篩選日本血吸蟲雌蟲雌雄合抱前后差異表達基因及功能初步鑒定
發(fā)布時間：2018-10-21 14:09
【摘要】：目的:血吸蟲病是血吸蟲感染引起的一種危害嚴重的人獸共患病。蟲卵不但是血吸蟲病的主要傳染源也是血吸蟲病最重要的致病因子。血吸蟲是雌雄異體的吸蟲,雌雄合抱的生理學過程對雌蟲的性成熟和雌蟲產(chǎn)卵的作用是至關(guān)重要的。本研究通過基因芯片技術(shù)篩選出血吸蟲雌雄合抱前后差異表達基因,探究參與雌雄合抱發(fā)生的分子機制,尋找出與雌蟲性成熟及產(chǎn)卵有關(guān)的基因。我們希望這項研究可以為血吸蟲病的治療和預防提供新的靶點。方法:收集血吸蟲尾蚴,以1000條/只感染新西蘭兔,分別與感染后16天、18天、24天采用灌注法收集蟲體,通過預冷PBS和雌雄分離等處理后作為樣品送至上海伯豪生物有限公司做基因芯片實驗。對芯片結(jié)果進行聚類分析篩選出合抱前后差異表達基因,并對這些差異基因進行生物信息學分析。從分析的結(jié)果中挑選從合抱初期到合抱后期表達量一直升高的13個基因和表達量下降的2個基因,利用熒光定量PCR驗證結(jié)果是否與基因芯片一致。結(jié)合生物信息學分析結(jié)果及熒光定量PCR結(jié)果,決定挑選fs800(AY810878.1)、cu-zn-sod(FN316039.1)、eggshell precursor protein(FN317063.1)這三種基因進行下一步功能鑒定的實驗。首先通過熒光定量PCR分析上述基因在血吸蟲不同發(fā)育階段的表達情況,再利用原位雜交技術(shù)對這三種基因進行定位。隨后用特異性Sjfs800 si RNA轉(zhuǎn)染28天合抱的血吸蟲,并在體外培養(yǎng)10天。通過q PCR在m RNA水平中測量干擾效應。通過激光共聚焦掃描顯微鏡觀察日本血吸蟲雌蟲卵黃腺的形態(tài)學改變并使用光學顯微鏡觀察日本血吸蟲雌蟲產(chǎn)卵數(shù)量的變化。結(jié)果:通過SAM對基因芯片篩選的結(jié)果進行差異聚類分析,去除重復的基因后,發(fā)現(xiàn)合抱后期與合抱前期相比差異表達的基因有198個,合抱后期與合抱初期相比差異表達的基因有132個,合抱初期與合抱前期相比差異表達的基因有26個。用熒光定量PCR對15個差異表達基因進行驗證,有13個與基因芯片的結(jié)果是一致的,說明利用基因芯片篩選差異表達基因基本可靠。對差異表達基因進行GO分析,結(jié)果顯示合抱初期相對與合抱前期差異表達基因參與生物學的過程主要集中在氧化還原的過程、含氮化合物的代謝過程及環(huán)狀化合物代謝過程。合抱后期相對于合抱前期差異表達基因參與生物學過程的基因主要集中在核苷酸代謝過程、碳水化合物代謝過程、磷代謝過程和轉(zhuǎn)運過程。合抱后期相對于合抱初期差異表達基因參與生物學過程主要集中在轉(zhuǎn)運過程、核苷酸代謝過程、碳水化合物代謝過程和磷代謝過程。KEGG分析差異表達基因編碼的蛋白參與的代謝通路,結(jié)果顯示合抱初期相對與合抱前期差異表達基因參與的代謝通路有7條,合抱后期相對與合抱前期差異表達基因參與代謝通路有56條,合抱后期相對與合抱初期差異表達的基因參與的代謝通路有47條。許多基因在TGF-β信號通路,Wnt信號通路、MAPK信號通路、PPAR信號通路中高表達,值得注意的是發(fā)現(xiàn)了在生殖發(fā)育相關(guān)信號通路中存在高表達基因。結(jié)合以上實驗結(jié)果及相關(guān)文獻的查閱,我們將目光聚集在合抱前期到合抱后期表達量一直升高的基因上,并最終選擇了fs800,cu-zn-sod,eggshell precursor protein這三種基因。分析蟲卵、尾蚴、16天雌蟲、24天雌蟲、42天雌蟲這五個階段fs800,cu-zn-sod,eggshell precursor protein的表達情況,其結(jié)果顯示隨著蟲體的不斷發(fā)育,這三者的表達量逐漸升高,均在42天達到高峰,表明這三者與蟲體發(fā)育分化有著密切關(guān)系。利用原位雜交技術(shù)分別在28天蟲體內(nèi)對三個基因進行定位。在雌蟲靠近卵巢位置的卵黃腺內(nèi)有fs800的m RNA的陽性信號,eggshell precursor protein的m RNA則主要在雌蟲卵模的位置發(fā)現(xiàn)有陽性信號,而cu-zn-sod的m RNA陽性信號則在雌雄蟲體內(nèi)均表達。應用Si RNA體外對28天雌雄合抱的成蟲進行RNAi,培養(yǎng)10天后,觀察發(fā)現(xiàn)相對于錯配組和空白組,實驗組fs800的表達量明顯下降,干擾效率達到60%,同時雌雄蟲的合抱率和雌蟲的產(chǎn)卵量也有一定的下降。激光共聚焦結(jié)果顯示相對于對照組,干擾組的雌蟲的卵黃腺內(nèi)未成熟的卵黃細胞增多,說明干擾了fs800后對雌蟲的卵黃腺的正常發(fā)育有一定影響。結(jié)論:本實驗通過基因芯片技術(shù)成功篩選出大量雌蟲合抱前后差異表達的基因。通過對這些基因的生物信息學分析,選取了fs800,cu-zn-sod,eggshell precursor protein進行基因定位,發(fā)現(xiàn)fs800定位于雌蟲的卵黃腺。隨后對fs800進行干擾,初步的實驗結(jié)果顯示fs800與雌雄合抱及雌蟲產(chǎn)卵有直接的關(guān)系,但具體的機制還有待進一步研究。
[Abstract]:Objective: The schistosomiasis is a kind of serious human animal disease caused by schistosome infection. Egg is not but the main source of schistosomiasis is also the most important pathogenic factor of schistosomiasis. Schistosoma japonicum is a paragonimus paragonimus, and the physiological process of male and female clasping plays an important role in the oviposition of female and female worms. This study screened the differentially expressed genes between male and female of Schistosoma japonicum by gene chip technology, explored the molecular mechanism involved in the genesis of male and female, and found genes related to female and female gametophyte and oviposition. We hope that this study will provide new targets for the treatment and prevention of schistosomiasis. Methods: A new Zealand rabbits infected with Schistosoma japonicum were collected by perfusion method on 16 days, 18 days and 24 days after infection, and the samples were sent to Shanghai Bohao Biotech Co., Ltd. to perform gene chip experiment by pre-cooling PBS and separation of male and female. By cluster analysis of chip results, the differentially expressed genes were screened by cluster analysis, and bioinformatic analysis of these genes was carried out. From the results of analysis, 13 genes and two genes whose expression level was decreased from the early stage to the late stage were selected, and whether the results were consistent with the gene chip by fluorescence quantitative PCR. Combined with the results of bioinformatic analysis and fluorescence quantitative PCR, we decided to select the three genes of f800 (AY810878. 1), accession number-zn-sod (FN316039. 1), eggshell precuritidine (FN317063.1). Firstly, the expression of the above genes at different developmental stages of schistosome was analyzed by fluorescence quantitative PCR, and the three genes were located by in situ hybridization technique. Mice were then transfected with specific pf800 si RNA for 28 days, and cultured in vitro for 10 days. Interference effects were measured in m RNA levels by q PCR. The morphological changes of the yellow glands of the female eggs of Schistosoma japonicum were observed by laser confocal scanning microscope and the number of eggs of Schistosoma japonicum was observed with an optical microscope. Results: The results showed that there were 198 genes which were differentially expressed in the late stage and early stage compared with the early stage, 132 of the genes differentially expressed in the late stage and the early stage were found. There were 26 genes differentially expressed in the early stage and early stage. Fifteen differential expression genes were verified by fluorescence quantitative PCR, and the results of 13 genes were consistent with the results of gene chip. The results indicated that the gene chip was used to screen the differentially expressed genes. The expression of differentially expressed genes was analyzed by GO. The results showed that the expression of differentially expressed genes in the early stage was mainly focused on the process of redox, the metabolic process of nitrogen-containing compounds and the metabolic process of cyclic compounds. The gene involved in the biological process is mainly involved in the metabolic process, carbohydrate metabolism, phosphorus metabolism and transport. In the late stage, the differentially expressed genes involved in the initial differential expression of genes involved in the process of transport, metabolic processes, carbohydrate metabolism and phosphorus metabolism. KEGG analyzed the metabolic pathway of protein involved in differentially expressed genes. The results showed that there were 7 metabolic pathways involved in the differentially expressed genes in the early stage of the combination, while 56 were involved in the metabolic pathway with respect to the differentially expressed genes in the late stage. 47 of the metabolic pathways involved in the gene involved in the early phase of the closure compared to the initial differentially expressed genes. Many genes are highly expressed in the signaling pathway, signal transduction pathway, MAPK signal pathway and p38 signaling pathway, and it is worth noting that there are high expression genes in reproductive development-related signaling pathway. In combination with the above experimental results and the reference of relevant literature, we focused our attention on the genes that have been elevated in the early stage to the late stage of fusion, and finally selected the three genes of fs800, p27-zn-sod, eggshell precuricum. The expression of f800, p27-zn-sod and eggshell precuritidine in the five stages of egg, female, 16-day female worm, 24-day female worm and 42-day female worm were analyzed. The results showed that the expression level of these three species increased gradually with the development of pollen tube, all of which reached the peak at 42 days. It is shown that these three are closely related to the development and differentiation of the pituitary gland. Three genes were located in 28-day insect by in situ hybridization technique. The positive signals of the f800 mRNA were found in the yolk glands near the ovary, and the m RNA of the eggshell precuritidine was found to be positive in the position of the female egg mold, while the m RNA positive signal of the n-zn-sod was expressed in both male and female worms. After 10 days of RNAi, the expression level of f800 in experimental group was obviously decreased, and the interference efficiency reached 60%. The results showed that the immature yolk cells in the yolk glands of the female worms of the interfering group were increased with respect to the control group, indicating that the normal development of the yolk glands of the female worm after the f800 was interfered with. Conclusion: The gene chip technology was used successfully to screen the differentially expressed genes of large numbers of female worms. Based on the bioinformatic analysis of these genes, the gene localization was carried out by f800, NCI-zn-sod, eggshell precuricum gene and found that f800 was located in the yolk gland of female worm. The results showed that f800 had a direct relationship with female and female oviposition, but the specific mechanism was still to be studied further.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R383.24
本文編號：2285328
[Abstract]:Objective: The schistosomiasis is a kind of serious human animal disease caused by schistosome infection. Egg is not but the main source of schistosomiasis is also the most important pathogenic factor of schistosomiasis. Schistosoma japonicum is a paragonimus paragonimus, and the physiological process of male and female clasping plays an important role in the oviposition of female and female worms. This study screened the differentially expressed genes between male and female of Schistosoma japonicum by gene chip technology, explored the molecular mechanism involved in the genesis of male and female, and found genes related to female and female gametophyte and oviposition. We hope that this study will provide new targets for the treatment and prevention of schistosomiasis. Methods: A new Zealand rabbits infected with Schistosoma japonicum were collected by perfusion method on 16 days, 18 days and 24 days after infection, and the samples were sent to Shanghai Bohao Biotech Co., Ltd. to perform gene chip experiment by pre-cooling PBS and separation of male and female. By cluster analysis of chip results, the differentially expressed genes were screened by cluster analysis, and bioinformatic analysis of these genes was carried out. From the results of analysis, 13 genes and two genes whose expression level was decreased from the early stage to the late stage were selected, and whether the results were consistent with the gene chip by fluorescence quantitative PCR. Combined with the results of bioinformatic analysis and fluorescence quantitative PCR, we decided to select the three genes of f800 (AY810878. 1), accession number-zn-sod (FN316039. 1), eggshell precuritidine (FN317063.1). Firstly, the expression of the above genes at different developmental stages of schistosome was analyzed by fluorescence quantitative PCR, and the three genes were located by in situ hybridization technique. Mice were then transfected with specific pf800 si RNA for 28 days, and cultured in vitro for 10 days. Interference effects were measured in m RNA levels by q PCR. The morphological changes of the yellow glands of the female eggs of Schistosoma japonicum were observed by laser confocal scanning microscope and the number of eggs of Schistosoma japonicum was observed with an optical microscope. Results: The results showed that there were 198 genes which were differentially expressed in the late stage and early stage compared with the early stage, 132 of the genes differentially expressed in the late stage and the early stage were found. There were 26 genes differentially expressed in the early stage and early stage. Fifteen differential expression genes were verified by fluorescence quantitative PCR, and the results of 13 genes were consistent with the results of gene chip. The results indicated that the gene chip was used to screen the differentially expressed genes. The expression of differentially expressed genes was analyzed by GO. The results showed that the expression of differentially expressed genes in the early stage was mainly focused on the process of redox, the metabolic process of nitrogen-containing compounds and the metabolic process of cyclic compounds. The gene involved in the biological process is mainly involved in the metabolic process, carbohydrate metabolism, phosphorus metabolism and transport. In the late stage, the differentially expressed genes involved in the initial differential expression of genes involved in the process of transport, metabolic processes, carbohydrate metabolism and phosphorus metabolism. KEGG analyzed the metabolic pathway of protein involved in differentially expressed genes. The results showed that there were 7 metabolic pathways involved in the differentially expressed genes in the early stage of the combination, while 56 were involved in the metabolic pathway with respect to the differentially expressed genes in the late stage. 47 of the metabolic pathways involved in the gene involved in the early phase of the closure compared to the initial differentially expressed genes. Many genes are highly expressed in the signaling pathway, signal transduction pathway, MAPK signal pathway and p38 signaling pathway, and it is worth noting that there are high expression genes in reproductive development-related signaling pathway. In combination with the above experimental results and the reference of relevant literature, we focused our attention on the genes that have been elevated in the early stage to the late stage of fusion, and finally selected the three genes of fs800, p27-zn-sod, eggshell precuricum. The expression of f800, p27-zn-sod and eggshell precuritidine in the five stages of egg, female, 16-day female worm, 24-day female worm and 42-day female worm were analyzed. The results showed that the expression level of these three species increased gradually with the development of pollen tube, all of which reached the peak at 42 days. It is shown that these three are closely related to the development and differentiation of the pituitary gland. Three genes were located in 28-day insect by in situ hybridization technique. The positive signals of the f800 mRNA were found in the yolk glands near the ovary, and the m RNA of the eggshell precuritidine was found to be positive in the position of the female egg mold, while the m RNA positive signal of the n-zn-sod was expressed in both male and female worms. After 10 days of RNAi, the expression level of f800 in experimental group was obviously decreased, and the interference efficiency reached 60%. The results showed that the immature yolk cells in the yolk glands of the female worms of the interfering group were increased with respect to the control group, indicating that the normal development of the yolk glands of the female worm after the f800 was interfered with. Conclusion: The gene chip technology was used successfully to screen the differentially expressed genes of large numbers of female worms. Based on the bioinformatic analysis of these genes, the gene localization was carried out by f800, NCI-zn-sod, eggshell precuricum gene and found that f800 was located in the yolk gland of female worm. The results showed that f800 had a direct relationship with female and female oviposition, but the specific mechanism was still to be studied further.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R383.24
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