LPS对鹅等级卵泡基质层TLR家族基因表达的影响

发布时间：2018-07-31 07:39

【摘要】：【目的】研究鹅卵泡基质层TLR家族基因表达谱及其对LPS不同处理时间后的反应性。【方法】在大群散养条件下,实时观察鹅产蛋过程,分别在产蛋后8和2 h以及产前4、16和28 h注射LPS,并在产蛋后8h屠宰,即LPS作用0、6、12、24和36 h,每个时间点各5只鹅。屠宰时,取卵巢,观察卵泡外观形态,并分离F1-F5级卵泡基质层。试验鹅自由饮水、自由采食、自然光照。LPS处理0、24和36 h的单个F1-F5级卵泡基质层或卵泡用于基因表达分析,而其他LPS处理的每只鹅等级卵泡基质层RNA等质量混合后用于基因表达分析。采用普通PCR方法检测10种鸟类TLR家族基因的在鹅等级卵泡基质层的表达谱,其中以脾脏组织为阳性对照,以不加c DNA模板为阴性对照。根据RT-PCR的表达谱结果,采用Real-time PCR检测不同等级卵泡基质层TLRs表达水平以及不同LPS处理时间对基质层TLRs表达水平的影响。对不同等级卵泡和不同LPS处理时间对基质层TLRs表达的数据,采用单因子方差分析,Duncan法多重比较;对变性卵泡与对照组基质层TLRs表达水平的数据,采用T检验分析。【结果】(1)已公开的10种鸟类TLR家族基因,如TLRs 1A、1B、2A、2B、3、4、5、7、15和21基因均在等级卵泡基质层组织中表达。(2)随等级卵泡生长,TLRs 2A和15表达水平逐渐升高;F1级卵泡基质层TLR2A表达水平显著高于F3、F4和F5级卵泡,TLR15表达水平显著高于F5级卵泡;其他TLR家族基因,如TLRs 1A、1B、2B、3、4、5、7和21在不同等级卵泡基质层中的表达水平差异不显著。(3)在LPS处理0、6和12 h时,等级卵泡外观形态无明显变化,但在LPS处理24 h时,3只鹅的等级卵泡呈深黄色胶冻状变性;在LPS处理36 h时,全部试验鹅等级卵泡呈深黄色胶冻状变性。(4)与对照组(LPS处理0 h)相比,TLRs 2A、4和5表达水平在LPS处理12和24 h时显著升高,TLR2B表达水平在LPS处理24 h时显著升高,TLRs 7和15表达水平在LPS处理6-24 h期间均显著升高,而TLRs 1A、1B、3和21表达水平在LPS处理6-24 h期间差异不显著。(5)与等级卵泡基质层相比,在LPS处理24和36 h的变性卵泡中,TLRs 1A、2A、2B、4、5、7和15表达水平显著升高,TLR3表达水平显著降低,而在LPS处理36 h的变性卵泡中,TLRs 1B和21表达水平显著升高。【结论】已发现的10种鸟类TLR家族基因均在种鹅等级卵泡基质层表达,且随LPS作用时间延长,等级卵泡形态结构发生变化,但TLRs表达水平逐渐升高。
[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.
【作者单位】：江苏省农业科学院畜牧研究所动物品种改良和繁育重点实验室;
【基金】：江苏省自然科学基金(BK20130718) 国家水禽产业技术体系(CARS-43-16)
【分类号】：S835

【相似文献】