【作者】 余燕；

【导师】 陈耀星；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 小肠是禽类消化与吸收的重要场所和黏膜屏障的第一道防线。前期研究发现不同波长光照射能够影响雏鸡小肠黏膜的机械屏障和免疫屏障,但对孵化后期鸡胚发育有何影响尚未见报道。鸡胚孵化后期是小肠可塑性最强的时期,此时小肠的良好发育也是出壳后雏鸡获得最大发育和生长的基础。因此,本实验在鸡胚孵化期间分别使用波长为400-700nm白光、480nm蓝光、560nm绿光和660nm红光进行人工照射并以黑暗作为对照,采用组织学、组织化学、免疫组织化学、qRT-PCR, ELISA和细胞培养等实验方法探索不同波长光照射对鸡胚肠道和法氏囊发育的影响并初步探讨其作用机制,为充分挖掘利用孵化期间给予光照以提高鸡胚出壳后的生长潜能和免疫功能提供理论依据。研究结果如下：1.孵化后期鸡胚小肠结构和功能的发育鸡胚孵化后期随着胚龄的增加,小肠绒毛高度、肌层厚度、PCNA的表达以及杯状细胞的密度都出现了显著的变化。到了E21胚龄,十二指肠、空肠和回肠的绒毛高度分别比E15增加150%-180%,肌层厚度是E15的1.2-1.7倍,PCNA平均光密度值仅为E15的38%-53%；杯状细胞由E15以硫酸化亚型为主到E18胚龄以中性杯状细胞为主,而到了E21胚龄唾液酸化亚型杯状细胞和MUC2阳性细胞大量出现,十二指肠、空肠和回肠杯状细胞密度分别是E18的3.3、4.5和7.1倍,其Muc2mRNA的表达水平分别是E15的26.63、11.67和7.75倍。肠上皮细胞间镶嵌连接减少,紧密连接、缝隙连接和桥粒发达。E15小肠蔗糖-异麦芽糖酶(SI)和小肽转运载体(PepTl) mRNA已经表达,随着胚龄的增加,十二指肠、空肠的SI mRNA表达和空肠、回肠的PepTl mRNA表达呈现先增加(E18)后下降(E21)趋势,而回肠SI mRNA表达持续下降,十二指肠PepTl mRNA的表达持续上升。随着胚龄的增加,空肠的抗氧化功能和回盲交界处T淋巴细胞的增殖能力持续增加,但B淋巴细胞增殖能力各胚龄之间没有明显差异。2.孵化后期鸡胚法氏囊形态结构和功能发育E15法氏囊淋巴滤泡开始出现,滤泡内淋巴细胞PCNA阳性率高达97.7%,并具有一定的抗氧化功能,但淋巴细胞对ConA和LPS刺激反应能力较差。随着胚龄的增加,法氏囊淋巴滤泡面积明显增大,但滤泡内淋巴细胞PCNA阳性率下降；E21法氏囊淋巴滤泡面积是E15的6倍,PCNA阳性率为75%,与E18相比差异不显著,但抗氧化能力进一步提高,受抗原刺激后,T淋巴细胞增殖能力进一步加强,而B淋巴细胞增殖能力仍然处于较低水平。3.白光对后期鸡胚小肠黏膜结构和功能的影响在E15和E18,白光组鸡胚小肠黏膜的形态结构和生理功能与黑暗下孵化的鸡胚相比均没有统计学上的差异。到E21时,白光组回肠绒毛高度、Muc2mRNA、小肠各段PepTl和SI mRNA的表达显著高于黑暗组,但空肠抗氧化能力之间差异不显著。ConA刺激后,白光组回盲交界处T淋巴细胞增殖能力显著高于黑暗组,而LPS刺激后,B淋巴细胞增殖能力差异不显著。4.白光对后期鸡胚法氏囊发育和功能的影响E15,白光组与黑暗组的法氏囊淋巴细胞滤泡面积、PCNA表达、抗氧化能力和淋巴细胞增殖能力均没有显著差异。到E21白光组CAT、SOD和T-AOC的活力显著高于黑暗组(p<0.05),而MDA含量黑暗组比白光组高24.9%(p<0.05)；抗原刺激后,白光组T淋巴细胞增殖能力显著高于黑暗组(p<0.05),而B淋巴细胞的增殖能力在两组之间无显著差异。5.单色光对后期鸡胚小肠黏膜形态结构和功能的影响在E15,不同单色光下孵化的鸡胚小肠黏膜的形态结构和生理功能与黑暗下孵化的相比均没有统计学上的差异。随着胚龄的增加,各光色组鸡胚黏膜的形态结构和生理功能的变化模式基本一致,但变化的幅度并不相同。E21时,绿光组的十二指肠、空肠和回肠绒毛高度及肌层厚度均显著高于黑暗组(p<0.05),空肠和回肠黏膜上皮PCNA表达量显著高于其它处理组(p<0.05)。绿光组十二指肠、空肠和回肠PepTl和SI mRNA的表达量显著高于红光组和黑暗组(p<0.05)。杯状细胞密度在绿光和蓝光组空肠和回肠中显著高于黑暗组和红光组,绿光组回肠中硫酸化细胞亚型和唾液酸化细胞亚型的比值(0.39±0.05)显著低于其它处理组。绿光组小肠各段Muc2mRNA和MUC2的表达显著高于黑暗组和红光组。绿光组抗氧化能力最强,显著高于其它处理组(p<0.05)。ConA刺激时,绿光组的回盲交界处淋巴细胞增殖指数最高,与红光组、黑暗组相比差异显著(p<0.05)；但LPS刺激下各处理组之间没有显著差异。6.单色光对法氏囊发育和功能的影响E15,红光组法氏囊淋巴细胞滤泡与其它光色组相比最小(p<0.05)。随着胚龄增加,各光色组法氏囊淋巴细胞滤泡的面积不断加大,滤泡内淋巴细胞PCNA阳性率降低,抗氧化功能不断加强。E21,绿光组和蓝光组滤泡面积分别为35499.0±1775.4μm2,26586.5±5419.0μm2,与黑暗组(23875.1±2388.4μm2)相比差异显著(p<0.05),红光组滤泡面积最小,为15288.1±307.2μm2,与其它光色组相比差异显著(p<0.05),滤泡内淋巴细胞PCNA阳性率绿光组最高,为77.9%,与红光组(71.7%)和黑暗组(73.2%)相比差异显著(p<0.05)。绿光组的CAT、SOD和T-AOC活力最大,与黑暗组相比差异显著(p<0.05),而MDA的含量黑暗组显著高于绿光组。ConA刺激,绿光组法氏囊T淋巴细胞增殖指数比红光组和黑暗组分别高12.1%和5.1%,绿光组与红光组和黑暗组相比差异显著(p<0.05)；而LPS刺激时,绿光组法氏囊B淋巴细胞的增殖率高于红光组和黑暗组(p<0.05),但与蓝光组相比差异不显著。7.光照对空肠组织中IL-6和TNF-a的影响与黑暗组相比,不同单色光和白光对E15鸡胚空肠组织中IL-6和TNF-a的影响并不显著。随着鸡胚的发育,E21时绿光组的1L-6和TNF-a含量在各组中均为最高,分别是329.21±19.69ng/L和374.62±23.35ng/L,与黑暗组(248.21±71.20ng/L;267.86±26.03ng/L)差异显著p<0.05)。红光组空肠组织IL-6最低,为238.01±11.16ng/L,但与黑暗组相比差异不显著。8.光照对后期鸡胚空肠中褪黑激素受体Mella、Mellb和Mel1c mRNA表达的影响在鸡胚孵化期间,不同单色光和白光照射对鸡胚空肠Mel1a、Mel1b和Mel1c mRNA具有一定的影响。随着鸡胚胚龄的增加,不同处理组Mel1a、Mel1b和Mel1c mRNA的表达均呈现增加趋势。E21胚龄时,鸡胚空肠绿光组Mel1a、Mel1b和Mel1c mRNA的表达均为最高,分别是黑暗组和红光组的1.5-1.7、1.1和1.2-2.5倍。Mel1a mRNA的表达与黑暗组、红光组相比差异显著(p<0.0400),绿光组Mel1c mRNA的表达比红光组显著增高(p=0.0228)9.光照对后期鸡胚法氏囊中褪黑激素受体Mel1a. Mel1b和Mel1c mRNA表达的影响随着鸡胚的发育,不同单色光和白光显著影响法氏囊中Mel1a、Mel1b和Mel1c mRNA的表达。绿光组和蓝光组Mel1a、Mel1b和Mel1c mRNA的表达随着胚龄的增加呈增高趋势,而白光组和黑暗组Mel1a、Mel1b和Mel1c mRNA的表达随着胚龄的增加呈现先增加后下降的趋势,红光组Mel1a、Mel1b和Mel1c mRNA的表达随着胚龄的增加呈现出持续下降的趋势。结论：(1) E15-E21期间是小肠黏膜消化吸收与免疫功能快速发育阶段,小肠不同类型的上皮细胞发生以及消化酶的表达具有一定的时空顺序。(2)鸡胚孵化期间不同波长光照射对小肠和法氏囊的发育与功能具有一定的影响,其中绿光能显著提高小肠和法氏囊的发育,促进鸡胚小肠和法氏囊功能的提早成熟,而红光具有一定的抑制作用。(3)光照通过影响褪黑激素受体的表达调节鸡胚肠道和法氏囊细胞因子水平和抗氧化功能进而影响孵化后期鸡胚小肠和法氏囊的发育。更多还原

【Abstract】 Small intestine is an important place to digest and absorb and the first defensive line of mucosal barrier for birds. Previous studies found different wavelength lights could influence the mucosal mechanical and immunological barriers in the small intestine of broilers, but little is known about the effects of different wavelength lights on the development of the small intestine in chicken embryos during the late incubation. However, the late incubation of chick embryo is the best period for plasticity of small intestine, meanwhile the perfect development of small intestine is good to get the maximum development and growth after posthatching. In order to explore the effects of different wavelength lights on the development of small intestine and bursa of chick embryos during the late period, chicken embryos were exposed to white light (400-700nm), blue light (480nm), green light (560nm), red light (660nm), and dark as a control, and were studied by the methods of histology, histochemistry, immunohistochemistry, qRT-PCR, ELISA and cell culture. The results would be provided a theoretical basis for excavation of improving the growth potential and immune function of just post-hatch chicks with lights stimulating during chicken embryos incubation. The results as followed:1. Development of morphological structure and physiological function of small intestine in chick embryos during the late incubationDuring the late-stage of chicken incubation, the length of villus, thickness of muscle layer, the expression of PCNA, and density of goblet cells changed significantly in the small intestine with the embryonic age increasing. Up to E21, the villus height and thickness of muscle layer of duodenum, jejunum and ileum were increased150-180%and1.2-1.7times compared with E15, respectively. The average optical density value of small intestinal mucosa PCNA in duodenum, jejunum and ileum was higher38-53%than that of E15. The sulfated goblet cells were the most at E15and neutral goblet cells are prodominent at E18, and at E21, a large number of sialylated subtype goblet cells emerged, the goblet cell density of duodenum, jejunum and ileum were higher3.3,4.5and7.1fold than those of E18, and the expressions of Muc2mRNA in duodenum, jejunum and ileum were higher26.63,11.67and7.75fold than those of E15, and MUC2positive cells emerged largely. At this time, mosaic connection between intestinal epithelial cells was decreased, and tight junctions, gap junctions and desmosomes were developed.SI and PepT1mRNA began to express on E15. With the increase of embryo age, the expression of SI mRNA in duodenum and jejunum, PepTl mRNA in jejunum and ileum increased on E18and then decreased on E21. While the expression of SI mRNA in ileum decreased continually and the expression of PepTl mRNA in duodenum increased continually. The antioxidant function of jejunum and the ability of T lymphocyte proliferation of ileocecal junction increased continually and there was no obvious difference in B lymphocyte proliferation of ileocecal junction among different embryo ages.2. Development of morphology and function of bursa of Fabricus during the late incubation of chick embryosThe lymphoid follicles of bursa began to appear on E15, and the PCNA positive rate of intrafollicilar lymphocytes was high, reaching97.7%. The bursa had some antioxidant function, but the ability of lymphocyte proliferation was low after stimulation by ConA and LPS. With the increase of embryo age, the area of lymphoid follicle of bursa increased rapidly and the PCNA positive rate of intrafollicilar lymphocytes decreased. Until E21, the area of lymphoid follicle of bursa was increased6times than that of E15, and the PCNA positive rate of intrafollicilar lymphocytes was similar as that of E18. However, the antioxidant function and the ability of T lymphocyte proliferation further improved, but ability of B lymphocyte proliferation had no obvious change after antigen simulation.3. Effect of white light on mucosa structure and physiological function of small intestine of chick embryos during the late incubationOn E15and E18, there were no significant difference between white group and dark group in the morphological structure and physiological function of small intestine. Until E21, villus height, Muc2, PepTl and SI mRNA expression in white group were higher than those of in dark group. However, there was no obvious difference in antioxidant ability between the two groups. The ability of T lymphocyte transformation was higher in white group than that of in dark group (p<0.05) after stimulation of ConA, while there was no significant difference between the two groups in the ability of B lymphocyte transformation after stimulation of LPS.4. Effect of white light on growth and physiological function of bursa of Fabricus in chick embryos during the late incubationOn E15, there was no significant difference between white and dark groups in follicular area of bursa, the expression of PCNA in follicle, the antioxidant ability and the ability of lymphocyte transformation. On E21, CAT, SOD activity and T-AOC was significantly higher in white group than that of in dark group (p<0.05), and content of MDA in jejunum was higher24.9%in white group than that of in dark group. After antigen stimulating, the ability of T lymphocyte proliferation was significantly higher than that of in dark group, while there was no significant difference between the two groups in the ability of B lymphocyte proliferation.5. Effect of monochromatic light on mucosa structure and physiological function of small intestine in chick embryos during the late incubationOn E15, there was no significant difference among different treatment groups in morphological structure and physiological function of small intestine in chicken embryos. With the increase of embryo age, the change pattern of small intestinal mucosal morphology and function were similar among different treatment groups, but the magnitude of the change was not the same in different treatment groups. Until E21, villus height and muscle thickness in the three segments of small intestine were higher in green light group than that of in dark group (p<0.05) The expression of intestinal mucosal PCNA in jejunum and ileum was higher significantly in green group than that of other treatment groups. The expression of PepTl and SI mRNA in duodenum, jejunum and ileum was higher in green group than that of in red and dark groups. Goblet cell density in jejunum and ileum of green and blue groups were higher than that of in dark and red groups. And the ratio of sulfated and sialylated goblet cells in ileum were lower in green group than that of other treatment groups. The expression of Muc2gene and MUC2of the three segments of small intestine was higher in green group than that of other treatment groups. The antioxidant function was higher in green group than other treatment groups. After simulation of ConA, lymphocyte proliferation index of ileocecal junction in green group was the highest, showing significant difference compared with that of red and dark groups (p<0.05). While simulated with LPS, there was no significant difference among different treatment groups.6. Effect of monochromatic light on development and physiological function of bursa in chick embryos during the late incubationOn E15, the area of follicle of bursa in red group was the smallest, which were significant difference compared with other treatment groups. With increasing of embryo age, the area of follicles of bursa in each treatment group increased, the expression of PCNA in follicle decreased and antioxidant function of bursa enhanced. Until E21, the area of follicular in green and blue groups were35499.0±1775.4and26586.5±5419.0, respectively, and there were significant difference compared with that of darkness group (23875.1±2388.4)(p<0.05). And the area of follicular in red group was the smallest,15288.1±307.2, which was significant difference among the other treatment groups (p<0.05). The positive rate of PCNA in follicle in green group was the maximum (77.9%), which was significant difference as compared to red group (71.7%) and dark group (73.2%)(p<0.05). The activity of CAT, SOD and T-AOC in green group was higher significantlythan that of dark group (p<0.05). While content of MDA in dark group was higher than that of green group. When stimulated with ConA, lymphocyte proliferation index in green group was higher by12.1%and5.1%than red and dark group. When stimulated with LPS, the ability of lymphocyte proliferation was higher in green group than red and black group. However, there was no difference was observed between green and blue group.7. Effect of light on IL-6and TNF-α in jejunum tissue of chick embryos during the late incubationOn E15, there was no significant difference among different treatment groups in the level of IL-6 and TNF-a in jejunum. With the increase of embryo age, on E21, the level of IL-6and TNF-a in jejunum in green group was329.21±19.69ng/L and374.62±23.35ng/L, respectively. There was significant difference between green and dark group (248.21±71.20ng/L;267.86±26.03ng/L)(p<0.05). The level of IL-6in jejunum in red group was the lowest (238.01±11.16ng/L), but there was no significant difference between red and dark groups (p>0.05).8. Effect of light on expression of Mella, Mellb and Mellc in jejunum in chick embryos during the late incubationVarious monochromatic lights and white light stimulus had a certain effect on the expression of Mel1a、Mel1b and Mellc mRNA in chicken embryo jejunum during the stage of incubation. With the age increasing, the expression of Mella, Mellb and Mellc mRNA in different treatment groups increased. On E21, the expressions of Mel1a、Mel1b and Mel1c mRNA of jejunum in chicken embryos were the highest in green group, it was1.5-1.7,1.1and1.2-2.5folds as high as that of dark and red groups. There was significant difference in Mella mRNA expression in green group compared with that of dark and red group (p<0.0400), and the expression of Mellc mRNA was higher significantly in green group than that of red light (p=0.0228).9. Effect of light on the expression of Mella, Mellb and Mellc in bursa of chick embryos during the late incubationWith the increase of embryo age, the expression of Mel1a, Mel1b and Mel1c in green and blue groups continued to increase. The expression of Mel1a, Mel1b and Mel1c on E21were higher8.1,16.3and4.4times that of E15in green group, and the expression of Mel1a, Mellb and Mel1c were higher7.7,2.7and4.4times than that of on E15in blue group. The expression of Mella, Mellb and Mel1c in white and dark groups increased on E18and then decreased on E21, which was decreased in red group from E15to E21.Conclusion:(1) The late stage of chicken embryos incubation (from E35to E21) was the stage of rapid development for morphological structure and function of small intestinal mucosa. The ontogeny and differentiation of epithelial cells in broiler embryos display temporal and spatial differences.(2) Lights had a certain effects on development and function of small intestine and bursa. Green light enhanced significantly the development of small intestinal mucosa and bursa, and promoted the maturity of small intestinal mucosa and bursa earlier during the late incubation.(3) The effects of light on the development and function of small intestine and bursa might be associated with the secretion of melatonin in chick embryos and further influencing the cytokine, antioxidant function and expression of melatonin receptor in jejunum and bursa.更多还原