Efficacy of recombinant human osteoprotegerin combined with tinidazole in the treatment of periodontitis mice and its correlation with serum RANKL and MCP-1 levels

Objective: To investigate the effect of recombinant human osteoprotegerin combined with tinidazole on mice with periodontitis and the effect on serum RANKL and MCP-1 levels. Methods: 80 SPF-cleaned mice were randomly divided into 4 groups, 20 each, model group, tinidazole group and recombinant human osteoprotegerin group were modeled by Kimura et al., and tinidazole group received tinidazole. After intragastric administration, the recombinant human osteoprotegerin group was injected with recombinant human osteoprotegerin in the periodontal pocket according to the tinidazole group. The periodontal changes of the four groups of mice were observed and recorded, and the gingival rating was performed. Epithelial tissue morphology was observed by hematoxylin-eosin (HE) staining. Serum levels of IL-4, IL-6, RANKL and MCP-1 were measured by enzyme-linked immunosorbent assay. Results:After the intervention, the model group developed severe inflammatory reactions, including redness, hemorrhage, and deep periodontal pockets. The teeth were significantly loosened. The mice in the tinidazole group and the recombinant human osteoprotegerin group recovered substantially, and the gingival rating of the recombinant human osteoprotegerin group was better than that. The tinidazole group and the model group (P<0.05). The results of HE staining showed that the model group had edema, vasodilation and a large amount of inflammatory infiltration. The epithelial structure of the mice in the tinidazole group and the recombinant human osteoprotegerin group was intact and arranged closely and orderly. After intervention, the IL-4 in the tinidazole group and the recombinant human osteoprotegerin group was significantly higher than the model group and IL-6 was significantly lower than the model group (P<0.05), and the recombinant human osteoprotegerin group IL-4 was significantly higher after the intervention. IL-6 was significantly lower in the tinidazole group than in the tinidazole group (P<0.05). After the intervention, the tinidazole group and the recombinant human osteoprotegerin group were significantly reduced, and the recombinant human osteoprotegerin group RAKNL and MCP-1 were significantly lower than the model group (P>0.05). Conclusion: Recombinant human osteoprotegerin combined with tinidazole has a better therapeutic effect on gums and teeth in mice with periodontitis, and can lower the levels of RAKNL and MCP-1 in serum, inhibit bone resorption and protect teeth.

内毒素对重组人成骨蛋白-1诱导人牙髓细胞活性的影响

目的：研究具核梭杆菌的内毒素（ＬＰＳ）对重组人成骨蛋白－１（ｒｈＯＰ－１）诱导牙髓细胞增殖、分化的影响。方法：采用噻唑盐比色测定（ＭＴＴ）、酶动力学方法和放射免疫技术。结果：在较低浓度ＬＰＳ作用时，可协同增加ｒｈＯＰ－１对牙髓细胞增殖和碱性磷酸酶（ＡＬＰａｓｅ）活性，但对骨钙素（ＢＧＰ）无明显影响，而高浓度ＬＰＳ则起抑制作用。结论：提示适度的ＬＰＳ在牙髓对龋病的防御反应过程中起着一定调节作用。

人重组骨形成蛋白-1对牙髓细胞生物学效应的研究

通过细胞培养技术、噻唑盐比色测定（ＭＴＴ）、酶动力学方法和放射免疫技术，了解人重组骨形成蛋白－１（ｒｈＯＰ－１）对体外培养的人牙髓细胞增殖、碱性磷酸酶活性及骨钙素表达的影响。结果表明，ｒｈＯＰ－１可促进牙髓细胞增殖，提高牙髓碱性磷酸酶活性及骨钙素的表达，在本实验剂量范围内呈浓度依赖关系，具有诱导牙髓细胞向成骨方向转化的趋势。

人成骨蛋白-1成熟肽基因5’端的修饰及其在大肠杆菌中的表达

目的：对人成骨蛋白－１（ＯＰ－１）成熟肽基因进行修饰、克隆并在大肠杆菌中表达、检测，为ＯＰ－１的进一步纯化、复性、诱骨活性的研究以及未来的临床应用奠定基础。方法：在不改变氨基酸的前提下，用ＰＣＲ的方法对ＯＰ－１的成熟肽Ｎ端序列加以修饰，将编码成熟肽的ｃＤＮＡ３’端０．４２ｋｂ基因片段克隆于温控型大肠杆菌表达载体ｐＢＶ２２０，受控于ＰＬＰＲ启动子。重组子以大肠杆菌ＤＨ５α为宿主细胞进行温度诱导表达。用鼠抗牛天然ＢＭＰｓ单抗对表达产物进行ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｉｎｇ确证。结果：工程菌经４２℃、５ｈ诱导后，在ＳＤＳ－ＰＡＧＥ上出现一条新的蛋白带，分子量在１．６×１０４ｕ左右，约占菌体总蛋白的１９．８％。主要以包涵体形式存在的表达产物经初步纯化后，可获得纯度较高的重组人成骨蛋白－１成熟肽（ｒｈＯＰ－１）。表达产物的ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｎｇ确证确均呈阳性反应。结论：表达产物即为目的蛋白，使ｈＯＰ－１成熟肽基因５’端的修饰及其在大肠杆菌中的表达在国内第一次获得成功。

重组人成骨蛋白-1的纯化及其生物学活性研究

目的 获得高纯度的重组人成骨蛋白-1（rhOP-1），探讨大肠杆菌表达rhOP-1的生物学活性。方法 表达rhOP-1工程菌经发酵后，裂菌、提取包涵体，层析法纯化蛋白并逐步降低尿素浓度透析复性。采用改良MTT法检测rhOP-1对NIH3T3细胞增殖和对硝基苯酚法（PNPP）检测rhOP-1对细胞碱性磷酸酶（ALP）活性的影响，同时在体内通过小鼠肌袋埋植实验测定rhOP-1活性。结果 经纯化的rhOP-1纯度可达98％。体外rhOP-1浓度在0．06～6μg／ml可明显促进细胞生长和ALP活性；体内rhOP-1埋植剂植入小鼠肌间隙2周后，可明显提高体内钙含量，且成骨趋势明显。结论 原核表达的rhOP-1具有良好的生物学活性。

重组人成骨蛋白-1复性条件的优化

目的优化重组人成骨蛋白-1(rhOP-1)包涵体蛋白的复性条件。方法将表达rhOP-1的大肠杆菌菌体在冰浴下超声裂解,分离提取包涵体,用8mol/L尿素溶解,纯化后进行梯度透析复性。利用TotalLab软件分析目的蛋白二聚体的含量,用体内法和体外法测定其生物学活性。结果经纯化后,目的蛋白纯度达97%以上。最佳复性条件为4℃,pH9.0,蛋白浓度为0.4～0.8mg/ml,尿素浓度为2mol/L,L-Arg浓度为0.4mol/L;目的蛋白复性率达75%以上,复性后蛋白具有较高的生物学活性。结论已确定了rhOP-1包涵体梯度透析复性的最佳条件。

可溶性重组人成骨蛋白-1的制备

目的制备可溶性重组人成骨蛋白-1(rhOP-1),并尝试制备注射制剂。方法rhOP-1工程菌经发酵表达,收集菌体,提取包涵体,用SP-FF阳离子层析柱纯化。取纯度达95%以上的rhOP-1蛋白,透析复性,加入L-精氨酸作为复性助溶剂,制备成冻干粉末,以不加助溶剂的rhOP-1为对照。将制备的冻干粉末复溶后,观察rhOP-1溶解性,并检测其活性。结果复性液中加入L-精氨酸的复性rhOP-1蛋白浓度为0.5mg/ml,回收率可达60%,冻干粉末复溶后,溶解性及rhOP-1活性良好。结论用此复性方法制备的rhOP-1溶液作为注射剂,有较好的应用前景。

CCDC134调控人牙髓干细胞成骨分化

目的探讨CCDC134(coiled⁃coil domain containing 134)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)成骨分化功能的调控作用。方法从牙髓组织中,分离培养hDPSCs,并分别以NC⁃CCDC134、shCCDC134、CCDC134慢病毒转染hDPSCs,分为空白对照组、阴性对照组、CCDC134下调组(shCCDC134)、CCDC134过表达组(CCDC134)。流式细胞术检测hDPSCs表面标志物Stro⁃1、CD105、CD34、CD45;甲苯胺蓝染色检测克隆形成;碱性磷酸酶(alkaline phosphatase,ALP)染色检测ALP表达;茜素红染色检测矿化结节形成;油红染色检测成脂能力;qPCR检测CCDC134、Runt相关转录因子2(Runt⁃related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)、骨形态发生蛋白⁃2(bone morphogenetic protein⁃2,BMP⁃2)、Smad家族成员1(mothers against decapentaplegic homolog 1,SMAD1)的mRNA水平表达;蛋白印迹法检测CCDC134、RUNX2、OCN、BMP⁃2、SMAD1蛋白表达水平。进一步以BMP⁃2信号激活剂(BMP⁃2)和抑制剂(Dorsomorphin)分别干预CCDC134下调及上调的hDPSCs(分组为:shCCDC134、shCCDC134+BMP⁃2、CCDC134、CCDC134+Dorsomorphin),细胞聚合体移植于裸鼠皮下2个月,HE染色法检测新骨形成。结果hDPSCs高表达间充质干细胞表面标志物,低表达造血干细胞表面标志物。与空白对照组相比,成骨诱导的hDPSCs中CCDC134的表达升高;与阴性对照组相比,shCCDC134组CCDC134的表达降低,CCDC134组的表达升高(P<0.05);shCCDC134组的矿化结节减少、成骨相关基因和蛋白表达降低(P<0.05),CCDC134组的指标升高(P<0.05);shCCDC134组的BMP⁃2/SMAD1信号通路的相关表达降低,CCDC134组表达升高(P<0.05)。与shCCDC134组相比,shCCDC134+BMP⁃2组成骨相关基因和蛋白表达升高、裸鼠皮下新骨形成增加(P<0.05),与CCDC134组相比,CCDC134+Dorsomorphin组以上指标降低(P<0.05)。结论CCDC134通过调控BMP⁃2/SMAD1信号通路促进hDPSCs成骨分化。