發(fā)布時(shí)間：2018-03-26 14:58

  本文選題：堿性成纖維細(xì)胞生長因子　切入點(diǎn)：成纖維細(xì)胞　出處：《天津醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:研究堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor,b FGF)對體外培養(yǎng)的人牙齦成纖維細(xì)胞(human gingival fibroblasts,h GFs)成骨分化能力、細(xì)胞增殖及凋亡的影響,探索b FGF在人牙齦成纖維細(xì)胞體外誘導(dǎo)成骨分化過程中的作用。方法:經(jīng)患者知情同意后,取就診于天津醫(yī)科大學(xué)口腔醫(yī)院頜面外科14-20歲志愿者在下頜無齲病或牙周病的阻生智齒拔除術(shù)中分離的新鮮健康牙齦組織,去除表面的血凝塊,用含雙抗(100 U/L青霉素,100μg/L鏈霉素)的磷酸鹽緩沖液(phosphate buffer solution,PBS)充分沖洗。(1)采用組織塊貼壁法體外培養(yǎng)人牙齦成纖維細(xì)胞,取第三代細(xì)胞進(jìn)行實(shí)驗(yàn),實(shí)驗(yàn)分組如下:組1為普通培養(yǎng)基組;組2為普通培養(yǎng)基+10μg/L b FGF組;組3為成骨誘導(dǎo)組;組4為成骨誘導(dǎo)+10μg/L b FGF組;(2)應(yīng)用四甲基偶氮唑藍(lán)比色法(methyl thiazolyl terazolium,MTT)檢測在不同培養(yǎng)條件下人牙齦成纖維細(xì)胞的增殖能力;(3)應(yīng)用吖啶橙/溴化乙錠(acridine orange/ethidium bromide,AO/EB)雙熒光染色法檢測在不同培養(yǎng)條件下b FGF對人牙齦成纖維細(xì)胞凋亡的影響;(4)應(yīng)用堿性磷酸酶染色法及茜素紅染色法檢測在不同培養(yǎng)條件下人牙齦成纖維細(xì)胞的成骨分化能力;(5)應(yīng)用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription-polymerase chain reaction,RT-PCR)檢測人牙齦成纖維細(xì)胞在不同培養(yǎng)條件下成骨相關(guān)基因:runt相關(guān)轉(zhuǎn)錄因子(runt-related transcription factor 2,Runx 2)、堿性磷酸酶(alkaline phosphatase,ALP)、膠原蛋白I(collagen I,Col I)的表達(dá)。(6)使用SAS V8統(tǒng)計(jì)分析軟件對所得數(shù)據(jù)進(jìn)行分析,計(jì)量資料數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示。細(xì)胞增殖及凋亡結(jié)果采用兩獨(dú)立樣本t檢驗(yàn),其余結(jié)果多組間比較采用方差分析(analysis of variance,ANONA)。檢驗(yàn)水準(zhǔn)α=0.05,P0.05時(shí)差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:(1)采用組織塊貼壁法成功培養(yǎng)出人牙齦成纖維細(xì)胞。原代細(xì)胞生長相對較緩慢,約5-8天有單個(gè)細(xì)胞從組織塊中爬出,傳代后細(xì)胞趨于一致的長梭形,生長旺盛;(2)MTT檢測顯示,在普通培養(yǎng)基和成骨誘導(dǎo)培養(yǎng)基中,10μg/L的b FGF均能促進(jìn)人牙齦成纖維細(xì)胞的增殖(P0.01);(3)AO/EB雙染色法顯示,隨著時(shí)間的延長各組中凋亡細(xì)胞數(shù)目增多;在培養(yǎng)第3天至第7天內(nèi),不論在普通培養(yǎng)基或者成骨誘導(dǎo)培養(yǎng)基中,10μg/L b FGF均能抑制人牙齦成纖維細(xì)胞的凋亡(P0.05);而在普通培養(yǎng)基培養(yǎng)的第9天至第11天,以及成骨誘導(dǎo)培養(yǎng)基培養(yǎng)的第11天,10μg/L b FGF表現(xiàn)出對細(xì)胞凋亡的促進(jìn)作用(P0.05);(4)在成骨誘導(dǎo)培養(yǎng)基中人牙齦成纖維細(xì)胞具有骨向分化能力,堿性磷酸酶染色呈陽性并形成鈣結(jié)節(jié);10μg/L b FGF對人牙齦成纖維細(xì)胞的堿性磷酸酶活性與礦化結(jié)節(jié)形成能力均無明顯影響;(5)PT-PCR結(jié)果顯示:細(xì)胞培養(yǎng)7天時(shí),Runx 2在成骨誘導(dǎo)條件下的表達(dá)水平高于普通培養(yǎng)基(P0.05),加入10μg/L b FGF對Runx 2的表達(dá)無影響;ALP與Col I在成骨誘導(dǎo)培養(yǎng)基中的表達(dá)量明顯高于在普通培養(yǎng)基中(P0.05),10μg/L b FGF的加入僅能促進(jìn)普通培養(yǎng)基中細(xì)胞ALP與Col I的表達(dá)水平(P0.05)。結(jié)論:(1)10μg/L b FGF能夠促進(jìn)人牙齦成纖維細(xì)胞的增殖,在培養(yǎng)的早期抑制細(xì)胞凋亡而晚期時(shí)促進(jìn)細(xì)胞凋亡;(2)10μg/L bFGF對人牙齦成纖維細(xì)胞的骨向分化無明顯影響。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R781

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳嵩;毛釗;;牙周組織工程中牙齦成纖維細(xì)胞的研究進(jìn)展[J];醫(yī)學(xué)研究生學(xué)報(bào);2013年03期

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本文編號：1668343