本文選題：弱精癥 + DGE　； 參考：《揚(yáng)州大學(xué)》2015年碩士論文

【摘要】：在家禽生產(chǎn)中,據(jù)不完全統(tǒng)計(jì),每年有5-12%的種公雞因產(chǎn)精能力低下遭到淘汰。中國(guó)的大部分地方雞品種更是存在繁殖效率低下的顯著弱點(diǎn),其公母配比高于專門(mén)化高產(chǎn)品種一倍以上,是產(chǎn)業(yè)化程度不高,良種繁育體系不健全的重要原因之一。為了探究公雞弱精癥的發(fā)病機(jī)理,本課題前期應(yīng)用數(shù)字基因表達(dá)譜技術(shù)(Digital Gene Expression,DGE)構(gòu)建了無(wú)精癥公雞與正常公雞的睪丸差異表達(dá)基因數(shù)據(jù)庫(kù)。本研究用生物信息學(xué)方法重新分析DGE數(shù)據(jù)庫(kù),挑選了與公雞弱精癥相關(guān)的6個(gè)候選基因(COX7B.PTGDS, hPGDS、SPAG6、WNT2、CCNF),從300只42周齡的北京油雞種公雞的大群體中,進(jìn)行了為期4周的精液品質(zhì)檢測(cè),篩出弱精及正常個(gè)體各15只。采用RT-qPCR對(duì)這6個(gè)候選基因在兩組個(gè)體的睪丸組織中的表達(dá)量進(jìn)行了相對(duì)定量檢測(cè),來(lái)驗(yàn)證DGE結(jié)果的可靠性。從驗(yàn)證的差異表達(dá)基因中挑選了CCNF (cyclin F),同時(shí)以雞精原干細(xì)胞-SSCs、睪丸支持細(xì)胞、DF1細(xì)胞為實(shí)驗(yàn)?zāi)Ｐ?進(jìn)行了三種細(xì)胞CCNF基因表達(dá)量的檢測(cè)；采用RNAi技術(shù),構(gòu)建的4條慢病毒靶向干擾載體在三種細(xì)胞上做了轉(zhuǎn)染效率和干擾效率的檢測(cè),篩選出最佳轉(zhuǎn)染效率時(shí)病毒滴度/MOI值和最優(yōu)干擾序列,應(yīng)用RT-qPC R技術(shù)檢測(cè)轉(zhuǎn)染后24 h、48 h、72 h、96 h模式細(xì)胞中CCNF基因的mRNA水平的表達(dá)量；用CCK8法檢測(cè)慢病毒載體siR-1侵染后模式細(xì)胞在不同時(shí)間點(diǎn)細(xì)胞周期的變化,為探索公雞弱精癥的發(fā)病機(jī)理的深入研究和最終尋找到致病潛在標(biāo)志物做了基礎(chǔ)鋪墊。結(jié)果表明：1. RT-qPCR對(duì)DGE中挑選的與弱精相關(guān)的6個(gè)候選基因的定量結(jié)果發(fā)現(xiàn),COX7B、PTGDS在弱精組個(gè)體睪丸的表達(dá)量顯著高于正常個(gè)體(P0.01), WNT2、hPGDS、CCNF在弱精組個(gè)體的表達(dá)量顯著低于正常個(gè)體(P0.05), SPAG6在兩組睪丸中的表達(dá)量沒(méi)有顯著差異,該結(jié)果與DGE的結(jié)果相吻合,說(shuō)明DGE結(jié)果可靠,確立了COX7B、PTGDS, WNT2、hPGDS、CCNF這5個(gè)基因可作為公雞弱精癥的重要候選基因。2.從前期初步驗(yàn)證的候選基因中挑選了CCNF基因,SSCs、睪丸支持細(xì)胞、DF1細(xì)胞的定量檢測(cè)結(jié)果顯示CCNF在這三種細(xì)胞的表達(dá)量相當(dāng),說(shuō)明CCNF基因具有廣泛表達(dá)特性。3.在三種模式細(xì)胞上的轉(zhuǎn)染效率檢測(cè)和干擾載體的篩選結(jié)果發(fā)現(xiàn),當(dāng)病毒液與培養(yǎng)基以1：19即滴度為5×106TU/mL、MOI=5,此條件下的慢病毒液侵染DF1和支持細(xì)胞效率可達(dá)60%,而在SSCs上的轉(zhuǎn)染效率只有20%,說(shuō)明慢病毒載體對(duì)SSCs侵染效果不佳。進(jìn)而在DF1和支持細(xì)胞上的CCNF靶向siRNA干擾的定量檢測(cè)結(jié)果顯示siR-1轉(zhuǎn)染兩種細(xì)胞后在72 h和96 hCCNF基因表達(dá)量極顯著下調(diào)(P0.01),說(shuō)明構(gòu)建的干擾載體能有效沉默這兩種細(xì)胞中CCNF基因的表達(dá)。4.在兩種細(xì)胞上四個(gè)時(shí)間點(diǎn)CCK8檢測(cè)細(xì)胞活力的結(jié)果發(fā)現(xiàn),空白組和陰性對(duì)照組細(xì)胞活力均沒(méi)有明顯變化；與空白組相比,在siR-1干擾的支持細(xì)胞中,72 h和96 h這兩個(gè)時(shí)間點(diǎn)的細(xì)胞活力極顯著下降(P0.01),而在siR-1干擾的DF1細(xì)胞中,72 h和96 h這兩個(gè)時(shí)間點(diǎn)的細(xì)胞活力顯著下降(P0.05)；兩種細(xì)胞在相同干擾條件下,支持細(xì)胞增值變化強(qiáng)于DF1細(xì)胞。結(jié)果說(shuō)明CCNF基因參與了細(xì)胞的增殖的調(diào)控,且隨著其表達(dá)量的降低,細(xì)胞增殖也會(huì)隨之下降或停滯。
[Abstract]:In poultry production, according to incomplete statistics, 5-12% cocks are eliminated every year because of low sperm production capacity. Most of the local chicken breeds in China have a significant weakness in the low reproductive efficiency, and the proportion of their male and female parents is more than twice that of the specialized high-yield varieties, which is an important reason for the low industrialization and the imperfect breeding system. In order to explore the pathogenesis of cock asthenospermia, Digital Gene Expression (DGE) was used to construct a database of differentially expressed genes in testis of azoospermia cocks and normal cocks in the earlier period of this study. This study reanalyzed the DGE database by bioinformatics, and selected 6 related to the cock asthenospermia. The candidate genes (COX7B.PTGDS, hPGDS, SPAG6, WNT2, CCNF) were tested for 4 weeks of semen quality, sifting weak sperm and 15 normal individuals from 300 42 weeks old Beijing Chicken Rooster. RT-qPCR was used to detect the expression of the 6 candidate genes in the testis of two groups of individuals. The reliability of the DGE results was verified. CCNF (cyclin F) was selected from the verifying differentially expressed genes. At the same time, three kinds of cell CCNF gene expression were detected with Chicken Spermatogonial Stem Cells -SSCs, testis support cells and DF1 cells as experimental models. The 4 lentivirus targeting interference vectors constructed by RNAi Technology were transformed on three kinds of cells. Detection of infection efficiency and interference efficiency, the /MOI value of virus titer and the optimal interference sequence were screened out at the best transfection efficiency. RT-qPC R technique was used to detect the expression of mRNA level of CCNF gene in 24 h, 48 h, 72 h, 96 h mode cells; and CCK8 method was used to detect the cell cycle of the lentivirus carrier after the infected mode cells at different time points. In order to explore the pathogenesis of the cock asthenospermia and to find the underlying marker for the disease, the results showed that the quantitative results of 1. RT-qPCR on the 6 candidate genes associated with the weak spermatogenesis in DGE showed that the expression of COX7B and PTGDS in the spermatozoa of the asthenosin group was significantly higher than that of the normal individuals (P0.01). The expression of WNT2, hPGDS and CCNF in the asthenospermia group was significantly lower than that of the normal individuals (P0.05). There was no significant difference in the expression of SPAG6 in the two groups of testis. The results were consistent with the results of DGE, indicating that the results of DGE were reliable, and COX7B, PTGDS, WNT2, hPGDS, and CCNF these 5 genes could be used as an important candidate for the cock asthenospermia. CCNF gene, SSCs, testis support cells were selected in the preliminary candidate genes, and the quantitative detection results of DF1 cells showed that the expression of CCNF in these three cells was equal, indicating that the CCNF gene has extensive expression characteristics of.3. in the detection of transfection efficiency on the three mode cells and screening results of the stem disturbance carrier, when the virus fluid and the culture are cultured. At 1:19, the titer was 5 x 106TU/mL, MOI=5, and the lentivirus infecting DF1 and supporting cell efficiency under this condition could reach 60%, while the transfection efficiency on SSCs was only 20%, indicating that the lentivirus carrier was not effective to SSCs infection. Then the quantitative detection results of CCNF targeting siRNA on DF1 and support cells showed siR-1 transfection of two cells. The expression of 72 h and 96 hCCNF genes was significantly down (P0.01), indicating that the constructed interference carrier could effectively silence the expression of CCNF gene in the two cells, and.4. detected cell viability at four time point CCK8 on two cells, and found that the cell vitality of the blank group and the negative control group had no significant changes; compared with the blank group, the cell viability was in Si. In the support cells of R-1 interference, the cell viability of 72 h and 96 h time points decreased significantly (P0.01), while the cell vitality of 72 h and 96 h in siR-1 interfered DF1 cells decreased significantly (P0.05); the two cells were stronger than DF1 cells under the same interference conditions. The result indicated that CCNF gene was involved. The regulation of cell proliferation, and with the decrease of its expression, cell proliferation will also decline or stagnate.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S858.31

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