【摘要】：目的在課題組前期對(duì)國(guó)人WS患者進(jìn)行突變篩查及致病基因功能研究的基礎(chǔ)上,利用Mitf RNA干擾果蠅模型,觀察果蠅表型及壽命變化,并利用表達(dá)譜芯片篩選Mitf RNAi時(shí)果蠅Wg/Wnt通路上表達(dá)量異常的基因,在體內(nèi)實(shí)驗(yàn)水平探索Mitf基因通過(guò)影響Wg/Wnt通路而導(dǎo)致WS的作用機(jī)制。 方法1、利用果蠅GAL4-UAS系統(tǒng),觀察Mitf RNA干擾果蠅的表型變化：將UAS-Mitf RNAi雄果蠅果蠅(實(shí)驗(yàn)組)和w1118雄果蠅果蠅(對(duì)照組)分別與elav-GAL4、ey-GAL4、DA-GAL4處女雌果蠅雜交,觀察子代果蠅表型變化；2、觀察雜交所得各組果蠅壽命變化：在實(shí)驗(yàn)組和對(duì)照組與各GAL4品系雜交得到的子代果蠅中,分別取健康雄果蠅,在相同條件下培養(yǎng),統(tǒng)計(jì)其死亡時(shí)間和個(gè)體數(shù),計(jì)算其最高壽限(90%死亡時(shí)間),半數(shù)死亡時(shí)間,繪制壽命曲線；3、利用Affymertrix GeneChip(?) Drosophila Genome2.0Array果蠅表達(dá)譜芯片篩選缺陷表型果蠅Wg/Wnt通路上表達(dá)量異常的基因。 結(jié)果1、實(shí)驗(yàn)組Mitf RNAiey-GAL4子代果蠅眼睛均較對(duì)照組w1118ey-GAL4子代果蠅眼睛變小,無(wú)明顯性別差異；實(shí)驗(yàn)組Mitf RNAielav-GAL4子代果蠅全部出現(xiàn)殘翅表型,而對(duì)照組w1118 elav-GAL4子代全部翅膀發(fā)育正常；實(shí)驗(yàn)組Mitf RNAiDA-GAL4子代果蠅與對(duì)照組Mitf RNAiDA-GAL4子代果蠅相比,未見(jiàn)明顯表型變化；2、實(shí)驗(yàn)組果蠅和對(duì)照組存活時(shí)間沒(méi)有明顯差異(p0.05),實(shí)驗(yàn)組果蠅較對(duì)照組半數(shù)死亡時(shí)間略有提前,最高壽限略短；3、通過(guò)分析表達(dá)譜芯片數(shù)據(jù),發(fā)現(xiàn)在殘翅果蠅Wg/Wnt通路上有15個(gè)基因表達(dá)量異常,其中上調(diào)的基因有4個(gè),分別為Wnt6, Apc, wdb,fz2；下調(diào)的基因有11個(gè),分別為：CkIIbeta2, Pka-C2, CG15800, Roclb, Ssl, Ste12DOR, skpF, sinah, skpD, CG32568, Apc2。 結(jié)論1、Mitf RNAi果蠅與ey-GAL4果蠅雜交出現(xiàn)小眼表型及Mitf單獨(dú)作用對(duì)果蠅壽命影響不顯著與哺乳動(dòng)物WS模型一致,進(jìn)一步證實(shí)了Mitf不僅在基因序列上與脊椎動(dòng)物有高度保守性,在基因功能上也有一定的保守性；2、Mitf RNAi果蠅與elav-GAL4果蠅雜交子代出現(xiàn)殘翅表型,與臨床上WS3和WS4表型類似,不僅提示了Mitf可能通過(guò)某種機(jī)制調(diào)控Wnt通路的功能,也說(shuō)明利用果蠅來(lái)研究WS是可行的；3、在Mitf RNAi時(shí)表達(dá)譜芯片篩選出15個(gè)表達(dá)量異常的基因,提示Mitf可能是通過(guò)上述基因來(lái)影響Wg/Wnt信號(hào)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R764.43;R-332

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