肝星状细胞和枯否细胞在肝纤维化过程中的相互作用

发布时间：2018-07-31 10:37

【摘要】：背景：病毒、酒精、寄生虫等多种病因都可以导致肝脏纤维化。去除或控制病因可以从一定程度上控制疾病的进展，但部分肝纤维化持续存在。了解肝纤维化的机制是控制肝纤维化进展及逆转肝纤维化的关键。肝纤维化是肝脏损伤修复失衡的结果。在这个过程中，主要涉及两个重要的细胞：肝星状细胞和枯否细胞。肝星状细胞是产生细胞外基质的主要细胞，它的激活和凋亡是肝纤维化的进展和逆转的核心环节。枯否细胞是肝脏固有免疫的重要组成部分，在肝脏的损伤和修复过程中发挥重要作用。同时，二者都位于肝脏的窦周隙。二者从功能上、空间上对肝纤维化的作用使二者成为研究的焦点。目的： 1.研究健康和纤维化的人体肝脏内的炎症情况与纤维化情况，以及肝星状细胞的活化情况和枯否细胞的聚集情况； 2.研究诱导过程中的巨噬细胞和诱导成功后的巨噬细胞对肝星状细胞细胞系LX-2细胞激活的影响，以及LX-2细胞对两种不同的巨噬细胞的激活及功能的影响。材料与方法： 1.肝脏组织和肝脏灌洗液来自于行肝移植手术的供体和受体，其中，硬化肝脏来自行肝移植手术的受体，正常肝脏来自于行肝移植手术的供体。由于从人肝组织中获得星状细胞和枯否细胞的数量有限，我们用人肝星状细胞的细胞系（LX-2细胞）来代替肝星状细胞；用外周血分离出来的单核细胞诱导出的巨噬细胞来代替枯否细胞。实验中用到的外周血购自长春市中心血站。 2.采用实时定量聚合酶链反应来观察肝脏的炎症状况（IL-1β, IL-6, IL-10,IL-12, TNF-α）和纤维化（α-SMA, Col1A1, TIMP-1）状况；用免疫组织化学方法来研究肝星状细胞（α-SMA）和枯否细胞（CD68）在肝脏内的差异。 3.实验中采用单核-巨噬集落刺激因子和巨噬细胞集落刺激因子来分别诱导M1和M2型巨噬细胞。用流式细胞术检测巨噬细胞的表型，用实时定量聚合酶链反应和酶联免疫吸附实验检测细胞因子的表达和分泌。 4.肝脏内的枯否细胞部分是从外周血中补充而来，诱导过程中的巨噬细胞与LX-2细胞的相互作用则模拟这个过程。肝脏内的枯否细胞部分是驻扎在肝脏血窦的固有细胞，，它的表型随着肝脏内环境的改变而改变，诱导成功后的巨噬细胞与LX-2细胞的相互作用则模拟这个过程。我们分别用诱导过程中的巨噬细胞和诱导成功后的巨噬细胞与LX-2细胞按照1:5的比例进行共培养。用流式细胞术检测巨噬细胞的表型，用实时定量聚合酶链反应和酶联免疫吸附试验检测细胞因子的表达和分泌。结果： 1.在纤维化肝脏中，促炎性基因（IL-1β, IL-6, IL-12, TNF-α）表达增高，抗炎性基因（IL-10）表达降低，纤维化相关基因表达（α-SMA, Col1A1, TIMP-1）也增高。多元回归相关性分析表明，促炎性基因的表达与纤维化相关基因的表达呈正相关，抗炎性基因的表达与纤维化相关基因的表达呈负相关，但与调节细胞外基质降解的酶MMP-2没有相关性。此外，在纤维化肝脏中，激活的星状细胞的数量和聚集的枯否细胞的数量较正常肝脏要多。 2. M1型巨噬细胞呈油煎蛋样，可以高表达和分泌促炎性细胞因子IL-6,IL-12和TNF-α,抗炎性细胞因子IL-10较低；而M2型巨噬细胞呈长梭状，高表达和分泌抗炎性细胞因子IL-10，促炎性细胞因子IL-6, IL-12, TNF-α则相反。 3.诱导过程中的巨噬细胞，无论是向M1型巨噬细胞诱导，还是向M2型巨噬细胞诱导，都可以促进LX-2细胞表达纤维化相关基因（α-SMA, Col1A1,TIMP-1）和调节纤维化的基因（MMP-2, MMP-9），且二者没有明显差异。LX-2细胞可以影响巨噬细胞的极化过程，无论是向M1型巨噬细胞极化，还是向M2型巨噬细胞极化，都表现出相似的表型和功能。 4.诱导成功后的巨噬细胞，无论是向M1型诱导，还是M2型诱导，都可以促进LX-2细胞表达纤维化相关基因（α-SMA, Col1A1, TIMP-1）和调节纤维化的基因（MMP-2, MMP-9），但M1型巨噬细胞表现出的促纤维化能力更强。LX-2细胞可以影响巨噬细胞的极化过程，无论是向M1型巨噬细胞极化，或是向M2型巨噬细胞极化，分泌IL-10和TNF-α的能力，较正常诱导的M1型和M2型巨噬细胞要强，其中，M1型巨噬细胞分泌更多的TNF-α，M2型分泌更多的IL-10。结论： 1.纤维化的肝脏中，枯否细胞、活化的星状细胞增多，有更多的细胞外基质并呈现促炎性反应的状态；纤维化相关基因α-SMA、Col1A1的表达与促炎性基因的表达存在正相关性，而与抗炎性基因的表达存在负相关性； 2.不同类型的巨噬细胞对LX-2细胞都有激活活用，促进其纤维化相关基因的表达，LX-2细胞可以促进诱导成功后巨噬细胞的原有功能。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R575.2

【参考文献】