摘要
目的探究血小板反应蛋白(TSP)-1对人牙周膜成纤维细胞(hPDLFs)表达白细胞介素(IL)-6和环氧化酶(COX)-2的影响。方法选取12只6周龄雄性SD大鼠随机均分为正常组和牙周炎组,丝线结扎法建立实验性牙周炎大鼠模型;Western blot检测大鼠牙龈组织中TSP-1的蛋白表达;qRT-PCR检测每组牙龈组织中TSP-1、IL-6、COX-2 mRNA表达。组织块法分离培养hPDLFs,构建携带TSP-1基因的重组腺病毒(Ad-TSP-1-GFP),同时以空载腺病毒(Ad-GFP)作为阴性对照,转染细胞;或在hPDLFs培养上清液中添加重组TSP-1蛋白(rTSP-1)。ELISA检测hPDLFs表达IL-6的蛋白水平;Western blot检测hPDLFs表达COX-2的蛋白水平;qRT-PCR检测IL-6、COX-2 mRNA表达水平。结果与正常组比较,实验性牙周炎大鼠牙龈组织中TSP-1蛋白和mRNA表达升高,且IL-6和COX-2 mRNA表达升高。与Ad-GFP组比较,Ad-TSP-1-GFP转染hPDLFs可明显提高细胞TSP-1蛋白和mRNA水平,同时可促进hPDLFs表达IL-6、COX-2蛋白和mRNA水平。与空白对照组比较,添加rTSP-1也可促进hPDLFs表达IL-6、COX-2蛋白和mRNA水平。结论TSP-1能够显著上调hPDLFs表达IL-6、COX-2的水平,进而加重牙周炎症症状。
Objective To explore the expressions of TSP-1 in the gingiva tissues of rat periodontitis model, and to verify the effect of TSP-1 on the expressions of interleukin-6(IL-6) and cyclooxygenase-2(COX-2) in primary cultured human periodontal ligament fibroblasts(hPDLFs). Methods 12 female SD rats were randomized into 2 groups, periodontitis group(ligature induced, n=6), control group(left unligated, n=6). Western blot was used to detect the protein level of TSP-1, and qRT-PCR was used to detect the mRNA level of TSP-1, IL-6 and COX-2 in the gingiva tissues in each group. After primary culturing hPDLFs with tissue block method, recombinant adenovirus(Ad-TSP-1-GFP) carrying TSP-1 gene was constructed and cells were transfected with no-load adenovirus(Ad-GFP) as a negative control, or add recombinant tsp-1 protein(rtsp-1) to the supernatant of hPDLFs culture. Il-6 expression level of hPDLFs was detected by ELISA. The expression of COX-2 in hPDLFs was detected by Western blot. mRNA expression levels of IL-6 and COX-2 were detected by qRT-PCR. Results Compared to the normal group, TSP-1 expressions in gingiva tissues were markedly increased in periodontitis group, and mRNA levels of IL-6 and COX-2 were synchronously elevated. Compared with Ad-GFP group, the level of TSP-1 was significantly higher in Ad-TSP-1-GFP group, meantime, the expression of IL-6 and COX-2 at both protein and mRNA levels in Ad-TSP-1-GFP group were also promoted.The results were confirmed by adding rTSP-1 into cell culture, since the Western blot and qRT-PCR analyses revealved a prominent upregulation of IL-6 and COX-2 after being induced by rTSP-1. Conclusion TSP-1 can significantly upregulate the expressions of IL-6 and COX-2 in hPDLFs, and then aggravate the symptoms of periodontitis.
作者
刘笑笑
沈振国
赵荣权
邢田
侯爱兵
Liu Xiaoxiao;Shen Zhenguo;Zhao Rongquan(College of Stomatology,Hospital of Stomatology,Key Lab of 0ral Diseases Research of Anlui Province,Anhui Medical University,Hefei 230031)
出处
《安徽医科大学学报》
CAS
北大核心
2020年第4期550-556,共7页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金(编号:1908085MH256)
安徽医科大学博士科研资助基金(编号:XJ201708)
国家级大学生创新创业训练计划项目(编号:201810366041)。
关键词
TSP-1
人牙周膜成纤维细胞
腺病毒
重组蛋白
牙周炎
TSP-1
human periodontal membrane fibroblast
adenovirus
recombinant protein
periodontitis
