【摘要】：目的:近年來,隨著脂肪干細(xì)胞(adipose-derived stem cells,ASCs)的發(fā)現(xiàn),基于組織再生原理的脂肪組織工程得到了極大發(fā)展,以脂肪干細(xì)胞為種子細(xì)胞的脂肪組織工程研究成為熱點(diǎn)。在脂肪組織工程構(gòu)建中,支架材料是關(guān)鍵的組成部分,組織工程中的支架材料需要滿足多個條件:具有良好的生物相容性,在特定的時間內(nèi)可以被降解等。纖維蛋白膠(fibringel)是模擬凝血反應(yīng),纖維蛋白原在凝血酶的作用下形成的具有一定物理強(qiáng)度的膠狀物。已經(jīng)有報道纖維蛋白膠作為支架材料用于多種組織工程的研究中,但是,纖維蛋白膠作為支架材料復(fù)合ASCs用于構(gòu)建組織工程脂肪尚缺乏足夠研究。本研究將檢測纖維蛋白膠對于ASCs的細(xì)胞毒性,進(jìn)一步豐富使用纖維蛋白膠制作脂肪組織支架的理論成果。方法:1、提取人脂肪源干細(xì)胞,分離培養(yǎng),取第三代細(xì)胞接種到96孔板,并行鏡下觀察。流式細(xì)胞術(shù)檢測ASCs表面抗原及ASCs成脂誘導(dǎo)和油紅O染色。2、等體積的50 mg/ml纖維蛋白原溶液和500 IU/ml凝血酶溶液制成纖維蛋白膠,并制成不同濃度的纖維蛋白膠浸潤提取液。并分為以下四組:陰性對照組、50%纖維蛋白膠浸提液組、100%纖維蛋白膠浸提液組、陽性對照組(最大酶活性釋放對照)。使用LDH檢測試劑盒檢測各組培養(yǎng)液中LDH的含量。3、24h和72h兩個時間點(diǎn)中50%纖維蛋白膠浸提液組、100%纖維蛋白膠浸提液組分別與陰性對照組比較,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差描述,將所有數(shù)據(jù)輸入到SPSS17.0軟件中進(jìn)行處理分析。通過單因素方差分析方法進(jìn)行組間對比,通過SNK-q檢驗(yàn)方法進(jìn)行組間對比,P(27)0.05證明具有顯著性差異。結(jié)果:1、光學(xué)鏡下第3代ASCs為典型的成纖維細(xì)胞樣形態(tài),呈細(xì)長梭形,有粗大的分支,核仁明顯。2、流式細(xì)胞術(shù)檢測ASCs表面抗原CD49d、CD105高表達(dá),CD34、C D45、CD106不表達(dá)或極低表達(dá)。3、ASCs在成脂誘導(dǎo)培養(yǎng)基中誘導(dǎo)兩周后油紅O染色陽性。4、在24h和72h兩個時間點(diǎn)中50%纖維蛋白膠浸提液組、100%纖維蛋白膠浸提液組分別與陰性對照組比較,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差描述,將所有數(shù)據(jù)輸入到SPSS17.0軟件中進(jìn)行處理分析。通過單因素方差分析方法進(jìn)行組間對比,通過SNK-q檢驗(yàn)方法進(jìn)行組間兩兩對比。計算結(jié)果顯示各組及對照組間均沒有顯著差異(P0.05),提示纖維蛋白膠浸提液并不增加ASCs的LDH釋放,纖維蛋白膠對于ASCs無明顯的細(xì)胞毒性。結(jié)論:不同濃度纖維蛋白膠對人脂肪源干細(xì)胞無明顯細(xì)胞毒性,纖維蛋白膠有望成為人脂肪源干細(xì)胞生長的支架材料。
[Abstract]:Objective: in recent years, with the discovery of adipose stem cells (adipose-derived stem cells,ASCs), adipose tissue engineering based on the principle of tissue regeneration has been greatly developed, adipose tissue engineering with adipose stem cells as seed cells has become a hot topic. In adipose tissue engineering, scaffold material is a key component. Scaffold materials in tissue engineering need to meet many conditions: good biocompatibility, can be degraded in a specific time, and so on. Fibrin gel (fibringel) is a kind of gelatin with physical strength formed by fibrinogen under the action of thrombin. It has been reported that fibrin glue has been used as scaffold material in various tissue engineering studies. However, the use of fibrin glue as scaffold material composite ASCs in the construction of tissue engineering fat has not been sufficiently studied. This study will detect the cytotoxicity of fibrin glue to ASCs and enrich the theoretical results of using fibrin glue as scaffold for adipose tissue. Methods: 1. Human adipose stem cells were isolated and cultured, the third generation cells were inoculated into 96 well plate and observed under microscope. Flow cytometry was used to detect the surface antigen of ASCs, lipogenesis induced by ASCs and oil red O staining. 2Fibrin glue was prepared from 50 mg/ml fibrinogen solution and 500 IU/ml thrombin solution of equal volume, and different concentrations of fibrin gel were obtained. They were divided into four groups: negative control group, 50% fibrin gel extract group, 100% fibrin gel extract group, and positive control group (maximum enzyme activity release control group). The LDH assay kit was used to detect the content of LDH in the culture medium of each group. The 50% fibrin gel extract group and the 100% fibrin gel extract group were compared with the negative control group at 24 h and 72 h, respectively. The data were described as mean 鹵standard deviation. Input all data into SPSS17.0 software for processing and analysis. The results showed that there was significant difference between the two groups by univariate ANOVA and, P (27 by SNK-q test. Results: 1. The third generation of ASCs was a typical fibroblast-like form with slender fusiform and coarse branching under optical microscope. 2The nucleolus was obvious. Flow cytometry was used to detect the high expression of ASCs surface antigen CD49d,CD105, but no or very low expression of CD34,C D45 CD106. 3ASCs were induced in adipogenic medium for two weeks and oil red O staining was positive. At 24 h and 72 h, 50% fibrin glue was detected. The extraction solution group and 100% fibrin gel extraction group were compared with the negative control group, respectively. The data are described as mean 鹵standard deviation, and all the data are input into SPSS17.0 software for processing and analysis. Single factor analysis of variance (ANOVA) and SNK-q test were used to compare the two groups. The results showed that there was no significant difference between each group and the control group (P0.05), suggesting that fibrin gel extract did not increase the LDH release of ASCs, and fibrin glue had no obvious cytotoxicity to ASCs. Conclusion: different concentrations of fibrin glue have no obvious cytotoxicity to human adipose stem cells, and fibrin glue is expected to be a scaffold material for human adipose stem cells to grow.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08

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