重组腺病毒介导siBMPR-Ⅱ抑制UHMWPE诱导破骨细胞分化成熟的作用
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摘要
背景
人工关节置换术在治疗类风湿性关节炎、骨关节炎、股骨头坏死以及老年性髋部骨折等方面，已经取得巨大成功，它能显著改善患者的运动功能和生存质量。人工关节松动失效是术后严重的并发症。近年研究发现，假体周围产生的磨损颗粒，诱导破骨细胞过度分化成熟，引起局部骨溶解是导致人工关节晚期无菌松动的最重要因素。防治术后无菌松动的关键即是抑制局部破骨细胞过度激活所引起的骨溶解效应。最近几年发现BMPs可直接或间接调控破骨细胞的分化成熟，使我们思考是否可以通过RNA干扰技术沉默BMP受体，干扰BMP通路来抑制破骨细胞的分化成熟，因为它可以作用于破骨前体细胞本身，也可调控成骨细胞对破骨细胞的影响，这将对抑制破骨细胞的分化成熟起到双管齐下的作用，这是其它靶点及信号通路所不具备的。
目的
构建重组腺病毒siRNA-BMPR-II，观察其在UHMWPE诱导溶骨的小鼠植骨气囊模型中，抑制破骨细胞分化成熟及骨溶解的作用。并通过体外培养原代成骨细胞与原代破骨前体细胞，观察siBMPR是否具有抑制破骨前体细胞向具有溶骨作用的成熟破骨细胞分化的间接或直接调控作用。
方法
1.将选定的siBMPR-II基因片段应用到改良的AdEasy系统中，将siBMPR-II基因片段克隆连接到pSES-HUS穿梭质粒上，构成pSES-HUS-siBMPR-II质粒。在基因测序后，正确的重组质粒被Pmel酶线性化，与骨架质粒pAdEasy-1一起转导到感受态细胞Escherichiacoli BJ5183中，构成pAd-siBMPR-II质粒。并通过脂质体2000转染到293T细胞中进行病毒包装。测定病毒滴度，获得携带siBMPR-II的重组腺病毒。
2.在体内研究中，首先建立小鼠植骨气囊聚乙烯颗粒（UHMWPE）诱导破骨细胞形成的溶骨模型。通过real-time PCR、Western blot、破骨细胞TRAP（抗酒石酸酸性磷酸酶）染色，相关蛋白免疫组织化学与免疫荧光等实验方法，检测各处理组局部破骨细胞数量及溶骨情况。观察siBMPR在体内是否能抑制UHMWPE颗粒引起的破骨细胞过度分化成熟，减少局部溶骨的作用。
3.体外实验：原代成骨细胞与原代破骨前体细胞培养体系的建立。 A:原代成骨细胞的分离培养与纯化。B:原代破骨前体细胞分离与纯化。
(1)在原代破骨前体细胞培养体系中:
通过检测经siBMPR处理过后破骨细胞形成的标志性基因及蛋白的表达，以及破骨细胞的特殊染色(抗酒石酸酸性磷酸酶染色)观察BMP信号通路直接调控破骨细胞分化成熟的作用。
(2)在原代成骨细胞培养体系中：
通过检测经siBMPR处理过后的成骨细胞RANKL、OPG的基因及蛋白表达情况，用以观察siBMPR作用于破骨细胞分化成熟是否是通过间接调控作用。
结果
1.成功构建表达siBMPR-II的重组腺病毒。并通过real-time PCR,Western blot检测,重组腺病毒siBMPR-II可明显减降破骨前体细胞BMPR-II基因与蛋白的表达。
2.证实在UHMWPE诱导溶骨的小鼠植骨气囊模型中，通过siRNA干扰BMPR-II的表达，可以明显抑制破骨细胞的分化成熟及骨的溶解。
3.通过体外培养原代成骨细胞与原代破骨前体细胞，观察得到siBMPR-II可以直接抑制破骨前体细胞向具有溶骨作用的成熟破骨细胞的分化，以及调控成骨细胞OPG/RANKL的表达，间接抑制破骨细胞的分化成熟。
结论
本课题揭示了在磨损颗粒引起的局部溶解模型中，局部注射重组腺病毒siBMPR-II可有效的起到抑制破骨细胞分化成熟与骨溶解的作用。同时也证实了BMPR-II信号通路可直接或间接调控破骨细胞的分化成熟。
Background
Total joint replacement is a common procedure that has provenhighly successful for the treatment of rheumatoid arthritis, ostseoarthritis,femoral head necrosis, hip fractures in the elderly and other diseases. Itreduces pain, restores joint function, and allows arthritis patients to returnto varied activities of daily living. Aseptic loosening(AL) is the single mostcommon complication of total joint arthroplasty. The long-term outcome oftotal joint replacement surgery remains compromised by weardebris-associated implant loosening. The critical factor may contribute toloosening is the adverse tissue response to wear debris. The particles arephagocytosed by macrophages, activate an inflammatory cascade, result ina bone resorption process and promote osteoclastogenesis at thebone-implant interface. A growing body of literature suggests that BMPsinfluence the formation and activity of osteoclasts, and BMP signalingplays an important role in the osteoclast formation. So we will employ anRNA interference approach by transfecting a small interfering RNA (siRNA) specific for BMPR in osteoblast and osteoclast, to inhibit BMPsignaling pathways, regulate osteoclast formation directly or indirectly, andreduce osteolysis. BMP signaling plays a two-pronged role which othersignaling pathways do not have.
Objective
To construct the recombinant adenovirus vector containing siRNAtargeting BMPR-II gene. In order to observe the effect ofadenovirus-mediated siRNA targeting BMPR-II on UHMWPE-inducedosteoclast formation, and the effects of BMPR-II signaling on osteoclastformation are mediated directly by osteoclast itself, as well as indirectly byaltered expression of RANKL and OPG in osteoblast.
Methods
1. To construct the recombinant adenovirus vector containing siRNAtargeting BMPR-II gene using modified AdEasy system, the designedsiRNA oligonucleotide fragments of BMPR-II gene were cloned intoshuttle plasmid pSES-HUS to construct pSES-HUS-siBMPR-II plasmid.Afterward, the correct recombinant was linearized by Pmel, followingco-transformation with the backbone vector pAdEasy-1in Escherichia coliBJ5183to construct pAd-siBMPR-II plasmid, and then transfected into293T cell line via Lipofectamine2000to package the adenovirus. Viralsupernatants were harvested. The viral titers were determined by infecting293T cells with serial dilutions of concentrated adenoviruses.
2. In vivo studies, firstly, we should build the air pouch model inwhich the differentiation and formation of osteoclasts are induced byUHMWPE, then test the effect of adenovirus-mediated siRNA targetingBMPR-II on UHMWPE-induced osteoclast formation by real-time PCR,Western blot, TRAP staining, immunohistochemistry, immunofluorescence,electron microscopy and so on.
3. In vitro studies, firstly, we should build the bone marrow cells andprimary osteoblasts culture system.
(1) In the bone marrow cells culture system, to test whether theadenovirus-mediated siRNA targeting BMPR-II can inhibit the osteoclastdifferentiation by real-time PCR, Western blot, TRAP staining and so on.
(2) In the primary osteoblasts culture system, to measure whether theadenovirus-mediated siRNA targeting BMPR-II can changed the gene andprotein expression of RANKL and OPG by real-time PCR, Western blot.
Results
1. Adenovirus-mediated siRNA targeting BMPR-II was builtsuccessfully. And adenovirus-mediated BMPR-II siRNA could successfullyinhibit BMPR-II expression in high efficiency both at mRNA and proteinlevels.
2. The air pouch model with UHMWPE was built successfully. Wefound that siBMPR-II inhibited differ-entiation of osteoclasts in theUHMWPE-stimulated pouches, including reduction of gene and protein expression levels of TRAP and RANK, and TRAP positive cells, decreasein the area of absorbing lacuna.
3. Furthermore, we revealed that loss of BMPR-II signaling inhibitsosteoclast differentiation directly, and acts on osteoblasts to reduceosteoclast formation through altered expression of RANKL and OPG,including down-regulation of the amount of TRAP positive cells inducedby RANKL, the gene and protein expression levels of osteoclast marker(TRAP and RANK) in the bone marrow cells culture system, and resultingin an increase of OPG mRNA and protein expression levels, a decrease ofRANKL expression in the primary osteoblasts culture system.
Conclusions
In the present study, we revealed that locally injection ofadenovirus-mediated siRNA targeting BMPR-II appears to be a feasibleand effective candidate to treat or prevent wear debris-associated osteolysis.Furthermore, we revealed that the effects of BMPR-II signaling onosteoclast formation are mediated directly by osteoclast itself, as well asindirectly by altered expression of RANKL and OPG in osteoblast.
人工关节置换术在治疗类风湿性关节炎、骨关节炎、股骨头坏死以及老年性髋部骨折等方面，已经取得巨大成功，它能显著改善患者的运动功能和生存质量。人工关节松动失效是术后严重的并发症。近年研究发现，假体周围产生的磨损颗粒，诱导破骨细胞过度分化成熟，引起局部骨溶解是导致人工关节晚期无菌松动的最重要因素。防治术后无菌松动的关键即是抑制局部破骨细胞过度激活所引起的骨溶解效应。最近几年发现BMPs可直接或间接调控破骨细胞的分化成熟，使我们思考是否可以通过RNA干扰技术沉默BMP受体，干扰BMP通路来抑制破骨细胞的分化成熟，因为它可以作用于破骨前体细胞本身，也可调控成骨细胞对破骨细胞的影响，这将对抑制破骨细胞的分化成熟起到双管齐下的作用，这是其它靶点及信号通路所不具备的。
目的
构建重组腺病毒siRNA-BMPR-II，观察其在UHMWPE诱导溶骨的小鼠植骨气囊模型中，抑制破骨细胞分化成熟及骨溶解的作用。并通过体外培养原代成骨细胞与原代破骨前体细胞，观察siBMPR是否具有抑制破骨前体细胞向具有溶骨作用的成熟破骨细胞分化的间接或直接调控作用。
方法
1.将选定的siBMPR-II基因片段应用到改良的AdEasy系统中，将siBMPR-II基因片段克隆连接到pSES-HUS穿梭质粒上，构成pSES-HUS-siBMPR-II质粒。在基因测序后，正确的重组质粒被Pmel酶线性化，与骨架质粒pAdEasy-1一起转导到感受态细胞Escherichiacoli BJ5183中，构成pAd-siBMPR-II质粒。并通过脂质体2000转染到293T细胞中进行病毒包装。测定病毒滴度，获得携带siBMPR-II的重组腺病毒。
2.在体内研究中，首先建立小鼠植骨气囊聚乙烯颗粒（UHMWPE）诱导破骨细胞形成的溶骨模型。通过real-time PCR、Western blot、破骨细胞TRAP（抗酒石酸酸性磷酸酶）染色，相关蛋白免疫组织化学与免疫荧光等实验方法，检测各处理组局部破骨细胞数量及溶骨情况。观察siBMPR在体内是否能抑制UHMWPE颗粒引起的破骨细胞过度分化成熟，减少局部溶骨的作用。
3.体外实验：原代成骨细胞与原代破骨前体细胞培养体系的建立。 A:原代成骨细胞的分离培养与纯化。B:原代破骨前体细胞分离与纯化。
(1)在原代破骨前体细胞培养体系中:
通过检测经siBMPR处理过后破骨细胞形成的标志性基因及蛋白的表达，以及破骨细胞的特殊染色(抗酒石酸酸性磷酸酶染色)观察BMP信号通路直接调控破骨细胞分化成熟的作用。
(2)在原代成骨细胞培养体系中：
通过检测经siBMPR处理过后的成骨细胞RANKL、OPG的基因及蛋白表达情况，用以观察siBMPR作用于破骨细胞分化成熟是否是通过间接调控作用。
结果
1.成功构建表达siBMPR-II的重组腺病毒。并通过real-time PCR,Western blot检测,重组腺病毒siBMPR-II可明显减降破骨前体细胞BMPR-II基因与蛋白的表达。
2.证实在UHMWPE诱导溶骨的小鼠植骨气囊模型中，通过siRNA干扰BMPR-II的表达，可以明显抑制破骨细胞的分化成熟及骨的溶解。
3.通过体外培养原代成骨细胞与原代破骨前体细胞，观察得到siBMPR-II可以直接抑制破骨前体细胞向具有溶骨作用的成熟破骨细胞的分化，以及调控成骨细胞OPG/RANKL的表达，间接抑制破骨细胞的分化成熟。
结论
本课题揭示了在磨损颗粒引起的局部溶解模型中，局部注射重组腺病毒siBMPR-II可有效的起到抑制破骨细胞分化成熟与骨溶解的作用。同时也证实了BMPR-II信号通路可直接或间接调控破骨细胞的分化成熟。
Background
Total joint replacement is a common procedure that has provenhighly successful for the treatment of rheumatoid arthritis, ostseoarthritis,femoral head necrosis, hip fractures in the elderly and other diseases. Itreduces pain, restores joint function, and allows arthritis patients to returnto varied activities of daily living. Aseptic loosening(AL) is the single mostcommon complication of total joint arthroplasty. The long-term outcome oftotal joint replacement surgery remains compromised by weardebris-associated implant loosening. The critical factor may contribute toloosening is the adverse tissue response to wear debris. The particles arephagocytosed by macrophages, activate an inflammatory cascade, result ina bone resorption process and promote osteoclastogenesis at thebone-implant interface. A growing body of literature suggests that BMPsinfluence the formation and activity of osteoclasts, and BMP signalingplays an important role in the osteoclast formation. So we will employ anRNA interference approach by transfecting a small interfering RNA (siRNA) specific for BMPR in osteoblast and osteoclast, to inhibit BMPsignaling pathways, regulate osteoclast formation directly or indirectly, andreduce osteolysis. BMP signaling plays a two-pronged role which othersignaling pathways do not have.
Objective
To construct the recombinant adenovirus vector containing siRNAtargeting BMPR-II gene. In order to observe the effect ofadenovirus-mediated siRNA targeting BMPR-II on UHMWPE-inducedosteoclast formation, and the effects of BMPR-II signaling on osteoclastformation are mediated directly by osteoclast itself, as well as indirectly byaltered expression of RANKL and OPG in osteoblast.
Methods
1. To construct the recombinant adenovirus vector containing siRNAtargeting BMPR-II gene using modified AdEasy system, the designedsiRNA oligonucleotide fragments of BMPR-II gene were cloned intoshuttle plasmid pSES-HUS to construct pSES-HUS-siBMPR-II plasmid.Afterward, the correct recombinant was linearized by Pmel, followingco-transformation with the backbone vector pAdEasy-1in Escherichia coliBJ5183to construct pAd-siBMPR-II plasmid, and then transfected into293T cell line via Lipofectamine2000to package the adenovirus. Viralsupernatants were harvested. The viral titers were determined by infecting293T cells with serial dilutions of concentrated adenoviruses.
2. In vivo studies, firstly, we should build the air pouch model inwhich the differentiation and formation of osteoclasts are induced byUHMWPE, then test the effect of adenovirus-mediated siRNA targetingBMPR-II on UHMWPE-induced osteoclast formation by real-time PCR,Western blot, TRAP staining, immunohistochemistry, immunofluorescence,electron microscopy and so on.
3. In vitro studies, firstly, we should build the bone marrow cells andprimary osteoblasts culture system.
(1) In the bone marrow cells culture system, to test whether theadenovirus-mediated siRNA targeting BMPR-II can inhibit the osteoclastdifferentiation by real-time PCR, Western blot, TRAP staining and so on.
(2) In the primary osteoblasts culture system, to measure whether theadenovirus-mediated siRNA targeting BMPR-II can changed the gene andprotein expression of RANKL and OPG by real-time PCR, Western blot.
Results
1. Adenovirus-mediated siRNA targeting BMPR-II was builtsuccessfully. And adenovirus-mediated BMPR-II siRNA could successfullyinhibit BMPR-II expression in high efficiency both at mRNA and proteinlevels.
2. The air pouch model with UHMWPE was built successfully. Wefound that siBMPR-II inhibited differ-entiation of osteoclasts in theUHMWPE-stimulated pouches, including reduction of gene and protein expression levels of TRAP and RANK, and TRAP positive cells, decreasein the area of absorbing lacuna.
3. Furthermore, we revealed that loss of BMPR-II signaling inhibitsosteoclast differentiation directly, and acts on osteoblasts to reduceosteoclast formation through altered expression of RANKL and OPG,including down-regulation of the amount of TRAP positive cells inducedby RANKL, the gene and protein expression levels of osteoclast marker(TRAP and RANK) in the bone marrow cells culture system, and resultingin an increase of OPG mRNA and protein expression levels, a decrease ofRANKL expression in the primary osteoblasts culture system.
Conclusions
In the present study, we revealed that locally injection ofadenovirus-mediated siRNA targeting BMPR-II appears to be a feasibleand effective candidate to treat or prevent wear debris-associated osteolysis.Furthermore, we revealed that the effects of BMPR-II signaling onosteoclast formation are mediated directly by osteoclast itself, as well asindirectly by altered expression of RANKL and OPG in osteoblast.
引文
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