本文選題：牙齦成纖維細胞 + 細胞培養(yǎng)��； 參考：《濱州醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：本實驗通過研究4種非貴金屬烤瓷合金(鎳鉻合金、鈷鉻合金、鈦合金、純鈦)浸提液對人牙齦成纖維細胞(human gingival fibroblasts,HGFs)活性及不同時間下(1h、2h、3h、6h)細胞產(chǎn)生炎性因子TNF-a水平的影響,比較4種非貴金屬烤瓷合金的生物相容性,為臨床選擇良好的烤瓷修復(fù)體提供理論依據(jù)。方法：實驗1、牙齦成纖維細胞原代培養(yǎng)。采用組織塊法原代培養(yǎng)人牙齦成纖維細胞,倒置纖維鏡下觀察細胞形態(tài)并對第三代細胞進行免疫學(xué)鑒定,采用血球計數(shù)法繪制細胞生長曲線。實驗2、MTT法檢測烤瓷合金浸提液對牙齦成纖維細胞活性的影響。將4種非貴金屬烤瓷合金試件(鎳鉻合金、鈷鉻合金、鈦合金、純鈦)分別浸透在含體積分?jǐn)?shù)為10% FBS的DMEM液中。浸提7天后分別作用于體外培養(yǎng)的人牙齦成纖維細胞48h,空白對照組為含體積分?jǐn)?shù)為10%FBS的DMEM液,MTT比色法測定各組細胞的吸光度值,并計算其相對增殖率。實驗3.ELISA法檢測不同時間下烤瓷合金浸提液對牙齦成纖維細胞中炎性因子TNF-a水平的影響。將4種烤瓷合金試件(鎳鉻合金、鈷鉻合金、鈦合金、純鈦)分別在含體積分?jǐn)?shù)為10% FBS的DMEM中浸提7天。用ELISA法測定人牙齦成纖維細胞與烤瓷合金浸提液作用1n、2h、3h、6h后各組烤瓷合金浸提液中牙齦成纖維細胞分泌的TNF-a的含量。結(jié)果：1、原代培養(yǎng)出牙齦成纖維細胞,鏡下觀察細胞呈梭形,具有長短不等的數(shù)個突起,核為卵圓形并靠近胞質(zhì)中央,細胞成螺旋狀、放射狀或柵欄狀。免疫組化鑒定Anti-Vimentin染色陽性,Anti-Cytokeratin染色陰性,為來源于中胚層的成纖維細胞。2、MTT結(jié)果顯示,烤瓷合金浸提液與牙齦成纖維細胞作用48h后,各組細胞的相對增殖率從小到大依次為鎳鉻組77.78%、鈦合金組79.01%、鈷鉻組85.19%和純鈦組98.77%,而且各組烤瓷金屬的細胞毒性均為0級或1級。3、ELISA結(jié)果顯示,烤瓷合金浸提液與牙齦成纖維細胞作用一段時間后,細胞培養(yǎng)上清液中的TNF-α呈現(xiàn)先上升后下降的趨勢,并且細胞在浸提液中培養(yǎng)3h時TNF-α達到高峰。此時鎳鉻合金、鉆鉻合金和鈦合金組的細胞培養(yǎng)上清液中TNF-α的含量會明顯升高,且該三組之間差異有顯著性(P≤0.05),而純鈦與空白組之間差異無顯著性(P0.05)。結(jié)論：1、采用組織塊培養(yǎng)法原代培養(yǎng)人牙齦成纖維細胞方法簡單易行,可為從細胞分子生物學(xué)水平研究口腔材料的生物相容性提供實驗基礎(chǔ)。2、鎳鉻合金與鈦合金的細胞毒性最大,其細胞毒性為1級；鈷鉻合金次之；純鈦的細胞毒性最小,其細胞毒性為0級。這4種烤瓷合金除純鈦材料外均對細胞有一定毒性,但其余3種合金也均在臨床允許的范圍內(nèi)。3、鎳鉻合金、鈷鉻合金、鈦合金可以促進人牙齦成纖維細胞分泌TNF-a,且對細胞增殖活性有顯著影響；而純鈦對人牙齦成纖維細胞分泌TNF-a無影響,對細胞增殖活性亦無影響。由此說明純鈦烤瓷冠的生物相容性良好,適合臨床廣泛應(yīng)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- 偽 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- 偽 reached the peak at 3 h after the cells were cultured in the extract. At this time, the content of TNF- 偽 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，

本文編號：2051902