肝星状细胞(HSCs)在肝纤维化的发生发展中起主导作用。激活的HSCs增殖并转化为肌成纤维细胞，表达α-平滑肌肌动蛋白(α-SMA)，合成、分泌以Ⅰ、Ⅲ型胶原为主的细胞外基质(ECM)的功能增强，同时胞浆中丰富的维生素A脂滴减少甚至消失，维甲酸(RA)水平也急剧下降，提示促脂肪细胞分化调控及维甲酸信号通路可能与维持HSCs的静息状态有关。维甲酸核内受体RXRα在核受体信号通路中具有核心作用，是促进细胞分化和抑制细胞增殖的重要调控因子，既可形成同二聚体，又是其它多种核受体发挥作用所必需形成的异二聚体中的配偶体。过氧化物酶体增生因子激活受体γ (PPARγ)是核内固醇类激素受体PPARs家族的一个亚型，可通过与RXRα结合形成异二聚体与特殊的PPAR反应元件(PPRE)结合调控多种促进细胞分化和脂质代谢基因转录。国外研究及本课题组前期的工作已证实RXRα和PPARγ基因表达下调及相伴随的RA信号转导削弱是HSCs激活的重要内在分子机制。通过质粒转染上调RXRα或PPARγ均可部分逆转激活HSCs的表型，调控RXRα及的PPARγ表达有助于肝纤维化的治疗。 基因表达调控的途径主要包括：①转录水平，DNA→mRNA，②转录后mRNA的降解，③翻译水平，mRNA→蛋白，④翻译后，蛋白的降解，泛素-蛋白酶体途径。至今转录后mRNA的降解及mRNA→蛋白质翻译水平调控的研究相对较少。MicroRNAs(miRNAs)是一类～22nt的内源性单链RNA分子，进化上高度保守，表达具有时空特异性。成熟miRNAs参与形成RISC，并通过互补序列识别靶mRNA，与其3’端非翻译区(UTR)结合，诱导转录后基因沉默。MiRNAs的发现为基因转录后水平调控的研究提供了全新的概念。理解MiRNAs在一些复杂疾病基本病理过程中的作用，有助于疾病的诊断、预后判断，并为治疗提供新的靶点。本课题选取与RXRα和PPARγ相关的miRNAs作为研究对象，旨在寻找与HSCs去脂肪细胞分化激活有关的miRNAs。 第一部分 大鼠原代HSCs激活与去脂肪细胞分化 目的：建立稳定可靠的大鼠HSCs原代分离培养技术，并验证原代HSCs体外培养从静止到激活，去脂肪细胞分化的全过程，检测HSCs激活过程中RXRα和PPARγ在mRNA和蛋白水平的表达，为后续实验建立基础。 方法：分离培养大鼠原代HSCs；通过UV，FITC激发波长观察HSCs胞浆中维生素A脂滴的自发荧光，SudanⅢ脂肪染色显示胞浆中的脂滴，免疫荧光和western blot检测desmin，α-SMA，collagen Ⅰ进行鉴定；荧光实时定量-PCR和western blot检测HSCs激活过程中RXRα和PPARγ表达变化； 结果：成功分离培养大鼠原代HSCs，细胞纯度高于90%。D3HSCs胞浆富含维A脂滴，处于静止状态；D10HSCs脂滴大部分丢失，表达α-SMA和collagen Ⅰ，处于激活状态。激活HSCs中RXRα和PPARγmRNA表达减低(分别为47.19%，**P＜ 0.01； 65.81%，**P＜0.01)，蛋白表达下降(分别为84.10%，**P＜0.01；73.03%， *P＜0.05)。 第二部分 大鼠原代HSCs激活过程中RXRα和PPARγ相关MicroRNAs的表达变化 目的：探索在HSCs激活过程中与RXRα和PPARγ相关的miRNAs表达是否发生了变化，并筛选出变化显著的miRNAs。 方法：根据Sanger miRBase target version 2.0和 Targetscan 3.1 选取与RXRα和PPARγ3’UTR同源，进化上高度保守的miRNAs，并通过荧光实时定量-PCR检测D3HSCs和D10HSCs中相关的miRNAs，以表达增高大于1.5倍或减低大于0.5倍为变化显著。 结果：建立了荧光实时定量-PCR检测miRNAs及pre-miRNAs的方法。经荧光实时定量-PCR筛选出与RXRα和PPARγ相关，在HSCs激活过程中变化显著的miRNAs15种，其中增高的9种，减低的6种。MiR-27a和miR-27b在激活的HSCs 中大量表达,增高显著(分别为2.49倍，P＜0.05和2.98倍，P＜0.05)，根据Targetscan 3.1数据库，RXRα和PPARγ的3’UTR均存在高度保守的miR-27互补结合位点，故将miR-27a和miR-27b作为进一步研究的对象。 第三部分 Anti-miR-27a和Anti-miR-27b对P2HSCs激活状态的影响 目的：观察anti-miR-27a，27b对P2HSCs细胞表型及增殖的影响和对RXRα和PPARγ表达的影响。 方法：采用脂质体基因转染技术将anti-miR-27a， 27b导入P2HSCs，SudanⅢ脂肪染色观察胞浆内脂滴形成，BrdU掺入法检测P2HSCs的增殖能力，并通过荧光实时定量-PCR检测和western blot检测α-SMA，collagen Ⅰ， RXRα和PPARγ的表达。 结果：Anti-miR-27a和anti-miR_27b共转染P2HSCs，可部分逆转激活P2HSCs的表型，胞浆中消失的脂滴重新出现，且SudanⅢ染色阳性细胞与anti-miR的浓度呈正相关(r=0.999，P＜0.05)； BrdU阳性细胞减少74.10%(*P＜0.05)， P2HSCs细胞增殖受到抑制；但collagen Ⅰ和α-SMA的表达无明显变化。Anti-miR-27a和anti-miR-27b共转染可在mRNA和蛋白水平增加P2HSCs中RXRα的表达(分别为1.69倍，**P＜ 0.01，2.30倍，**P＜0.01)，但对PPARγ的表达无显著影响。Anti-miR-27a和anti-miR-27b单独分别转染无上述效应。 第四部分 MicroRNA-27a，MicroRNA-27b 与 RXRα3’UTR 的相互作用 目的：通过荧光素酶报告系统进一步证实miR-27a和27b确实可以与RXRα mRNA 3’UTR的miR-27识别位点结合，并抑制其表达。 方法：psiCHECK-2/RXRα3’UTR报告质粒和pcDNA6.2-GW/EmGFP-mir-27a 或pcDNA6.2-GW/EmGFP-mir-27b表达质粒共转染293T细胞，检测Renilla荧光素酶的活性。 结果：Veritas Microplate Luminometer检测miR-27a和miR-27b转染组Renilla 荧光素酶的活性分别减低39.65%(**P＜0.01)和49.35%(**P＜ 0.01)，证明miR-27a和miR-27b可通过与RXRαmRNA的3’UTR相互作用抑制RXRα的表达。 结论 体外原代培养自动激活的大鼠HSCs中miR-27a和miR-27b表达增加，通过anti-miR抑制miR-27a和miR-27b可部分逆转激活HSCs的表型，胞浆中消失的脂滴重新出现，细胞增殖受到抑制。MiR-27a和miR-27b可能部分通过抑制靶基因RXRα的表达而非PPARγ的表达，参与了HSCs去脂肪细胞分化激活的过程。 关键词：miR-27a，miR-27b，肝星状细胞，RXRα，PPARγ，肝纤维化，脂肪形成，增殖 中图分类号：R364.3+2，R657.3+1，Q254，Q952.1

 Introduction Hepatic stellate cells (HSCs) play an essential role in the formation of liver fibrosis. During acute and chronic liver injury, HSCs are activated and transdiffemtiate into proliferative, proinflammatory, fibrogenic myofibroblastlike cells, begin to express alpha-smooth muscle actin (α-SMA) and excrete more extra cellular matrix mainly composed of collagen Ⅰ. At the same time, the cytoplasimc vitamin A riched lipid droplets reduced accompanied with diminished retinoic acid (RA) content in activated HSCs, suggesting the importance of adipogenic transcriptional regulator and retinoic acid signaling pathway in the maintenance of the quiescent HSCs phenotype. RA is a well identified regulator in cell differentiation and proliferation. Two families of RA receptors are known: the retinoic acid nuclear receptors (RARα, β and γ) and the retiniod X receptors (RXRα, β and γ). RXRα can form homodimers or serve as obligate heterodimeric partners for the other members of the steroid/thyroid hormone receptor superfamily such as vitamin D receptor, peroxisome proliferator- activated receptor (PPAR) and liver X receptor (LXR) and play a central role in nuclear receptor signaling pathway. RXRα: PPARγ heterodimers bind to peroxisome proliferator response elements (PPREs) to control the transcription of a number of genes involved in cell differentiation and lipid metabolism. Transfection of RXRα or PPARγ expression vector can revert characteristic biochemical and morphological changes of culture activated HSCs, suggesting the importance of RXRα and PPARγ in the maintenance of the quiescent HSCs phenotype. Efficient regulation of the expression of RXRα and PPARγ may be of therapeutic benefit for liver fibrosis. There are four levels in the regulation of gene expression: 1. transcription level, DNA→ mRNA; 2. post-transcription level, mRNA degradation; 3. translation level, mRNA→ protein; 4. post-translation level, protein degradation. Till now, the mechanism underlying mRNAs degradation and the regulation from mRNAs to protein are largely unknow. MicroRNAs (miRNAs) are a newly identified class of endogenous small (～22 nt) regulatory RNAs, they are evolutionarily conserved, exhibit tissue-specific or developmental-stage-specific expression, and play essential roles in regulating cell differentiation, proliferation, and apoptosis. Coupling with RNA induced silencing complex (RISC), miRNAs can bind to the complementary sequence in the 3' untranslated region of target mRNAs and cause either mRNAs degradation or translational suppression resulting in gene suppression at a post-transcriptional level. Identification of abnormally expressed miRNAs in pathologic state would be helpful to further understand the mechanism of disease and modulation of their activity may be of therapeutic benefit. We started from the miRNAs related to either RXRα or PPARγ and tried to find out the miRNAs that involved in HSCs activation. Part Ⅰ The Activation and Trans-differentiation of Primary Cultured Rat HSCs Aims: Set up a stable and reliable technique for isolation and culture of rat HSCs; verify the trans-differentiation of primary cultured rat HSCs; examine the expression of RXRα and PPARγ during HSCs activation. Methods: Isolation and culture of rat HSCs in vitro; the cytoplasmic vitamin A riched lipid droplets are observed under UV and FITC fields and by Sudan Ⅲ staining; desmin, α-SMA and collagen Ⅰ are detected by immunofluorescence and western blot; RXRα and PPARγ are assessed by real-time PCR and western blots. Results: Primary HSCs were isolated from SD rats, the purities were higher than 90%. At day 10 since seeding, primary cultured HSCs activated, cytoplasmic lipid droplets decreased and began to express α-SMA and collagen Ⅰ. The expression of RXRα and PPARγ mRNA dropped by 47.19%(**P＜0.01), and 65.81%(**P＜0.01) individually in activated HSCs, the expression of protein declined by 84.10%(**P < 0.01) and 73.03%( *P＜0.05) respectively. Part Ⅱ The Expression of MicroRNAs Related to RXRα and PPARγ During HSCs Activation Aims: To explore if there is a change in the expression of miRNAs related to RXRα and PPARγ during HSCs activation, and to identify the significantly altered miRNAs. Methods: MiRNAs that predicted to target either RXRα or PPARγ and conserved across various species were selected according to Sanger miRBase Targets version 2.0 and Targetscan3.1. The expression of mature miRNAs related to RXRα and PPARγ were evaluated in quiescent (D3 in culture) and activated HSCs (D10 in culture) by real-time PCR. The miRNAs that increased＞ 1.5folds or decreased＞0.5folds were regarded as significantly changed. Results: Among the 31 miRNAs finally detected, 9 were significantly up regulated and 6 were down regulated in activated HSCs. MiR-27a and miR-27b were chosen for further investigation, because they are abundantly expressed and significantly up regulated (increased 2.49 folds, P＜0.05 and 2.98 folds, P＜0.05 respectively) in activated HSCs. and are predicted to target both RXRα and PPARγ. Part Ⅲ The Effects of Anti-miR-27a and Anti-miR-27b on HSCs Activation Aims: To evaluate the effects of anti-miR-27a and 27b on the phenotype and proliferation of activated HSCs, and their effects on the expression of RXRα and PPARγ. Methods: Anti-miR-27a and 27b were transiently transfected into passage two HSCs, the cytoplasmic lipid droplets were visualized by sudanⅢ staining, cell proliferation rate were evaluated by BrdU incorporation assay, the expression of α-SMA, collagen Ⅰ,RXRα and PPARγ were determined by real-time PCR and western blot. Results: Co-transfection of anti-miR-27a and 27b can partly reverse the phenotype of activated HSCs, in some P2HSCs lipid droplets reappeared and Sudan Ⅲ positive-staining cells were correlated to the concentration of anti-miR-27a and 27b(r=0.999, P＜0.05), implying the dose dependent effect. BrdU incorporation assay showed that the labeling index (LI) was decreased by 74.1% in HSCs co-transfected with anti-miR-27a and 27b (*P＜0.05). RXRα was up regulated by 1.69 folds (**P＜ 0.01) at mRNA level and 2.3 folds (**P＜0.01) at protein level in the HSCs co-transfected by anti-miR-27a and 27b. The expression of PPARγ, α-SMA and collagen Ⅰ were not affected. Part Ⅳ The Interaction Between MicroRNA-27a, MicroRNA-27b and RXRα 3'UTR Aims: To comfirm that miR-27a and miR-27b can bind the 3'UTR of RXRα and inhibit its translation. Methods: Luciferase assays were performed in 293T cells that co-transfected by psiCHECK-2/ RXRα 3'UTR reporter vector with either pcDNA6.2-GW/EmGFP-mir-27a or pcDNA6.2-GW/EmGFP-mir-27b miRNA expression vectors. Results: Renilla luciferase activity was decreased by 39.65% (**P＜0.01) in cells co-transfected with RXRα 3'UTR reporter vector and miR-27a expression vector and by 49.35 (**P＜0.01) in cells co-transfected with RXRα 3'UTR reporter vector and miR-27b expression vector respectively. These facts suggest that miR-27a and miR-27b can bind and interact with the 3'UTR of RXRα and inhibit translation of the chimeric transcript. Conclusions 1.MiR-27a and miR-27b are both up-regulated and abundantly expressed in primary culture activated HSCs, 2. Co-transfection of anti-miR-27a and anti-miR-27b can partly inverse the phenotype of activated HSCs: the disappeared lipid droplets emerged again and proliferation was repressed, but the expression of collagen Ⅰ and α-SMA were not changed. 3. Co-transfection of anti-miR-27a and 27b can increased the RXRα expression at both mRNA and protein level in activated HSCs, but have no effect on PPARγ. So the effect of miR-27a and miR-27b on HSCs activation is partially caused by the down regulation of RXRα, some other target genes could also participate in the course, and need further investigation. Keywords: miR-27a, miR-27b, HSCs, RXRα, PPARγ, liver fibrosis, adipogenesis, proliferation Chinese Library Classification Number: R364.3+2, R657.3+1, Q254, Q952.1