【摘要】：【目的】研究鵝卵泡基質(zhì)層TLR家族基因表達(dá)譜及其對(duì)LPS不同處理時(shí)間后的反應(yīng)性。【方法】在大群散養(yǎng)條件下,實(shí)時(shí)觀察鵝產(chǎn)蛋過(guò)程,分別在產(chǎn)蛋后8和2 h以及產(chǎn)前4、16和28 h注射LPS,并在產(chǎn)蛋后8h屠宰,即LPS作用0、6、12、24和36 h,每個(gè)時(shí)間點(diǎn)各5只鵝。屠宰時(shí),取卵巢,觀察卵泡外觀形態(tài),并分離F1-F5級(jí)卵泡基質(zhì)層。試驗(yàn)鵝自由飲水、自由采食、自然光照。LPS處理0、24和36 h的單個(gè)F1-F5級(jí)卵泡基質(zhì)層或卵泡用于基因表達(dá)分析,而其他LPS處理的每只鵝等級(jí)卵泡基質(zhì)層RNA等質(zhì)量混合后用于基因表達(dá)分析。采用普通PCR方法檢測(cè)10種鳥類TLR家族基因的在鵝等級(jí)卵泡基質(zhì)層的表達(dá)譜,其中以脾臟組織為陽(yáng)性對(duì)照,以不加c DNA模板為陰性對(duì)照。根據(jù)RT-PCR的表達(dá)譜結(jié)果,采用Real-time PCR檢測(cè)不同等級(jí)卵泡基質(zhì)層TLRs表達(dá)水平以及不同LPS處理時(shí)間對(duì)基質(zhì)層TLRs表達(dá)水平的影響。對(duì)不同等級(jí)卵泡和不同LPS處理時(shí)間對(duì)基質(zhì)層TLRs表達(dá)的數(shù)據(jù),采用單因子方差分析,Duncan法多重比較;對(duì)變性卵泡與對(duì)照組基質(zhì)層TLRs表達(dá)水平的數(shù)據(jù),采用T檢驗(yàn)分析。【結(jié)果】(1)已公開的10種鳥類TLR家族基因,如TLRs 1A、1B、2A、2B、3、4、5、7、15和21基因均在等級(jí)卵泡基質(zhì)層組織中表達(dá)。(2)隨等級(jí)卵泡生長(zhǎng),TLRs 2A和15表達(dá)水平逐漸升高;F1級(jí)卵泡基質(zhì)層TLR2A表達(dá)水平顯著高于F3、F4和F5級(jí)卵泡,TLR15表達(dá)水平顯著高于F5級(jí)卵泡;其他TLR家族基因,如TLRs 1A、1B、2B、3、4、5、7和21在不同等級(jí)卵泡基質(zhì)層中的表達(dá)水平差異不顯著。(3)在LPS處理0、6和12 h時(shí),等級(jí)卵泡外觀形態(tài)無(wú)明顯變化,但在LPS處理24 h時(shí),3只鵝的等級(jí)卵泡呈深黃色膠凍狀變性;在LPS處理36 h時(shí),全部試驗(yàn)鵝等級(jí)卵泡呈深黃色膠凍狀變性。(4)與對(duì)照組(LPS處理0 h)相比,TLRs 2A、4和5表達(dá)水平在LPS處理12和24 h時(shí)顯著升高,TLR2B表達(dá)水平在LPS處理24 h時(shí)顯著升高,TLRs 7和15表達(dá)水平在LPS處理6-24 h期間均顯著升高,而TLRs 1A、1B、3和21表達(dá)水平在LPS處理6-24 h期間差異不顯著。(5)與等級(jí)卵泡基質(zhì)層相比,在LPS處理24和36 h的變性卵泡中,TLRs 1A、2A、2B、4、5、7和15表達(dá)水平顯著升高,TLR3表達(dá)水平顯著降低,而在LPS處理36 h的變性卵泡中,TLRs 1B和21表達(dá)水平顯著升高�！窘Y(jié)論】已發(fā)現(xiàn)的10種鳥類TLR家族基因均在種鵝等級(jí)卵泡基質(zhì)層表達(dá),且隨LPS作用時(shí)間延長(zhǎng),等級(jí)卵泡形態(tài)結(jié)構(gòu)發(fā)生變化,但TLRs表達(dá)水平逐漸升高。
[Abstract]:[objective] to study the gene expression profile of TLR family in follicular stromal layer of goose and its reactivity to LPS for different time. [methods] the process of goose laying was observed in real time under the condition of large group of free-feeding. LPS was injected at 8 and 2 hours after laying and at 16 and 28 hours before birth, and slaughtered at 8 h after laying, I. e., the treatment of LPS for 24 and 36 hours, with 5 geese at each time point. During slaughter, the ovaries were taken, the appearance of follicles was observed, and the F1-F5 grade follicle stroma was isolated. The test geese drank freely, fed freely, and were treated with natural light. LPs for 24 and 36 h for single F1-F5 follicle stroma or follicle for gene expression analysis. The other LPS treatments were used for gene expression analysis in each goose grade follicle matrix layer, such as RNA and other mass mixing. The expression profiles of 10 bird TLR family genes in goose grade follicle stroma were detected by ordinary PCR method. Spleen tissue was used as positive control and c DNA template was used as negative control. According to the results of RT-PCR expression profile, Real-time PCR was used to detect the expression level of TLRs in follicular stroma and the effect of different LPS treatment time on TLRs expression in stroma. The data of TLRs expression in stromal layer of follicles with different grades and different treatment time of LPS were compared by single factor analysis of variance (ANOVA) and TLRs expression level in stroma of degenerative follicle and control group. T-test analysis. [results] (1) TLR family genes of 10 species of birds, For example, the expression of TLR15 and 21 genes in the stroma of grade F _ 1 follicles were significantly higher than those in grade F _ 1 follicles. (2) the expression of TLR15 in the stromal layer of F _ 1 follicles was significantly higher than that in grade F _ 5 follicles with the increase of the expression level of TLRs _ 2A and 15 in the follicular stroma of grade F _ 1. The expression level of TLR15 was significantly higher than that of the follicles of grade F _ (5) and F _ (3) F _ (4) or F _ (5) grade follicles. The expression levels of other TLR family genes, such as TLRs 1A1A1A1BO2BO2BO2BO2BO2BO2BO3O7 and 21, were not significantly different in different grades of follicular stroma. (3) there was no significant change in appearance of grade follicles at 0h and 12h after LPS treatment, and there was no significant difference in the appearance of grade follicles in different grades of follicular stroma. But when treated with LPS for 24 h, the grade follicles of 3 geese showed dark yellow gelatinized denaturation, and when treated with LPS for 36 h, the grade follicles of three geese showed dark yellow gelatinized denaturation. (4) compared with the control group (LPS treatment for 0 h), the expression levels of TLR2An4 and 5 increased significantly at 12 and 24 h of LPS treatment. The expression level of TLR2B increased significantly at 24 h after LPS treatment. (4) compared with the control group (LPS treatment for 0 h), the expression levels of TLR2A4 and 5 increased significantly at 12 and 24 h of LPS treatment. 15 expression level increased significantly during 6-24 h of LPS treatment. However, there was no significant difference between the expression levels of TLRs 1A 1 B3 and 21 during 6-24 h of LPS treatment. (5) compared with the grade follicular stroma layer, the expression levels of TLRs 1A1A1A2A2AO2BO2BP4 and 15 increased significantly in the degenerative follicles treated with LPS for 24 and 36 h, and the expression level of TLR3 was significantly lower than that in the stroma of grade follicles, and the expression level of TLR3 was significantly decreased in the degenerated follicles treated with LPS. However, the expression levels of TLRs 1B and 21 were significantly increased in the denatured follicles treated with LPS for 36 h. [conclusion] all the TLR family genes of 10 species of birds were expressed in the stroma of graded follicles of goose, and the expression of TLRs was prolonged with the time of LPS treatment. The morphological structure of grade follicles changed, but the expression of TLRs increased gradually.
【作者單位】： 江蘇省農(nóng)業(yè)科學(xué)院畜牧研究所動(dòng)物品種改良和繁育重點(diǎn)實(shí)驗(yàn)室;
【基金】：江蘇省自然科學(xué)基金(BK20130718) 國(guó)家水禽產(chǎn)業(yè)技術(shù)體系(CARS-43-16)
【分類號(hào)】：S835

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