中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
33期
5257-5265
,共9页
刘国印%王瑞%董磊%张俊峰%赵建宁
劉國印%王瑞%董磊%張俊峰%趙建寧
류국인%왕서%동뢰%장준봉%조건저
组织构建%骨组织工程%磨损微粒%成骨细胞%内质网应激反应%细胞凋亡%骨溶解%无菌性松动%国家自然科学基金
組織構建%骨組織工程%磨損微粒%成骨細胞%內質網應激反應%細胞凋亡%骨溶解%無菌性鬆動%國傢自然科學基金
조직구건%골조직공정%마손미립%성골세포%내질망응격반응%세포조망%골용해%무균성송동%국가자연과학기금
osteoblasts%endoplasmic reticulum%Caspases
背景：磨损微粒能够在体外诱导成骨细胞凋亡，但是发生骨溶解的骨组织中是否也存在成骨细胞的凋亡以及骨组织中的成骨细胞凋亡信号通过何种途径进行传导目前尚不清楚。<br> 目的：分析内质网应激反应在骨溶解骨组织中成骨细胞凋亡和骨溶解发生发展中的作用。<br> 方法：制备磨损微粒诱导骨溶解动物模型。实验分为4组：空白对照组只接受PBS的刺激；磨损微粒组只接受纳米合金粉末悬液的刺激；内质网应激阳性对照组接受纳米合金粉末+毒胡萝卜素的刺激；内质网应激抑制组接受纳米合金粉末悬液及造模后当时、造模后1，2，3和5 d分别腹腔注射4-苯基丁酸。通过甲苯胺蓝染色、苏木精-伊红染色和碱性磷酸酶染色观察骨溶解的病理变化；分析骨溶解颅骨组织中成骨细胞分化成熟情况；Western Blot 方法检测骨溶解颅骨组织内内质网应激反应标志蛋白的表达变化；TUNEL 和 Caspase-3免疫组织化学方法检测骨溶解颅骨组织内成骨细胞的凋亡情况。<br> 结果与结论：磨损微粒能够在体外诱导小鼠颅骨骨溶解的发生、加重炎症细胞的浸润以及抑制成骨细胞分化成熟，同时磨损微粒还可以上调成骨细胞内质网应激反应标志蛋白以及促进骨溶解骨组织中成骨细胞的凋亡。经内质网应激抑制剂(4-苯基丁酸)的治疗后，骨溶解症状明显缓解，骨侵蚀和炎症浸润显著降低，成骨细胞的分化成熟得到改善，凋亡的成骨细胞急剧减少，内质网应激标志蛋白的表达逐渐减弱。表明内质网应激反应参与骨溶解的形成并在骨溶解的发生发展中发挥重要作用。提示内质网应激可作为一种新的治疗靶点，为临床逆转或治疗骨溶解和无菌性松动提供新的思路和方法。
揹景：磨損微粒能夠在體外誘導成骨細胞凋亡，但是髮生骨溶解的骨組織中是否也存在成骨細胞的凋亡以及骨組織中的成骨細胞凋亡信號通過何種途徑進行傳導目前尚不清楚。<br> 目的：分析內質網應激反應在骨溶解骨組織中成骨細胞凋亡和骨溶解髮生髮展中的作用。<br> 方法：製備磨損微粒誘導骨溶解動物模型。實驗分為4組：空白對照組隻接受PBS的刺激；磨損微粒組隻接受納米閤金粉末懸液的刺激；內質網應激暘性對照組接受納米閤金粉末+毒鬍蘿蔔素的刺激；內質網應激抑製組接受納米閤金粉末懸液及造模後噹時、造模後1，2，3和5 d分彆腹腔註射4-苯基丁痠。通過甲苯胺藍染色、囌木精-伊紅染色和堿性燐痠酶染色觀察骨溶解的病理變化；分析骨溶解顱骨組織中成骨細胞分化成熟情況；Western Blot 方法檢測骨溶解顱骨組織內內質網應激反應標誌蛋白的錶達變化；TUNEL 和 Caspase-3免疫組織化學方法檢測骨溶解顱骨組織內成骨細胞的凋亡情況。<br> 結果與結論：磨損微粒能夠在體外誘導小鼠顱骨骨溶解的髮生、加重炎癥細胞的浸潤以及抑製成骨細胞分化成熟，同時磨損微粒還可以上調成骨細胞內質網應激反應標誌蛋白以及促進骨溶解骨組織中成骨細胞的凋亡。經內質網應激抑製劑(4-苯基丁痠)的治療後，骨溶解癥狀明顯緩解，骨侵蝕和炎癥浸潤顯著降低，成骨細胞的分化成熟得到改善，凋亡的成骨細胞急劇減少，內質網應激標誌蛋白的錶達逐漸減弱。錶明內質網應激反應參與骨溶解的形成併在骨溶解的髮生髮展中髮揮重要作用。提示內質網應激可作為一種新的治療靶點，為臨床逆轉或治療骨溶解和無菌性鬆動提供新的思路和方法。
배경：마손미립능구재체외유도성골세포조망，단시발생골용해적골조직중시부야존재성골세포적조망이급골조직중적성골세포조망신호통과하충도경진행전도목전상불청초。<br> 목적：분석내질망응격반응재골용해골조직중성골세포조망화골용해발생발전중적작용。<br> 방법：제비마손미립유도골용해동물모형。실험분위4조：공백대조조지접수PBS적자격；마손미립조지접수납미합금분말현액적자격；내질망응격양성대조조접수납미합금분말+독호라복소적자격；내질망응격억제조접수납미합금분말현액급조모후당시、조모후1，2，3화5 d분별복강주사4-분기정산。통과갑분알람염색、소목정-이홍염색화감성린산매염색관찰골용해적병리변화；분석골용해로골조직중성골세포분화성숙정황；Western Blot 방법검측골용해로골조직내내질망응격반응표지단백적표체변화；TUNEL 화 Caspase-3면역조직화학방법검측골용해로골조직내성골세포적조망정황。<br> 결과여결론：마손미립능구재체외유도소서로골골용해적발생、가중염증세포적침윤이급억제성골세포분화성숙，동시마손미립환가이상조성골세포내질망응격반응표지단백이급촉진골용해골조직중성골세포적조망。경내질망응격억제제(4-분기정산)적치료후，골용해증상명현완해，골침식화염증침윤현저강저，성골세포적분화성숙득도개선，조망적성골세포급극감소，내질망응격표지단백적표체축점감약。표명내질망응격반응삼여골용해적형성병재골용해적발생발전중발휘중요작용。제시내질망응격가작위일충신적치료파점，위림상역전혹치료골용해화무균성송동제공신적사로화방법。
BACKGROUND:Wear particles-induced osteoblasts apoptosis in vitro has been documented in many studies. However, the apoptosis of osteoblasts in osteolytic bone tissue and the selective mechanisms involved in the pathogenesis of osteolysis have been studied rarely. <br> OBJECTIVE:To investigate the influence of endoplasmic reticulum (ER) stress on the apoptosis of osteoblasts in osteolytic bone tissue and osteolysis progression. <br> METHODS:The mouse model of osteolysis was induced with wear particles placed onto the calvaria. The experiment was divided into four groups:blank control group (PBS stimulation);wear particle group (nano-al oy powder suspension stimulation);ER stress positive control group (nano-al oy powder+thapsin stimulation);and ER stress inhibitor group (nano-al oy powder+sodium 4-phenylbutyrate stimulation). The histopathologic change of osteolysis was assessed by hematoxylin-eosin, toluidine blue and alkaline phosphatase staining. Osteoblast proliferation and differentiation in osteolytic craniums were measured. The expression of ER stress markers in osteolytic craniums was examined by western blot analysis. Osteoblast apoptosis was analyzed by TUNEL staining and immunohistochemistry of Caspase-3 in osteolytic craniums. <br> RESULTS AND CONCLUSION:Wear particles were capable of inducing osteolysis, aggravating the infiltration of inflammatory cells, and inhibiting the differentiation of osteoblasts in osteolytic craniums. Meanwhile wear particles upregulated the ER stress markers and promote the apoptosis in osteolytic craniums. Blocking ER stress with sodium 4-phenylbutyrate dramatical y reduced the severity of osteolysis, significantly reduced bone invasion and inflammatory infiltration, promoted the differentiation of osteoblasts, and dramatical y reduced the apoptosis. Along with apoptosis, the expression of ER stress marker was decreased. The present study suggests that the ER stress may be crucial for osteolysis and represent a potential therapeutic target in the prevention and treatment of patients with total joint replacement who are at high risk of early aseptic loosening development.
