不同烤瓷合金对人牙龈成纤维细胞毒性和炎性损伤的影响

发布时间：2018-06-22 06:13

  本文选题：牙龈成纤维细胞 + 细胞培养　； 参考：《滨州医学院》2014年硕士论文

【摘要】：目的：本实验通过研究4种非贵金属烤瓷合金(镍铬合金、钴铬合金、钛合金、纯钛)浸提液对人牙龈成纤维细胞(human gingival fibroblasts,HGFs)活性及不同时间下(1h、2h、3h、6h)细胞产生炎性因子TNF-a水平的影响,比较4种非贵金属烤瓷合金的生物相容性,为临床选择良好的烤瓷修复体提供理论依据。方法：实验1、牙龈成纤维细胞原代培养。采用组织块法原代培养人牙龈成纤维细胞,倒置纤维镜下观察细胞形态并对第三代细胞进行免疫学鉴定,采用血球计数法绘制细胞生长曲线。实验2、MTT法检测烤瓷合金浸提液对牙龈成纤维细胞活性的影响。将4种非贵金属烤瓷合金试件(镍铬合金、钴铬合金、钛合金、纯钛)分别浸透在含体积分数为10% FBS的DMEM液中。浸提7天后分别作用于体外培养的人牙龈成纤维细胞48h,空白对照组为含体积分数为10%FBS的DMEM液,MTT比色法测定各组细胞的吸光度值,并计算其相对增殖率。实验3.ELISA法检测不同时间下烤瓷合金浸提液对牙龈成纤维细胞中炎性因子TNF-a水平的影响。将4种烤瓷合金试件(镍铬合金、钴铬合金、钛合金、纯钛)分别在含体积分数为10% FBS的DMEM中浸提7天。用ELISA法测定人牙龈成纤维细胞与烤瓷合金浸提液作用1n、2h、3h、6h后各组烤瓷合金浸提液中牙龈成纤维细胞分泌的TNF-a的含量。结果：1、原代培养出牙龈成纤维细胞,镜下观察细胞呈梭形,具有长短不等的数个突起,核为卵圆形并靠近胞质中央,细胞成螺旋状、放射状或栅栏状。免疫组化鉴定Anti-Vimentin染色阳性,Anti-Cytokeratin染色阴性,为来源于中胚层的成纤维细胞。2、MTT结果显示,烤瓷合金浸提液与牙龈成纤维细胞作用48h后,各组细胞的相对增殖率从小到大依次为镍铬组77.78%、钛合金组79.01%、钴铬组85.19%和纯钛组98.77%,而且各组烤瓷金属的细胞毒性均为0级或1级。3、ELISA结果显示,烤瓷合金浸提液与牙龈成纤维细胞作用一段时间后,细胞培养上清液中的TNF-α呈现先上升后下降的趋势,并且细胞在浸提液中培养3h时TNF-α达到高峰。此时镍铬合金、钻铬合金和钛合金组的细胞培养上清液中TNF-α的含量会明显升高,且该三组之间差异有显著性(P≤0.05),而纯钛与空白组之间差异无显著性(P0.05)。结论：1、采用组织块培养法原代培养人牙龈成纤维细胞方法简单易行,可为从细胞分子生物学水平研究口腔材料的生物相容性提供实验基础。2、镍铬合金与钛合金的细胞毒性最大,其细胞毒性为1级；钴铬合金次之；纯钛的细胞毒性最小,其细胞毒性为0级。这4种烤瓷合金除纯钛材料外均对细胞有一定毒性,但其余3种合金也均在临床允许的范围内。3、镍铬合金、钴铬合金、钛合金可以促进人牙龈成纤维细胞分泌TNF-a,且对细胞增殖活性有显著影响；而纯钛对人牙龈成纤维细胞分泌TNF-a无影响,对细胞增殖活性亦无影响。由此说明纯钛烤瓷冠的生物相容性良好,适合临床广泛应用。
[Abstract]:Objective: to study the effects of four kinds of non-precious metal porcelain alloy (Ni-Cr alloy, cobalt-chromium alloy, titanium alloy, pure titanium) on the activity of (human gingival fibroblast HGFs and the level of TNF-a produced by human gingival fibroblast cells at different time (1h, 2h, 3h, 6h). The biocompatibility of four kinds of non-precious metal ceramic alloys was compared to provide theoretical basis for clinical selection of porcelain-ceramic restorations. Methods: in experiment 1, gingival fibroblasts were cultured in primary culture. The primary cultured human gingival fibroblasts were cultured by tissue block method. The morphology of the cells was observed under the inverted fibroscope and the third generation cells were identified by immunology. The cell growth curve was drawn by blood cell count method. The effect of porcelain alloy extract on gingival fibroblast activity was detected by MTT assay. Four non-precious metal ceramic alloy specimens (Ni-Cr, Co-Cr, Ti, pure titanium) were immersed in DMEM solution containing 10% FBS by volume. Human gingival fibroblasts cultured in vitro were treated for 48 hours after 7 days of extraction. The absorbance of the cells was measured by MTT colorimetric assay in 10 S DMEM solution, and the relative proliferation rate was calculated. 3. The effect of porcelain alloy extract on the level of TNF-a in gingival fibroblasts was detected by Elisa. Four kinds of porcelain alloy specimens (Ni-Cr, Co-Cr, Ti, pure titanium) were extracted in DMEM containing 10% FBS for 7 days, respectively. The contents of TNF-a secreted by gingival fibroblasts in each group of porcelain alloy extractions were determined by Elisa. Results the gingival fibroblasts were cultured in primary culture. The cells were spindle-shaped and had several protuberances of different lengths. The nucleus was oval and close to the center of the cytoplasm, and the cells were spiral, radial or palisade. Anti-Vimentin positive staining and Anti-Cytokeratin staining were identified by immunohistochemistry. The results of MTT assay of fibroblasts derived from mesoderm showed that the ceramic alloy extract acted with gingival fibroblasts for 48 h. The relative proliferation rate of each group was 77.78% in nickel chromium group, 79.01% in titanium alloy group, 85.19% in cobalt chromium group and 98.7777% in pure titanium group, and the cytotoxicity of porcelain fused metal in each group was grade 0 or grade 1. 3 Elisa results showed that the relative proliferation rate of each group was 77.78%, 79.01% in titanium alloy group, 85.19% in cobalt chromium group and 98.7777% in pure titanium group. The TNF- 伪 in the supernatant of cell culture increased first and then decreased after a period of interaction between porcelain alloy extract and gingival fibroblasts, and the TNF- 伪 reached the peak at 3 h after the cells were cultured in the extract. At this time, the content of TNF- 伪 in the supernatant of Nickel-Chromium alloy, drilling-chromium alloy and titanium alloy group increased significantly, and there was significant difference between the three groups (P 鈮，

本文编号：2051902