LDH法测定纤维蛋白胶对人脂肪干细胞的细胞毒性
发布时间：2018-10-11 15:01
【摘要】：目的:近年来,随着脂肪干细胞(adipose-derived stem cells,ASCs)的发现,基于组织再生原理的脂肪组织工程得到了极大发展,以脂肪干细胞为种子细胞的脂肪组织工程研究成为热点。在脂肪组织工程构建中,支架材料是关键的组成部分,组织工程中的支架材料需要满足多个条件:具有良好的生物相容性,在特定的时间内可以被降解等。纤维蛋白胶(fibringel)是模拟凝血反应,纤维蛋白原在凝血酶的作用下形成的具有一定物理强度的胶状物。已经有报道纤维蛋白胶作为支架材料用于多种组织工程的研究中,但是,纤维蛋白胶作为支架材料复合ASCs用于构建组织工程脂肪尚缺乏足够研究。本研究将检测纤维蛋白胶对于ASCs的细胞毒性,进一步丰富使用纤维蛋白胶制作脂肪组织支架的理论成果。方法:1、提取人脂肪源干细胞,分离培养,取第三代细胞接种到96孔板,并行镜下观察。流式细胞术检测ASCs表面抗原及ASCs成脂诱导和油红O染色。2、等体积的50 mg/ml纤维蛋白原溶液和500 IU/ml凝血酶溶液制成纤维蛋白胶,并制成不同浓度的纤维蛋白胶浸润提取液。并分为以下四组:阴性对照组、50%纤维蛋白胶浸提液组、100%纤维蛋白胶浸提液组、阳性对照组(最大酶活性释放对照)。使用LDH检测试剂盒检测各组培养液中LDH的含量。3、24h和72h两个时间点中50%纤维蛋白胶浸提液组、100%纤维蛋白胶浸提液组分别与阴性对照组比较,数据以均数±标准差描述,将所有数据输入到SPSS17.0软件中进行处理分析。通过单因素方差分析方法进行组间对比,通过SNK-q检验方法进行组间对比,P(27)0.05证明具有显著性差异。结果:1、光学镜下第3代ASCs为典型的成纤维细胞样形态,呈细长梭形,有粗大的分支,核仁明显。2、流式细胞术检测ASCs表面抗原CD49d、CD105高表达,CD34、C D45、CD106不表达或极低表达。3、ASCs在成脂诱导培养基中诱导两周后油红O染色阳性。4、在24h和72h两个时间点中50%纤维蛋白胶浸提液组、100%纤维蛋白胶浸提液组分别与阴性对照组比较,数据以均数±标准差描述,将所有数据输入到SPSS17.0软件中进行处理分析。通过单因素方差分析方法进行组间对比,通过SNK-q检验方法进行组间两两对比。计算结果显示各组及对照组间均没有显著差异(P0.05),提示纤维蛋白胶浸提液并不增加ASCs的LDH释放,纤维蛋白胶对于ASCs无明显的细胞毒性。结论:不同浓度纤维蛋白胶对人脂肪源干细胞无明显细胞毒性,纤维蛋白胶有望成为人脂肪源干细胞生长的支架材料。
[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 卤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 卤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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08
[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 卤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 卤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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08
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