内质网应激相关蛋白MANF在脾脏中的表达

发布时间：2018-06-24 11:33

本文选题：MANF + 脾脏　；参考：《安徽医科大学》2012年硕士论文

【摘要】：内质网应激（endoplasmic reticulum stress，ER stress）是真核细胞的一种保护性应激反应，通过激活未折叠蛋白反应（unfolded protein response，UPR）通路来减少细胞内蛋白的异常聚集，从而起到细胞保护作用。ER应激与很多因素所致疾病的发生、发展密切相关。越来越多的证据表明，ER应激及其UPR通路可能参与机体的炎症免疫反应。脾脏是机体细胞免疫和体液免疫中心，参与先天免疫和获得性免疫。脾脏能够清除循环中的衰老红细胞，并能有效去除血源性细菌和细胞残片；加之它的T、B淋巴细胞的高度有序分布，使得脾脏成为抗菌最重要的器官。MANF (mesencephalic astrocyte-derived neurotrophic factor, MANF)基因是我们从30000个基因中筛选出的对ER应激最敏感的基因，它是一种分泌性蛋白，ER应激能诱导其表达上调。故我们推测，MANF可能选择性表达于脾细胞中，在脾脏的发育和浆细胞分化中发挥重要的作用。目的：本研究的目的是观察大鼠出生后不同发育阶段脾脏的结构变化、浆细胞的发育与分化以及MANF蛋白在脾细胞中选择性的表达情况，为研究MANF的生物学活性与免疫调节的关系提供实验基础。方法：选取不同发育时期鼠的脾脏，并制备组织标本。分别采用HE染色、免疫组织化学及免疫荧光技术，观察大鼠不同时期脾脏的结构及CD138（浆细胞）、CD68（巨噬细胞）阳性细胞的发育特点以及出生后不同时期MANF在脾脏中的表达情况；使用T、B淋巴细胞的表面标记物CD3及CD20，观察MANF在成年鼠及外伤切除的人脾脏中各类淋巴细胞中差异性的表达特点。用弗氏完全佐剂（Freund'scomplete adjuvant，FCA）制备大鼠佐剂性关节炎（adjuvant arthritis，AA）模型，观察MANF在炎性大鼠脾脏中的表达情况。结果： 1.大鼠出生后不同时期脾脏的发育 1.1脾脏结构的发育大鼠在出生后1~4wk，脾脏的白髓和红髓发育不成熟，没有完整的白髓结构和血管结构，而出现大量的细胞团；6wk时白髓有雏形，脾脏的白髓和红髓中出现血管样结构；8wk时白髓具有典型的结构特征，如中央动脉周围有大量的T细胞形成的PALS及B细胞组成的滤泡。 1.2CD68阳性细胞的发育大鼠出生后1wk，脾脏组织中即有大量的、散在分布的、呈分枝状的CD68阳性的细胞；而在出生6wk后，巨噬细胞呈圆形或阿米巴状，细胞内CD68的表达量在12-22wk时明显增加。 1.3CD138阳性细胞的发育大鼠在出生后1~4wk，脾脏组织中无CD138阳性的浆细胞；6wk后，脾脏的白髓和红髓中少量的CD138阳性细胞；8wk时白髓中有大量的CD138阳性细胞，此时CD138的表达量达到峰值，而后随着月龄的增加CD138的表达有缓慢下降趋势。 1.4MANF的表达大鼠在出生后1~4wk，MANF在脾脏中广泛表达，尤其是在成团细胞的中MANF也有表达；6~12wk后，MANF在散在的细胞中表达，且定位于胞浆。这些MANF阳性细胞多数分布在边缘区和红髓中；少数分布在动脉周围淋巴鞘中。而22wk后，MANF则弥散分布于胞浆中。免疫荧光双标发现，1-3wk时MANF不在CD68阳性细胞中表达。在出生后4wk时在少量的CD68细胞中有MANF表达，此后表达MANF的CD68细胞逐渐增多。由于CD138阳性细胞在出生后6wk时才出现，此时部分CD138阳性细胞中有MANF表达；8wk后CD138阳性细胞与MANF共染细胞开始逐步增加，至10wk达到峰值，而后随着年龄的增加MANF+CD138+数量有缓慢下降。 2. MANF在成熟脾脏中的选择性表达在成熟大鼠和人的脾脏中，MANF阳性细胞主要出现在红髓和边缘区，不存在于白髓中，且MANF的表达主要定位于胞浆中，而不是细胞核中。MANF阳性细胞的分布与CD138和CD68阳性细胞的分布一致，该结果提示MANF在成熟脾细胞中可能在浆细胞和巨噬细胞中表达。为了证实该结果，我们进一步进行免疫荧光双标。结果发现，MANF不在CD20阳性的淋巴细胞中表达，只有极少数的CD3阳性细胞中有MANF的表达，而MANF主要在CD68和CD138阳性细胞中表达。该结果提示MANF的表达可能与ER应激有关。 3. UPR通路蛋白在MANF阳性细胞中的表达在成年人的脾细胞中，，大多数表达BIP、CHOP、ATF6和XBP1的细胞中有MANF表达，而仅有少数BIP、CHOP、ATF6或XBP1阳性细胞不表达MANF；在CD68阳性的细胞中有BIP和CHOP表达，但无XBP1的表达；在CD138阳性细胞中有BIP和XBP1的表达，而无CHOP的表达。上述结果提示，CD138阳性的浆细胞中存在ER应激，MANF的表达可能与ER应激有关。 4. MANF在AA大鼠脾脏中表达在AA大鼠，免疫系统被激活，脾脏中的淋巴细胞相应也被激活。我们的研究发现，在AA大鼠的脾脏中，MANF的表达量明显增多，并集中在脾脏边缘区，提示炎症也诱导MANF在脾细胞中的表达。结论(1)脾脏中浆细胞的分化滞后于巨噬细胞，出生早期机体主要以非特异性免疫为主。(2) MANF在脾细胞中选择性表达，其主要在浆细胞和巨噬细胞中表达，这可能与这些细胞的分化和功能有关。（3）浆细胞和巨噬细胞中均存在ER应激，而浆细胞中UPR通路被激活，MANF的表达与ER应激有关；（4）免疫性关节炎症大鼠脾脏中MANF表达上调，提示炎症能诱导MANF表达。
[Abstract]:Endoplasmic reticulum stress (endoplasmic reticulum stress, ER stress) is a protective stress response to eukaryotic cells. By activating the unfolded protein reaction (unfolded protein response, UPR) pathway to reduce the abnormal aggregation of intracellular proteins, the development of cell protection is closely related to the development of the disease caused by.ER stress and many factors. More and more evidence suggests that ER stress and its UPR pathway may be involved in the immune response to the body. The spleen is the body's cell immunity and humoral immune center, and participates in the innate and acquired immunity. The spleen can remove the aging red cells in the circulation and effectively remove the blood derived bacteria and cell fragments; in addition, its T, B drenching The highly ordered distribution of the Ba cells makes the spleen become the most important organ of.MANF (mesencephalic astrocyte-derived neurotrophic factor, MANF), which is the most sensitive gene that we have screened from 30000 genes for ER stress. It is a secretory protein, and ER stress can induce its up-regulated expression. Therefore, we speculate that MANF can be used. It can be selectively expressed in spleen cells, and plays an important role in the development of spleen and plasma cell differentiation.
Objective:
The aim of this study was to observe the structural changes of the spleen at different developmental stages of the rat, the development and differentiation of plasma cells and the selective expression of MANF protein in the spleen cells, which provided an experimental basis for the study of the relationship between the biological activity of MANF and the immunoregulation.
Method:
HE staining, immunohistochemistry and immunofluorescence technique were used to observe the spleen of rats at different developmental stages and to observe the structure of spleen and CD138 (plasma cell), the development characteristics of CD68 (macrophage) positive cells and the expression of MANF in the spleen at different periods of birth at different stages of the rats, and the use of T, B The differential expression of MANF in the lymphocytes of adult and traumatic human spleens was observed by CD3 and CD20. The expression of adjuvant arthritis (adjuvant arthritis, AA) in rats was prepared by Freund'scomplete adjuvant (FCA), and the expression of MANF in the spleen of inflammatory rats was observed.
Result:
The development of spleen at different periods after birth of 1. rats
1.1 the development of the structure of the spleen
1~4wk after birth, the white pulp and red pulp of the spleen developed immature, without a complete white pulp structure and vascular structure, and a large number of cell masses; the white pulp had a embryonic form at 6wk, the white pulp of the spleen and the blood vessel in the red pulp; the white pulp had a typical structural characteristic at 8wk, such as the formation of a large number of T cells around the central artery. Follicles composed of PALS and B cells.
Development of 1.2CD68 positive cells
After the birth of the rat 1wk, there were a large number of splenic tissues, scattered and branched CD68 positive cells, and the macrophages were round or amoeba after birth 6wk, and the expression of CD68 in the cells increased significantly at 12-22wk.
Development of 1.3CD138 positive cells
There was no CD138 positive plasma cells in 1~4wk after birth in rats. After 6wk, a small amount of CD138 positive cells in the white pulp and red pulp of the spleen were found, and there were a large number of CD138 positive cells in the white pulp at 8wk, and the expression of CD138 reached the peak at this time, and then the expression of CD138 decreased slowly with the increase of the age of the month.
Expression of 1.4MANF
After birth 1~4wk, MANF is widely expressed in the spleen, especially in the medium MANF of the cells; after 6~12wk, MANF is expressed in scattered cells and located in the cytoplasm. Most of these MANF positive cells are distributed in the marginal and red pulp; a few are distributed in the peri arterial lymphatic sheath. And MANF, after 22wk, distributes in diffuse distribution. In the cytoplasm.
MANF was not expressed in CD68 positive cells at 1-3wk. MANF expression was found in a small number of CD68 cells at postnatal 4wk, and thereafter the CD68 cells expressed in MANF increased gradually. The CD138 positive cells appeared at the 6wk of the postnatal 6wk, and there were MANF expressions in some CD138 positive cells. The CO staining cells began to increase gradually, reaching the peak value at 10wk, and then slowly decreased with the increase of MANF+CD138+ age.
Selective expression of 2. MANF in mature spleen
In the mature rat and human spleen, MANF positive cells mainly appear in the red pulp and the marginal area, not in the white pulp, and the expression of MANF is mainly located in the cytoplasm, but not the distribution of.MANF positive cells in the nucleus is consistent with the distribution of CD138 and CD68 positive cells. This result suggests that MANF may be in the plasma cell and in the mature spleen cells. The results showed that MANF was not expressed in CD20 positive lymphocytes, and only a few of the CD3 positive cells were expressed in MANF, while MANF was expressed in CD68 and CD138 positive cells. The results suggest that the expression of MANF may be associated with ER stress.
Expression of 3. UPR pathway protein in MANF positive cells
In the adult spleen cells, most of the cells expressing BIP, CHOP, ATF6 and XBP1 have MANF expression, but only a few BIP, CHOP, ATF6 or XBP1 positive cells do not express MANF; there are BIP and expressions in CD68 positive cells, but there is no expression; there is no expression in the positive cells. It is suggested that there is ER stress in CD138 positive plasma cells, and the expression of MANF may be related to ER stress.
Expression of 4. MANF in the spleen of AA rats
In AA rats, the immune system was activated and the lymphocytes in the spleen were activated. Our study found that in the spleen of AA rats, the expression of MANF was significantly increased and concentrated in the marginal region of the spleen, suggesting that the inflammation also induced the expression of MANF in the spleen cells.
Conclusion (1) the differentiation of the plasma cells in the spleen lagged behind the macrophages, and the early body was mainly non-specific. (2) MANF was selectively expressed in the spleen cells, mainly expressed in the plasma cells and macrophages, which may be related to the differentiation and function of these cells. (3) ER stress is present in plasma cells and macrophages. The UPR pathway is activated in cells, and the expression of MANF is related to ER stress. (4) the expression of MANF in spleen of rats with immune arthritis is upregulated, suggesting that inflammation can induce MANF expression.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】