LPS对鹅等级卵泡基质层TLR家族基因表达的影响

Time Course Effect of Lipopolysaccharide on Toll-Like Receptors Expression in the Goose Follicular Stroma

【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。

【Objective】 This study aims to investigate the transcriptional profiling of Toll-like receptors（TLRs） and their responses to lipopolysaccharide with different treatment times in the goose follicular stroma. 【Method】 The laying process was monitored in a flock of Yangzhou geese. The geese were injected intravenously with LPS（1.5mg/kg body weight） 8 and 2 h after oviposition, and 4, 16 and 28 h before oviposition. All experimental geese were slaughtered 8 h after oviposition. Therefore, time course of LPS was achieved, namely at 0, 6, 12, 24 and 36 h after injection of LPS. Five geese at each time point were selected. After slaughtering, the ovaries were collected, the follicle morphology was observed, and the stroma of the first largest follicles（F1）, F2, F3, F4 and F5 was isolated. The animals were provided with feed and water ad libitum, and maintained under natural photoperiod. For the geese 0, 24 and 36 h of after LPS treatment, all samples were used for gene expression analysis. For the other geese, RNA samples of follicular stroma of each goose were mixed with equal concentrations for gene expression analysis. RT-PCR was performed to examine the transcriptional profiling of 10 types of avian TLRs in follicular stroma. The spleen tissue was used as the positive control, and the sample without c DNA sample was used as the negative control. The expression levels of TLRs in follicular stroma among different hierarchical follicles and among different time points were tested using Real-time PCR. For data on the time course of LPS and different hierarchical follicles, the significance of differences was analyzed using the one-way ANOVA followed by Duncan＇s multiple range tests. For data between stroma in control group and DF, statistical analysis was carried out using independent-samples T test. 【Result】 All 10 reported TLRs in poultry, namely TLRs 1A, 1B, 2A, 2B, 3, 4, 5, 7, 15 and 21 were expressed in goose stroma of hierarchical follicles. The expression level of TLR2 A exhibited a tendency to increase with follicular growth. The TLR2 A expression in F1 was higher than in F3, F4 and F5, and the TLR15 expression in F1 was higher than in F5. There were no significant effects of follicle sizes on the expression of TLRs 1A, 1B, 2B, 3, 4, 5, 7 and 21 in stroma. The morphology and colour of ovarian follicles were not changed at 0, 6 and 12 h after administration of LPS. However, the hierarchical follicles of three birds after 24 h and all birds after 36 h became an irregular ellipse or circle in shape and deep yellow in colour. Compared with the control（LPS treatment 0 h）, the expression of TLRs 2A, 4 and 5 was significantly increased at 12 and 24 h after LPS treatment, the expression of TLR2 B was significantly increased at 24 h, and the expression of TLRs 7 and 15 was significantly increased during the 6 to 24 h period. LPS stimulation did not significantly affect the expression of TLRs 1A, 1B, 3 and 21 during the 6 to 24 h period. Compared with the control, the expression of TLRs 1A, 2A, 2B, 4, 5, 7 and 15 was significantly increased in the denatured hierarchical follicles at 24 and 36 h, while the expression of TLRs 1B and 21 was significantly increased in the denatured hierarchical follicles at 36 h. 【Conclusion】 All the 10 members of avian TLR families are expressed in goose follicular stroma. Furthermore, with prolonged LPS treatment, the morphology of hierarchical follicles is changed, but the TLRs expression levels are still increased.