修回日期: 2007-12-26
接受日期: 2008-01-15
在线出版日期: 2008-02-08
目的: 探讨注射用血栓通对肝星状细胞LX02基因表达的影响及其机制.
方法: 将10-3 mg/L的注射用血栓通与人肝星状细胞LX02共同孵育48 h, 提取mRNA, 并逆转录成cDNA. 与芯片杂交, 根据杂交信号强弱筛选相关基因.
结果: 41条肝星状细胞基因差异表达, 其中表达上调的基因21条, 下调基因20条, 这些基因主要与能量代谢、细胞黏附、DNA结合、磷酸化、甲基化等相关, 还有部分基因功能不详.
结论: 经注射用血栓通作用后, 肝星状细胞基因表达发生变化, 其可能通过调控这些基因的表达实现了对肝星状细胞功能的抑制作用.
引文著录: 李国力, 魏红山, 邱爽, 曾辉. 注射用血栓通对肝星状细胞基因表达的影响. 世界华人消化杂志 2008; 16(4): 417-421
Revised: December 26, 2007
Accepted: January 15, 2008
Published online: February 8, 2008
AIM: To explore the effects of panax notoginseng saponins (PNS) on gene expression of cultured human hepatic stellate cells (HSCs) and its mechanism.
METHODS: PNS (10-3 mg/L) and HSCs (LX02 cells) were cultured for 48 h. Total RNA was isolated from HSCs, reverse transcrpted into cDNA and hybridized with GeneChip. The expression of mRNA was examined with the human GeneChip analysis system.
RESULTS: Differential expression was found in 41 genes of HSCs, of which, 20 were down-regulated and 21 up-regulated. Some differentially expressed genes were associated with metabolism, cell adherence, DNA binding phosphorylation and methylation. The function of some genes was unknown.
CONCLUSION: Panax notoginseng saponins can change the gene expression in HSCs by regulating their expression and inhibiting the function of HSCs.
- Citation: Li GL, Wei HS, Qiu S, Zeng H. Effect of panax notoginseng saponins on gene expression of cultured human hepatic stellate cells. Shijie Huaren Xiaohua Zazhi 2008; 16(4): 417-421
- URL: https://www.wjgnet.com/1009-3079/full/v16/i4/417.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v16.i4.417
三七总皂甙中的人参皂甙Rg1、Rb1和三七皂甙R1三种组分的含量达80%以上, 其具有较好的扩血管和改善微循环的作用. 近年来研究表明, 三七总皂甙对肝星状细胞生物功能具有抑制作用[1], 提示其对肝纤维化可能具有一定疗效. 注射用血栓通是用特殊工艺从三七主根中提取出三七总皂甙制成, 其特点是纯度高、生物活性高、水溶性好和毒性低. 我们以基因芯片技术对注射用血栓通作用的肝星状细胞LX02基因表达进行分析, 初步探讨注射用血栓通对肝星状细胞功能抑制的机制.
肝星状细胞株LX02为徐列明教授惠赠, 细胞培养相关试剂及总RNA提取试剂TRIzol均购自Invitrogen公司, 注射用血栓通为广西梧桐制药有限公司产品.
1.2.1 细胞培养: 在25 cm2培养瓶中常规培养LX02细胞, 细胞生长至对数期时分别将三七总皂甙及溶剂DMEM加入细胞培养液中, 使终浓度达到10-3 mg/L(根据MTT实验提示三七总皂甙在此浓度时对肝星状细胞增殖抑制最明显), 72 h后收获细胞约1×107, 按每5×106个细胞加入1 mL TRIzol试剂, 立即于液氮中保存.
1.2.2 总RNA提取和mRNA纯化: 使用TRIzol试剂一步法提取三七总皂甙和DMEM处理的LX02细胞总RNA(分别标记为实验组和对照组), 样品经分光光度计检测吸光度A值, 并行热稳定实验, 于-20 ℃和70 ℃保温1 h后, 经琼脂糖凝胶电泳检测28S、18S条带变化.
1.2.3 探针标记: 总RNA逆转录标记cDNA探针并纯化, Cy3-dUTP标记对照组细胞mRNA(5 μg), Cy5-dUTP标记实验组细胞mRNA(5 μg). 乙醇沉淀后溶解在20 μL 5×SSC+0.2% SDS杂交液中.
1.2.4 芯片制备: 芯片包含的4096个cDNA由上海博星基因芯片有限公司提供, 包括原癌基因和抑癌基因、免疫调节相关基因、细胞凋亡和应激反应蛋白相关基因、信号转导相关基因等. 以通用引物进行PCR扩增, PCR产物长度为1000-3000 bp. 靶基因以0.5 g/L溶解于3×SSC溶液中, 用Cartesian公司的Cartesian 7500点样仪及TeleChem公司的硅烷化玻片进行点样. 玻片经水合(2 h)、室温干燥(0.5 h), UV交联, 再分别用0.2% SDS、水及0.2%的硼氢化钠溶液处理10 min, 晾干备用.
1.2.5 杂交及洗涤: 将基因芯片和杂交探针在95 ℃水浴变性5 min, 将混合探针加在基因芯片上, 置于60 ℃杂交15-17 h. 依次以2×SSC+0.2% SDS、0.1%×SSC+0.2% SDS、0.1% SSC洗涤10 min, 室温晾干.
1.2.6 检测与分析: 用General Scanning公司的ScanArray3000扫描芯片. 为了监控芯片杂交技术体系的整个过程, 用预先选定的内参照基因(24条管家基因, 每个基因点2个点, 共48个点)对Cy3和Cy5的原始提取信号进行均衡和修正. 用ImaGene3.0软件分析Cy3、Cy5两种荧光信号的强度, 计算Cy5/Cy3比值. 阳性结果判断: Cy5/Cy3>2.0, 红色荧光, 显示表达增强; Cy5/Cy3<0.5, 为绿色荧光, 显示表达减弱; 黄色代表表达水平无差异.
总RNA的吸光度A260/A280>1.80, 热稳定实验70 ℃保温1 h与-20 ℃1 h电泳条带比较, 显示28 S条带无明显降解, 电泳结果证实已抽提高纯度的总RNA(图1). mRNA主要集中于900-4000 bp的连续条带.
实验组探针标记Cy5(红色), 对照组探针标记Cy3(绿色), 红绿颜色的差异就显示该基因在实验组和对照组中基因表达水平上的差异, 黄色代表表达水平无差异(图2, 图3).
本研究共筛选出41条差异表达的基因, 其中20条基因表达下调, 21条基因表达上调, 差异表达的基因中有6条基因功能未知(表1).
序号 | 编码基因 | Cy5/Cy3 |
1 | FOLH1: folate hydrolase (prostate-specific membrane antigen) 1 | 0.148 |
2 | Homo sapiens cDNA clone IMAGE: 4331862 | 0.274 |
3 | Homo sapiens cDNA clone IMAGE: 5767167 | 0.311 |
4 | COASY: Coenzyme A synthase | 0.327 |
5 | SLC1A6: solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6 | 0.332 |
6 | SUPT3H: suppressor of Ty 3 homolog | 0.342 |
7 | APEH: N-acylaminoacyl-peptide hydrolase | 0.350 |
8 | PECAM1: platelet/endothelial cell adhesion molecule (CD31 antigen) | 0.351 |
9 | INSIG1: insulin induced gene 1 | 0.352 |
10 | SHROOM2: shroom family member 2 | 0.360 |
11 | RUSC2: RUN and SH3 domain containing 2 | 0.362 |
12 | Homo sapiens cDNA clone UI-1-BC1p-asi-c-08-0-UI 3' | 0.364 |
13 | RBM14: RNA binding motif protein 14 | 0.365 |
14 | RAB5C: member RAS oncogene family | 0.368 |
15 | ZNF508: zinc finger protein 508 | 0.370 |
16 | TEX264: testis expressed 264 | 0.379 |
17 | Homo sapiens, clone IMAGE: 3914313 | 0.381 |
18 | ILK: integrin-linked kinase | 0.382 |
19 | HCN1: hyperpolarization activated cyclic nucleotide-gated potassium channel 1 | 0.385 |
20 | SNTA1: syntrophin, alpha 1 (dystrophin-associated protein A1, 59kDa, acidic component) | 0.387 |
21 | GLMN: glomulin, FKBP associated protein | 2.441 |
22 | ATPase, H+ transporting, lysosomal 56/58 kDa, V1 subunit B2 | 2.447 |
23 | MAN2B2: mannosidase, alpha, class 2B, member 2 | 2.453 |
24 | CFTS3: cleavage and polyadenylation factor subunit | 2.458 |
25 | TTC35: tetratricopeptide repeat domain 35 | 2.470 |
26 | PLCB4: phospholipase C, beta 4 | 2.480 |
27 | FOXN2: forkhead box N2 | 2.491 |
28 | MTR: 5-methyltetrahydrofolate-homocysteine methyltransferase | 2.529 |
29 | ICAM1: intercellular adhesion molecule 1 (CD54), human rhinovirus receptor | 2.561 |
30 | Homo sapiens cDNA clone UI-E-CK1-afi-a-04-0-UI | 2.599 |
31 | ATPase, H+ transporting, lysosomal accessory protein 2 | 2.606 |
32 | SEPT4: septin 4 | 2.622 |
33 | KCNJ10: potassium inwardly-rectifying channel, subfamily J, member 10 | 2.622 |
34 | CD302 molecule | 2.643 |
35 | ABHD2: abhydrolase domain containing 2 | 2.657 |
36 | phosphorylase, glycogen | 2.680 |
37 | MEGF9: multiple EGF-like-domains 9 | 2.700 |
38 | Homo sapiens cDNA clone IMAGE: 5755160 | 2.750 |
39 | ID1: inhibitor of DNA binding 2, dominant negative helix-loop-helix protein | 2.889 |
40 | CYP2J: cytochrome P450, family 2, subfamily J, polypeptide 2 | 2.932 |
41 | OPCML: opioid binding protein/cell adhesion molecule-like | 3.540 |
活化的肝星状细胞能大量增殖, 分泌大量胶原成分[2], 是肝纤维化、肝硬化时细胞外基质的主要来源和细胞学基础[4]. 魏红山et al在国内首次发现肝纤维化大鼠肝星状中血管紧张素Ⅰ的表达, 提示肾素-血管紧张素系统在主导循环系统疾病之外, 对肝纤维化的发病也有一定影响, 动物实验证明依那普利等循环系统疾病的治疗药对肝纤维化也具有较好的治疗作用[5].
近年来研究表明, 常用于循环系统疾病的中药提取物三七总皂甙在实验性肝损伤和肝纤维化中也具有一定的防治作用[6-7], 提示三七总皂甙在肝脏疾病方面具有一定的应用前景.
注射用血栓通是用特殊工艺从三七主根中提取出三七总皂甙制成, 其特点是纯度高、生物活性高、水溶性好. 注射用血栓通作用的LX02细胞共有41条基因表达发生差异变化, 其中21条基因表达上调, 20条基因表达下调. 表达下调的基因包括与合成代谢, 细胞增殖、抑制凋亡等信号传导相关的基因: FOLH1为叶酸吸收所必需[8]; COASY是辅酶A合成酶, 参与细胞的能量代谢和多种生物合成过程[9]; SLC1A6是谷氨酸和天冬氨酸的转运载体[10]; SUPT3H是STAGA乙酰基转移酶复合体的重要成分, STAGA与DNA复制纠错, 损伤修复相关[11], Myc蛋白在与靶基因启动子结合时需要STAGA的辅助[12]; APEH能够从肽段中水解末端酰基化的氨基酸, 该反应对水解活细胞中氧化损伤的蛋白十分重要, 与机体抗氧化损伤密切相关, 在多种肿瘤中都能够监测到此基因位点的缺失[13]; PECAM1即CD31, 研究显示CD31在某些肿瘤表面表达升高, 以往研究显示其可以抑制线粒体依赖的凋亡和Bax介导的凋亡[14]. 封闭肿瘤细胞表面的CD31能够诱导细胞凋亡, 从而抑制细胞增殖[15]; RAB5C属于RAS癌基因家族, 与细胞增殖及肿瘤形成密切相关[16]; ILK位于细胞增殖等信号传递的上游, 在TGF-β1介导的肝星状细胞增殖过程中非常重要[17]; HCN1能够介导肌细胞等细胞的兴奋性动作电位, 注射用血栓通作用后肝星状细胞表达该基因下调, 提示肝星状细胞活化表型减少[18]. 表达上调的基因中: OPCML: 最近研究发现其表达能抑制肿瘤生长、抑制细胞增殖[19]; CYP2J: 属于细胞色素P450家族, 参与药物和甾体类激素的代谢, 其表达升高有助于维持细胞稳定状态, 降低对激素的反应[20]; ID1具有抑制细胞表型转化的作用, 能拮抗TGF-β1促分化、增殖的作用[21]; SEPT4能够促进多种途径诱导的凋亡过程[22].
总之, 通过基因芯片筛选, 经注射用血栓通作用后肝星状细胞基因表达发生变化, 提示注射用血栓通可能通过调控这些基因的表达实现了其对肝星状细胞功能的抑制作用. 目前实验结果的验证工作正在进行, 这些基因在肝星状细胞中的具体生物功能还需进一步研究.
自从发现肝星状细胞表达血管受体Ⅰ及血管紧张素促肝纤维化发生的现象以来, 越来越多的研究表明肝纤维化和其他系统硬化性疾病有相似的发病机制, 由此本文研究了对缓解动脉硬化有良好效果的中成药注射用血栓通对肝纤维化也有一定疗效, 本实验旨在进一步研究注射用血栓通抗肝纤维化的分子机制.
潘兴华, 副主任医师, 成都军区昆明总医院病理实验科
2005年以来, 王文兵et al对三七总皂甙在肝纤维、肝损伤、诱导肝星状细胞凋亡等方面的研究作了相关的报道.
本文主要应用基因芯片技术初步筛选了中药成分三七总皂甙对肝星状细胞的基因表达.
三七总皂甙: 是从药用植物三七提取的化学混合物, 其成分中的活性成分人参皂甙Rg1、Rb1和三七皂甙R1三种组分的含量达80%以上, 其具有较好的扩血管和改善微循环的作用.
本文内容和撰写基本符合要求, 具有一定的科学价值.
编辑: 李军亮 电编: 吴鹏朕
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