丝素蛋白支架联合外泌体复合物，利用扫描电镜技术检测丝蛋白支架的结构。利用苏木精`-伊红（HE）染色和番红`-快绿染色检测新西兰大白兔软骨缺损模型中软骨结构。结果第2代BMSCs表面蛋白CD44和CD29表达分别为60.2%和58.3%，CD34和CD45表达分别为3.4%和2.6%。慢病毒Aggrecan過表达载体转染效率为（95±2）%。扫描电镜观察显示，外泌体为直径40～100 nm双层膜的膜状结构。Western Blot检测显示，外泌体可以表达分子标志物CD81和CD63。HE染色和番红`-快绿染色结果显示，Aggrecan过表达载体修饰BMSCs分泌的外泌体联合丝素蛋白支架对软骨缺损的修复作用明显优于BMSC组和阴性对照组（F=19.298、12.548，P<0.05）。结论Aggrecan过表达载体修饰BMSCs分泌的外泌体联合丝素蛋白支架对软骨缺损的修复具有明显的促进作用，可为骨性关节炎治疗提供新的思路与方法。
[关键词]外泌体;间充质基质细胞;丝素蛋白支架;骨关节炎
[ABSTRACT]ObjectiveTo investigate the effect of exosome derived from Aggrecan overexpression vector`-modified bone marrow mesenchymal stem cells （BMSCs） combined with silk fibroin scaffold in the repair of cartilage defect in New Zealand white rabbits. MethodsRabbit`-derived BMSCs were cultured. Flow cytometry was used to measure the expression of CD44， CD29， CD34， and CD45 on the surface of the second`-generation BMSCs. Lentivirus Aggrecan`-overexpression plasmid was transfected into BMSCs， the exosomes were extracted by ultracentrifugation， and Western blot was used to measure the expression of the biomar`-kers CD81 and CD63. The silk fibroin scaffold`-exosome complexes were constructed， and scanning electron microscopy was used to observe the structure of silk fibroin scaffolds. HE staining and safranine`-fast green staining were used to observe cartilage structure in New Zealand white rabbits with cartilage defect. ResultsThe expression rates of CD44 and CD29 on the surface of the se`-cond`-generation BMSCs were 60.2% and 58.3%， respectively， and the expression rates of CD34 and CD45 were 3.4% and 2.6%， respectively. The transfection efficiency of lentiviral Aggrecan`-overexpression vector was （95±2）%. Scanning electron microscopy showed that the exosome had a double`-membrane structure with a diameter of 40-100 nm. Western blot showed that exosomes expressed the molecular markers CD81 and CD63. HE staining and saffron`-fast green staining showed that exosomes secreted by Aggrecan overexpression vector`-modified BMSCs combined with silk fibroin scaffold had a better effect in repairing cartilage defect than the BMSC group and the negative control group （F=19.298 and 12.548，P<0.05）. Conclusionexosomes secreted by Aggrecan overexpression vector`-modified BMSCs combined with silk fibroin scaffold can significantly promote the repair of cartilage defect， which provides new ideas and methods for the treatment of osteoarthritis in clinical practice. [3]OSAWA Y， SEKI T， TAKEGAMI Y， et al. Cementless total hip arthroplasty for osteonecrosis and osteoarthritis produce similar results at ten years follow`-up when matched for age and gender[J]. International Orthopaedics， 2018，42（7， SI）：1683`-1688.
[4]HUI Tianqian， ZHOU Yachuan， WANG Tingyu， et al. Activation of beta`-catenin signaling in aggrecan`-expressing cells in temporomandibular joint causes osteoarthritis`-like defects[J]. International Journal of Oral Science， 2018，10（2）：13`-17.
[5]ZHANG Zhao， LI Xiaofei， HUANG Heng， et al. Cross`-coupling effects of silencing of cyclooxygenase`-2 （COX`-2）/aggrecanase`-1 and over`-expressed insulin`-like growth factor 1 （IGF`-1） in an osteoarthritis animal model[J]. Medical Science Monitor， 2017，23（7）：5302`-5310.
[6]馮笑，韩絮，张亚，等. 与软骨细胞共培养促进BMSCs分化为软骨细胞[J]. 江苏大学学报（医学版）， 2017，27（5）：403`-407.
[7]阿勒泰别克·闹乎旦，张玉玲，白广超，等. Wnt因子联合BMP`-7诱导BMSCs向成骨分化的研究[J]. 石河子大学学报（自然科学版）， 2017，35（1）：113`-118.
[8]薄建成，张海宁，杜青峰，等. 介导pcDNA`-IGF`-1质粒转染骨髓间充质干细胞两种方法比较[J]. 青岛大学医学院学报， 2016，52（5）：505`-507，511.
[9]王颖，张雷，周咏，等. miR`-21诱导犬BMSCs骨向分化的体外实验研究[J]. 安徽医科大学学报， 2017，52（9）：1318`-1323.
[10]徐亮，陶树清，文刚，等. RhoA/ROCK信号通路在骨质疏松大鼠BMSCs成骨分化中的研究[J]. 中国骨质疏松杂志， 2017，23（11）：1415`-1419.
[11]亓俊华，吴梅，徐祥，等. TNF`-α对小鼠骨髓间充质干细胞免疫抑制作用影响[J]. 青岛大学医学院学报， 2015，51（2）：169`-171，174.
[12]黄勇，吴华拉，马琳，等. γ`-分泌酶抑制剂阻断Notch信号通路对BMSCs向肺泡上皮细胞分化的影响[J]. 基因组学与应用生物学， 2017，36（5）：1727`-1731.
[13]陈能，邵云峰，刘傥，等. 带部分松质骨小牛皮质骨复合骨髓间充质干细胞植入兔体内成骨及骨形态发生蛋白2的表达[J]. 中国组织工程研究， 2017，21（17）：2684`-2689.
[14]刘天丹，张保朝，郝明亮. 胶原蛋白`-明胶复合支架材料修复周围神经缺损[J]. 中国组织工程研究， 2017，21（2）：286`-290.
[15]陶旋，李强，李诗鹏，等. 慢病毒介导人骨形态发生蛋白2/骨髓间充质干细胞/脱钙骨基质修复股骨大段缺损[J]. 中国组织工程研究， 2018，22（9）：1338`-1343.
[16]张志勇，于光屹，陶丹丹，等. 应用骨髓间充质干细胞复合关节软骨脱细胞基质修复兔软骨缺损的实验研究[J]. 中国医药指南， 2017，15（22）：53`-54.
[17]姜良斌，韦标方，冯志，等. 人脱细胞羊膜与骨髓间充质干细胞复合体修复关节软骨缺损[J]. 中国组织工程研究， 2017，21（26）：4113`-4118.
[18]SONG Jialin， ZHENG Wei， CHEN Wei， et al. Lentivirus`-mediated microRNA`-124 gene`-modified bone marrow mesenchymal stem cell transplantation promotes the repair of spinal cord injury in rats[J]. Experimental and Molecular Medicine， 2017，49（5）：1`-9.
[19]AN Ke， LIU Huiping， ZHONG Xiaolong， et al. hTERT`-Immortalized bone mesenchymal stromal cells expressing rat galanin via a single tetracycline`-inducible lentivirus system[J]. Stem Cells Int， 2017， 2017：6082684. [20]WANG Bangjun， LIAN Kai， LI Jun， et al. Restoration of osteogenic differentiation by overexpression of cannabinoid receptor 2 in bone marrow mesenchymal stem cells isolated from osteoporotic patients[J]. Experimental and Therapeutic Me`-dicine， 2018，15（1）：357`-364.
[21]DU Xiufan， HUANG Fangli， ZHANG Shujiang， et al. Carboxymethylcellulose with phenolic hydroxyl microcapsules enclosinggene`-modified BMSCs for controlled BMP`-2 release in vitro[J]. Artificial Cells Nanomedicine And Biotechnology， 2017，45（8）：1`-14.
[22]LU Yao， GAO Hui， ZHANG Man， et al. Glial cell line`-derived neurotrophic factor`-transfected placenta`-derived versus bone marrow`-derived mesenchymal cells for treating spinal cord injury[J]. Medical Science Monitor， 2017，23（1）：1800`-1811.
[23]ZENG Yanling， ZHENG Hao， CHEN Qiuru， et al. Bone marrow`-derived mesenchymal stem cells overexpressing MiR`-21 efficiently repair myocardial damage in rats[J]. Oncotarget， 2017，8（17）：29161`-29173.
[24]SUN Bingyin， ZHAO Baoxiang， ZHU Jieying， et al. Role of TGF`-beta 1 expressed in bone marrow`-derived mesenchymal stem cells in promoting bone formation in a rabbit femoral defect model[J]. International Journal of Molecular Medicine， 2018，42（2）：897`-904.
[25]ZHAO Na， ZENG Li， LIU Yang， et al. DLX3 promotes bone marrow mesenchymal stem cell proliferation through H19/miR`-675 axis[J]. Clinical Science， 2017，131（22）：2721`-2735.
[26]KAMERKAR S， LEBLEU V S， SUGIMOTO H， et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J]. Nature， 2017，546（7659）：498`-503.
[27]PAN Lei， LIANG Wei， FU Min， et al. Exosomes`-mediated transfer of long noncoding RNA ZFAS1 promotes gastric can`-cer progression[J]. Journal of Cancer Research and Clinical Oncology， 2017，143（6）：991`-1004.
[28]GUO Shangchun， TAO Shicong， YIN Wenjing， et al. Exosomes derived from platelet`-rich plasma promote the re`-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model[J]. Theranostics， 2017，7（1）：81`-96.
[29]TAO Shicong， YUAN Ting， ZHANG Yuelei， et al. Exosomes derived from miR`-140`-5p`-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model[J]. Theranostics， 2017，7（1）：180`-195.
[关键词]外泌体;间充质基质细胞;丝素蛋白支架;骨关节炎
[ABSTRACT]ObjectiveTo investigate the effect of exosome derived from Aggrecan overexpression vector`-modified bone marrow mesenchymal stem cells （BMSCs） combined with silk fibroin scaffold in the repair of cartilage defect in New Zealand white rabbits. MethodsRabbit`-derived BMSCs were cultured. Flow cytometry was used to measure the expression of CD44， CD29， CD34， and CD45 on the surface of the second`-generation BMSCs. Lentivirus Aggrecan`-overexpression plasmid was transfected into BMSCs， the exosomes were extracted by ultracentrifugation， and Western blot was used to measure the expression of the biomar`-kers CD81 and CD63. The silk fibroin scaffold`-exosome complexes were constructed， and scanning electron microscopy was used to observe the structure of silk fibroin scaffolds. HE staining and safranine`-fast green staining were used to observe cartilage structure in New Zealand white rabbits with cartilage defect. ResultsThe expression rates of CD44 and CD29 on the surface of the se`-cond`-generation BMSCs were 60.2% and 58.3%， respectively， and the expression rates of CD34 and CD45 were 3.4% and 2.6%， respectively. The transfection efficiency of lentiviral Aggrecan`-overexpression vector was （95±2）%. Scanning electron microscopy showed that the exosome had a double`-membrane structure with a diameter of 40-100 nm. Western blot showed that exosomes expressed the molecular markers CD81 and CD63. HE staining and saffron`-fast green staining showed that exosomes secreted by Aggrecan overexpression vector`-modified BMSCs combined with silk fibroin scaffold had a better effect in repairing cartilage defect than the BMSC group and the negative control group （F=19.298 and 12.548，P<0.05）. Conclusionexosomes secreted by Aggrecan overexpression vector`-modified BMSCs combined with silk fibroin scaffold can significantly promote the repair of cartilage defect， which provides new ideas and methods for the treatment of osteoarthritis in clinical practice. [3]OSAWA Y， SEKI T， TAKEGAMI Y， et al. Cementless total hip arthroplasty for osteonecrosis and osteoarthritis produce similar results at ten years follow`-up when matched for age and gender[J]. International Orthopaedics， 2018，42（7， SI）：1683`-1688.
[4]HUI Tianqian， ZHOU Yachuan， WANG Tingyu， et al. Activation of beta`-catenin signaling in aggrecan`-expressing cells in temporomandibular joint causes osteoarthritis`-like defects[J]. International Journal of Oral Science， 2018，10（2）：13`-17.
[5]ZHANG Zhao， LI Xiaofei， HUANG Heng， et al. Cross`-coupling effects of silencing of cyclooxygenase`-2 （COX`-2）/aggrecanase`-1 and over`-expressed insulin`-like growth factor 1 （IGF`-1） in an osteoarthritis animal model[J]. Medical Science Monitor， 2017，23（7）：5302`-5310.
[6]馮笑，韩絮，张亚，等. 与软骨细胞共培养促进BMSCs分化为软骨细胞[J]. 江苏大学学报（医学版）， 2017，27（5）：403`-407.
[7]阿勒泰别克·闹乎旦，张玉玲，白广超，等. Wnt因子联合BMP`-7诱导BMSCs向成骨分化的研究[J]. 石河子大学学报（自然科学版）， 2017，35（1）：113`-118.
[8]薄建成，张海宁，杜青峰，等. 介导pcDNA`-IGF`-1质粒转染骨髓间充质干细胞两种方法比较[J]. 青岛大学医学院学报， 2016，52（5）：505`-507，511.
[9]王颖，张雷，周咏，等. miR`-21诱导犬BMSCs骨向分化的体外实验研究[J]. 安徽医科大学学报， 2017，52（9）：1318`-1323.
[10]徐亮，陶树清，文刚，等. RhoA/ROCK信号通路在骨质疏松大鼠BMSCs成骨分化中的研究[J]. 中国骨质疏松杂志， 2017，23（11）：1415`-1419.
[11]亓俊华，吴梅，徐祥，等. TNF`-α对小鼠骨髓间充质干细胞免疫抑制作用影响[J]. 青岛大学医学院学报， 2015，51（2）：169`-171，174.
[12]黄勇，吴华拉，马琳，等. γ`-分泌酶抑制剂阻断Notch信号通路对BMSCs向肺泡上皮细胞分化的影响[J]. 基因组学与应用生物学， 2017，36（5）：1727`-1731.
[13]陈能，邵云峰，刘傥，等. 带部分松质骨小牛皮质骨复合骨髓间充质干细胞植入兔体内成骨及骨形态发生蛋白2的表达[J]. 中国组织工程研究， 2017，21（17）：2684`-2689.
[14]刘天丹，张保朝，郝明亮. 胶原蛋白`-明胶复合支架材料修复周围神经缺损[J]. 中国组织工程研究， 2017，21（2）：286`-290.
[15]陶旋，李强，李诗鹏，等. 慢病毒介导人骨形态发生蛋白2/骨髓间充质干细胞/脱钙骨基质修复股骨大段缺损[J]. 中国组织工程研究， 2018，22（9）：1338`-1343.
[16]张志勇，于光屹，陶丹丹，等. 应用骨髓间充质干细胞复合关节软骨脱细胞基质修复兔软骨缺损的实验研究[J]. 中国医药指南， 2017，15（22）：53`-54.
[17]姜良斌，韦标方，冯志，等. 人脱细胞羊膜与骨髓间充质干细胞复合体修复关节软骨缺损[J]. 中国组织工程研究， 2017，21（26）：4113`-4118.
[18]SONG Jialin， ZHENG Wei， CHEN Wei， et al. Lentivirus`-mediated microRNA`-124 gene`-modified bone marrow mesenchymal stem cell transplantation promotes the repair of spinal cord injury in rats[J]. Experimental and Molecular Medicine， 2017，49（5）：1`-9.
[19]AN Ke， LIU Huiping， ZHONG Xiaolong， et al. hTERT`-Immortalized bone mesenchymal stromal cells expressing rat galanin via a single tetracycline`-inducible lentivirus system[J]. Stem Cells Int， 2017， 2017：6082684. [20]WANG Bangjun， LIAN Kai， LI Jun， et al. Restoration of osteogenic differentiation by overexpression of cannabinoid receptor 2 in bone marrow mesenchymal stem cells isolated from osteoporotic patients[J]. Experimental and Therapeutic Me`-dicine， 2018，15（1）：357`-364.
[21]DU Xiufan， HUANG Fangli， ZHANG Shujiang， et al. Carboxymethylcellulose with phenolic hydroxyl microcapsules enclosinggene`-modified BMSCs for controlled BMP`-2 release in vitro[J]. Artificial Cells Nanomedicine And Biotechnology， 2017，45（8）：1`-14.
[22]LU Yao， GAO Hui， ZHANG Man， et al. Glial cell line`-derived neurotrophic factor`-transfected placenta`-derived versus bone marrow`-derived mesenchymal cells for treating spinal cord injury[J]. Medical Science Monitor， 2017，23（1）：1800`-1811.
[23]ZENG Yanling， ZHENG Hao， CHEN Qiuru， et al. Bone marrow`-derived mesenchymal stem cells overexpressing MiR`-21 efficiently repair myocardial damage in rats[J]. Oncotarget， 2017，8（17）：29161`-29173.
[24]SUN Bingyin， ZHAO Baoxiang， ZHU Jieying， et al. Role of TGF`-beta 1 expressed in bone marrow`-derived mesenchymal stem cells in promoting bone formation in a rabbit femoral defect model[J]. International Journal of Molecular Medicine， 2018，42（2）：897`-904.
[25]ZHAO Na， ZENG Li， LIU Yang， et al. DLX3 promotes bone marrow mesenchymal stem cell proliferation through H19/miR`-675 axis[J]. Clinical Science， 2017，131（22）：2721`-2735.
[26]KAMERKAR S， LEBLEU V S， SUGIMOTO H， et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J]. Nature， 2017，546（7659）：498`-503.
[27]PAN Lei， LIANG Wei， FU Min， et al. Exosomes`-mediated transfer of long noncoding RNA ZFAS1 promotes gastric can`-cer progression[J]. Journal of Cancer Research and Clinical Oncology， 2017，143（6）：991`-1004.
[28]GUO Shangchun， TAO Shicong， YIN Wenjing， et al. Exosomes derived from platelet`-rich plasma promote the re`-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model[J]. Theranostics， 2017，7（1）：81`-96.
[29]TAO Shicong， YUAN Ting， ZHANG Yuelei， et al. Exosomes derived from miR`-140`-5p`-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model[J]. Theranostics， 2017，7（1）：180`-195.