建立小鼠胚胎干细胞实验模型用于aFGF发育毒性的评价

发布时间：2018-03-02 16:47

本文选题：重组人酸性成纤维生长因子　切入点：小鼠胚胎干细胞实验　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：建立小鼠胚胎干细胞实验(EST)模型,验证该模型检测胚胎毒性的有效性,探讨aFGF的胚胎发育毒性及其对小鼠胚胎干细胞分化的影响。方法：体外培养小鼠胚胎干细胞,通过形态学观察、核型分析和碱性磷酸酶(AKP)染色等方法用于ES的鉴定,并按照欧洲替代试验方法验证中心(ECVAM)推荐的发育毒性评价方法,验证所建立的EST模型评价药物发育毒性的有效性；MTT法检测aFGF对ES细胞和BALB/c 3T3细胞增值的影响,RT-PCR半定量分析法检测aFGF对未分化基因Sox-2表达的影响,评价aFGF的胚胎毒性：并进一步采用RT-PCR方法测定不同剂量aFGF对ES细胞分化为外、中、内三个胚层过程中不同组织特异性分子标记物基因表达的影响,探讨aFGF较大剂量使用时,是否具有直接或潜在的胚胎发育毒性及致畸性,并用细胞免疫荧光检测分化的三个胚层特异性蛋白的表达量,以验证细胞分化的完整性。结果： (1)成功建立EST模型,5-Fu、DPH和Penicillin G细胞毒性检测结果表明,其胚胎毒性依次为强、弱和无,与临床药物毒性相一致。(2)aFGF(14-154)在0.01-100μg/mL范围内对3T3细胞和ES细胞呈不同程度的促增殖作用：当其大于100μg/ml时对ES细胞和3T3细胞均有不同程度的抑制作用：而在0.01-100μg/mL范围内对ES细胞的分化已经产生抑制作用。计算aFGF(14-154)对两种细胞增值及其对ES细胞分化的影响,结果显示分别为：IC50 3T3=263.786μg/mL, IC50 ES=393.12μg/mL,ID50 ES=6.42μg/mL,毒性评价结果为aFGF(14-154)具有弱胚胎毒性。(3)aFGF(14-154)在较低剂量下对外胚层标记物GFAP、Oligo2和Nestin基因表达有促进作用,并且都在1μg/mL aFGF时达到最高值,超过该剂量则促进作用下降；对中胚层各典型标记物BMP4、MHC和MyoD基因表达作用趋势不一致；而对内胚层标记物GATA6、TTR和ALB基因表达呈明显抑制作用。结论：依据本实验评价模型,aFGF的胚胎毒性判定为弱胚胎毒性,aFGF(14-154)对三个胚层特异性基因的表达的影响呈浓度依赖性,aFGF在同一剂量下对三个胚层的发育作用显示不一致甚至相反,其作用的不平衡性可能与其弱胚胎毒性相关；其中对内胚层标记物GATA6、TTR和ALB基因表达的抑制作用在判断aFGF的胚胎毒性中可能具有重要科学意义。
[Abstract]:Objective:. The mouse embryonic stem cell (est) model was established to verify the effectiveness of the model in detecting embryonic toxicity and to explore the embryonic developmental toxicity of aFGF and its effect on the differentiation of mouse embryonic stem cells. Methods:. Mouse embryonic stem cells were cultured in vitro and used for the identification of es by morphological observation, karyotype analysis and alkaline phosphatase (ALP) AKP staining. The developmental toxicity evaluation method recommended by ECVAM was verified according to the European substitution test. To verify the effectiveness of the established EST model in evaluating the developmental toxicity of drugs; the effect of aFGF on the proliferation of es cells and BALB/c 3T3 cells was detected by MTT assay. RT-PCR semi-quantitative analysis was used to detect the effect of aFGF on the expression of undifferentiated gene Sox-2. To evaluate the embryotoxicity of aFGF, and to determine the effects of different doses of aFGF on the expression of specific molecular markers in different tissues during the differentiation of es cells into outer, middle and mesoderm by RT-PCR method, and to explore the effects of different doses of aFGF on the expression of genes. Whether it has direct or potential embryotoxicity and teratogenicity, the expression of three specific proteins in the differentiated embryo layer was detected with cellular immunofluorescence to verify the integrity of cell differentiation. Results:. 1) EST model was successfully established. The results of cytotoxicity test showed that the embryotoxicity was strong, weak and no. In the range of 0.01-100 渭 g / mL, the proliferation of 3T3 cells and es cells was promoted to varying degrees: when it was more than 100 渭 g / ml, it inhibited es cells and 3T3 cells in varying degrees; and in the range of 0.01-100 渭 g / mL, it inhibited es cells and 3T3 cells to different degrees in the range of 0.01-100 渭 g / mL. The effects of aFGF14-154) on the proliferation of two types of cells and on the differentiation of es cells were calculated. The results showed that: IC50 3T 3N 263.786 渭 g / mL, IC50 ES=393.12 渭 g / mLN ID50 ES=6.42 渭 g / mL, toxicity evaluation showed that aFGF3T3T3T3T3T3FGF14154) could promote the expression of the ectodermal marker GFAP-Oligo2 and Nestin at a low dose, and reached the highest value at 1 渭 g / mL aFGF. When the above dose exceeded, the expression of BMP4MHC and MyoD genes in mesoderm was not consistent, but the expression of GATA6TCR and ALB in mesoderm was significantly inhibited. Conclusion:. According to the experimental evaluation model, the embryotoxicity of aFGF was determined to be weak embryotoxicity. The effect of FGF14-154) on the expression of three specific genes in the embryo layer was concentration-dependent. The developmental effects of aaFGF on the development of the three embryo layers at the same dose were inconsistent or even opposite. The imbalance of its action may be related to its weak embryotoxicity, and the inhibition of the expression of aFGF markers GATA6TTR and ALB may be of great scientific significance in judging the embryotoxicity of aFGF.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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