碱性成纤维细胞生长因子对人牙龈成纤维细胞增殖、凋亡及成骨分化的影响

发布时间：2018-03-26 14:58

本文选题：碱性成纤维细胞生长因子　切入点：成纤维细胞　出处：《天津医科大学》2015年硕士论文

【摘要】：目的:研究碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)对体外培养的人牙龈成纤维细胞(human gingival fibroblasts,h GFs)成骨分化能力、细胞增殖及凋亡的影响,探索b FGF在人牙龈成纤维细胞体外诱导成骨分化过程中的作用。方法:经患者知情同意后,取就诊于天津医科大学口腔医院颌面外科14-20岁志愿者在下颌无龋病或牙周病的阻生智齿拔除术中分离的新鲜健康牙龈组织,去除表面的血凝块,用含双抗(100 U/L青霉素,100μg/L链霉素)的磷酸盐缓冲液(phosphate buffer solution,PBS)充分冲洗。(1)采用组织块贴壁法体外培养人牙龈成纤维细胞,取第三代细胞进行实验,实验分组如下:组1为普通培养基组;组2为普通培养基+10μg/L b FGF组;组3为成骨诱导组;组4为成骨诱导+10μg/L b FGF组;(2)应用四甲基偶氮唑蓝比色法(methyl thiazolyl terazolium,MTT)检测在不同培养条件下人牙龈成纤维细胞的增殖能力;(3)应用吖啶橙/溴化乙锭(acridine orange/ethidium bromide,AO/EB)双荧光染色法检测在不同培养条件下b FGF对人牙龈成纤维细胞凋亡的影响;(4)应用碱性磷酸酶染色法及茜素红染色法检测在不同培养条件下人牙龈成纤维细胞的成骨分化能力;(5)应用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)检测人牙龈成纤维细胞在不同培养条件下成骨相关基因:runt相关转录因子(runt-related transcription factor 2,Runx 2)、碱性磷酸酶(alkaline phosphatase,ALP)、胶原蛋白I(collagen I,Col I)的表达。(6)使用SAS V8统计分析软件对所得数据进行分析,计量资料数据采用均数±标准差(x±s)表示。细胞增殖及凋亡结果采用两独立样本t检验,其余结果多组间比较采用方差分析(analysis of variance,ANONA)。检验水准α=0.05,P0.05时差异有统计学意义。结果:(1)采用组织块贴壁法成功培养出人牙龈成纤维细胞。原代细胞生长相对较缓慢,约5-8天有单个细胞从组织块中爬出,传代后细胞趋于一致的长梭形,生长旺盛;(2)MTT检测显示,在普通培养基和成骨诱导培养基中,10μg/L的b FGF均能促进人牙龈成纤维细胞的增殖(P0.01);(3)AO/EB双染色法显示,随着时间的延长各组中凋亡细胞数目增多;在培养第3天至第7天内,不论在普通培养基或者成骨诱导培养基中,10μg/L b FGF均能抑制人牙龈成纤维细胞的凋亡(P0.05);而在普通培养基培养的第9天至第11天,以及成骨诱导培养基培养的第11天,10μg/L b FGF表现出对细胞凋亡的促进作用(P0.05);(4)在成骨诱导培养基中人牙龈成纤维细胞具有骨向分化能力,碱性磷酸酶染色呈阳性并形成钙结节;10μg/L b FGF对人牙龈成纤维细胞的碱性磷酸酶活性与矿化结节形成能力均无明显影响;(5)PT-PCR结果显示:细胞培养7天时,Runx 2在成骨诱导条件下的表达水平高于普通培养基(P0.05),加入10μg/L b FGF对Runx 2的表达无影响;ALP与Col I在成骨诱导培养基中的表达量明显高于在普通培养基中(P0.05),10μg/L b FGF的加入仅能促进普通培养基中细胞ALP与Col I的表达水平(P0.05)。结论:(1)10μg/L b FGF能够促进人牙龈成纤维细胞的增殖,在培养的早期抑制细胞凋亡而晚期时促进细胞凋亡;(2)10μg/L bFGF对人牙龈成纤维细胞的骨向分化无明显影响。
[Abstract]:Objective: To study the effect of basic fibroblast growth factor (basic fibroblast growth factor, B FGF) on cultured human gingival fibroblasts (human gingival fibroblasts, H GFs) osteogenic differentiation, proliferation and apoptosis of cells, to explore the B FGF induced osteogenic differentiation process in human fibroblasts in vitro gums. Methods: the patients after informed consent, were hospitalized in Stomatological Hospital Affiliated to Tianjin Medical University and maxillofacial surgery at the age of 14-20 volunteers in the mandibular dental caries or periodontal disease impacted wisdom teeth extraction separation in fresh and healthy gingival tissue, the removal of blood clots, with double antibody (100 U/L penicillin, 100 g/L streptomycin) phosphate buffer liquid (phosphate buffer solution, PBS). The full flush (1) of cultured human gingival fibroblasts by tissue adherent method in vitro, take the third generation cells for experiment, experimental groups as follows: group 1, P Through the medium group; group 2 was +10 g/L B ordinary medium FGF group; group 3 bone induction group; group 4 was +10 g/L B osteogenic induction group FGF; (2) using four methyl thiazolyl tetrazolium colorimetric method (methyl thiazolyl terazolium, MTT) detection in different culture conditions. Gingival fibroblast proliferation; (3) the application of acridine orange / ethidium bromide (acridine orange/ethidium, bromide, AO/EB) double fluorescent staining under different culture conditions of B FGF on human gingival fibroblast apoptosis; (4) using alkaline phosphatase staining and alizarin red staining were detected in different culture conditions osteogenic differentiation of human gingival fibroblasts; (5) using a reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) detection of human gingival fibroblast osteoblast related genes in different culture conditions: runt related transcription factor (run T-related transcription factor 2, Runx 2), alkaline phosphatase (alkaline phosphatase, ALP I (collagen), collagen I, Col I). The expression of SAS (6) using V8 statistical analysis software to analyze the data, the mean and standard deviation of measurement data using (x + s) cell proliferation and said. Apoptosis results using two independent samples t test, the results were compared by analysis of variance (analysis of, variance, ANONA). A =0.05 level test, the difference was statistically significant P0.05. Results: (1) by tissue adherent method of human gingival fibroblasts in primary cell growth is relatively. Slowly, about 5-8 days to climb out of single cells from the tissue. After passage cells tend to be fusiform, consistent with the vigorous growth; (2) MTT showed that in normal medium and osteogenic induction medium, 10 g/L B FGF could promote human gingival fibroblasts The proliferation (P0.01); (3) showed that AO/EB double staining method, with the extension of time in each group, the number of apoptotic cells increased; after cultured third days to seventh days, regardless of osteogenic induction medium in normal medium or in 10 g/L B FGF could inhibit human gingival fibroblast apoptosis (P0.05 in the ordinary culture medium); Ninth days and eleventh days of culture, and osteogenic induction medium for eleventh days training, 10 g/L B FGF showed the apoptosis promoting effect (P0.05); (4) in osteogenic medium in human gingival fibroblast cells have osteogenic differentiation ability. Alkaline phosphatase staining was positive and the formation of calcium nodules; 10 g/L B FGF on human gingival fibroblasts alkaline phosphatase activity and mineralized nodule formation were not affected; (5) the result of PT-PCR showed that the cells cultured for 7 days, 2 Runx in osteogenic induction condition expression level is higher than that of ordinary medium (P 0.05), adding 10 g/L B FGF had no effect on the expression of Runx 2; ALP and Col I expression in osteogenic induction medium was significantly higher than that in normal medium (P0.05), adding 10 g/L B FGF can only promote the expression level of ALP cells with Col medium I (P0.05). Conclusion: (1) 10 g/L B FGF can promote the proliferation of human gingival fibroblasts, cultured in the early and late apoptosis and promote cell apoptosis; (2) 10 g/L bFGF had no obvious effect on differentiation of human gingival fibroblasts and bone.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R781

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