MITF导致Waardenburg综合征的果蝇模型研究
发布时间：2018-08-04 17:13
【摘要】：目的在课题组前期对国人WS患者进行突变筛查及致病基因功能研究的基础上,利用Mitf RNA干扰果蝇模型,观察果蝇表型及寿命变化,并利用表达谱芯片筛选Mitf RNAi时果蝇Wg/Wnt通路上表达量异常的基因,在体内实验水平探索Mitf基因通过影响Wg/Wnt通路而导致WS的作用机制。 方法1、利用果蝇GAL4-UAS系统,观察Mitf RNA干扰果蝇的表型变化：将UAS-Mitf RNAi雄果蝇果蝇(实验组)和w1118雄果蝇果蝇(对照组)分别与elav-GAL4、ey-GAL4、DA-GAL4处女雌果蝇杂交,观察子代果蝇表型变化；2、观察杂交所得各组果蝇寿命变化：在实验组和对照组与各GAL4品系杂交得到的子代果蝇中,分别取健康雄果蝇,在相同条件下培养,统计其死亡时间和个体数,计算其最高寿限(90%死亡时间),半数死亡时间,绘制寿命曲线；3、利用Affymertrix GeneChip(?) Drosophila Genome2.0Array果蝇表达谱芯片筛选缺陷表型果蝇Wg/Wnt通路上表达量异常的基因。 结果1、实验组Mitf RNAiey-GAL4子代果蝇眼睛均较对照组w1118ey-GAL4子代果蝇眼睛变小,无明显性别差异；实验组Mitf RNAielav-GAL4子代果蝇全部出现残翅表型,而对照组w1118 elav-GAL4子代全部翅膀发育正常；实验组Mitf RNAiDA-GAL4子代果蝇与对照组Mitf RNAiDA-GAL4子代果蝇相比,未见明显表型变化；2、实验组果蝇和对照组存活时间没有明显差异(p0.05),实验组果蝇较对照组半数死亡时间略有提前,最高寿限略短；3、通过分析表达谱芯片数据,发现在残翅果蝇Wg/Wnt通路上有15个基因表达量异常,其中上调的基因有4个,分别为Wnt6, Apc, wdb,fz2；下调的基因有11个,分别为：CkIIbeta2, Pka-C2, CG15800, Roclb, Ssl, Ste12DOR, skpF, sinah, skpD, CG32568, Apc2。 结论1、Mitf RNAi果蝇与ey-GAL4果蝇杂交出现小眼表型及Mitf单独作用对果蝇寿命影响不显著与哺乳动物WS模型一致,进一步证实了Mitf不仅在基因序列上与脊椎动物有高度保守性,在基因功能上也有一定的保守性；2、Mitf RNAi果蝇与elav-GAL4果蝇杂交子代出现残翅表型,与临床上WS3和WS4表型类似,不仅提示了Mitf可能通过某种机制调控Wnt通路的功能,也说明利用果蝇来研究WS是可行的；3、在Mitf RNAi时表达谱芯片筛选出15个表达量异常的基因,提示Mitf可能是通过上述基因来影响Wg/Wnt信号通路功能的。
[Abstract]:Objective on the basis of screening mutation and the function of pathogenic gene in Chinese WS patients, we observed the phenotype and longevity of Drosophila melanogaster by using Mitf RNA interference model. The expression microarray was used to screen the abnormal expression genes in the Wg/Wnt pathway of Drosophila melanogaster during Mitf RNAi, and to explore the mechanism of WS caused by the influence of Mitf gene on Wg/Wnt pathway in vivo. Methods 1. The phenotypic changes of fruit fly interfered by Mitf RNA were observed by GAL4-UAS system. The male Drosophila UAS-Mitf RNAi (experimental group) and the male fruit fly w1118 (control group) were crossed with the virgin female Drosophila elav-GAL4ey-GAL4Da-GAL4, respectively, and the phenotypic changes of the offspring were observed. 2. The changes of life span of fruit flies in each group were observed. The healthy male Drosophila melanogaster was selected from the progeny of the experimental group and the control group and each GAL4 strain, and cultured under the same conditions, the death time and the number of individuals were counted. Calculate the maximum lifetime (90% of the time of death), half of the time of death, draw the life curve 3, use Affymertrix GeneChip (? Drosophila Genome2.0Array drosophila expression microarray was used to screen abnormal expression genes in the Wg/Wnt pathway of Drosophila melanogaster. Results 1. The eyes of Mitf RNAiey-GAL4 progeny in experimental group were smaller than those of w1118ey-GAL4 progeny in control group, and there was no significant gender difference between experimental group and control group, while in experimental group, the residual wing phenotype was found in all Mitf RNAielav-GAL4 progeny, while in control group, all wings developed normally in w1118 elav-GAL4 progeny. There were no significant phenotypic changes in the experimental group compared with the control group (Mitf RNAiDA-GAL4 progeny). There was no significant difference in the survival time between the experimental group and the control group (p0.05), and the half death time of the experimental group was slightly earlier than that of the control group. The maximum lifespan was a little short. By analyzing the expression microarray data, 15 genes were found to be abnormal in the Wg/Wnt pathway of Drosophila melanogaster, among which 4 genes were up-regulated (Wnt6, Apc, wdbfz2), and 11 genes were down-regulated (Wnt6, APC, wdbfz2). They are: CkIIbeta2, Pka-C2, CG15800, Roclb, sll, Ste12DOR. skpF, sinah, skpD, CG32568, Apc2. Conclusion (1) the microocular phenotype of Mitf RNAi Drosophila and ey-GAL4 Drosophila hybrids and the effect of Mitf alone on the life span of Drosophila melanogaster are not significantly different from those of WS model in mammals, which further proves that Mitf is not only highly conserved with vertebrates in gene sequence. There is also a certain conserved gene function in the hybrid progenies of Drosophila RNAi and elav-GAL4, which is similar to WS3 and WS4 phenotypes in clinical practice, suggesting that Mitf may regulate the function of Wnt pathway through some mechanism. It is also suggested that it is feasible to study WS by using Drosophila, and 15 genes with abnormal expression were screened by expression microarray during Mitf RNAi, suggesting that Mitf may affect the function of Wg/Wnt signaling pathway through the above genes.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43;R-332
本文编号：2164552
[Abstract]:Objective on the basis of screening mutation and the function of pathogenic gene in Chinese WS patients, we observed the phenotype and longevity of Drosophila melanogaster by using Mitf RNA interference model. The expression microarray was used to screen the abnormal expression genes in the Wg/Wnt pathway of Drosophila melanogaster during Mitf RNAi, and to explore the mechanism of WS caused by the influence of Mitf gene on Wg/Wnt pathway in vivo. Methods 1. The phenotypic changes of fruit fly interfered by Mitf RNA were observed by GAL4-UAS system. The male Drosophila UAS-Mitf RNAi (experimental group) and the male fruit fly w1118 (control group) were crossed with the virgin female Drosophila elav-GAL4ey-GAL4Da-GAL4, respectively, and the phenotypic changes of the offspring were observed. 2. The changes of life span of fruit flies in each group were observed. The healthy male Drosophila melanogaster was selected from the progeny of the experimental group and the control group and each GAL4 strain, and cultured under the same conditions, the death time and the number of individuals were counted. Calculate the maximum lifetime (90% of the time of death), half of the time of death, draw the life curve 3, use Affymertrix GeneChip (? Drosophila Genome2.0Array drosophila expression microarray was used to screen abnormal expression genes in the Wg/Wnt pathway of Drosophila melanogaster. Results 1. The eyes of Mitf RNAiey-GAL4 progeny in experimental group were smaller than those of w1118ey-GAL4 progeny in control group, and there was no significant gender difference between experimental group and control group, while in experimental group, the residual wing phenotype was found in all Mitf RNAielav-GAL4 progeny, while in control group, all wings developed normally in w1118 elav-GAL4 progeny. There were no significant phenotypic changes in the experimental group compared with the control group (Mitf RNAiDA-GAL4 progeny). There was no significant difference in the survival time between the experimental group and the control group (p0.05), and the half death time of the experimental group was slightly earlier than that of the control group. The maximum lifespan was a little short. By analyzing the expression microarray data, 15 genes were found to be abnormal in the Wg/Wnt pathway of Drosophila melanogaster, among which 4 genes were up-regulated (Wnt6, Apc, wdbfz2), and 11 genes were down-regulated (Wnt6, APC, wdbfz2). They are: CkIIbeta2, Pka-C2, CG15800, Roclb, sll, Ste12DOR. skpF, sinah, skpD, CG32568, Apc2. Conclusion (1) the microocular phenotype of Mitf RNAi Drosophila and ey-GAL4 Drosophila hybrids and the effect of Mitf alone on the life span of Drosophila melanogaster are not significantly different from those of WS model in mammals, which further proves that Mitf is not only highly conserved with vertebrates in gene sequence. There is also a certain conserved gene function in the hybrid progenies of Drosophila RNAi and elav-GAL4, which is similar to WS3 and WS4 phenotypes in clinical practice, suggesting that Mitf may regulate the function of Wnt pathway through some mechanism. It is also suggested that it is feasible to study WS by using Drosophila, and 15 genes with abnormal expression were screened by expression microarray during Mitf RNAi, suggesting that Mitf may affect the function of Wg/Wnt signaling pathway through the above genes.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43;R-332
【相似文献】
相关期刊论文 前10条
1 张宝林,张青俊;Waardenburg综合征1例[J];临床耳鼻咽喉科杂志;1996年01期
2 王映芬;宋止仪;;Waardenburg综合征一例[J];湛江医学院学报;1985年02期
3 肖秀贞,廉毅鲜,王丽英;Waardenburg综合征1例[J];实用儿科临床杂志;1997年01期
4 孙彦，孙文海，王岩青，蒋光峰，张福刚;Waardenburg综合征2例[J];康复与疗养杂志;1994年04期
5 王栾第,泮力,霍江红;Waardenburg综合征1例[J];中国优生与遗传杂志;1997年04期
6 陈连军,方栩,方丽;Waardenburg综合征1例[J];临床皮肤科杂志;2000年06期
7 王秋菊,侯军华,张立文,曹菊阳,郗昕,马志中,杨贵舫,张素珍,顾瑞,杨伟炎;Waardenburg综合征的遗传学分析[J];听力学及言语疾病杂志;2003年01期
8 刘小伟,庞国祥;Waardenburg综合征Ⅰ型一例[J];中华眼科杂志;2004年01期
9 阎洪禄;Waardenburg氏综合征一家系二例[J];眼科研究;1990年01期
10 刘福民，，王秀英，叶月仙，方彭，高之睦;Waardenburg综合征（附三家系11例报告）[J];中国优生与遗传杂志;1995年03期
本文编号：2164552
本文链接：https://www.wllwen.com/yixuelunwen/yank/2164552.html
最近更新
教材专著
