本文選題：秋水仙堿 + 斑馬魚幼魚�。� 參考：《廣西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：1.維持斑馬魚模式集中養(yǎng)殖與繁育系統(tǒng)穩(wěn)定運(yùn)行，為實(shí)驗(yàn)開展提供優(yōu)質(zhì)胚胎、幼魚和成魚。2.整體水平上探討秋水仙堿對(duì)斑馬魚幼魚體內(nèi)SOD、Na+-K+-ATPase活性和MDA、GSH含量的影響3.觀察秋水仙堿對(duì)斑馬魚幼魚體內(nèi)抗氧化酶表達(dá)相關(guān)基因bcl2、gstp2、ucp2、nqo1、sod1的表達(dá)情況。4.局部水平上研究秋水仙堿對(duì)斑馬魚幼魚體內(nèi)肝臟Shh、Gli3蛋白表達(dá)以及肝臟脂肪含量的影響。 方法：1.在斑馬魚模式生物集中繁育系統(tǒng)穩(wěn)定運(yùn)行基礎(chǔ)上，獲取胚胎，培育幼魚和成魚用于實(shí)驗(yàn)。2.藥物暴露實(shí)驗(yàn)按如下方法進(jìn)行：預(yù)實(shí)驗(yàn)基礎(chǔ)上確定幼魚藥物暴露72h最大不致死濃度（MNLC），將3dpf斑馬魚幼魚隨機(jī)分為五組，給藥濃度分別為0.2MNLC、0.5MNLC、1.0MNLC、LC10，以Holt Buffer為空白對(duì)照組，藥物作用72h，進(jìn)行各項(xiàng)指標(biāo)測(cè)定：（1）抗氧化酶活性及抗氧化物含量測(cè)定。將幼魚充分研磨，制成組織勻漿，離心，取上清，測(cè)定幼魚體內(nèi)超氧化物歧化酶（SOD）、鈉-鉀-三磷酸腺苷酶（Na+-K+-ATPase）活性和谷胱甘肽（GSH）、丙二醛（MDA）含量。（2）抗氧化酶相關(guān)基因表達(dá)情況。利用RT-PCR方法測(cè)定藥物作用72h后斑馬魚幼魚體內(nèi)抗氧化酶表達(dá)相關(guān)基因bcl2、gstp2、ucp2、nqo1、sod1的表達(dá)情況。（3）肝組織病理變化以及Shh、Gli3蛋白表達(dá)情況測(cè)定。4%多聚甲醛磷酸液4℃固定24h，脫水，石蠟包埋，切片，對(duì)斑馬魚幼魚進(jìn)行HE染色、油紅染色，并采用免疫組化方法分析斑馬魚幼魚體內(nèi)Shh、Gli3蛋白表達(dá)變化情況。 結(jié)果：1.斑馬魚模式生物集中繁育體系能夠?yàn)閷?shí)驗(yàn)開展提供優(yōu)質(zhì)胚胎，繁育幼魚和成魚用于實(shí)驗(yàn)。2.秋水仙堿對(duì)6dpf斑馬魚幼魚的MNLC為20.6063μg mL-1，LC10為26.0615μg mL-1，LC50為34.7536μg mL-1。與空白對(duì)照組相比，隨著秋水仙堿濃度增大，斑馬魚幼魚SOD和Na+-K+-ATPase活性均降低；MDA含量隨著濃度增大而升高；GSH含量隨著濃度增大，含量降低。3.與空白對(duì)照組相比，隨著秋水仙堿濃度增加，各給藥組bcl2-mRNA、gstp2-mRNA、nqo1-mRNA、sod1-mRNA表達(dá)量減少。而ucp2-mRNA表達(dá)量與對(duì)照組相比則呈上升趨勢(shì)。4.與空白對(duì)照組比較，隨著秋水仙堿濃度增加，HE染色結(jié)果顯示，，高劑量組的幼魚肝組織變性，空泡增多，細(xì)胞間距增加。油紅染色結(jié)果顯示斑馬魚肝臟顏色加深，提示肝臟中脂肪含量增加；免疫組化法測(cè)定斑馬魚肝臟中Shh、Gli3蛋白表達(dá)量，隨著藥物濃度增加，兩種蛋白含量表達(dá)增加，提示秋水仙堿對(duì)肝臟損傷作用可能跟激活Hedgehog信號(hào)通路上相關(guān)成分有關(guān)。 結(jié)論：1.斑馬魚模式生物集中繁育體系穩(wěn)定運(yùn)行，可為藥物安全性評(píng)價(jià)提供胚胎、幼魚、成魚三種時(shí)期的實(shí)驗(yàn)對(duì)象，擴(kuò)大了應(yīng)用斑馬魚進(jìn)行藥物安全性評(píng)價(jià)的應(yīng)用范圍。2.以斑馬魚幼魚為實(shí)驗(yàn)對(duì)象，研究秋水仙堿對(duì)斑馬魚抗氧化系統(tǒng)的影響，表現(xiàn)為SOD和Na+-K+-ATPase活性受到抑制，GSH含量降低，MDA含量顯著增加，導(dǎo)致幼魚體內(nèi)氧化還原狀態(tài)失調(diào)。秋水仙堿對(duì)斑馬魚幼魚毒性作用可能與此相關(guān)。秋水仙堿對(duì)斑馬魚幼魚肝臟毒性的機(jī)制可能與激活Hedgehog信號(hào)通路有關(guān)。
[Abstract]:Objective: 1. to maintain the stable operation of the centralized aquaculture and breeding system of zebrafish, to provide high quality embryos, young fish and adult.2. on the overall level of the fish, the effect of colchicine on SOD, Na+-K+-ATPase activity and MDA, GSH content in zebrafish young fish 3. to observe the expression of colchine on the expression of antioxidant enzymes in zebrafish young fish Expression of gene BCL2, gstp2, UCP2, NQO1, SOD1, the effect of colchicine on the liver Shh, Gli3 protein expression and liver fat content in the liver of zebrafish young fish was studied at.4. level.
Methods: 1. on the basis of the stable operation of the zebrafish model biological centralized breeding system, obtaining embryos, cultivating young fish and adult fish for experimental.2. drug exposure experiments were carried out as follows: the maximum unlethal concentration of 72h (MNLC) was determined on the basis of pre experiment, and the young fish of 3dpf zebrafish were randomly divided into five groups, and the concentration of drug delivery was respectively 0.2MNLC, 0.5MNLC, 1.0MNLC, LC10, Holt Buffer as the blank control group, the drug action 72h, and the determination of various indexes: (1) antioxidant enzyme activity and antioxidant content determination. Young fish are fully lapping, tissue homogenate, centrifugation, Torikami Kiyo, determination of infant body superoxide dismutase (SOD), sodium potassium ATPase (Na+-K+-ATPas) E) activity and glutathione (GSH), malondialdehyde (MDA) content. (2) expression of antioxidant enzymes related genes. The expression of antioxidant enzymes related genes BCL2, gstp2, UCP2, NQO1, SOD1 in juvenile zebrafish after 72h was determined by RT-PCR method. (3) pathological changes of liver tissue and Shh, Gli3 protein expression determination of polymethylene Aldehyde phosphate solution was fixed at 4 C for 24h, dehydrated, paraffin embedded and sliced to stain the young zebrafish fish by HE staining and oil red staining. The changes of Shh and Gli3 protein expression in the young fish of zebrafish were analyzed by immunohistochemical method.
Results: 1. zebrafish model biological concentration breeding system can provide high quality embryos for experimental development, breeding young fish and adult fish for experimental.2. colchicine to 6dpf zebrafish young fish MNLC 20.6063 g mL-1, LC10 26.0615 mu g mL-1, LC50 34.7536 mu g mL-1. compared with the blank control group, with colchicine concentration increases, zebra fish The activity of SOD and Na+-K+-ATPase decreased in young fish, and the content of MDA increased with the concentration, and the content of GSH increased with the concentration, and the content of.3. decreased as the concentration of colchicine increased, with the increase of colchicine concentration, the expression of bcl2-mRNA, gstp2-mRNA, nqo1-mRNA, and sod1-mRNA decreased as the concentration of colchicine increased, while the expression of ucp2-mRNA was higher than that of the control group. Compared with the blank control group, with the increase of colchicine concentration, the results of HE staining showed that the liver tissue denatured, the vacuoles increased and the cell spacing increased in the high dose group. The oil red staining results showed that the liver color of zebrafish was deepened and the fat content in the liver was increased, and the immunohistochemical method was used to determine Shh, Gli3 protein in the liver of zebrafish. The expression of the two proteins increased as the concentration of the drug increased, suggesting that colchicine may be associated with the activation of the related components on the Hedgehog signaling pathway.
Conclusion: 1. zebrafish model biological centralized breeding system is stable operation, which can provide the experimental object of three periods of embryo, young fish and adult fish for the evaluation of drug safety. The application scope of the drug safety evaluation of zebrafish with zebrafish was expanded by.2., and the effects of colchicine on the anti oxidation system of zebrafish were studied. The activity of SOD and Na+-K+-ATPase was inhibited, the content of GSH decreased and the content of MDA increased significantly, which resulted in the maladjustment of redox state in the young fish. The toxicity of colchicine to zebrafish young fish may be related to this. The mechanism of colchicine's toxicity to the liver of zebrafish can be related to the activation of the Hedgehog signaling pathway.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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