發(fā)布時(shí)間：2018-06-24 11:33

  本文選題：MANF + 脾臟��； 參考：《安徽醫(yī)科大學(xué)》2012年碩士論文

【摘要】：內(nèi)質(zhì)網(wǎng)應(yīng)激（endoplasmic reticulum stress，ER stress）是真核細(xì)胞的一種保護(hù)性應(yīng)激反應(yīng)，通過激活未折疊蛋白反應(yīng)（unfolded protein response，UPR）通路來減少細(xì)胞內(nèi)蛋白的異常聚集，從而起到細(xì)胞保護(hù)作用。ER應(yīng)激與很多因素所致疾病的發(fā)生、發(fā)展密切相關(guān)。越來越多的證據(jù)表明，ER應(yīng)激及其UPR通路可能參與機(jī)體的炎癥免疫反應(yīng)。脾臟是機(jī)體細(xì)胞免疫和體液免疫中心，參與先天免疫和獲得性免疫。脾臟能夠清除循環(huán)中的衰老紅細(xì)胞，并能有效去除血源性細(xì)菌和細(xì)胞殘片；加之它的T、B淋巴細(xì)胞的高度有序分布，使得脾臟成為抗菌最重要的器官。MANF (mesencephalic astrocyte-derived neurotrophic factor, MANF)基因是我們從30000個(gè)基因中篩選出的對(duì)ER應(yīng)激最敏感的基因，它是一種分泌性蛋白，ER應(yīng)激能誘導(dǎo)其表達(dá)上調(diào)。故我們推測，MANF可能選擇性表達(dá)于脾細(xì)胞中，在脾臟的發(fā)育和漿細(xì)胞分化中發(fā)揮重要的作用。 目的： 本研究的目的是觀察大鼠出生后不同發(fā)育階段脾臟的結(jié)構(gòu)變化、漿細(xì)胞的發(fā)育與分化以及MANF蛋白在脾細(xì)胞中選擇性的表達(dá)情況，為研究MANF的生物學(xué)活性與免疫調(diào)節(jié)的關(guān)系提供實(shí)驗(yàn)基礎(chǔ)。 方法： 選取不同發(fā)育時(shí)期鼠的脾臟，并制備組織標(biāo)本。分別采用HE染色、免疫組織化學(xué)及免疫熒光技術(shù)，觀察大鼠不同時(shí)期脾臟的結(jié)構(gòu)及CD138（漿細(xì)胞）、CD68（巨噬細(xì)胞）陽性細(xì)胞的發(fā)育特點(diǎn)以及出生后不同時(shí)期MANF在脾臟中的表達(dá)情況；使用T、B淋巴細(xì)胞的表面標(biāo)記物CD3及CD20，觀察MANF在成年鼠及外傷切除的人脾臟中各類淋巴細(xì)胞中差異性的表達(dá)特點(diǎn)。用弗氏完全佐劑（Freund'scomplete adjuvant，F(xiàn)CA）制備大鼠佐劑性關(guān)節(jié)炎（adjuvant arthritis，AA）模型，觀察MANF在炎性大鼠脾臟中的表達(dá)情況。 結(jié)果： 1.大鼠出生后不同時(shí)期脾臟的發(fā)育 1.1脾臟結(jié)構(gòu)的發(fā)育 大鼠在出生后1~4wk，脾臟的白髓和紅髓發(fā)育不成熟，沒有完整的白髓結(jié)構(gòu)和血管結(jié)構(gòu)，而出現(xiàn)大量的細(xì)胞團(tuán)；6wk時(shí)白髓有雛形，脾臟的白髓和紅髓中出現(xiàn)血管樣結(jié)構(gòu)；8wk時(shí)白髓具有典型的結(jié)構(gòu)特征，如中央動(dòng)脈周圍有大量的T細(xì)胞形成的PALS及B細(xì)胞組成的濾泡。 1.2CD68陽性細(xì)胞的發(fā)育 大鼠出生后1wk，脾臟組織中即有大量的、散在分布的、呈分枝狀的CD68陽性的細(xì)胞；而在出生6wk后，巨噬細(xì)胞呈圓形或阿米巴狀，細(xì)胞內(nèi)CD68的表達(dá)量在12-22wk時(shí)明顯增加。 1.3CD138陽性細(xì)胞的發(fā)育 大鼠在出生后1~4wk，脾臟組織中無CD138陽性的漿細(xì)胞；6wk后，脾臟的白髓和紅髓中少量的CD138陽性細(xì)胞；8wk時(shí)白髓中有大量的CD138陽性細(xì)胞，此時(shí)CD138的表達(dá)量達(dá)到峰值，而后隨著月齡的增加CD138的表達(dá)有緩慢下降趨勢。 1.4MANF的表達(dá) 大鼠在出生后1~4wk，MANF在脾臟中廣泛表達(dá)，尤其是在成團(tuán)細(xì)胞的中MANF也有表達(dá)；6~12wk后，MANF在散在的細(xì)胞中表達(dá)，且定位于胞漿。這些MANF陽性細(xì)胞多數(shù)分布在邊緣區(qū)和紅髓中；少數(shù)分布在動(dòng)脈周圍淋巴鞘中。而22wk后，MANF則彌散分布于胞漿中。 免疫熒光雙標(biāo)發(fā)現(xiàn)，1-3wk時(shí)MANF不在CD68陽性細(xì)胞中表達(dá)。在出生后4wk時(shí)在少量的CD68細(xì)胞中有MANF表達(dá)，此后表達(dá)MANF的CD68細(xì)胞逐漸增多。由于CD138陽性細(xì)胞在出生后6wk時(shí)才出現(xiàn)，此時(shí)部分CD138陽性細(xì)胞中有MANF表達(dá)；8wk后CD138陽性細(xì)胞與MANF共染細(xì)胞開始逐步增加，至10wk達(dá)到峰值，而后隨著年齡的增加MANF+CD138+數(shù)量有緩慢下降。 2. MANF在成熟脾臟中的選擇性表達(dá) 在成熟大鼠和人的脾臟中，MANF陽性細(xì)胞主要出現(xiàn)在紅髓和邊緣區(qū)，不存在于白髓中，且MANF的表達(dá)主要定位于胞漿中，而不是細(xì)胞核中。MANF陽性細(xì)胞的分布與CD138和CD68陽性細(xì)胞的分布一致，該結(jié)果提示MANF在成熟脾細(xì)胞中可能在漿細(xì)胞和巨噬細(xì)胞中表達(dá)。為了證實(shí)該結(jié)果，我們進(jìn)一步進(jìn)行免疫熒光雙標(biāo)。結(jié)果發(fā)現(xiàn)，MANF不在CD20陽性的淋巴細(xì)胞中表達(dá)，只有極少數(shù)的CD3陽性細(xì)胞中有MANF的表達(dá)，而MANF主要在CD68和CD138陽性細(xì)胞中表達(dá)。該結(jié)果提示MANF的表達(dá)可能與ER應(yīng)激有關(guān)。 3. UPR通路蛋白在MANF陽性細(xì)胞中的表達(dá) 在成年人的脾細(xì)胞中，，大多數(shù)表達(dá)BIP、CHOP、ATF6和XBP1的細(xì)胞中有MANF表達(dá)，而僅有少數(shù)BIP、CHOP、ATF6或XBP1陽性細(xì)胞不表達(dá)MANF；在CD68陽性的細(xì)胞中有BIP和CHOP表達(dá)，但無XBP1的表達(dá)；在CD138陽性細(xì)胞中有BIP和XBP1的表達(dá)，而無CHOP的表達(dá)。上述結(jié)果提示，CD138陽性的漿細(xì)胞中存在ER應(yīng)激，MANF的表達(dá)可能與ER應(yīng)激有關(guān)。 4. MANF在AA大鼠脾臟中表達(dá) 在AA大鼠，免疫系統(tǒng)被激活，脾臟中的淋巴細(xì)胞相應(yīng)也被激活。我們的研究發(fā)現(xiàn)，在AA大鼠的脾臟中，MANF的表達(dá)量明顯增多，并集中在脾臟邊緣區(qū)，提示炎癥也誘導(dǎo)MANF在脾細(xì)胞中的表達(dá)。 結(jié)論(1)脾臟中漿細(xì)胞的分化滯后于巨噬細(xì)胞，出生早期機(jī)體主要以非特異性免疫為主。(2) MANF在脾細(xì)胞中選擇性表達(dá)，其主要在漿細(xì)胞和巨噬細(xì)胞中表達(dá)，這可能與這些細(xì)胞的分化和功能有關(guān)。（3）漿細(xì)胞和巨噬細(xì)胞中均存在ER應(yīng)激，而漿細(xì)胞中UPR通路被激活，MANF的表達(dá)與ER應(yīng)激有關(guān)；（4）免疫性關(guān)節(jié)炎癥大鼠脾臟中MANF表達(dá)上調(diào)，提示炎癥能誘導(dǎo)MANF表達(dá)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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相關(guān)期刊論文 前2條

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