本文選題：星形膠質(zhì)細(xì)胞 + 炎癥��； 參考：《山東大學(xué)》2009年博士論文

【摘要】： 近幾十年來,中樞神經(jīng)系統(tǒng)疾病(如創(chuàng)傷、神經(jīng)退行性疾病、腦血管病等)的發(fā)病率逐年增加,其致殘率和死亡率較高,而成體中樞神經(jīng)系統(tǒng)自身的再生能力較弱,目前臨床只能對(duì)癥治療,尚無有效的治療方法。近年來,干細(xì)胞(stemcells)移植療法逐漸成為治療中樞神經(jīng)系統(tǒng)疾病的一種有前途的治療措施。 干細(xì)胞是一類具有自我更新、自我復(fù)制能力和多向分化潛能的細(xì)胞,在合適的條件下,可以分化為不同類型的具有特定形態(tài)、特定分子標(biāo)志和特殊功能的成熟細(xì)胞。骨髓基質(zhì)干細(xì)胞(bone marrow stromal stem cells,BMSCs)是來源于骨髓的一種干細(xì)胞。BMSCs可從自體骨髓取材、來源充足、取材方便,是治療性干細(xì)胞移植中比較理想的種子細(xì)胞。 近年來研究發(fā)現(xiàn),BMSCs不僅可分化為成骨細(xì)胞、軟骨細(xì)胞、成肌細(xì)胞等間充質(zhì)來源的細(xì)胞,在合適的培養(yǎng)條件下,BMSCs還可橫向分化為神經(jīng)細(xì)胞;并在不同的體內(nèi)微環(huán)境或體外誘導(dǎo)條件下,分化為不同遞質(zhì)類型的神經(jīng)細(xì)胞。因此,BMSCs有望成為干細(xì)胞移植治療神經(jīng)系統(tǒng)疾病的理想種子細(xì)胞,具有廣闊的臨床應(yīng)用前景。 星形膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)內(nèi)數(shù)量最多的一類細(xì)胞,也是中樞神經(jīng)系統(tǒng)微環(huán)境的主要構(gòu)成成分。研究發(fā)現(xiàn),星形膠質(zhì)細(xì)胞不僅對(duì)神經(jīng)元提供營(yíng)養(yǎng)、支持、保護(hù)、隔離作用,在神經(jīng)元的發(fā)育、軸突再生、突觸形成和信號(hào)傳遞以及干細(xì)胞的分化等方面也發(fā)揮著重要作用。 實(shí)驗(yàn)證明,中樞神經(jīng)系統(tǒng)損傷或發(fā)生神經(jīng)退行性疾病后,炎癥反應(yīng)非常明顯,星形膠質(zhì)細(xì)胞的激活并分泌一系列炎性細(xì)胞因子是這種炎癥反應(yīng)的重要標(biāo)志。這種炎癥反應(yīng)所形成的微環(huán)境對(duì)神經(jīng)組織中固有的神經(jīng)干細(xì)胞或移植的骨髓基質(zhì)干細(xì)胞會(huì)產(chǎn)生什么影響,至今仍不得而知。 本研究利用體外建立的星形膠質(zhì)細(xì)胞炎癥模型,研究了激活的星形膠質(zhì)細(xì)胞對(duì)骨髓基質(zhì)干細(xì)胞增殖及分化的影響,并對(duì)其作用機(jī)制進(jìn)行了初步探討。 一、骨髓基質(zhì)干細(xì)胞的分離、培養(yǎng)和體外擴(kuò)增 由于骨髓中細(xì)胞成分復(fù)雜,且BMSCs在骨髓中的含量很少,約10~5個(gè)骨髓單個(gè)核細(xì)胞中僅含2～5個(gè)BMSCs,因而BMSCs的分離、培養(yǎng)、純化和體外擴(kuò)增的難度較大。 根據(jù)BMSCs密度比較低和容易貼壁生長(zhǎng)的特點(diǎn),首先用密度為1.077g/ml的淋巴細(xì)胞分離液將大部分的造血細(xì)胞去除,然后通過貼壁培養(yǎng)、定期換液去除懸浮生長(zhǎng)的造血細(xì)胞,使用低糖DMEM培養(yǎng)液(含10%FBS)培養(yǎng)擴(kuò)增。原代培養(yǎng)7天左右,BMSCs可形成明顯的細(xì)胞集落;細(xì)胞生長(zhǎng)至80%～90%融合時(shí)用0.25%胰蛋白酶-0.02%EDTA消化,傳代培養(yǎng)。傳至3代時(shí),細(xì)胞基本純化,形態(tài)趨于一致,呈長(zhǎng)梭形。由于BMSCs缺乏專一的特異性抗原標(biāo)志,本實(shí)驗(yàn)應(yīng)用流式細(xì)胞儀檢測(cè)了多種細(xì)胞表面抗原,以便確認(rèn)BMSCs。檢測(cè)結(jié)果顯示:幾乎所有的培養(yǎng)細(xì)胞,其表面抗原CD29和CD90呈陽性,說明培養(yǎng)的細(xì)胞為較純的BMSCs。 二、炎性星形膠質(zhì)細(xì)胞條件培養(yǎng)基對(duì)BMSCS增殖及分化的影響 在成功培養(yǎng)BMSCs的基礎(chǔ)上,為模擬中樞神經(jīng)系統(tǒng)體內(nèi)炎性微環(huán)境下星形膠質(zhì)細(xì)胞細(xì)胞對(duì)BMSCs增殖及分化的影響,我們首先分別收集加入脂多糖(LPS)培養(yǎng)12、36、72h后的星形膠質(zhì)細(xì)胞上清液作為炎性星形膠質(zhì)細(xì)胞條件培養(yǎng)基(astrocyte conditioned medium,ACM,分別稱為12h ACM、36h ACM、72h ACM),同時(shí)收集無LPS的星形膠質(zhì)細(xì)胞上清液作為正常星形膠質(zhì)細(xì)胞條件培養(yǎng)基(normal ACM,N-ACM),然后將收集的條件培養(yǎng)基加入BMSCs的培養(yǎng)中,觀察細(xì)胞的增殖及神經(jīng)分化情況。 細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示,和N-ACM相比,炎性星形膠質(zhì)細(xì)胞條件培養(yǎng)基(12hACM、36h ACM、72h ACM)可明顯促進(jìn)BMSCs的增殖,36h ACM的促增殖作用明顯強(qiáng)于12h及72h ACM兩組。神經(jīng)分化結(jié)果表明,和N-ACM相比,炎性條件培養(yǎng)基可使BMSCs向神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞分化率明顯升高,36h ACM組的神經(jīng)元陽性細(xì)胞比例明顯高于12h ACM和72h ACM兩組;而神經(jīng)膠質(zhì)細(xì)胞的分化率,12h ACM、36h ACM、72h ACM三組無明顯差異。 三、炎性星形膠質(zhì)細(xì)胞條件培養(yǎng)基調(diào)節(jié)BMSCs增殖及分化的機(jī)制研究 第二部分的實(shí)驗(yàn)結(jié)果表明,炎性星形膠質(zhì)細(xì)胞可促進(jìn)BMSCs的增殖及神經(jīng)分化,炎性星形膠質(zhì)細(xì)胞的這種促進(jìn)作用可能是通過分泌的各種因子而實(shí)現(xiàn)的。本實(shí)驗(yàn)重點(diǎn)檢測(cè)了白細(xì)胞介素-6(IL-6)。以往的實(shí)驗(yàn)證實(shí),作為一種多功能的炎性細(xì)胞因子,IL-6在各種中樞神經(jīng)系統(tǒng)創(chuàng)傷或疾病中的表達(dá)明顯增加,并在神經(jīng)發(fā)育及再生等過程中發(fā)揮重要作用。為明確IL-6在炎性星形膠質(zhì)細(xì)胞誘導(dǎo)的BMSCs增殖及神經(jīng)分化過程中的作用,我們首先檢測(cè)了炎性星形膠質(zhì)細(xì)胞合成和分泌IL-6的情況。ELISA檢測(cè)結(jié)果顯示,炎性星形膠質(zhì)細(xì)胞培養(yǎng)基中IL-6的含量明顯增多,且36h ACM中IL-6的含量明顯高于12h和72h ACM,RT-PCR結(jié)果與此相一致。當(dāng)在培養(yǎng)基中添加特異性IL-6抗體以阻斷IL-6的作用時(shí),BMSCs的增殖明顯減少,BMSCs向星形膠質(zhì)細(xì)胞方向分化的趨勢(shì)也明顯減弱,但BMSCs向神經(jīng)元方向分化明顯增多,說明IL-6可促進(jìn)BMSCs的增殖及向星形膠質(zhì)細(xì)胞方向分化,而抑制BMSCs向神經(jīng)元方向分化。 結(jié)論: 炎癥激活的星形膠質(zhì)細(xì)胞能明顯促進(jìn)骨髓基質(zhì)干細(xì)胞的增殖及向神經(jīng)方向分化,且其作用強(qiáng)度與作用時(shí)間密切相關(guān),炎性星形膠質(zhì)細(xì)胞分泌的炎性細(xì)胞因子IL-6參與了這一過程。
[Abstract]:In recent decades, the incidence of central nervous system diseases (such as trauma, neurodegenerative disease, cerebrovascular disease, etc.) has increased year by year, its rate of disability and mortality is high, but the regeneration ability of the adult central nervous system is weak, and there is no effective treatment in clinical treatment. In recent years, stem cell (stemcells) transplantation therapy This method has gradually become a promising treatment for central nervous system diseases.
Stem cells, a class of cells with self renewal, self replicating and pluripotent differentiation potential, can differentiate into different types of mature cells with specific morphologic, specific molecular markers and special functions under suitable conditions. Bone marrow stromal cells (bone marrow stromal stem cells, BMSCs) are a dry fine derived from bone marrow. Cell.BMSCs can be extracted from autologous bone marrow, with sufficient sources and convenient materials. It is an ideal seed cell in therapeutic stem cell transplantation.
In recent years, it has been found that BMSCs can not only differentiate into osteoblasts, chondrocytes, myoblasts and other mesenchymal stem cells. Under appropriate culture conditions, BMSCs can also differentiate into neural cells transversely, and differentiate into different neurotransmitter types in different microenvironment or in vitro induced conditions. Therefore, BMSCs is expected to become a potential source. The ideal seed cells for stem cell transplantation in the treatment of nervous system diseases have broad clinical application prospects.
Astrocytes are the largest number of cells in the central nervous system, and are also the main components of the central nervous system microenvironment. It is found that astrocytes not only provide nutrition, support, protection, isolation, neuron development, axonal regeneration, synapse formation and signal transmission, and stem cell differentiation in neurons. And it also plays an important role.
The experimental results show that the inflammatory reaction is very obvious after the central nervous system is damaged or the neurodegenerative disease is occurring. The activation and secretion of a series of inflammatory cytokines from astrocytes is an important sign of this inflammatory reaction. The microenvironment formed by this inflammatory reaction is an inherent neural stem cell in the nerve fabric or the bone marrow base of the transplant. The effect of mesenchymal stem cells is still unknown.
In this study, the effect of activated astrocytes on the proliferation and differentiation of bone marrow stromal cells was studied by using a model of astrocyte inflammation established in vitro, and the mechanism of its action was preliminarily discussed.
Isolation, culture and in vitro expansion of bone marrow stromal cells
Because the cell composition of bone marrow is complex and the content of BMSCs in bone marrow is few, about 2~5 BMSCs in 10~5 bone marrow mononuclear cells is only contained, so the separation, culture, purification and in vitro amplification of BMSCs are difficult.
According to the characteristics of low density and easy adherent growth of BMSCs, the most of the hematopoietic cells were removed by the lymphocyte separation solution with density of 1.077g/ml, and then the hemopoietic cells were removed by adhesion culture, and the suspension growing hematopoietic cells were removed regularly, and the low sugar DMEM culture solution (including 10% FBS) was used to cultivate and expand. The BMSCs could be formed for about 7 days in the primary culture. The cell colonies were obvious; when the cells grew to 80% ~ 90% fusion, the cells were digested with 0.25% trypsin -0.02%EDTA and were cultured. When the cells were passed to the 3 generation, the cells were basically purified, and the morphology tended to be a long shuttle. Because of the lack of specific antigen markers of BMSCs, a variety of cell surface antigens were detected by flow cytometry in order to confirm BMSCs. The results showed that almost all the cultured cells showed positive surface antigen CD29 and CD90, indicating that the cultured cells were more pure BMSCs..
Two, the effect of conditioned medium of inflammatory astrocytes on proliferation and differentiation of BMSCS
On the basis of the successful culture of BMSCs, in order to simulate the effect of astrocyte cells on the proliferation and differentiation of BMSCs in the inflammatory microenvironment of the central nervous system, we first collected astrocyte supernatant after adding lipopolysaccharide (LPS) to culture 12,36,72h as an inflammatory astrocyte conditioned medium (astrocyte conditione). D medium, ACM, called 12h ACM, 36h ACM, 72h ACM), and collects the astrocyte supernatant without LPS as a normal astrocyte conditioned medium (normal ACM,), and then the conditioned medium is added to the cultured culture to observe the cell proliferation and neural differentiation.
The results of cell proliferation assay showed that the inflammatory astrocyte conditioned medium (12hACM, 36h ACM, 72h ACM) could significantly promote the proliferation of BMSCs, and the proliferation promoting effect of 36h ACM was significantly stronger than that of 12h and 72h ACM two. The cell differentiation rate was significantly higher. The percentage of neurons positive cells in 36h ACM group was significantly higher than that of 12h ACM and 72h ACM two groups, while the differentiation rate of neuroglia cells, 12h ACM, 36h ACM, 72h ACM three groups were not significantly different.
Three, conditioned medium of inflammatory astrocytes regulate BMSCs proliferation and differentiation.
The experimental results in the second part suggest that inflammatory astrocytes can promote the proliferation and nerve differentiation of BMSCs. This promotion of inflammatory astrocytes may be achieved through various factors secreted. This experiment focuses on interleukin -6 (IL-6). Previous experiments have proved that it is a multifunctional inflammatory cell. Factors, the expression of IL-6 in various central nervous system trauma or diseases is significantly increased and plays an important role in the process of nerve development and regeneration. In order to clarify the role of IL-6 in the process of BMSCs proliferation and nerve differentiation induced by inflammatory astrocytes, we first detected the synthesis and secretion of IL-6 in inflammatory astrocytes. The results of.ELISA detection showed that the content of IL-6 in the inflammatory astrocyte culture medium increased significantly, and the content of IL-6 in 36h ACM was significantly higher than that of 12h and 72h ACM, and RT-PCR results were in accordance with this phase. The trend of differentiation was also markedly weakened, but the differentiation of BMSCs to neurons was significantly increased, indicating that IL-6 could promote the proliferation of BMSCs and differentiate into astrocytes, and inhibit the differentiation of BMSCs into neurons.
Conclusion:
Inflammation activated astrocytes can obviously promote the proliferation and differentiation of bone marrow stromal cells to the nerve, and their action intensity is closely related to the time of action. Inflammatory cytokine IL-6 secreted by inflammatory astrocytes is involved in this process.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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