本文選題：羥基喜樹堿 + 絲裂霉素��； 參考：《南京醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:通過體外培養(yǎng)人眼Tenon’s囊成纖維細(xì)胞(Human Tenon’s capsule fibroblasts,HTFs),觀察羥基喜樹堿(HCPT)對HTFs增殖、移行及細(xì)胞周期的影響,探討其可能的作用機(jī)制,并與絲裂霉素C(MMC)對比。 方法:1、HTFs的體外培養(yǎng)和鑒定:取正常供體新鮮的Tenon’s囊組織,采用組織塊培養(yǎng)法,進(jìn)行成纖維細(xì)胞的體外培養(yǎng),并用光鏡、免疫熒光法觀察鑒定; 2、MTT法檢測不同濃度的羥基喜樹堿(0.031、0.062、0.125、0.25、0.5、1、2、4mg/l)、絲裂霉素(0.0031、0.0062、0.0125、0.025、0.05、0.1、0.2、0.4mg/l)作用24h、48h、72h后HTFs的A值變化,觀察兩種藥物對HTFs的抑制作用,并進(jìn)行對比; 3、流式細(xì)胞儀測定羥基喜樹堿及絲裂霉素C對HTFs細(xì)胞周期的影響; 4、采用劃痕法檢測羥基喜樹堿及絲裂霉素C對HTFs遷移的抑制能力; 5、通過臺盼藍(lán)染色法鑒定羥基喜樹堿、絲裂霉素C對HTFs增殖、遷移的抑制作用是否由藥物的細(xì)胞毒性引起; 6、瓊脂糖凝膠電泳觀察細(xì)胞凋亡結(jié)果; 7、Realtime PCR檢測羥基喜樹堿、絲裂霉素C作用24后HTFs的Smad7 mRNA基因表達(dá)的變化,探討羥基喜樹堿抑制HTFs增殖的可能機(jī)制。 結(jié)果:1、體外成功培養(yǎng)正常HTFs,細(xì)胞長梭形,胞漿豐富,胞核大,生長能力強(qiáng),呈漩渦狀或羽毛狀生長;HTFs波形蛋白染色陽性、角蛋白染色陰性,證實(shí)所培養(yǎng)的細(xì)胞為成纖維細(xì)胞,而非上皮細(xì)胞; 2、MTT法顯示不同濃度的HCPT組、MMC組作用24h、48h、72h后HTFs細(xì)胞的增值率均低于空白對照組,差異具有統(tǒng)計(jì)學(xué)意義;HCPT(0.031—0.125mg/l)、MMC(0.0031—0.0125mg/l)對HTFs的抑制率不顯著增高,HCPT(0.25—4mg/l)、MMC(0.025—0.4mg/l)對HTFs的抑制率顯著增高,呈現(xiàn)劑量依賴性和時間依賴性;HCPT作用24h、48h、72h的IC50分別為2.24mg/l、0.76mg/l、0.39mg/l,MMC作用24h、48h、72h的IC50分別為0.34mg/l、0.24mg/l、0.07mg/l,MMC對HTF增殖的抑制效應(yīng)約要強(qiáng)于HCPT; 3、流式細(xì)胞儀檢測HCPT(0、0.25、1、4mg/l)、MMC(0、0.025、0.1、0.4mg/l)對HTFs細(xì)胞周期的影響,結(jié)果顯示HCPT主要影響HTFs的G2期和S期,MMC主要將HTFs阻滯于G1期; 4、劃痕法檢測不同濃度的HCPT、MMC作用24h、48h、72h后對HTFs遷移能力的影響,結(jié)果顯示HCPT、MMC作用后HTFs的遷移能力受到抑制,這種抑制作用呈劑量依賴性而與時間無顯著相關(guān); 5、臺盼藍(lán)染色法檢測HCPT(0、0.25、0.5、1、2、4mg/l)、MMC(0、0.0025、0.05、0.1、0.2、0.4)作用24h后HTFs的活細(xì)胞率,結(jié)果顯示HCPT組、MMC組活細(xì)胞率和空白對照組比較均無統(tǒng)計(jì)學(xué)差異; 6、0.4mg/lMMC、4mg/lHCPT作用HTFs24h后,提取出的DNA經(jīng)過1%瓊脂糖凝膠電泳,呈現(xiàn)細(xì)胞凋亡后特異性梯狀條帶; 7、Realtime PCR檢測0.4mg/lMMC、4mg/lHCPT作用HTFs24h后,Smad7 mRNA表達(dá)水平顯著上調(diào)。 結(jié)論:1、羥基喜樹堿和絲裂霉素C對體外培養(yǎng)HTFs的增殖、遷移有明顯抑制作用,這種抑制作用呈劑量和時間依賴性。該兩種藥物對HTFs增殖、遷移的抑制效應(yīng):MMC約為HCPT的10倍左右,HCPT的安全性要高于MMC; 2、羥基喜樹堿主要影響HTFs的G2和S期,絲裂霉素C主要將HTFs阻滯于G1期(靜止期); 3、羥基喜樹堿、絲裂霉素C抑制HTFs增殖、遷移的作用和藥物的細(xì)胞毒性無關(guān); 4、羥基喜樹堿及絲裂霉素C均能誘導(dǎo)HTFs凋亡; 5、羥基喜樹堿抑制HTFs增殖的機(jī)制可能是:通過上調(diào)Smad7 mRNA的表達(dá)阻斷TGF—β信號通路從而抑制HTFs增殖和遷移。
[Abstract]:Objective: To observe the effect of Hydroxycamptothecin (HCPT) on the proliferation, migration and cell cycle of Tenon 's cysts (Human Tenon' s capsule fibroblasts, HTFs) in vitro, and to explore the possible mechanism of the action of HTFs, and compare with C (MMC) pairs of mitomycin.
Methods: 1, in vitro culture and identification of HTFs: fresh Tenon 's cyst tissue of normal donor, tissue culture method was used to culture the fibroblasts in vitro, and the identification was observed by light microscopy and immunofluorescence. 2, MTT method was used to detect the different concentrations of Hydroxycamptothecin (0.031,0.062,0.125,0.25,0.5,1,2,4mg/l) and Mitomycin (0.0031,0.0062,0.01). 25,0.025,0.05,0.1,0.2,0.4mg/l) change the A value of HTFs after 24h, 48h, 72h, observe the inhibitory effect of two drugs on HTFs, and compare them. 3, the effect of hydroxycamptothecin and mitomycin C on the HTFs cell cycle is measured by flow cytometry; 4, the inhibition ability of hydroxyl camptothecin and mitomycin C to HTFs migration is detected by the scratch method; 5 A trypan blue staining method was used to identify hydroxyl camptothecin and mitomycin C on HTFs proliferation and the inhibition of migration was caused by cytotoxicity of drugs; 6, agarose gel electrophoresis was used to observe the results of cell apoptosis; 7, Realtime PCR was used to detect the changes in the expression of Smad7 mRNA gene in HTFs after C action of mitomycin C, and to explore hydroxyl camptothecin The possible mechanism to inhibit the proliferation of HTFs.
Results: 1, the normal HTFs was successfully cultured in vitro, the cells grew spindle shaped, the cytoplasm was rich, the nucleus was large, the growth ability was strong, the cells were whirlpool or featherlike growth, and HTFs vimentin was positive, and the keratin staining was negative, which proved that the cultured cells were fibroblasts, not epithelial cells.
2, MTT method showed different concentrations of HCPT group. The increment rate of HTFs cells in group MMC was lower than that of blank control group after 24h, 48h and 72h. The difference was statistically significant. HCPT (0.031 0.125mg/l), MMC (0.0031 - 0.0125mg/l) did not significantly increase the inhibition rate of HTFs. (0.25 - 0.025), the inhibition rate increased significantly. The effects of HCPT on 24h, 48h, and 72h are 2.24mg/l, 0.76mg/l, 0.39mg/l, MMC, 24h, 48h, respectively.
3, the effect of HCPT (0,0.25,1,4mg/l) and MMC (0,0.025,0.1,0.4mg/l) on the cycle of HTFs cells was detected by flow cytometry. The results showed that HCPT mainly affected the G2 phase and S phase of HTFs, and MMC mainly blocked the HTFs in G1 stage.
4, the effect of different concentrations of HCPT and MMC on the migration of HTFs after 24h, 48h and 72h was detected. The results showed that the migration ability of HTFs was inhibited after the action of HCPT and MMC, and this inhibitory effect was dose-dependent but had no significant correlation with time.
5, trypan blue staining was used to detect the living cell rate of HCPT (0,0.25,0.5,1,2,4mg/l) and MMC (0,0.0025,0.05,0.1,0.2,0.4) after 24h, and the results showed that there was no significant difference in the rate of living cells between the HCPT group and the MMC group compared with the blank control group.
After 6,0.4mg/lMMC and 4mg/lHCPT were applied to HTFs24h, the extracted DNA was shown by 1% agarose gel electrophoresis, showing a specific ladder like band after apoptosis.
7, Realtime PCR detected 0.4mg/lMMC, 4mg/lHCPT after HTFs24h, Smad7 mRNA expression level was significantly up-regulated.
Conclusion: 1, hydroxycamptothecin and mitomycin C have obvious inhibitory effect on the proliferation and migration of HTFs in vitro. This inhibitory effect is dosed and time dependent. The inhibitory effects of the two drugs on HTFs proliferation and migration are about 10 times as much as HCPT, and the safety of HCPT is higher than that of MMC.
2, HCPT mainly affected the G2 and S phases of HTFs. Mitomycin C mainly blocked HTFs in G1 phase (quiescent stage).
3, hydroxycamptothecin and mitomycin C inhibited HTFs proliferation.
4, hydroxycamptothecin and mitomycin C both induce HTFs apoptosis.
5, the mechanism of HCPT inhibiting the proliferation of HTFs is probably to inhibit the proliferation and migration of HTFs by up regulating the expression of Smad7 mRNA and blocking the TGF - beta signaling pathway.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R779.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 沈瑞雄,邵國興,王禾,石炳毅,王強(qiáng);羥基喜樹堿誘導(dǎo)大鼠腎臟移植免疫耐受機(jī)制的探討[J];第四軍醫(yī)大學(xué)學(xué)報;2003年08期

2 袁丹;容如濱;喻宗沅;李忠銘;;抗腫瘤藥物10-羥基喜樹堿的研究與應(yīng)用進(jìn)展[J];化學(xué)與生物工程;2007年12期

3 羅莉霞,李平華,彭惠,駱云鵬,湯為學(xué);羥基喜樹堿抑制體外培養(yǎng)兔晶體上皮細(xì)胞的生長[J];基礎(chǔ)醫(yī)學(xué)與臨床;2001年04期

4 沈瑞雄,石炳毅,佟建秋,莫春柏,周文強(qiáng),邵國興;羥基喜樹堿對異基因大鼠心臟移植物中細(xì)胞因子mRNA的影響[J];解放軍醫(yī)學(xué)雜志;2002年10期

5 沈瑞雄,石炳毅,莫春柏,佟建秋,周文強(qiáng),邵國興;羥基喜樹堿誘導(dǎo)大鼠心臟移植免疫耐受機(jī)制的探討[J];解放軍醫(yī)學(xué)雜志;2002年10期

6 李莉;廖立新;陳剛?cè)?余東平;;羥基喜樹堿對人增生性瘢痕成纖維細(xì)胞膠原蛋白分泌的影響[J];江西醫(yī)學(xué)院學(xué)報;2009年10期

7 袁衛(wèi)如,毛玲娥;喜樹堿倍氯米松復(fù)合劑外用治療銀屑病臨床觀察[J];臨床皮膚科雜志;1998年06期

8 孫鈺;曹曉建;汪雷;孫思鑫;;羥基喜樹堿和絲裂霉素C預(yù)防椎板切除術(shù)后硬膜外瘢痕粘連的研究[J];南京醫(yī)科大學(xué)學(xué)報(自然科學(xué)版);2007年11期

9 陳君毅;孫興懷;;轉(zhuǎn)染Smad7基因的人Tenon囊成纖維細(xì)胞Smad2及Ⅰ型膠原蛋白表達(dá)的改變(簡報)[J];分子細(xì)胞生物學(xué)報;2006年03期

10 楊力,郭樹忠;成纖維細(xì)胞與創(chuàng)傷修復(fù)的生物學(xué)過程[J];中國臨床康復(fù);2002年04期

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