摘要
小型哺乳动物在冬眠过程中自然产生的免疫学变化可用于研究免疫系统的抑制作用。而理解它的机制对于在不同临床条件下如心肺转流术,移植,外伤,麻醉等提高保存器官的功能有重要医学相关性。因此本论文研究了冬眠动物血液,脾脏和褐色脂肪组织(BAT)中的白细胞总量和不同类型白细胞在冬眠的各个时期受冬眠作用的影响和.离体培养的脾细胞的吞噬功能并测定相关的淋巴细胞运输调节因子的1‐磷酸鞘氨醇(S1P)在血清中的含量。实验结果显示,在冬眠的麻痹阶段,先天性免疫系统根据功能调节。相反的,适应性免疫系统的主要调节机制是细胞数量的减少,并与易位因子S1P水平变化一致。本研究为冬眠过程中的免疫学变化,先天性免疫系统和适应性免疫系统的作用和功能以及其调节机制提供了理论依据。
Immunological changes that occur naturally in small mammals during their hibernation process may provide directions to suppress the immune system. Understanding its nature and mechanisms is of major medical relevance for improving the preservation of organ function in different clinical situations such as cardiopulmonary by-pass, transplantation, trauma, transport, anesthesia etc.
Hibernators are known to grossly decrease the number of circulating white blood cells (WBC) during torpor, the phase of hibernation in which body temperature and metabolism are substantially reduced. In this study, it was determined whether all WBC types are equally affected during hibernation by making a class distinction of different types of WBC in hamster blood, spleen and brown adipose tissue (BAT) during subsequent phases of hibernation. Also, the phagocytosis function of isolated and in vitro cultured splenocytes was explored. Finally, the involvement of sphingosine-1-phosphate (S1P) as a lymphocyte trafficking regulator was studied by measuring S1P levels in serum. For the above mentioned experiments, two animal models of hibernation have been used: the deep torpor model, whose body temperature equals environmental temperature during torpor (about 70C), and the daily torpor model, with a torpor body temperature of 220C that makes it more similar to clinical situations.
Changes in the numbers of circulating WBC were determined in the deep (Syrian hamster) and the daily (Djungarian hamster) torpor models of hibernation by automated hematology analysis and Giemsa staining of blood smears. In addition, PCR quantification of CD markers expressed by specific types of WBC was performed on spleen and BAT after development of the necessary functional primers confirmed by sequencing. The phagocytosis function of isolated splenocytes was studied by measurement of their proliferation capacity following incubation with the T-cell mitogen phyothemaggluitn (PHA). High performance liquid chromatography-tandem mass spectrometry was used to measure serum levels of sphingosine-1-phosphate during hibernation.
In the deep torpor model of hibernation, blood analysis revealed a significant decrease (up to 98%) in the levels of circulating WBC during torpor, followed by a rapid normalization after arousal. This modulation pattern is, to some extent, similar in spleen and BAT. PCR quantification showed an increase in spleen mRNA levels of CD16b (neutrophils) and a significant decrease in spleen mRNA levels of TLR4 and MYD88, both signaling molecules of the phagocytosis pathway. In BAT, mRNA levels of signaling molecules were unaffected, but mRNA level of CD 56 (natural killer specific marker) increased during torpor and arousal phases. Also, the significant decrease of spleen and BAT mRNA levels of CD3e (T-lymphocytes), CD4 (helper T-lymphocytes) and CD20 (B-lymphocytes) during torpor was mirrored by the decrease in levels of sphingosine-1-phosphate, the primary trafficking regulator of B and T-lymphocytes.
Remarkably, the number of circulating WBC during hibernation phases in daily torpor was affected in a similar way as in deep torpor. Also, variations in levels of S1P during hibernation correspond with the modulation pattern previously found in the deep torpor model.
Our results suggest that, during the torpor phase of hibernation, the innate immune system is subject to a regulation by function. In contrast, the primary regulation mechanism of the adaptive immune system is a decrease in numbers of cells and corresponds to levels of the translocation factor S1P. This study provides useful insights in the immunological changes during hibernation, the role and function of the innate and adaptive immune system and their underlying regulation mechanisms.
引文
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