【作者】 张洁；

【导师】 郭长军； 陈树国；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2008， 硕士

【摘要】 目的:探讨镍铬烤瓷合金、钴铬烤瓷合金和金铂烤瓷合金,以及镍铬烤瓷合金表面镀金对体外培养的人牙龈成纤维细胞(Human gingival fibroblast,HGF)分泌免疫细胞因子白细胞介素-6(Interleukin-6,IL-6)、生长与增殖活性以及附着生长能力等生物学行为的影响。从而评价其生物相容性,为临床烤瓷合金的选择提供参考。方法:1于临床取得正常牙龈组织,采用组织块贴壁法进行人牙龈成纤维细胞的原代培养,运用细胞免疫组织化学染色方法通过波形丝蛋白、角蛋白染色鉴定细胞来源。2用不同烤瓷合金提浸液分别对人牙龈成纤维细胞进行培养,以未处理的DMEM培养液作为阴性对照。通过酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)抗体加心法测定细胞培养上清液中IL-6的分泌量;通过四甲基偶氮唑盐微量酶反应比色法(Methylthiazoletrazolium,MTT)检测烤瓷合金提浸液对人牙龈成纤维细胞生长与增殖的影响。3以盖玻片接种细胞作为阴性对照,在不同烤瓷合金表面分别接种人牙龈成纤维细胞。采用MTT法检测人牙龈成纤维细胞在不同烤瓷合金表面的附着生长与增殖活性;扫描电镜观察细胞形态与排列附着情况。结果:1细胞免疫组织化学染色,波形丝蛋白染色阳性、角蛋白染色阴性,证实细胞符合中胚层来源的成纤维细胞特点。2不同烤瓷合金提浸液培养人牙龈成纤维细胞,ELISA结果显示:6小时细胞培养上清液中IL-6的浓度均值分别为:对照组:133.75±12.50pg/ml,镍铬烤瓷合金组(A):180.00±10.36 pg/ml,钴铬烤瓷合金组(B):168.50±10.08 pg/ml,镍铬烤瓷合金镀金组(C):145.00±8.90 pg/ml,金铂烤瓷合金组(D):138.75±10.31 pg/ml。A、B组与对照组之间有显著性差异(P<0.05);C、D组与对照组之间无显著性差异(P>0.05);A组与B组、C组与D组比较,无显著性差异(P>0.05);A组与C组、A组与D组、B组与C组、B组与D组两两比较,有显著性差异(P<0.05)。24小时对照组与A、B、C、D组细胞上清液中IL-6的浓度均值分别为:189.50±10.12pg/ml,228.75±8.54pg/ml,223.75±7.50pg/ml , 200.50±9.57pg/ml , 196.50±10.79pg/ml。A、B组与对照组之间有显著性差异(P<0.05);C、D组与对照组之间无显著性差异(P>0.05);A组与B组、C组与D组比较,无显著性差异(P>0.05);A组与C组、A组与D组、B组与C组、B组与D组两两比较,有显著性差异(P<0.05)。3不同烤瓷合金提浸液培养人牙龈成纤维细胞,MTT比色实验结果显示:与对照组相比,6小时镍铬烤瓷合金组(A),钴铬烤瓷合金组(B),镍铬烤瓷合金镀金组(C),金铂烤瓷合金组(D)细胞相对增殖率均值分别为:113.07±4.16%,110.50±4.55%,102.57±4.36%,99.00±3.17%。A、B组与对照组之间有显著性差异(P<0.05);C、D组与对照组之间无显著性差异(P>0.05);A组与B组、C组与D组比较无显著性差异(P>0.05);A组与C组、A组与D组、B组与C组、B组与D组两两比较,有显著性差异(P<0.05)。与对照组相比,24小时A、B、C、D组细胞相对增殖率分别为:78.89±6.03%,82.33±4.44%,95.36±4.17%,97.92±5.83%。A、B组与对照组之间有显著性差异(P<0.05);C、D组与对照组之间无显著性差异(P>0.05);A组与B组、C组与D组比较,无显著性差异(P>0.05);A组与C组、A组与D组、B组与C组、B组与D组两两比较,有显著性差异(P<0.05)。4不同烤瓷合金表面人牙龈成纤维细胞的MTT比色结果显示:与对照组相比,镍铬烤瓷合金组(A),钴铬烤瓷合金组(B),镍铬烤瓷合金镀金组(C),金铂烤瓷合金组(D)细胞相对增殖率分别为:69.39±3.71%,75.00±5.03%,86.72±3.55%,89.27±3.88%。A、B、C、D组与对照组之间均有显著性差异(P<0.05);A组与B组、C组与D组比较,无显著性差异(P>0.05);A组与C组、A组与D组、B组与C组、B组与D组两两比较,有显著性差异(P<0.05)。5扫描电镜观察人牙龈成纤维细胞黏附行为:与对照组相比,镍铬烤瓷合金(A),钴铬烤瓷合金(B),镍铬烤瓷合金镀金(C),金铂烤瓷合金(D)表面附着细胞数目少,显示细胞极性,沿材料表面沟纹方向排列。A、B、C组细胞形态变化明显。D组细胞形态变化不明显。结论:1体外成功培养了人牙龈成纤维细胞。2镍铬烤瓷合金和钴铬烤瓷合金提浸液对人牙龈成纤维细胞的增殖显示先促进后抑制作用;并且使细胞IL-6分泌水平升高。镍铬烤瓷合金镀金与金铂烤瓷合金提浸液不影响细胞细胞的增殖活性和IL-6的分泌水平。3镍铬烤瓷合金、钴铬烤瓷合金、镍铬烤瓷合金镀金和金铂烤瓷合金均抑制人牙龈成纤维细胞的附着生长,影响细胞排列与形态。4金铂烤瓷合金与镍铬烤瓷合金镀金对人牙龈成纤维细胞生物学行为的影响较小,镍铬与钴铬烤瓷合金对细胞的生物学行为影响较大。更多还原

【Abstract】 Objective: This study is to investigate the effect of Ni-Cr, Co-Cr and Au-Pt ceramic alloys and Ni-Cr ceramic alloys with gold plating on the secretion of interleukin-6 (IL-6) by human gingival firoblasts (HGFs), and on the proliferation and attachment of HGF.Methods:1 Specimens of healthy gingival tissue were obtained from clinical operations, and primary culture of HGFs was performed using the tissue culture method. In order to identify the origin of the cultured cells, we performed immunocytochemical staining including vimentin and cytokeratin staining.2 With untreated DMEM medium as negative control, we cultured HGFs in the eluates of various types of ceramic alloys separately. The amount of IL-6 secreted in the medium supermatant was measured by enzyme-linked immunosorbent assay (ELISA). In addition, we examined the influence of the eluates of various ceramic alloys on the growth and proliferation of HGFs. 3 HGFs were seeded onto the surface of various types of ceramic alloys as well as onto the cover slips, which served as negative control. Then we examined the adhesive growth and proliferating activity of human gingival firoblasts by Methylthiazoletrazolium (MTT) assay, and observe the cell material attachment with scanning electron microscope (SEM).Results:1 Immunocytochemistry result revealed that vimentin staining was positive and cytokeratin staining was negative. Therefore, the cultured cells were proved to be in accordance with the properties of fibroblasts from the mesoderm.2 The result of ELISA measuring the secretion of IL-6 by HGFs, which is cultivated in eluates of various types of ceramic alloys, is listed as follows. At 6 hours, the average concentration level of IL-6 secreted in the medium supermatant for control group, Ni-Cr ceramic alloys (A), Co-Cr ceramic alloys (B), Ni-Cr ceramic alloys with gold plating (C) and Au-Pt ceramic alloys (D) is 133.75±12.50pg/ml,180.00±10.36pg/ml,168.50±10.08pg/ml,145.00±8.90pg/ml,and 138.75±10.31pg/ml respectively. Group A or group B is significantly different from the control (P<0.05). However, group C or group D is not significantly different from the control (P>0.05). Additionally, Group A is significantly different from group C or group D (P<0.05). Similarly, Group B is significantly different from group C or group D (P<0.05). Both of the differences between group A and B, and group C and D are not significant (P>0.05).At 24 hours, the average concentration level of IL-6 secreted in the medium supermatant for control group and group A, group B, group C, group D is 189.50±10.12 pg/ml,228.75±8.54 pg/ml,223.75±7.50 pg/ml,200.50±9.57 pg/ml,and 196.50±10.79 pg/ml respectively. Group A or group B is significantly different from the control (P<0.05). However, group C or group D is not significantly different from the control (P>0.05). Additionally, Group A is significantly different from group C or group D (P<0.05). Similarly, Group B is significantly different from group C or group D (P<0.05). Both of the differences between group A and B, and group C and D are not significant (P>0.05).3 The result of MTT assay measuring the proliferating activity of HGFs, which is cultivated in eluates of various types of ceramic alloys, is listed as follows.At 6 hours, the average proliferating activity of HGFs for control group, Ni-Cr ceramic alloys (A), Co-Cr ceramic alloys (B), Ni-Cr ceramic alloys with gold plating (C) and Au-Pt ceramic alloys (D) is 113.07±4.16%,110.50±4.55%, 102.57±4.36%,and 99.00±3.17% respectively. Group A or group B is significantly different from the control (P<0.05). However, group C or group D is not significantly different from the control (P>0.05). Additionally, Group A is significantly different from group C or group D (P<0.05). Similarly, Group B is significantly different from group C or group D (P<0.05). Both of the differences between group A and B, and group C and D are not significant (P>0.05).At 24 hours, the average proliferating activity of HGFs for control group and group A, group B, group C, group D is 78.89±6.03%, 82.33±4.44%, 95.36±4.17%, and 97.92±5.83% respectively. Group A or group B is significantly different from the control (P<0.05). However, group C or group D is not significantly different from the control (P>0.05). Additionally, Group A is significantly different from group C or group D (P<0.05). Similarly, Group B is significantly different from group C or group D (P<0.05). But no significant difference was observed between group A and group B, group C and group D (P>0.05).4 The result of MTT assay measuring the proliferating activity of HGFs, which is cultivated onto the surface of various types of ceramic alloys, showed that the average proliferating activity of HGFs for control group, Ni-Cr ceramic alloys (A), Co-Cr ceramic alloys (B), Ni-Cr porcelain alloys with gold plating (C) and Au-Pt porcelain alloys (D) is 69.39±3.71%, 75.00±5.03%, 86.72±3.55%, and 89.27±3.88% respectively. Each of the group A, group B, group C, and group D is significantly different from the control (P<0.05). Additionally, Group A is significantly different from group C or group D (P<0.05). Similarly, Group B is significantly different from group C or group D (P<0.05). But no significant difference was observed between group A and group B, group C and group D (P>0.05).5 The SEM result indicates that the number of HGFs attached to the surface of Ni-Cr ceramic alloys (A), Co-Cr ceramic alloys (B), Ni-Cr ceramic alloys with gold plating (C) and Au-Pt ceramic alloys (D) were less than that of HGFs attached to the surface of the control group. The alignment of the cells along the rills of the materials showed polarity. The morphological change of cells between group A, B, C and the control is significant. The morphological change of cells between group D and the control is not significant.Conclusion:1 The human gingival fibroblasts (HGFs) were cultured successfully in vitro.2 The eluates of Ni-Cr and Co-Cr ceramic alloys can first promote the proliferation of HGFs, and then suppress it. Besides, they can stimulate the secretion of IL-6. But the eluates of Ni-Cr ceramic alloys with gold plating and Au-Pt porcelain alloys did not affect the proliferation of HGFs and the secretion of IL-6.3 Ni-Cr, Co-Cr, Ni-Cr and Au-Pt ceramic alloys and Ni-Cr ceramic alloys with gold plating can suppress the growth of adhesive HGFs, and influence the cells alignment and morphology.4 Au-Pt ceramic alloy and Ni-Cr ceramic alloy with gold plating have no obvious influnce on the biological behavior of HGFs, while Ni-Cr and Co-Cr ceramic alloys have significant influnce on that.更多还原