【摘要】：背景： 病毒、酒精、寄生蟲等多種病因都可以導(dǎo)致肝臟纖維化。去除或控制病因可以從一定程度上控制疾病的進(jìn)展，但部分肝纖維化持續(xù)存在。了解肝纖維化的機(jī)制是控制肝纖維化進(jìn)展及逆轉(zhuǎn)肝纖維化的關(guān)鍵。 肝纖維化是肝臟損傷修復(fù)失衡的結(jié)果。在這個(gè)過程中，主要涉及兩個(gè)重要的細(xì)胞：肝星狀細(xì)胞和枯否細(xì)胞。肝星狀細(xì)胞是產(chǎn)生細(xì)胞外基質(zhì)的主要細(xì)胞，它的激活和凋亡是肝纖維化的進(jìn)展和逆轉(zhuǎn)的核心環(huán)節(jié)�？莘窦�(xì)胞是肝臟固有免疫的重要組成部分，在肝臟的損傷和修復(fù)過程中發(fā)揮重要作用。同時(shí)，二者都位于肝臟的竇周隙。二者從功能上、空間上對(duì)肝纖維化的作用使二者成為研究的焦點(diǎn)。 目的： 1.研究健康和纖維化的人體肝臟內(nèi)的炎癥情況與纖維化情況，以及肝星狀細(xì)胞的活化情況和枯否細(xì)胞的聚集情況； 2.研究誘導(dǎo)過程中的巨噬細(xì)胞和誘導(dǎo)成功后的巨噬細(xì)胞對(duì)肝星狀細(xì)胞細(xì)胞系LX-2細(xì)胞激活的影響，以及LX-2細(xì)胞對(duì)兩種不同的巨噬細(xì)胞的激活及功能的影響。 材料與方法： 1.肝臟組織和肝臟灌洗液來自于行肝移植手術(shù)的供體和受體，其中，硬化肝臟來自行肝移植手術(shù)的受體，正常肝臟來自于行肝移植手術(shù)的供體。由于從人肝組織中獲得星狀細(xì)胞和枯否細(xì)胞的數(shù)量有限，我們用人肝星狀細(xì)胞的細(xì)胞系（LX-2細(xì)胞）來代替肝星狀細(xì)胞；用外周血分離出來的單核細(xì)胞誘導(dǎo)出的巨噬細(xì)胞來代替枯否細(xì)胞。實(shí)驗(yàn)中用到的外周血購自長春市中心血站。 2.采用實(shí)時(shí)定量聚合酶鏈反應(yīng)來觀察肝臟的炎癥狀況（IL-1β, IL-6, IL-10,IL-12, TNF-α）和纖維化（α-SMA, Col1A1, TIMP-1）狀況；用免疫組織化學(xué)方法來研究肝星狀細(xì)胞（α-SMA）和枯否細(xì)胞（CD68）在肝臟內(nèi)的差異。 3.實(shí)驗(yàn)中采用單核-巨噬集落刺激因子和巨噬細(xì)胞集落刺激因子來分別誘導(dǎo)M1和M2型巨噬細(xì)胞。用流式細(xì)胞術(shù)檢測巨噬細(xì)胞的表型，用實(shí)時(shí)定量聚合酶鏈反應(yīng)和酶聯(lián)免疫吸附實(shí)驗(yàn)檢測細(xì)胞因子的表達(dá)和分泌。 4.肝臟內(nèi)的枯否細(xì)胞部分是從外周血中補(bǔ)充而來，誘導(dǎo)過程中的巨噬細(xì)胞與LX-2細(xì)胞的相互作用則模擬這個(gè)過程。肝臟內(nèi)的枯否細(xì)胞部分是駐扎在肝臟血竇的固有細(xì)胞，，它的表型隨著肝臟內(nèi)環(huán)境的改變而改變，誘導(dǎo)成功后的巨噬細(xì)胞與LX-2細(xì)胞的相互作用則模擬這個(gè)過程。我們分別用誘導(dǎo)過程中的巨噬細(xì)胞和誘導(dǎo)成功后的巨噬細(xì)胞與LX-2細(xì)胞按照1:5的比例進(jìn)行共培養(yǎng)。用流式細(xì)胞術(shù)檢測巨噬細(xì)胞的表型，用實(shí)時(shí)定量聚合酶鏈反應(yīng)和酶聯(lián)免疫吸附試驗(yàn)檢測細(xì)胞因子的表達(dá)和分泌。 結(jié)果： 1.在纖維化肝臟中，促炎性基因（IL-1β, IL-6, IL-12, TNF-α）表達(dá)增高，抗炎性基因（IL-10）表達(dá)降低，纖維化相關(guān)基因表達(dá)（α-SMA, Col1A1, TIMP-1）也增高。多元回歸相關(guān)性分析表明，促炎性基因的表達(dá)與纖維化相關(guān)基因的表達(dá)呈正相關(guān)，抗炎性基因的表達(dá)與纖維化相關(guān)基因的表達(dá)呈負(fù)相關(guān)，但與調(diào)節(jié)細(xì)胞外基質(zhì)降解的酶MMP-2沒有相關(guān)性。此外，在纖維化肝臟中，激活的星狀細(xì)胞的數(shù)量和聚集的枯否細(xì)胞的數(shù)量較正常肝臟要多。 2. M1型巨噬細(xì)胞呈油煎蛋樣，可以高表達(dá)和分泌促炎性細(xì)胞因子IL-6,IL-12和TNF-α,抗炎性細(xì)胞因子IL-10較低；而M2型巨噬細(xì)胞呈長梭狀，高表達(dá)和分泌抗炎性細(xì)胞因子IL-10，促炎性細(xì)胞因子IL-6, IL-12, TNF-α則相反。 3.誘導(dǎo)過程中的巨噬細(xì)胞，無論是向M1型巨噬細(xì)胞誘導(dǎo)，還是向M2型巨噬細(xì)胞誘導(dǎo)，都可以促進(jìn)LX-2細(xì)胞表達(dá)纖維化相關(guān)基因（α-SMA, Col1A1,TIMP-1）和調(diào)節(jié)纖維化的基因（MMP-2, MMP-9），且二者沒有明顯差異。LX-2細(xì)胞可以影響巨噬細(xì)胞的極化過程，無論是向M1型巨噬細(xì)胞極化，還是向M2型巨噬細(xì)胞極化，都表現(xiàn)出相似的表型和功能。 4.誘導(dǎo)成功后的巨噬細(xì)胞，無論是向M1型誘導(dǎo)，還是M2型誘導(dǎo)，都可以促進(jìn)LX-2細(xì)胞表達(dá)纖維化相關(guān)基因（α-SMA, Col1A1, TIMP-1）和調(diào)節(jié)纖維化的基因（MMP-2, MMP-9），但M1型巨噬細(xì)胞表現(xiàn)出的促纖維化能力更強(qiáng)。LX-2細(xì)胞可以影響巨噬細(xì)胞的極化過程，無論是向M1型巨噬細(xì)胞極化，或是向M2型巨噬細(xì)胞極化，分泌IL-10和TNF-α的能力，較正常誘導(dǎo)的M1型和M2型巨噬細(xì)胞要強(qiáng)，其中，M1型巨噬細(xì)胞分泌更多的TNF-α，M2型分泌更多的IL-10。 結(jié)論： 1.纖維化的肝臟中，枯否細(xì)胞、活化的星狀細(xì)胞增多，有更多的細(xì)胞外基質(zhì)并呈現(xiàn)促炎性反應(yīng)的狀態(tài)；纖維化相關(guān)基因α-SMA、Col1A1的表達(dá)與促炎性基因的表達(dá)存在正相關(guān)性，而與抗炎性基因的表達(dá)存在負(fù)相關(guān)性； 2.不同類型的巨噬細(xì)胞對(duì)LX-2細(xì)胞都有激活活用，促進(jìn)其纖維化相關(guān)基因的表達(dá)，LX-2細(xì)胞可以促進(jìn)誘導(dǎo)成功后巨噬細(xì)胞的原有功能。
[Abstract]:Background:
A variety of causes such as virus, alcohol, and parasite can cause liver fibrosis. Removal or control of the cause can control the progress of the disease to a certain extent, but some liver fibrosis persists. Understanding the mechanism of liver fibrosis is the key to control the progress of liver fibrosis and reverse the liver fibrosis.
Liver fibrosis is the result of the imbalance of the repair of liver injury. In this process, it mainly involves two important cells: hepatic stellate cells and Kupffer cells. Hepatic stellate cells are the main cells that produce extracellular matrix, its activation and apoptosis are the progress of liver fibrosis and the reversal of nuclear heart. Kupffer cells are the weight of liver inherent immunity. The part, which plays an important role in liver damage and repair, is located in the peri sinus of the liver. The function of the two is the focus of the two research on the function of the liver and the role of the two in the liver fibrosis.
Objective:
1. To study the inflammation and fibrosis in healthy and fibrotic human liver, as well as the activation of hepatic stellate cells and Kupffer cell aggregation.
2. the effect of macrophages and induced macrophages on the activation of LX-2 cells in the hepatic stellate cell line and the effect of LX-2 cells on the activation and function of two different macrophages in the induction process.
Materials and methods:
1. the liver tissue and liver lavage fluid from the donor and receptor of the liver transplantation is derived from the recipient of the liver transplantation, and the liver is derived from the recipient of the liver transplantation, and the normal liver is derived from the donor of the liver transplantation. The cell line of human hepatic stellate cells (LX-2) is used because the number of stellate and Kupffer cells from the human liver tissue is limited. Cells instead of hepatic stellate cells; macrophages induced by mononuclear cells isolated from peripheral blood to replace Kupffer cells. The peripheral blood used in the experiment was purchased from the central Changchun blood station.
2. the liver inflammatory conditions (IL-1 beta, IL-6, IL-10, IL-12, TNF- alpha) and fibrosis (alpha -SMA, Col1A1, TIMP-1) were observed by real-time quantitative polymerase chain reaction (PCR), and the difference between hepatic stellate cells (alpha -SMA) and Kupffer cells (CD68) in the liver was studied by immunohistochemistry.
3. the mononuclear macrophage colony stimulating factor and macrophage colony stimulating factor were used to induce M1 and M2 macrophages respectively. The phenotype of macrophages was detected by flow cytometry, and the expression and secretion of cytokines were detected by real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay.
4. the Kupffer cells in the liver are partly supplemented from the peripheral blood, and the interaction of macrophages and LX-2 cells in the induction process simulates this process. The Kupffer cells in the liver are the inherent cells in the hepatic sinusoids, whose phenotype changes with the changes in the liver environment and induces the successful macrophages and L The interaction of X-2 cells simulated the process. We co cultured the macrophages in the induction process and the macrophages induced by the induction and the LX-2 cells according to the proportion of 1:5. The phenotype of macrophages was detected by flow cytometry, and the cytokine was detected by real time quantitative polymerase chain reaction and ELISA. Expression and secretion.
Result:
1. in fibrotic liver, the expression of pro-inflammatory genes (IL-1, IL-6, IL-12, TNF- a) is higher, the expression of anti-inflammatory gene (IL-10) is reduced, and the expression of fibrosis related genes (alpha -SMA, Col1A1, TIMP-1) is also increased. Multiple regression correlation analysis shows that the expression of pro-inflammatory genes is positively related to the expression of fibrosis related genes, and the anti inflammatory genes are related. Expression has a negative correlation with the expression of fibrosis related genes, but has no correlation with the enzyme MMP-2 that regulates the degradation of extracellular matrix. In addition, the number of activated stellate cells and the number of Kupffer cells gathered in the fibrotic liver are more than those of the normal liver.
2. M1 type macrophages are fried eggs, which can express and secrete proinflammatory cytokines IL-6, IL-12 and TNF- alpha, and lower anti-inflammatory cytokines IL-10, while M2 macrophages are spindle shaped, high expression and secretion of anti-inflammatory cytokines IL-10, and proinflammatory cytokines IL-6, IL-12, TNF- a are the opposite.
3. the macrophages in the induction process, whether induced by M1 type macrophages or induced by M2 type macrophages, can promote LX-2 cells to express fibrosis related genes (alpha -SMA, Col1A1, TIMP-1) and the gene regulating fibrosis (MMP-2, MMP-9), and there is no significant difference between the two and the.LX-2 cells that can affect the polarization process of macrophages. Whether they are polarized to M1 macrophages or polarized to M2 macrophages, they exhibit similar phenotypes and functions.
4. induced macrophages, whether induced by M1 type or M2 type, can promote LX-2 cells to express fibrosis related genes (alpha -SMA, Col1A1, TIMP-1) and regulation of fibrosis genes (MMP-2, MMP-9), but M1 type macrophages show a stronger fibrinolytic activity and.LX-2 cells can affect the polarization process of macrophages. Whether it is polarized to M1 type macrophages or polarization of M2 type macrophages, the ability to secrete IL-10 and TNF- alpha is stronger than normal induced M1 and M2 type macrophages, of which, M1 type macrophages secrete more TNF- A and M2 type more IL-10..
Conclusion:
1. in the liver of fibrosis, Kupffer cells, activated stellate cells increase, and there are more extracellular matrix and proinflammatory response. The expression of fibrosis related gene alpha -SMA, Col1A1 is positively correlated with the expression of pro-inflammatory genes, but has negative correlation with the expression of anti inflammatory genes.
2. different types of macrophages are activated to activate LX-2 cells and promote the expression of their fibrosis related genes. LX-2 cells can promote the original function of macrophages after the induction of success.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R575.2

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