本文選題：重組人酸性成纖維生長(zhǎng)因子　切入點(diǎn)：小鼠胚胎干細(xì)胞實(shí)驗(yàn)　出處：《暨南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 建立小鼠胚胎干細(xì)胞實(shí)驗(yàn)(EST)模型,驗(yàn)證該模型檢測(cè)胚胎毒性的有效性,探討aFGF的胚胎發(fā)育毒性及其對(duì)小鼠胚胎干細(xì)胞分化的影響。 方法： 體外培養(yǎng)小鼠胚胎干細(xì)胞,通過(guò)形態(tài)學(xué)觀察、核型分析和堿性磷酸酶(AKP)染色等方法用于ES的鑒定,并按照歐洲替代試驗(yàn)方法驗(yàn)證中心(ECVAM)推薦的發(fā)育毒性評(píng)價(jià)方法,驗(yàn)證所建立的EST模型評(píng)價(jià)藥物發(fā)育毒性的有效性；MTT法檢測(cè)aFGF對(duì)ES細(xì)胞和BALB/c 3T3細(xì)胞增值的影響,RT-PCR半定量分析法檢測(cè)aFGF對(duì)未分化基因Sox-2表達(dá)的影響,評(píng)價(jià)aFGF的胚胎毒性：并進(jìn)一步采用RT-PCR方法測(cè)定不同劑量aFGF對(duì)ES細(xì)胞分化為外、中、內(nèi)三個(gè)胚層過(guò)程中不同組織特異性分子標(biāo)記物基因表達(dá)的影響,探討aFGF較大劑量使用時(shí),是否具有直接或潛在的胚胎發(fā)育毒性及致畸性,并用細(xì)胞免疫熒光檢測(cè)分化的三個(gè)胚層特異性蛋白的表達(dá)量,以驗(yàn)證細(xì)胞分化的完整性。 結(jié)果： (1)成功建立EST模型,5-Fu、DPH和Penicillin G細(xì)胞毒性檢測(cè)結(jié)果表明,其胚胎毒性依次為強(qiáng)、弱和無(wú),與臨床藥物毒性相一致。(2)aFGF(14-154)在0.01-100μg/mL范圍內(nèi)對(duì)3T3細(xì)胞和ES細(xì)胞呈不同程度的促增殖作用：當(dāng)其大于100μg/ml時(shí)對(duì)ES細(xì)胞和3T3細(xì)胞均有不同程度的抑制作用：而在0.01-100μg/mL范圍內(nèi)對(duì)ES細(xì)胞的分化已經(jīng)產(chǎn)生抑制作用。計(jì)算aFGF(14-154)對(duì)兩種細(xì)胞增值及其對(duì)ES細(xì)胞分化的影響,結(jié)果顯示分別為：IC50 3T3=263.786μg/mL, IC50 ES=393.12μg/mL,ID50 ES=6.42μg/mL,毒性評(píng)價(jià)結(jié)果為aFGF(14-154)具有弱胚胎毒性。(3)aFGF(14-154)在較低劑量下對(duì)外胚層標(biāo)記物GFAP、Oligo2和Nestin基因表達(dá)有促進(jìn)作用,并且都在1μg/mL aFGF時(shí)達(dá)到最高值,超過(guò)該劑量則促進(jìn)作用下降；對(duì)中胚層各典型標(biāo)記物BMP4、MHC和MyoD基因表達(dá)作用趨勢(shì)不一致；而對(duì)內(nèi)胚層標(biāo)記物GATA6、TTR和ALB基因表達(dá)呈明顯抑制作用。 結(jié)論： 依據(jù)本實(shí)驗(yàn)評(píng)價(jià)模型,aFGF的胚胎毒性判定為弱胚胎毒性,aFGF(14-154)對(duì)三個(gè)胚層特異性基因的表達(dá)的影響呈濃度依賴性,aFGF在同一劑量下對(duì)三個(gè)胚層的發(fā)育作用顯示不一致甚至相反,其作用的不平衡性可能與其弱胚胎毒性相關(guān)；其中對(duì)內(nèi)胚層標(biāo)記物GATA6、TTR和ALB基因表達(dá)的抑制作用在判斷aFGF的胚胎毒性中可能具有重要科學(xué)意義。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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