CN105944086B - Il-8 alone or combined with other cytokines in the treatment of liver fibrosis in a mammal - Google Patents

Il-8 alone or combined with other cytokines in the treatment of liver fibrosis in a mammal Download PDF

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CN105944086B
CN105944086B CN201610414306.0A CN201610414306A CN105944086B CN 105944086 B CN105944086 B CN 105944086B CN 201610414306 A CN201610414306 A CN 201610414306A CN 105944086 B CN105944086 B CN 105944086B
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余艳春
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浙江生创精准医疗科技有限公司
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Abstract

本发明涉及白介素‑8(IL‑8)单独或与其他细胞因子联合在治疗肝纤维化中的用途。 The present invention relates to interleukin -8 (IL-8) alone or combined with other cytokines in the treatment of liver fibrosis. 白介素‑8(IL‑8)单独或与其他细胞因子联合能够改善肝纤维化状况。 Interleukin -8 (IL-8) alone or combined with other cytokines can improve the health of hepatic fibrosis. 本发明更好地治愈肝纤维化或为治疗肝纤维化提供另一种可行的选择,提高患者的预后或者生活质量。 The present invention better cure for liver fibrosis or provide another viable option for the treatment of liver fibrosis and improve the prognosis or quality of life of patients.

Description

IL-8单独或与其他细胞因子联合在治疗肝纤维化中的用途 IL-8 alone or combined with other cytokines in the treatment of liver fibrosis in a mammal

技术领域 FIELD

[0001] 本发明属于生物制药领域,具体涉及白介素-8 (IL-8)单独或与其他细胞因子联合在治疗肝纤维化中的用途。 [0001] The present invention belongs to the biological pharmacy field, particularly relates to interleukin -8 (IL-8) alone or combined with other cytokines in the treatment of liver fibrosis.

背景技术 Background technique

[0002] 肝脏疾病严重影响人类的健康和生活质量,是人类面临的重大传染性疾病之一。 [0002] liver disease, a serious impact on human health and quality of life, is one of the major infectious diseases facing humanity. 每年中国有数以万计的患者死于各类肝病,因其并发症较多且死亡率较高,肝病的治疗引起了全世界的广泛关注和研究。 China has tens of thousands of patients each year die from all types of liver disease, because of its more complications and a higher mortality rate, treatment of liver disease caused widespread concern and research worldwide. 肝病主要包括肝损伤,爆发性肝衰竭,肝纤维化,肝硬化,肝细胞癌,胆汁淤积性肝,自身免疫性肝病,酒精性脂肪肝和非酒精性脂肪肝等。 Liver diseases including liver damage, fulminant hepatic failure, liver fibrosis, cirrhosis, hepatocellular carcinoma, cholestatic liver, autoimmune liver disease, fatty liver, alcoholic and non-alcoholic fatty liver. 肝纤维化是一种应对各种各样急/慢性刺激物(包括酒精,病毒感染,药物,毒素,胆汁和代谢物等)的伤口损伤反应,其是各种各样慢性肝损伤的必经之路和共同结果。 Liver fibrosis is a response to a variety of acute and wound damage response / chronic irritants (including alcohol, virus infections, drugs, toxins, bile and metabolites, etc.), which is a wide variety of chronic liver injury must pass through Road and a common result. 伴随着刺激物的持续损伤, 导致重新构建肝功能的速度跟不上胶原纤维合成和积累的速度,引起了细胞外基质(ECM, extracellular matrix)的积累,因过度地积累而产生持续的生理和病理变化。 With the continuing damage stimulus, resulting in re-construction of the velocity of liver function can not keep collagen synthesis and accumulation speed, causing the accumulation of extracellular matrix (ECM, extracellular matrix), because the accumulation of excessively generated and sustained physiological changes in pathology. 这些变化主要包括炎症细胞的募集,肝细胞的坏死,内皮屏障的损伤,肌成纤维细胞的激活和最终疤痕的形成。 These changes include the recruitment of inflammatory cells, and necrosis, endothelial barrier damage hepatocytes, and muscle activation to form the final scar fibroblasts. 持续的肝纤维化将破坏正常的肝脏结构,形成许多的肝小结,改变血液循环,最终引发肝硬化甚至演变成肝细胞癌。 Ongoing liver fibrosis will disrupt normal liver structure, the formation of many of liver nodules, changes in blood circulation, and ultimately lead to cirrhosis or hepatocellular carcinoma evolved into.

[0003] 正常的肝实质包含上皮细胞(肝细胞)和非实质细胞(网状内皮细胞,肝星状细胞和Kupffer细胞)。 [0003] normal liver parenchyma containing epithelial cells (hepatocytes) and non-parenchymal cells (reticuloendothelial cells, hepatic stellate cells and Kupffer cells). 正常的肝脏结构:血窦从肝细胞中以低密度的基底膜样的基质固定于Disse内,这样可以增加新陈代谢的物质和营养交换。 Normal liver structure: sinusoids to hepatocytes from low density basement membrane-like substrate is fixed to the Disse, which can increase the nutritional and metabolic exchange material. 当发生损伤时,肝星状细胞被激活,并分泌大量的ECM,导致隔膜明显增厚。 When damage occurs, hepatic stellate cells are activated and secrete large amounts of the ECM, leading to membrane thickening. 纤维化的肝脏结构:损伤达到一定的程度,ECM大量聚集在Disse内,导致内皮细胞窗孔样结构和肝脏微绒毛损失,而且门静脉血流量和肝细胞新陈代谢交换发生损害,并引起门静脉血压过高。 Liver Fibrosis structure: damage reaches a certain level, ECM large number gathered in the Disse, leading to endothelial cell fenestrae microvilli-like structure and liver damage, liver and portal blood flow and cell metabolism swap damage occurred, and cause high blood pressure portal .

[0004] 当前治疗肝纤维化最有效的方法是原位肝移植(0LT,orthotopic I iver transplantation),但是因肝脏捐献者的严重不足,使得OLT的应用受到了极大的限制,因此新的治疗手段是迫切解决肝脏供体短缺的一种方式。 [0004] The current liver fibrosis is the most effective treatment for orthotopic liver transplantation (0LT, orthotopic I iver transplantation), but because of a serious shortage of liver donors, making use of OLT has been greatly restricted, so the new treatment means is a way to solve urgent liver donor shortage.

[0005] 干细胞生物学已经变成了最热门的生物医学研究领域之一。 [0005] stem cell biology has become one of the hottest areas of biomedical research of. MSCs作为一类主要的成体干细胞,许多的临床前实验和临床试验关注MSCs在疾病方面的研究价值和应用前景。 MSCs as a major class of adult stem cells, many of preclinical and clinical trials focus on MSCs research value and application prospect in the disease. MSCs最初发现于骨髓,随后在许多组织与器官中均发现相似特性的MSCs,包括脂肪,肌肉, 结缔组织,脐带血,月经血,子宫内膜,羊膜,脾脏,心,牙齿,肺,胰腺,肝,脑,肾脏和外周血。 MSCs was originally found in the bone marrow, followed in many tissues and organs were found in MSCs similar characteristics, including fat, muscle, connective tissue, umbilical cord blood, menstrual blood, endometrial, amniotic membrane, spleen, heart, teeth, lungs, pancreas, liver, brain, kidneys and peripheral blood.

[0006] 目前,人们利用MSCs治疗肝纤维化的研究越来越深入,而且临床应用也正在进行中。 [0006] At present, people use MSCs more in-depth treatment of liver fibrosis, and clinical applications are in progress. 例如,中国专利申请201010551722.8公开了人脐带间充质干细胞抗肝纤维化注射液及其制备方法,该注射液由1 〇% -50 %人血白蛋白原液和49.5 % -89.5 %的勃脉力A液、0.5 % 肝素钙组成,其中人血白蛋白原液的浓度为10%,Iml注射液中含有人脐带间充质干细胞6 X 105-7X IO5个;其制备方法包括:提供间充质干细胞保存液,预冷、备用,该间充质干细胞保存液包括人血白蛋白原液和勃脉力A液、肝素钙;提供人脐带间充质干细胞并将其加入到间充质干细胞保存液中;通过重悬的方式,调整加入到间充质干细胞保存液中的人脐带间充质干细胞数量为6 X 105-7 X IO5个细胞/lml。 For example, Chinese Patent Application No. 201010551722.8 discloses a fibrosis injection and its preparation method of human umbilical cord mesenchymal stem cells against the injection of 1 billion% -50% stock solution of human albumin and 49.5% -89.5% of Plasmalyte a solution consisting of 0.5% heparin, human serum albumin concentration of the stock solution was 10%, Iml injection containing human umbilical cord mesenchymal stem cells a 6 X 105-7X IO5; preparation method comprising: providing inter mesenchymal stem cells storage solution, pre-cooled, standby, the mesenchymal stem cell preservation solution comprising human albumin stock solution and Plasmalyte A solution heparin; providing human umbilical cord mesenchymal stem cells and added to the mesenchymal stem cell preservation solution ; resuspended by way was added to adjust the mesenchymal mesenchymal stem cells of human umbilical cord preservation solution charged to the number of stem cells 6 X 105-7 X IO5 cells / lml. 中国专利申请201510024090.2公开了一种用于治疗肝纤维化的干细胞制剂及其制备方法,所述的治疗肝纤维化的干细胞制剂,是由人经血间充质干细胞与趋化因子CXCL8和生理盐水配制而成。 Chinese Patent Application No. 201510024090.2 discloses a stem cell preparation and a preparation method for the treatment of liver fibrosis, the treatment of liver fibrosis stem cell preparation is formulated from blood Human mesenchymal stem cells to the chemokine CXCL8 and saline made.

[0007] 但是,人们关于MSCs的易感性有些担忧,因为有研究已经提出MSCs自发地发生恶变。 [0007] However, it is about the susceptibility of MSCs bit worried, because studies have raised malignant MSCs occur spontaneously. 虽然MSCs向恶性转变的风险较低,最近公布的数据表明,人MSCs可能会显示基因组的突变,并表现基因水平的不稳定,在高通道端粒缺失(>170),微卫星不稳定,参与DNA修复基因表达下调和异质性点突变等。 Although MSCs to lower the risk of malignant transformation, recently published data show that the human genome mutations MSCs may display, and gene expression levels of instability, loss of telomeres in high-channel (> 170), microsatellite instability, is involved in DNA repair genes were down-regulated and heterogeneous point mutations. 虽然人MSC的恶性变换没有在临床试验中报道,也可能是后续跟踪时间相对较短,而肿瘤的发生可能需要较长时间才能出现。 Although malignant transformation of human MSC has not been reported in clinical trials, it could be relatively short follow-up time, and tumorigenesis may take longer to appear.

发明内容 SUMMARY

[0008] 为了更好地治愈肝纤维化或为治疗肝纤维化提供另一种可行的选择,提高患者的预后或者生活质量,本发明的发明人经过大量、坚持不懈的筛选,发现白介素-8 (IL-8)单独或者与其他细胞因子联合能够用于治疗肝纤维化。 [0008] In order to cure fibrosis or provides another viable option for the treatment of hepatic fibrosis, or improve the quality of life prognosis, the present inventors through large, persistent screening, interleukin-8 found (IL-8) alone or combined with other cytokines can be useful in the treatment of liver fibrosis. 而且,与其他细胞因子的联合具有显著较优或者协同作用。 Moreover, in combination with other cytokines or Jiaoyou significant synergy.

[0009] 本发明的一个方面涉及一种用于治疗肝纤维化的药物,其含有白介素-8 (IL-8)。 [0009] One aspect of the invention relates to a medicament for treating liver fibrosis, comprising interleukin -8 (IL-8). 任选地,本发明的药物还含有药学上可接受的载体。 Optionally, the pharmaceutical of the present invention further comprises a pharmaceutically acceptable carrier.

[0010] 优选地,本发明的用于治疗肝纤维化的药物还含有其他细胞因子,例如选自由骨保护素(OPG)、单核细胞趋化蛋白I (MCP-I)、HGF (肝细胞生长因子,hepatocyte growth factor)、GR0 (growth-regulated oncogene,肿瘤生长相关因子)和白介素-6 (IL-6)的细胞因子组成的组的细胞因子中的1个、2个、3个、4个或5个。 Drug [0010] Preferably, the present invention for the treatment of liver fibrosis further contain other cytokines, such as selected from the group consisting of osteoprotegerin (of OPG), monocyte chemoattractant protein-I (MCP-I), HGF (hepatocyte growth factor, hepatocyte growth factor), cytokines group GR0 (growth-regulated oncogene, tumor growth associated factor) and interleukin -6 (IL-6) cytokine consisting of 1, 2, 3, 4 or 5.

[0011] 本发明的第二方面提供了白介素-8 (IL-8)在制备用于治疗肝纤维化的药物中的用途。 [0011] A second aspect of the present invention provides Interleukin -8 (IL-8) for the manufacture of a medicament in the treatment of liver fibrosis. 任选地,用于治疗肝纤维化的药物还含有药学上可接受的载体。 Optionally, the medicament is for the treatment of liver fibrosis further comprising a pharmaceutically acceptable carrier. 优选地,用于治疗肝纤维化的药物还含有其他细胞因子,例如选自由骨保护素(OPG)、单核细胞趋化蛋白I (MCP- 1)、HGF (肝细胞生长因子,hepatocyte growth factor)、GR0 (growth-regulated oncogene,肿瘤生长相关因子)和白介素-6 (IL-6)的细胞因子组成的组的细胞因子中的I 个、2个、3个、4个或5个。 Preferably, the medicament is for treatment of liver fibrosis further contain other cytokines, such as selected from the group consisting of osteoprotegerin (of OPG), monocyte chemoattractant protein-I (MCP- 1), HGF (hepatocyte growth factor, hepatocyte growth factor ), cytokines group GR0 (growth-regulated oncogene, tumor growth associated factor) and interleukin -6 (IL-6) cytokine consisting of I, 2, 3, 4 or 5.

[0012] 本发明的第三方面提供了治疗受试者中肝纤维化的方法,其包括向所述受试者中施用有效量的白介素-8 (IL-8)或者将白介素-8 (IL-8)联合选自由骨保护素(OPG)、单核细胞趋化蛋白I (MCP-1)、HGF (肝细胞生长因子,hepatocyte growth factor)、GR0 (growth-regulated oncogene,肿瘤生长相关因子)和白介素-6 (IL-6)的细胞因子组成的组的细胞因子中的1个、2个、3个、4个或5个施用于所述受试者。 [0012] In a third aspect the present invention provides a method of treating liver fibrosis, comprising administering an effective amount of interleukin -8 (IL-8) or interleukin -8 (IL subject to the -8) selected from the group consisting of joint osteoprotegerin (OPG), monocyte chemoattractant protein-I (MCP-1), HGF (hepatocyte growth factor, hepatocyte growth factor), GR0 (growth-regulated oncogene, tumor growth factor-related) group of cytokines and cytokine interleukin -6 (IL-6) consisting of 1, 2, 3, 4 or 5 is administered to said subject.

[0013] 对于本发明所述的受试者,其优选为哺乳动物,更优选为人。 [0013] For the subject of the present invention, which is preferably a mammal, more preferably a human.

[0014] 白细胞介素-8 (interleukin-8,IL-8),又称趋化因子CXCL8(CXCL8),是一种炎性趋化性因子。 [0014] Interleukin -8 (interleukin-8, IL-8), also known as chemokine CXCL8 (CXCL8), is an inflammatory chemotactic factors. IL-8通过受体CXCRl和CXCR2等产生多种生物学功能,包括促炎性细胞趋化、促血管生成、促有丝分裂和诱导细胞增殖等。 IL-8 production by a variety of biological functions CXCRl and CXCR2 receptor and the like, including proinflammatory chemokines, pro-angiogenic, mitogenic induction of cell proliferation and the like. 研究发现IL-8与多种疾病尤其是肿瘤的发生发展关系密切,特异性地阻断IL-8及其受体CXCR1/2有望成为多种疾病的潜在治疗策略。 The study found IL-8 with a variety of diseases, especially cancer is closely related to the development of specifically block IL-8 and its receptor CXCR1 / 2 is expected to be a potential therapeutic strategy for many diseases. 也有很多报道称IL-8能够诱导促炎症与促进纤维化。 There are many reports that IL-8 can induce pro-inflammatory and promote fibrosis. 本领域技术人员可以对白介素-8作出任何修饰,前提是所述修饰不负面影响其活性。 Those skilled in the art can Interleukin -8 make any modification, provided that the modifications do not adversely affect its activity. 例如,可以对细胞因子进行修饰或装载在其他载体上,以提高其在体内的半衰期;或者可以与已知的穿透肽连接,以促进本发明化合物的透皮吸收或者越过血脑屏障等。 For example, modifications may be made to a cytokine or other load on the support, to increase their half-life in vivo; known or can be connected to the penetrating peptide, in order to facilitate transdermal absorption or the compounds of the invention cross the blood-brain barrier. 总之,本领域技术人员可对本发明的所述的细胞因子进行各种修饰以提高递送效率或用于其他目的并保持其活性。 In short, one skilled in the art that various modifications may be made to the cytokine of the present invention to improve delivery efficiency, or for other purposes and to maintain its activity. 这类修饰也是在本发明的范围之内的。 Such modifications are within the scope of the present invention.

[0015] 在本发明中,除各种细胞因子的活性成分外,本发明的方法、用途和产品还可以包含合适的药学上可接受的载体,包括促进活性成分加工成制剂(例如适于注射或输注的制剂)的赋形剂和助剂。 [0015] In the present invention, in addition to the active ingredient of various cytokines, the method of the present invention, the use of the product and also may comprise suitable pharmaceutically acceptable carriers, including the promotion of the active ingredients into preparations (e.g. suitable for injection or infusion formulation) auxiliaries and excipients.

[0016] 适于注射或输注的制剂可包括水性和非水性无菌注射液和水性和非水性无菌混悬剂,所述无菌注射液可任选地包含抗氧化剂、缓冲剂、抑菌剂和能使制剂与目的接收者的血液等压的溶质,所述无菌混悬剂可包括悬浮剂和增稠剂。 [0016] Formulations suitable for injection or infusion can include aqueous and non-aqueous sterile injection solutions and aqueous and non-aqueous sterile suspensions, optionally a sterile injectable solution may contain antioxidants, buffers, suppressing bacteriostats and solutes pressure can formulation such as blood of the intended recipient, the sterile suspensions may include suspending agents and thickening agents. 所述制剂可存在于单位剂量或多剂量容器中,例如密封的安瓿,并且可以保存在冻结干燥的(冻干)条件,在立即使用前仅需要加入无菌液体载体,例如注射用水。 The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only immediately before addition of the sterile liquid carrier, for example water for injections.

[0017] 本发明的各种细胞因子活性成分任选地可与固体赋形剂相组合,且任选地磨碎所得到的混合物,并且需要时,在加入合适的助剂后,加工颗粒的混合物,以获得所需剂型。 [0017] The various cytokines active ingredient of the present invention may optionally be combined with a solid excipient, the resulting mixture was milled and optionally, and if necessary, after adding suitable auxiliaries and processing the granule mixture, to obtain the desired dosage. 合适的赋形剂特别是填充剂例如糖,包括乳糖、蔗糖、甘露醇或山梨糖醇;纤维素或淀粉制剂、 明胶、黄蓍胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠和/或聚乙烯吡咯烷酮(PVP)。 Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose or starch preparations, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). 需要时,可以加入崩解剂,例如交联聚乙烯吡咯烷酮、琼脂或海藻酸或其盐例如海藻酸钠。 If desired, disintegrating agents may be added, such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0018] 在本发明中,施用各细胞因子的量可为能治疗或者协同治疗受试者中肝纤维化或抑制肝纤维化细胞增殖的任何量,其可以是相当于约0.01-15.Omg白介素-8 (IL-8),优选0 · 2-2 · Omg白介素-8 (IL-8)和0 · 01-20 · Omg选自由骨保护素(OPG)、单核细胞趋化蛋白1 (MCP-1)、HGF (肝细胞生长因子,hepatocyte growth factor)、GR0 (growth-regulated oncogene,肿瘤生长相关因子)和白介素-6 (IL-6)的细胞因子组成的组的细胞因子。 Volume [0018] In the present invention, each administration of cytokines may be co-therapy or to treat a subject an amount of fibrosis or inhibit any proliferation of the liver cells, which may be equivalent to about 0.01-15.Omg interleukin -8 (IL-8), preferably 0 · 2-2 · Omg interleukin -8 (IL-8), and 0 · 01-20 · Omg selected from the group consisting of osteoprotegerin (OPG), monocyte chemoattractant protein 1 ( MCP-1), HGF (hepatocyte growth factor, hepatocyte growth factor), cytokines group GR0 (growth-regulated oncogene, tumor growth associated factor) and interleukin -6 (IL-6) is a cytokine.

[0019] 更优选地,本发明的药物的剂量单位包括约l-4mg白介素-8 (IL-8)和0.01-20.Omg 选自由骨保护素(OPG)、单核细胞趋化蛋白I (MCP-I)、HGF (肝细胞生长因子,hepatocyte growth factor)、GR0 (growth-regulated oncogene,肿瘤生长相关因子)和白介素_6 (IL-6)的细胞因子组成的组的细胞因子。 [0019] More preferably, the pharmaceutical dosage unit of the present invention comprise from about l-4mg Interleukin -8 (IL-8) and selected from the group consisting of 0.01-20.Omg osteoprotegerin (of OPG), monocyte chemoattractant protein-I ( Cytokines group MCP-I), HGF (hepatocyte growth factor, hepatocyte growth factor), GR0 (growth-regulated oncogene, tumor growth associated factor) and interleukin _6 (IL-6) is a cytokine. 最优选地,剂量单位包括约2-3mg的白介素-8 (IL-8)和2.0-6. Omg选自由骨保护素、单核细胞趋化蛋白I (MCP-I)、HGF (肝细胞生长因子, hepatocyte growth factor)、GR0 (growth-regulated oncogene,肿瘤生长相关因子)和白介素-6 (IL-6)的细胞因子组成的组的细胞因子。 Most preferably, the dosage unit including interleukin -8 (IL-8) and about 2-3mg 2.0-6. Omg selected from the group consisting of osteoprotegerin, monocyte chemoattractant protein-I (MCP-I), HGF (hepatocyte growth factor, hepatocyte growth factor), cytokines group GR0 (growth-regulated oncogene, tumor growth associated factor) and cytokines interleukin -6 (IL-6) is composed.

[0020] 在本发明中,施用的有效量以及合适的单位剂量的测定在本领域技术人员的能力内,特别是根据本文提供的公开内容的启示下。 [0020] In the present invention, an effective amount administered as well as suitable assay unit dose within the capability of those skilled in the art, particularly in accordance with the teachings of the disclosure provided herein under.

[0021] 根据本发明,本发明的药学药物可以以任意有效剂量施用给药受试者。 [0021] According to the present invention, the pharmaceutical drug of the present invention may be administered in any effective dosage administered subject. 优选地,本发明的药物可以以多次剂量给药,例如从约2至约15次剂量,更优选约4-10次剂量,最优选约6次剂量。 Preferably, the pharmaceutical of the present invention may be administered in multiple doses, e.g., from about 2 to about 15 doses, more preferably about 4 to 10 doses, and most preferably about 6 doses. 在特别优选的实施方案,在给药过程中,以每三周给药约一次的频率将本发明的药物给药至受试者,例如注射、输注或口服。 In a particularly preferred embodiment, during administration, at a frequency of about once every three weeks of administration of the medicament of the present invention is administered to a subject, e.g. injection, infusion or orally. 在特别优选的实施方案,给药为通过静脉注射施用。 In a particularly preferred embodiment, the administration is administration by intravenous injection.

[0022] 应当理解本发明的药物可以按用于通过任意适宜的途径给药的任意适宜的方式配制。 [0022] It should be appreciated that medicament according to the present invention may be formulated for any suitable manner by any suitable route of administration.

[0023] 本发明的药物的剂量单位是基于常规进行给药受试者。 [0023] The pharmaceutical dosage unit of the present invention is based on conventional administration subject. 例如,剂量单位可以基于给药频率为每日一次、每周一次、每月一次等确定。 For example, the dosage unit may be based on the frequency of administration to once daily, weekly, monthly and the like is determined. 剂量单位也可基于以两次/周、三次/周等确定。 Dosage units may also be based at twice / week, three times / week, etc. OK.

[0024] 如本文所使用的,“包含”与“包括”、“含有”或“特征在于”同义,并且是包括在内的或开放性的,并且不排除另外的未陈述的元件或方法步骤。 [0024] As used herein, "comprising" and "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional elements or method unrecited step. 术语“包含”在本文中的任何表述,特别是在描述本发明的方法、用途或产品时,应理解为包括基本上由所述组分或元件或步骤组成和由所述组分或元件或步骤组成的那些产品、方法和用途。 The term "comprising" herein is any expression, particularly in describing the inventive method, use or product, it should be understood that consist essentially of the components or elements or steps and of the element or component or to include those product steps, methods and uses. 本文示例性描述的本发明适当地可以在不存在本文未具体公开的任何一种或多种元件、一种或多种限制的情况下进行实践。 Suitably the present invention illustratively described herein may be practiced, one or more of any element not specifically disclosed herein does not limit the presence of one or more.

[0025] 本发明的药物中可包含涉及该药学产品的说明书,且该说明书可以含有如下内容:适应症(例如肝纤维化)、施用剂量(例如上述所示例性说明的)以及可能产生的副作用等等。 [0025] The medicaments of the invention may comprise instructions relating to the pharmaceutical product, and this specification may contain the following: indication (e.g. liver fibrosis), the dosage (e.g., the above described exemplary) and possible side effects and many more.

[0026] 本文已采用的术语和表述用作描述性而不是限制性术语,并且在此种术语和表述的使用中不预期排除所示和所述特征或其部分的任何等价物,但应认识到各种修饰在请求保护的本发明的范围内是可能的。 [0026] The terms and expressions of description and not as limiting terms have been employed herein, and not intended to exclude the features shown and described or any portion of equivalents of the use of such terms and expressions, it should be appreciated that various modifications within the scope of the claimed invention are possible. 因此,应当理解尽管本发明已通过优选实施方案和任选特征具体公开,但本领域技术人员可以采用本文公开的概念的修饰和变化,并且此类修饰和变化被视为在如由附加权利要求定义的本发明的范围内。 Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art can employ modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered such as by the appended claims within the scope of the invention as defined.

[0027] 为更清楚地说明本发明,现结合如下实施例进行详细说明,但这些实施例仅仅是对本发明的示例性描述,并不能解释为对本申请的限制。 [0027] The present invention is more clearly described, in conjunction with the following examples now be described in detail, but these embodiments are merely described exemplary embodiments of the present invention, and should not be construed as limiting the present disclosure.

附图说明 BRIEF DESCRIPTION

[0028] 图1:细胞上清和细胞ELISA定量检测MCP-1,IL-6,HGF,GR0,IL-8和0PG。 [0028] Figure 1: ELISA quantification of cell and cell supernatants detected MCP-1, IL-6, HGF, GR0, IL-8 and 0PG. ELISA定量检测LX-2组,MenSC组和共培养组(co-culture组)培养72h后细胞上清液以及细胞的蛋白表达量。 ELISA quantitative detection of LX-2 group, and the cell supernatant protein expression after 72h MenSC cells co-cultured group and the group (co-culture group) culture. ㈧,细胞上清液蛋白量。 (Viii), the amount of protein in cell supernatants. ⑶,细胞的蛋白表达量。 ⑶, protein expression in cells. 分析及作图采用GraphPad Prism 5 软件。 Analysis and mapping using GraphPad Prism 5 software. 数值代表平均值土SD (η = 4)。 Values ​​represent the average of soil SD (η = 4).

具体实施方式 Detailed ways

[0029] 本发明结合附图和实施例作进一步的说明。 [0029] The present invention in conjunction with the drawings and embodiments described further.

[0030] 实施例1:肝纤维化小鼠模型的建立及鉴定 [0030] Example 1: Establishment and identification of liver fibrosis model mice

[0031] 本发明使用本领域常用的通过CCl4损伤小鼠肝脏而建立的肝纤维化小鼠模型。 [0031] Hepatic fibrosis mouse model of the present invention used in this field established by CCl4 liver injury in mice.

[0032] 将6-8周ICR小鼠(购自上海斯莱克(SLAC)实验动物有限责任公司)腹腔注射CC14, 按照ImL CClVkg小鼠体重注射。 [0032] 6-8 weeks ICR mice (purchased from Shanghai SLAC (SLAC) Experimental Animal Co., Ltd.) by intraperitoneal injection of CC14, according ImL CClVkg weight injected mice. CC14以10%的比例溶解于橄榄油(olive oil)中,用ImL微型注射器(BD Biosciences)每周注射两次并持续注射4周,这样就建立了小鼠的早期肝纤维化模型。 CC14 at a ratio of 10% was dissolved in olive oil (olive oil) in ImL weekly injections twice a micro syringe (BD Biosciences) and continued for 4 weeks, thus establishing early hepatic fibrosis model mice. 通过摘取小鼠肝脏,对其进行sirius red染色,检验肝脏组织病理情况;同时检测小鼠的血清肝功能指标,包括ALT,AST,ALB,ALP和TBIL。 Removal by mouse liver, its sirius red staining, liver pathology testing; Serum levels of liver function in mice, including ALT, AST, ALB, ALP and TBIL. 通过以上指标,验证小鼠纤维化模型是否构建成功。 Through the above indicators, verify whether the mouse fibrosis model was successfully constructed.

[0033] 实施例2 = MenSCs的分离和体外培养 [0033] Example isolated and culture in vitro of 2 = MenSCs

[0034] 在经期健康女性自愿者月经周期时,利用Divacup (Kitchener,0N)收集女性经血样品。 [0034] In healthy female volunteers during menstruation menstrual cycle, women collect blood samples using the Divacup (Kitchener, 0N). 在获取样品后的2天之内,将样品小心存于4°C。 Within 2 days after obtaining the sample, the sample will feel in the small 4 ° C. 取出这些样品之后,后续操作程序均在细胞房中进行处理。 After removal of these samples, the subsequent procedure was performed in a cell processing room. 将收集到的经血进行离心(lOOOrpm,10min,4°C),利用Ficoll-Paque 分离得到液体中的单核细胞(混合型),随后将细胞样品进行检测,检测上清液中细菌数量, 待各项指标达标后进行细胞体外增殖培养。 The collected blood by centrifugation (lOOOrpm, 10min, 4 ° C), using a Ficoll-Paque separated from monocytes obtained liquid (mixed type), followed by detecting the cell sample, the number of bacteria detected in the supernatant to be indicators of cell proliferation in vitro cultured compliance. 细胞分离之后用PBS洗涤3-5次(尽可能弃去杂质),然后将分离纯化的细胞转到加有DMEM/F12培养基(添加了l^penicillin/ streptomycin、I %amphotericin B和15%FBS)的细胞培养瓶(Corning)中继续贴壁培养。 After the cells were washed with PBS 3-5 times separated (discarded as impurities), and then isolated and purified cells added to DMEM / F12 medium (added l ^ penicillin / streptomycin, I% amphotericin B and 15% FBS ) in cell culture flasks (Corning) in adherent culture continued. 将分离后的细胞培养一周左右,每隔2天换1次液,同时吸弃未能正常贴壁的悬浮细胞/死细胞。 The isolated cells cultured for one week, for a second fluid every 2 days, and fails to normally aspirated suspension of adherent cells / dead cells. 随后,每隔3天更换新鲜Chang培养基,选取形态分布均一、生长状态良好的MenSCs进行扩大培养。 Subsequently, Chang changed every 3 days with fresh medium, uniform distribution of selected morphology, good growth were expanded state MenSCs. 当细胞密度达到80 %左右时,用PBS洗涤3次,而后加入0 · 25 % Trypsin-EDTA (5-IOmin)进行细胞消化,将消化后的细胞进行离心并按照1:3的比例进行细胞扩大培养(加入新鲜培养基)。 When the cell density reached about 80%, for cell digestion were washed 3 times with PBS, followed by addition of 0 · 25% Trypsin-EDTA (5-IOmin), the cells were digested by centrifugation and 1: for cell expansion Comparative Example 3 culture (added to fresh medium). 所有培养的细胞均放置于3 7 °C恒温和5 % C 0 2的饱和湿度培养箱(ThermoFisher)中。 All cultured cells were placed in a 3 7 ° C temperature and saturated humidity incubator (ThermoFisher) 5% C 0 2 in. 经过约5代的扩大培养,其形态已经基本趋于稳定和均一,并可以见到细胞呈现典型的梭形并呈旋涡状排列。 After expansion for about 5 generations, its shape has been stabilized and substantially uniform, and can see the cells showed a typical spindle form and arranged in spiral shape.

[0035] 为了进一步验证MenSCs的特性,利用流式细胞仪对MenSCs进行细胞表面标记分子的鉴定,检测的分子包括CD29,CD34,CD45,CD73,CD90,CD105,CDl 17和HLA-DR。 [0035] To further verify the characteristics of MenSCs of MenSCs molecules identified cell surface markers by flow cytometry, detection molecules include CD29, CD34, CD45, CD73, CD90, CD105, CDl 17, and HLA-DR. 结果显示MenSCs能够高表达CD29,CD73,CD90和CD105 (均>90%),低表达CD45,几乎不表达CD34, CDl 17和HLA-DR (均〈1%)。 The results show high expression can be MenSCs CD29, CD73, CD90 and of CD105 (both> 90%), low expression of CD45, almost no expression of CD34, CDl 17, and HLA-DR (all <1%).

[0036] 本文实施例中所使用的MenSCs为第5到第8代次的细胞(P5-P8),除非特别指出。 [0036] MenSCs used in the examples herein for the first embodiment of the fifth to eighth generations of cells (P5-P8), unless otherwise indicated.

[0037] 实施例3:表达绿色荧光蛋白(GFP)的MenSCs (GFP+MenSCs)的构建和筛选 [0037] Example 3: Construction and Screening MenSCs (GFP + MenSCs) expressing green fluorescent protein (GFP) of

[0038] P2-P3的MenSCs培养于T75培养瓶(Corning)中,当细胞贴壁生长到大约50%时吸弃培养基,用PBS洗涤3次,添加含有6yg/mL的polybrene (汉恒生物科技有限公司)以及GFP-PURO (汉恒生物科技有限公司)病毒液(Μ0Ι = 50;MOI ,multiplicity of infection)的IOmLChang氏完全培养基(杭州易文赛生物科技有限公司)。 [0038] P2-P3 is MenSCs cultured in T75 flasks (Corning), the medium was aspirated when the cells grew to about 50%, washed three times with PBS, containing added 6yg / mL of Polybrene (Han Heng Biological Technology Co., Ltd.) and GFP-PURO (Han Heng biotechnology Limited) virus solution (Μ0Ι = 50; MOI, multiplicity of infection) of IOmLChang's complete medium (Hangzhou Evans Biosciences Co., Ltd.). 1天后更换新鲜培养基,待细胞培养2-3天后再次更换新鲜培养基,并用2yg/mL的puromycin (Sigma)进行细胞筛选。 Replaced with fresh media after one day, to be replaced with fresh cell culture medium was again 2-3 days, and cells were screened with 2yg / mL of puromycin (Sigma). 对筛选后的细胞(GFP+MenSCs)进行GFP阳性检测以及细胞表面标记分子(包括⑶29,CD34,⑶45, CD73,CD90,CD105,CDl 17和HLA-DR)的表达与鉴定。 Cells (GFP + MenSCs) for the screening and detection of GFP positive cell surface marker molecules (including ⑶29, CD34, ⑶45, CD73, CD90, CD105, CDl 17, and HLA-DR) expression and identification.

[0039] 取筛选两周后的部分细胞进行鉴定,可以看出,与正常MenSCs相比,GFP+MenSCs并没有发生明显的形态变化,而通过GFP荧光直观观察,发现几乎全部的细胞(>99%)已经感染上了慢病毒,这样的结果通过流式细胞进行了进一步确认。 [0039] Filter section taken two weeks after the cells were identified, it can be seen, compared to normal MenSCs, GFP + MenSCs no obvious morphological changes, and by visual observation of GFP fluorescence, found that almost all of the cells (> 99 %) have been infected with lentivirus, this result was further confirmed by flow cytometry.

[0040] 为了验证GFP+MenSCs的表面标志物是否发生变化,检测了和MenSCs相对应的表面分子。 [0040] In order to verify the GFP + MenSCs whether the changed surface markers, and detecting the corresponding MenSCs surface molecules. 结果显示与正常MenSCs相比,GFP+MenSCs并没有发生明显的表面标记分子改变,而且表面标记分子特性和MenSCs—致。 The results show that compared to normal MenSCs, GFP + MenSCs no obvious change in surface marker molecules, and the surface properties and MenSCs- labeled molecules induced. 这进一步表明Puro-GFP慢病毒对于经血干细胞的形态及表面标记分子并无明显的影响。 This further indicates Puro-GFP lentivirus for dry blood cell morphology and surface labeled molecule is not significantly affected.

[0041] 实施例4:活体成像检测GFP+MenSCs移植小鼠后的组织分布 [0041] Example 4: in vivo imaging detector GFP + mice transplanted tissue distribution of MenSCs

[0042] 为了验证MenSCs具有向肝损伤部位趋化的功能,将稳定筛选的GFP+MenSCs移植到小鼠体内,一共分为5个组别: [0042] In order to verify MenSCs liver injury site having the function of chemokines, stabilized GFP + MenSCs screened transplanted into mice, divided into a total of five groups:

[0043] (1)正常对照组(normal control):对小鼠不做任何处理; [0043] (1) normal control group (normal control): mice without any treatment;

[0044] (2) GFP+MenSCs移植到正常小鼠(normal mice) 1 周组(命名为GFP+IW in NM); [0044] (2) GFP + MenSCs transplanted into normal mice (normal mice) 1 week group (designated GFP + IW in NM);

[0045] (3) GFP+MenSCs移植到肝纤维化模型小鼠(liver fibrosismice) 1周组(命名为GFP+Iff in LFM); [0045] (3) GFP + MenSCs transplanted into a mouse model of liver fibrosis (liver fibrosismice) 1 week group (designated GFP + Iff in LFM);

[0046] ⑷GFP+MenSCs移植到正常小鼠2周组(命名为GFP+2W in NM); [0046] ⑷GFP + MenSCs transplanted to 2 weeks (designated GFP + 2W in NM) normal mice;

[0047] (5)6??+]^1^〇8移植到肝纤维化模型小鼠2周组(命名为6??+21丨111^]\〇。 [0047] (5) + 6 ??] ^ ^ 1 to 2 weeks 〇8 transplanted liver fibrosis model mice (designated as 6 + 21 ?? ^ Shu 111] \ square.

[0048] 上述各组别的小鼠均采用麻醉处死,然后利用手术剪刀和眼科镊子将主要的几个器官进行分离,这些脏器包括肝(liver),肺(lung),脾(spleen),肾(kidney)和心(heart)。 [0048] The mice are used in each of the groups were anesthetized and sacrificed, and the use of an ophthalmic surgical scissors and tweezers were separated several major organs, these organs including the liver (liver), lung (Lung), spleen (spleen), renal (kidney) and cardiac (heart). 分离得到的器官用PBS清洗3次(每次3min),这样就能完全的去除残留的血块和杂货(减少或去除活体成像产生的干扰)。 Isolated organ was washed three times (3min) with PBS, and this can completely remove residual clot and groceries (reducing or removing the interference generated by in vivo imaging). 每个组平行处理6只小鼠(n = 6)。 Each set of parallel processing 6 mice (n = 6). 这些分离得到的器官在IVIS SPECTRUM小鼠活体成像系统(Caliper Life Sciences)下拍照,并用相关软件(Living Image 4.3. Isoftware)对焚光强度进行统计分析。 The isolated mouse organs at IVIS SPECTRUM vivo Imaging System (Caliper Life Sciences) photographed and using software (Living Image 4.3. Isoftware) for burning light intensity for statistical analysis.

[0049] 结果发现,正常对照组几乎不表达GFP蛋白,GFP+1W/2W in匪组和GFP+1W/2W in LFM组能够在肝脏中表达GFP蛋白。 [0049] The results showed almost no expression in normal control group of GFP, GFP + 1W / 2W in bandit group and GFP + 1W / 2W in LFM group capable of expressing GFP protein in the liver. GFP+IW/2W in LFM组的荧光强度明显高于对应时间点的GFP+1W/2W in NM组。 GFP + IW / 2W in LFM fluorescence intensity was significantly higher than the corresponding point in time GFP + 1W / 2W in NM group. 更重要的是,GFP+1W/2W in LFM组中大部分MenSCs迀移到了肝脏部位。 More importantly, GFP + 1W / 2W in LFM group most MenSCs Gan moved to the liver area. 这些结果显示MenSCs能够定向迀移到损伤部位。 These results show that MenSCs can be oriented Gan moved to the site of injury. 此外,荧光强度分析软件进一步确认了该结果。 Furthermore, fluorescence intensity analysis software is further confirmed this result.

[0050] 实施例5:肝组织CK-18免疫荧光检测 [0050] Example 5: liver CK-18 immunofluorescence

[0051] 在干细胞移植后的1周和2周,分别取MenSCs组(MenSCs 2W)和GFP+MenSCs组(GFP+ MenSCs IW和GFP+MenSCs 2W)小鼠的肝脏,每个组别有6个样品(n = 6)。 [0051] 1 week and 2 weeks after the stem cell transplantation, were taken MenSCs group (MenSCs 2W), and GFP + MenSCs group (GFP + MenSCs IW and GFP + MenSCs 2W) in liver of mice, six samples per group (n = 6). 将收集的肝组织用锡箱纸包起来,快速冻存于-80 °C冰箱。 The collected liver tissue is wrapped in a tin box, quick frozen and stored at -80 ° C freezer. 具体的免疫荧光操作步骤如下: Immunofluorescence specific steps are as follows:

[0052] (1)迅速从_80°C冰箱取出冰冻组织并用Tissue-Tek OCT包埋剂(Sakura)将样品进行包埋; [0052] (1) rapidly removed from _80 ° C Frozen tissue samples were embedded and refrigerator with Tissue-Tek OCT embedding medium (Sakura);

[0053] (2)将样品放置于冰冻切片机(CryoStar NX50,Thermo)切片,厚度为4-6μπι; [0053] (2) The sample is placed in a freezing microtome (CryoStar NX50, Thermo) slice thickness 4-6μπι;

[0054] (3)在室温放置30min,然后加入4°C预冷的丙酮個定样品IOmin),接着用PBS清洗3次(每次5min); [0054] (3) stand at room temperature 30min, 4 ° C followed by addition of acetone precooled a given sample IOmin), followed by washing with PBS three times (5min);

[0055] (4)阻断内源性过过氧化氢(H2O2)酶:加入3%的H2O2 (约IOmin),然后用蒸馏水进行水洗3min,之后再用PBS(0.01M PH=7.5)清洗切片三次(每次约5min); [0055] (4) through the block endogenous hydrogen peroxide (H2O2) Enzyme: 3% by H2O2 was added (about IOmin), 3min and then rinsed with distilled water, and then after PBS (0.01M PH = 7.5) Sections were washed three times (each about 5min);

[0056] (5)封闭:用5%的正常山羊血清(用PBS进行稀_,然后在室温孵育20min,接着再用PBS清洗切片三次(每次约5min); [0056] (5) Blocking: with 5% normal goat serum (diluted _ performed with PBS, and then incubated for 20min at room temperature and then the sections were washed with PBS three times (about 5min each);

[0057] (6)滴加适当比例稀释的特异性抗人CK-18—抗,4°C孵育过夜,接着再用PBS清洗切片三次(每次约5min); [0057] (6) was added dropwise an appropriate dilution ratio of specific anti-anti-human CK-18-, 4 ° C overnight incubation, the sections were washed with PBS and then three times (about 5min each);

[0058] (7)滴加适当比例稀释的Cy3标记的羊抗鼠二抗(稀释比例1:400),室温孵育60min; [0058] (7) was added dropwise an appropriate dilution ratio of Cy3-labeled goat anti-mouse secondary antibody (dilution ratio 1: 400), incubated for 60min at room temperature;

[0059] ⑶用DAPI复染20min以标记细胞核,然后用PBS清洗切片三次海次约5min); [0059] ⑶ 20min counterstained with DAPI to label cell nuclei, sections were then washed three times with PBS sea about 5min);

[0060] (9)封片,用荧光显微镜(U-HGLGPS光源,Olympus)观察并拍照。 [0060] (9) were mounted, with a fluorescence microscope (U-HGLGPS light source, an Olympus) and photographed.

[0061] 结果发现,MenSCs 2W组几乎不表达GFP;相比于MenSCs 2W组,GFP+MenSCs组能够明显的表达GFP,并且GFP+MenSCs 2W的荧光强度高于GFP+MenSCs IW组。 [0061] It was found, MenSCs 2W hardly expressed the GFP group; MenSCs 2W compared with the group, GFP + MenSCs group can significantly expressing GFP, and GFP + MenSCs 2W fluorescence intensity of GFP + MenSCs IW is higher than the set. 400倍放大的图片更加清晰的展示了这一结果(图略)。 400 times larger image more clearly shows this result (figure omitted). 这些进一步验证了MenSCs具有向损伤部位趋化的作用。 These further validate the role of MenSCs tends to have the injury site of. 特异性抗人CK-18的表达用于检测MenSCs移植到小鼠肝脏后,MenSCs是否有部分细胞分化成肝样细胞。 After the expression of specific anti-human CK-18 for detecting MenSCs liver transplanted into mice, MenSCs whether some cells into hepatocyte-like cells. 结果表明,MenSCs并未分化成肝样细胞。 The results showed that, MenSCs did not differentiate into hepatocyte-like cells.

[0062] 实施例6:移植经血干细胞改善小鼠的肝纤维化 [0062] Example 6: blood stem cell transplantation to improve liver fibrosis in mice

[0063] 实施例6.1 :小鼠尾静脉移植 Transplantation tail vein of mice: [0063] 6.1 cases embodiment

[0064] 使细胞浓度为5 X IO6AiU用ImL微型注射器通过尾静脉注射到已经构建好的纤维化模型小鼠中(每只小鼠移植约5\105细胞/10(^〇。将6??116113〇8/^6113〇8移植到肝纤维化模型小鼠命名为GFP+MenSCs组/MenSCs组,同时将只注射等量PBS的肝纤维化模型小鼠命名为PBS组。为了防止发生小鼠纤维化自发性恢复的情况,在干细胞移植的两周中,小鼠持续注射CC14。 [0064] The cells at a concentration of 5 X IO6AiU with ImL microsyringe by tail vein injection into prebuilt fibrosis model mice (5 per mouse transplanted about \ 105 cells / 10 (^ square. The 6 ?? 116113〇8 / ^ 6113〇8 transplanted into a mouse model of liver fibrosis GFP + MenSCs named group / MenSCs group, while the liver fibrosis model mice injected with an equal amount of PBS was designated as PBS group. mice to prevent fibrosis where spontaneous recovery, stem cell transplantation in two weeks, the mice were injected continuously CC14.

[0065] 实施例6.2: SR染色 SR staining: [0065] 6.2 cases embodiment

[0066] 在干细胞移植到小鼠后的第1周和第2周,分别麻醉并脱颈处死小鼠,分别取MenSCs组(MenSCs IW和MenSCs 2W)和PBS组(PBS IW和PBS 2W)小鼠的肝脏,每个组别有6个样品(n = 6)。 [0066] In the first week and two weeks after the stem cells transplanted into the mice were anesthetized and were sacrificed mice were taken MenSCs group (MenSCs IW and MenSCs 2W) and PBS group (PBS PBS IW and 2W) small rat liver, the six samples per group (n = 6). 具体的组织处理步骤如下: Particular tissue processing steps are as follows:

[0067] (1)固定:组织加入到10%的中性福尔马林中,固定大概1天; [0067] (1) Fixed: tissue was added to a 10% neutral formalin fixed about one day;

[0068] ⑵脱水:取出固定好的组织,将其依次放入梯度酒精进行脱水(分别是70%酒精Ih,80%酒精Ih,95%酒精Ih和100%酒精Ih); [0068] ⑵ dehydration: Remove the mounting well tissue, which in turn is placed in a gradient of dehydrated alcohol (70% alcohol, respectively Ih, Ih 80% ethanol, 95% ethanol and 100% ethanol Ih Ih);

[0069] (3)透明:先用二甲苯I浸泡lOmim,然后用二甲苯II浸泡20min; [0069] (3) Transparency: soaked with xylene lOmim I, II and then soaked with xylene 20min;

[0070] ⑷浸蜡:先用软蜡在60 °C下I h,然后用硬蜡在60 °C下2h; [0070] ⑷ waxing: first with a soft wax at 60 ° C I h, and then hard wax at 60 ° C 2h;

[0071] (5)包埋:硬蜡包埋; [0071] (5) embedding: embedding hard wax;

[0072] (6)切片:在石蜡切片机(HM325型轮转式)上进行组织切片,切片的厚度大概为5μ m; [0072] (6) sections: tissue sections were performed on paraffin microtome (HM325 rotary type), the slice thickness approximately 5μ m;

[0073] ⑵烤片:在60°C条件下,烤约4h。 [0073] ⑵ baking sheet: at 60 ° C under conditions bake about 4h.

[0074] 将干燥的石蜡切片放置于二甲苯中,依次在梯度酒精中(100%酒精3min,95%酒精3min,80%酒精3min,70%酒精3min)进行脱錯处理。 [0074] The dried xylene paraffin sections placed sequentially performed error removal process in graded alcohols (100% ethanol 3min, 95% alcohol 3min, 80% alcohol 3min, 70% ethanol 3min). 用事先配好的0.1%SR(0. Ig SR溶于IOOmL picric acid中)进行孵育30min,使用蒸馏水冲洗1-2次,使用梯度酒精脱水,中性树脂进行封片,利用显微镜(Olympus 1X83)进行图像采集。 With advance with a good 0.1% SR (0. Ig SR dissolved IOOmL picric acid) is incubated 30min, washed with distilled water 1-2 times, using a gradient alcohol dehydration, neutral resin were mounted, with a microscope (Olympus 1X83) image acquisition. 利用IPP 6software (Image Pro Plus 6,Media Cybernetics)分析胶原纤维面积(胶原面积/总的面积)。 Using the IPP 6software (Image Pro Plus 6, Media Cybernetics) analysis (collagen area / total area) collagen area.

[0075] MenSCs移植到纤维化小鼠1周和2周后,观察并检测胶原纤维面积。 [0075] MenSCs fibrosis transplanted into mice one week and two weeks later, and the detection of collagen fibers were observed area. 结果表明,尽管MenSCs IW组(1.9%)的胶原积累面积低于PBS IW组(2.4%),但其在统计学上并没有显著性差异;相反,MenSCs 2W组(2.2%)的胶原纤维面积相比于I3BS 2W组(3.4%)有明显减少, 说明肝纤维化在MenSCs移植2周后病理指标上得到了改善。 The results showed that, although the area of ​​collagen accumulation MenSCs IW group (1.9%) lower than the PBS IW group (2.4%), but no statistically significant differences; contrary, MenSCs 2W group (2.2%) of the area of ​​collagen fiber I3BS 2W compared to group (3.4%) significantly decreased, indicating liver fibrosis improved in 2 weeks after transplantation MenSCs pathological parameters.

[0076] 实施例6.3:小鼠血清肝功能指标检测 [0076] Example 6.3: Detection of mouse serum liver function

[0077] 在干细胞移植到小鼠后的第1周和第2周,分别麻醉并脱颈处死小鼠,分别收集MenSCs组(MenSCs IW和MenSCs 2W)和PBS组(PBS IW和PBS 2W)小鼠的血清,每个组别有6个样品(n = 6)。 [0077] In the first week and two weeks after the stem cells transplanted into the mice were anesthetized and were sacrificed mice were collected MenSCs group (MenSCs IW and MenSCs 2W) and PBS group (PBS PBS IW and 2W) small mouse serum, the six samples per group (n = 6). 具体的血清收集步骤如下: Specific serum collected following steps:

[0078] (1)将麻醉的小鼠平稳放置实验台上,使用眼科手术镊子进行小鼠眼球取血,倾斜小鼠,让其血液自然流出,放入到1.5mL的EP管中; [0078] (1) The anesthetized mouse is placed stationary bench, ophthalmic surgical tweezers eye bled mice, mice inclined, let the natural flow of blood, placed in a 1.5mL EP tube in;

[0079] (2)将倾斜管壁,常温放置90min; [0079] (2) the inclined wall, placed at room temperature 90min;

[0080] (3)轻柔操作,放入离心机中离心:4°C,4000rpm,15min; [0080] (3) Operation gently placed centrifuge centrifuge: 4 ° C, 4000rpm, 15min;

[0081] ⑷用IOOyL枪头缓慢吸取上清液(注意不要碰到下层的血块); [0081] ⑷ tip slowly with IOOyL supernatant (careful not to touch the lower clot);

[0082] (5)将上清液冰浴2min,然后-20°C冻存备用; [0082] (5) The supernatant ice bath 2min, -20 ° C and then frozen for later use;

[0083] 利用南京建成试剂盒,检验血清学中肝功能常见指标(ALT,AST,ALB,ALP和TBIL)。 [0083] using the kit built in Nanjing, serological tests in common indicators of liver function (ALT, AST, ALB, ALP and TBIL).

[0084] MenSCs细胞移植到纤维化小鼠1周和2周后,分别取样检测血清ALT,AST,ALB,ALP 和TBIL (表1)。 [0084] MenSCs fibrosis cells into mice after 1 and 2 weeks, respectively sampled serum ALT, AST, ALB, ALP and TBIL (Table 1). 结果表明,除了ALT之外,MenSCs IW组(相比于I3BS IW组)的血清学指标并没有显著性差异;相反,MenSCs 2W组(相比于PBS 2W组)的血清学指标有较显著的降低(ALB除外),说明肝纤维化在生理指标上得到了改善。 The results showed that, in addition to ALT, serological MenSCs IW group (group compared to I3BS IW) and no significant differences; contrary, MenSCs 2W serological group (group compared to the PBS 2W) are more significant reduction (except ALB), liver fibrosis has been described improvement in physiological indices.

[0085] 表1小鼠常见肝功能血清指标检测 [0085] The common liver function serum markers detected in mice TABLE 1

[0086] [0086]

Figure CN105944086BD00101

[0087] 注:ALT,丙氨酸氨基转移酶;AST,天冬氨酸氨基转移酶;ALB,白蛋白;ALP,碱性磷酸酶;TBIL,总胆红素。 [0087] Note: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; ALP, Alkaline Phosphatase; TBIL, total bilirubin. 代表P〈0 · 05,“#”代表P〈0 · 01。 On behalf of P <0 · 05, "#" represents the P <0 · 01.

[0088] 实施例6.4: HSCs活化指标及促纤维化因子检测 [0088] Example 6.4: HSCs fibrogenic factors and activation markers detected

[0089] 实施例6.4.1 :qRT_PCR检测HSCs活化相关基因表达 [0089] Example 6.4.1 embodiment: qRT_PCR detecting the activation of HSCs gene expression

[0090] 实时荧光定量PCR (Quantitative real-time PCR,qRT-PCR)技术检测MenSCs移植后肝脏中HSCs活化相关标志基因α-SMA和促纤维化因子TGF-βΙ的表达变化。 [0090] Real-time quantitative PCR (Quantitative real-time PCR, qRT-PCR) was detected after liver transplantation MenSCs Activated HSCs expression of a marker gene, and α-SMA profibrotic factor in the TGF-βΙ.

[0091] 在a-SMA表达中,肝纤维化模型组的表达量(6.6倍,相对于注射了橄榄油的正常小鼠组,下同)相比于注射了橄榄油的正常小鼠组有极显著上升;将MenSCs移植至肝纤维化小鼠模型后一周(7.1倍)的表达量相比于将PBS移植至肝纤维化小鼠模型后1周的PBS IW组(8.1倍)有显著下降趋势,同样,将MenSCs移植至肝纤维化小鼠模型后2周的MenSCs 2W组(9.1倍)的表达量相比于将PBS移植至肝纤维化小鼠模型后2周的PBS 2W组(12.3倍)也有显著下调趋势。 [0091] In a-SMA expression, the expression of liver fibrosis model group (6.6 times with respect to the group of normal mice injected olive oil, hereinafter the same) as compared to normal mice injected with a group of olive oil significantly increased; one week the amount of expression (7.1-fold) after the MenSCs transplanted liver fibrosis in mice compared to the PBS after the transplanted liver fibrosis in mice one week IW PBS group (8.1-fold) decreased significantly trends Similarly, a set of 2W expression (9.1-fold) after 2 weeks of MenSCs MenSCs hepatic fibrosis mouse model transplanted at 2 weeks compared to the PBS group 2W (12.3 after the PBS mice transplanted liver fibrosis times) is also a significant downward trend. 类似地,在TGF-βΙ表达中,肝纤维化模型组的表达量(2.2倍)相比于注射了橄榄油的正常小鼠组有极显著上升;将MenSCs移植至肝纤维化小鼠模型后1周的MenSCs IW组和将PBS移植至肝纤维化小鼠模型后1周的PBS IW组的表达并没有显著性差异,然而,将MenSCs移植至肝纤维化小鼠模型后两周后(4.6倍)的表达相比于将PBS移植至肝纤维化小鼠模型后2周的PBS 2W组(3倍)有显著的下调。 After MenSCs transplanted mice liver fibrosis; Similarly, TGF-βΙ expression, expression (2.2-fold) hepatic fibrosis model group as compared to normal mice injected olive oil group had significant increased 1 week MenSCs IW group and PBS was transplanted into an expression 1 week after the PBS group IW hepatic fibrosis in mice and no significant difference, however, after MenSCs transplanted liver fibrosis in mice after two weeks (4.6 fold) in expression compared to PBS mice transplanted liver fibrosis after 2 weeks 2W group PBS (3 times) significantly down-regulated.

[0092] 实施例6.4.2:免疫组织化学检测a-SMA和TGF-βΙ的蛋白表达水平 [0092] Example 6.4.2: Immunohistochemical detection of a-SMA and protein expression of TGF-βΙ

[0093] 在经血干细胞移植到小鼠后的第1周和第2周,分别麻醉并脱颈处死小鼠,分别取MenSCs组(MenSCs IW和MenSCs 2W)和PBS组(PBS IW和PBS 2W)小鼠的肝脏,每个组别有6个样品(η = 6)。 [0093] In the first week and two weeks menstrual blood stem cell transplanted into the mice were anesthetized and were sacrificed mice were taken MenSCs group (MenSCs IW and MenSCs 2W) and PBS group (PBS PBS IW and 2W) mouse liver, six samples per group (η = 6). 将肝脏组织制作成干燥的石蜡切片(参照2.1.3.3.2.2),利用EnVi s ion (Dako) 二步法来检测α-SM和TGF-βΙ的表达情况。 The dried made into liver tissue paraffin sections (see 2.1.3.3.2.2) using EnVi s ion (Dako) two step to detect the expression of α-SM, and the TGF-βΙ. 免疫组织化学具体的操作步骤如下: Immunohistochemistry specific steps are as follows:

[0094] (1)脱錯:先用二甲苯I浸泡lOmim,然后用二甲苯II浸泡IOmin; [0094] (1) off the error: soaked with xylene lOmim I, II and then soaked with xylene IOmin;

[0095] (2)将干燥的石蜡切片放置于二甲苯中,依次在梯度酒精中(100%酒精3min,95% 酒精3min,80%酒精3min,70%酒精3min)进行脱錯处理,然后水洗2min; [0095] (2) The dried xylene paraffin sections placed sequentially performed error removal process in graded alcohols (100% ethanol 3min, 95% alcohol 3min, 80% alcohol 3min, 70% ethanol 3min), and then washed with water 2min;

[0096] ⑶抗原修复:切片放入到已经预热好的0.01M(mol/L)柠檬酸缓冲液(PH = 6.0) 中,将其煮沸(约20min),然后取出切片让其自然的冷却; [0096] ⑶ antigen retrieval: slices into the already preheated 0.01M (mol / L) citric acid buffer (PH = 6.0), which was boiled (about 20min), and then allowed to naturally cool remove the slices ;

[0097] (4)阻断内源性过过氧化氢(H2O2)酶:加入3%的H2O2 (约IOmin),然后用蒸馏水进行水洗3min,之后再用PBS(0.01M PH=7.5)清洗切片三次(每次约5min); [0097] (4) through the block endogenous hydrogen peroxide (H2O2) Enzyme: 3% by H2O2 was added (about IOmin), 3min and then rinsed with distilled water, and then after PBS (0.01M PH = 7.5) Sections were washed three times (each about 5min);

[0098] (5)滴加α-SMA和TGF-βΙ—抗,工作浓度为1:100,将切片放置在湿盒中,于4°C冰箱内孵育过夜; [0098] (5) solution of α-SMA and anti-TGF-βΙ- working concentration of 1: 100, the sections were placed in a humidified chamber at 4 ° C in a refrigerator overnight incubation;

[0099] (6) PBS清洗切片三次(每次约5min),然后滴加HRP共辄的抗鼠或抗兔IgG二抗(EnVision工作液),将其放置于室温30min; [0099] (6) PBS the sections were washed three times (about 5min each), followed by dropwise addition of anti-mouse HRP co noir or anti-rabbit IgG secondary antibody (an EnVision working solution), which was left at room temperature for 30 min;

[0100] (7) PBS清洗切片三次海次约5min),然后加入DAB显色液孵育20min; [0100] (7) PBS the sections were washed three times sea about 5min), followed by addition of DAB color was incubated for 20min;

[0101] ⑶蒸馏水洗,终止显色; [0101] ⑶ of distilled water, color development was stopped;

[0102] (9)用Harris苏木精来复染切片30s,然后用蒸馏水清洗切片(约lOmin,是为了进行兰化); [0102] (9) with Harris hematoxylin counterstain slice to 30s, and then the sections were washed with distilled water (about lOmin, Portland is for the purpose);

[0103] (10)依次在梯度酒精中(100%酒精3min,95%酒精3min,80%酒精3min,70%酒精3min)处理,然后用二甲苯透明,接着用中性树脂进行封片; [0103] (10) sequentially in graded alcohols (100% ethanol 3min, 95% alcohol 3min, 80% alcohol 3min, 70% ethanol 3min) followed by treatment with xylene, were mounted with neutral resin, followed by;

[0104] (11)瞭干,在显微镜(1X83,Olympus)下观察并拍照。 [0104] (11) dry is observed and photographed under a microscope (1X83, Olympus).

[0105] 分别收集四个组(CCl4组,Vehicle组,MenSCs组和PBS组)小鼠的肝组织,利用免疫组化检测α-SMA和TGF-βΙ的蛋白表达情况。 [0105] were collected four group (CCl4 group, Vehicle group, MenSCs group and PBS group) on liver, protein expression using immunohistochemistry and α-SMA of TGF-βΙ. 纤维化模型组命名为CCl4组;将只注射橄榄油的正常组小鼠命名为Vehicle组;将MenSCs移植到肝纤维化模型小鼠命名为MenSCs组;以及将PBS注射到肝纤维化模型小鼠命名为PBS组。 Fibrosis model group named group CCl4; normal mice were injected with the olive oil is named Vehicle group; MenSCs transplanted into the liver fibrosis model mice named MenSCs group; and PBS was injected into the hepatic fibrosis model mice named for the PBS group. 在Vehicle组中a-SMA的表达仅限于血管平滑肌细胞和中央静脉周围;而在CCU组中a-SMA能够表达于窦周间隙,表明HSCs被激活了。 Vehicle group in the a-SMA expression was limited to blood vessels and smooth muscle cells surrounding central vein; in CCU group capable of being expressed in a-SMA sinus gap indicates the activated HSCs. MenSCs移植到纤维化小鼠1周和2周后,MenSCs IW组(相比于PBS IW组)和MenSCs 2W组(相比于PBS 2W组)的a-SMA表达明显受到了抑制,说明MenSCs移植能够降低HSCs的激活。 MenSCs fibrosis in mice transplanted to 1 week and 2 weeks after, MenSCs IW group (group compared to the PBS IW) and a-SMA expression (compared to the PBS group 2W) 2W of the group was inhibited significantly MenSCs described transplant MenSCs It can reduce the activation of HSCs. 类似地,在Vehicle组中TGF-βΙ表达于细胞浆中且几乎全部表达,而在CCl4组中TGF-βΙ的表达也是全部表达于所有细胞中,但是其表达量明显增强。 Similarly, in the Vehicle group, TGF-βΙ expressed in the cytoplasm and almost all of the expression in the expression of TGF-βΙ CCl4 group is also expressed in all cells all, but its expression was significantly enhanced. MenSCs移植到纤维化小鼠1周和2周后, MenSCs IW组的TGF-βΙ表达与PBS IW组相比,并没有显著性差异,而MenSCs 2W组(相比于PBS 2W组)的TGF-βΙ表达强度明显减弱。 MenSCs fibrosis in mice transplanted to 1 week and 2 weeks after, MenSCs IW group of TGF-βΙ expression group compared with PBS IW, and no significant difference, and MenSCs 2W group (group compared to the PBS 2W) of TGF- βΙ expression intensity decreased significantly.

[0106] 实施例6.4.3:Western blot检测a-SMA的蛋白表达 [0106] Example 6.4.3: Western blot detection of protein expression of a-SMA

[0107] 麻醉处死小鼠后,分别收集四个组(CC14组,Vehicle组,MenSCs组和PBS组,各组的命名同实施例6.4.2)小鼠的肝脏组织,取米粒大小的肝脏,用小型匀浆器破碎组织。 [0107] Mice were sacrificed after anesthesia, four groups were collected (CC14 group, Vehicle group, MenSCs group and PBS group, each group designated with the embodiment of Example 6.4.2) of mouse liver tissue, the liver of the grain size, crushed with a small tissue homogenizer. 首先加入500yL的蛋白裂解液(按照比例RIPA裂解液:PMSF= 100 :1进行配制),然后在冰上进行裂解(约60min),接着4°C离心(12000rpm,5min),将离心所得的上清液转移到1.5mL的EP管中。 500yL added first protein lysate (according to the ratio of RIPA lysis buffer: 1 for preparation: PMSF = 100), and then lysed (approximately 60min) on ice, followed by 4 ° C centrifugation (12000 rpm, 5min), the resultant centrifugal the supernatant was transferred to a 1.5mL EP tube. 利用BCA试剂盒测定肝组织样品的蛋白浓度,做好记录并保存于-80°C冰箱备用。 Protein concentrations were determined using liver tissue samples BCA kit, make a record and stored at -80 ° C refrigerator spare.

[0108] 每个组别取20yg蛋白上清液,western blot实验检测a-SMA蛋白表达。 [0108] each group taking 20yg supernatant protein, western blot assay expression of a-SMA. 具体步骤如下: Specific steps are as follows:

[0109] (1)蛋白上清液按照1:4的比例混合于4X loading buffer (Life Technologies) 中,将混合后的样品放入到98°C金属加热仪(加热5min),使样品中的蛋白质得到充分的变性; [0109] (1) The supernatant protein ratio of 1: 4 mixed in 4X loading buffer (Life Technologies), the samples were placed in a 98 ° C mixed metal heating apparatus (heating 5min), a sample of protein denaturation full;

[0110] (2)把10%预制胶(Life Technologies)放入电泳槽中,将样品加入新鲜的电泳缓冲液(Life Technologies)中跑胶,以电压为120V的强度进行电泳(大约70min,以蓝色指示剂到达预制胶3/4处为宜); [0110] (2) The 10% precast gels (Life Technologies) into the electrophoresis tank, the samples were added to fresh electrophoresis buffer (Life Technologies) in the gel was run, at a voltage of 120V was electrophoresed intensity (about 70min to Ready gel blue indicator reaches 3/4 appropriate);

[0111] (3)用专门的切胶器进行切胶,然后依次铺上三层海绵,四层过滤滤纸,切下来的凝胶,PVDF膜(Millipore),四层过滤滤纸,三层海绵,随后将配制好的转膜缓冲液倒入western blot电泳槽(Life Technologies)中,接着将其转入4°C冰箱中且以恒定的电流(200mA)进行转膜Ih; [0111] (3) is cut with a special cut plastic gel, and then successively covered sponge three, four filter paper, cut out of the gel, PVDF membrane (Millipore), four filter paper, sponge three, then the prepared transfer buffer poured western blot electrophoresis tank (Life Technologies), followed by 4 ° C which was transferred to a refrigerator and a constant current (200mA) for Ih is wiped film;

[0112] (4)用0.5%的BSA溶液(生工生物科技有限公司),并放置于摇床上室温平摇封闭Ih左右; [0112] (4) with 0.5% BSA solution (Sangon Biological Technology Co., Ltd.), and placed on a shaker at room temperature of about Ih is closed pan;

[0113] (5)用TBST洗膜2次海次洗膜约10min,置于摇床上进行),然后加入一抗于4°C孵育过夜; [0113] (5) washing the membrane with TBST Wash 2 times sea views film of about 10min, placed on a shaker for), and then primary antibody was added and incubated overnight at 4 ° C;

[0114] ⑶一抗回收,用TBST洗膜3次海次洗膜约10min,置于摇床上进行); [0114] ⑶ antibody recovered, washed 3 times sea membrane was washed with TBST film of about 10min, placed on a shaker for);

[0115] ⑵加入相应的二抗,然后在室温下放置于摇床中孵育Ih; [0115] ⑵ appropriate secondary antibody was added, then placed on a shaker decentralized incubated Ih at room temperature;

[0116] (8)用TBST洗膜3次(每次洗膜约IOmin,置于摇床上进行),加入ECL化学发光液(Bio-Rad),在全自动凝胶成像系统(Tanon-4500智能转换型)下进行扫膜并拍照。 [0116] (8) The membrane was washed with TBST 3 times (each wash for approximately IOmin film, placed on a shaker for), was added ECL chemiluminescence (Bio-Rad), the automatic gel imaging system (Tanon-4500 Intelligent scavenged film transfer type) and photographed under.

[0117] 其中,转膜缓冲液的配方:甘氨酸为2.9g,Tris为5.8g,SDS为0.37g,甲醇200mL,加CldH2O定容至IOOOmL; TBST的配方:Tris-base为24.4g和NaCl为80g,用浓HCl调整PH至7.4 (?財旨示剂),然后加0.05%的1¥661120姐8111&amp;)〇 [0117] wherein, in formula transfer buffer: glycine 2.9g, Tris is 5.8g, SDS was 0.37g, 200 mL methanol, added to the volume CldH2O IOOOmL; TBST formula: Tris-base of 24.4g of NaCl and 80g, adjusted with concentrated HCl to PH 7.4 (? purpose financial agent shown), and then adding 0.05% 1 ¥ 661120 sister 8111 & amp;) square

[0118] 结果表明,Vehicle组几乎不表达a-SMA,CCl4组的表达量呈显著上升;MenSCs IW 组的蛋白表达量相比于PBS IW组有显著下降趋势,同样地,MenSCs 2W组的蛋白表达量相比于PBS 2W组也有显著下调趋势。 [0118] The results show that, Vehicle group is hardly expressed a-SMA, the expression of CCl4 group was significantly increased; protein expression MenSCs IW group compared to the PBS IW group significantly decreased, in the same manner, MenSCs 2W group of proteins expression compared to the PBS 2W group also has a significant downward trend.

[0119] 为了进一步调查经血干细胞是如何改善肝纤维化,本发明的发明人进行了一下实施例。 [0119] To further investigate how to improve the blood stem cells of liver fibrosis, the present inventors conducted a bit embodiment.

[0120] 实施例7:MenSCs和LX-2细胞Transwell模型的建立 7 [0120] Example: to establish MenSCs and LX-2 Transwell cell model

[0121] 为了研究MenSCs对LX-2细胞的影响,利用Transwell小室(滤膜直径:24mm,滤膜孔径:0.4ym;Crning),将本研究分为3个组别,即LX-2组(n = 4),2X IO5ZVell的LX-2细胞铺于Transwell小室的下层;MenSC组(n = 4),2X 105/well的MenSCs铺于Transwell小室的上层; 共培养组(co-culture组)(n = 4),2X 105/well的LX-2细胞铺于Transwell小室的下层并且2 X 105/well的MenSCs铺于Transwell小室的上层。 [0121] To study the effect MenSCs on LX-2 cells, using the Transwell chamber (filter diameter: 24mm, membrane pore size: 0.4ym; Crning), the present study was divided into three groups, i.e., LX-2 group ( n = 4), 2X IO5ZVell of LX-2 cells were plated in the lower Transwell chamber of; MenSC group (n = 4), 2X 105 / well of MenSCs plated on the upper Transwell chamber; a co-culture group (co-culture group) ( n = 4), 2X 105 / well of LX-2 cells were plated in the lower chambers of the Transwell and 2 X 105 / well is plated in MenSCs the upper Transwell chamber. 每个小室均加3mL的DMEM培养基(10% FBS)。 3mL of DMEM medium (10% FBS) was added to each cell.

[0122] 实施例8: MenSCs对LX-2细胞增殖的影响 8 [0122] Example: Effect MenSCs on LX-2 cell proliferation

[0123] 为了研究MenSCs对LX-2细胞增殖的影响,采CCK-8 (Do jindo)试剂盒检测LX-2组和共培养组(co-culture组)中LX-2细胞增殖情况,具体的实验步骤如下: [0123] To study the effect MenSCs on LX-2 cell proliferation, mining CCK-8 (Do jindo) kit LX-2 group and the proliferation of co-culture group (co-culture group) in LX-2 cells, specific experimental steps:

[0124] (I) LX-2组(n = 4)和共培养组(co-culture组)(n = 4)共培养24h,48h和72h; [0124] (I) LX-2 group (n = 4) and co-culture group (co-culture group) (n = 4) were cultured 24h, 48h, and 72h;

[0125] (2)按照说明书,分别向其底层加入一定量的CCK-8试剂(同时设定空白对照组; [0125] (2) according to the instructions, to which were added a quantity of the underlying CCK-8 reagent (while setting the control group;

[0126] (3)将培养板在培养箱内孵育2h (视情况l_3h); [0126] (3) The plates were incubated for 2h (optionally l_3h) in an incubator;

[0127] (4)用多功能酶标仪(SpectraMax M5 ,Molecular Devices)测定在450nm处的OD 值; [0127] (4) with a multi-functional microplate reader (SpectraMax M5, Molecular Devices) at OD values ​​measured at 450nm;

[0128] (5)计算,将所得到的OD值减去相应的空白对照。 [0128] (5) calculated, OD value obtained by subtracting the corresponding blank. 所得结果即为该组别的OD450。 The result is the OD450 of the groups.

[0129] 2 · 2 · 4 · IMenSCs抑制LX-2细胞增殖 [0129] 2 · 2 · 4 · IMenSCs LX-2 cell proliferation inhibition

[0130] 在共培养实验进行之前,验证了LX-2细胞。 [0130] Prior to co-culture experiments carried out to verify the LX-2 cells. 通过细胞免疫荧光染色技术,发现LX-2 细胞几乎全部表达α-SMA (>95 %),同时放大400倍后发现细胞生长状态良好,基本骨架清晰正常。 By immunofluorescence staining technique, we found that almost all of LX-2 cells express α-SMA (> 95%), while the 400-fold amplification was found good cell growth state, normal clear basic skeleton. 说明LX-2细胞已经处于激活状态,能够作为后续的共培养实验。 LX-2 cells has been described in an active state, it can be used as the subsequent co-culture experiments.

[0131] 在共培养实验中,观测了LX-2组和共培养组(co-culture组)在共培养24h,48h和72h之后,LX-2细胞的细胞形态和数目。 [0131] In the co-culture experiments, observations of LX-2 group and the group co-cultivation (co-culture group) 24h, 48h, and 72h after, shape and number of cells in the LX-2 cells were co-cultured. 发现,虽然在细胞培养24h后,LX-2组和共培养组(co-culture组)中的LX-2细胞数目差异不显著;在细胞共培养48h和72h后,共培养组(C0-culture组)中的LX-2细胞的数目明显少于LX-2组。 Found though, after cultured for 24h in a cell, the number of differences LX-2 cells LX-2 group and the co-culture group (co-culture group) is not significant; co-cultured cells after 48h and 72h, co-culture group (C0-culture number of LX-2 cells in the group) is significantly less than the LX-2 group. 更进一步,用CCK-8实验检测并验证了LX-2细胞增殖情况,发现48h和72h的共培养后,共培养组(co-culture组)中的OD值明显低于LX-2组,也就是说共培养使得LX-2细胞的生长和增殖明显受到抑制。 Still further, by CCK-8 assay and verify the LX-2 cell proliferation, 48h and 72h after co-culture found co-culture group (co-culture group) significantly lower than the OD values ​​in LX-2 group, and that co-culture such that the growth and proliferation of LX-2 cells was significantly inhibited.

[0132] 实施例9:MenSCs对LX-2细胞周期的影响 9 [0132] Example: Effect MenSCs on LX-2 cell cycle

[0133] 为了研究MenSCs对LX-2细胞周期的影响,利用细胞周期试剂盒(Sigma)检测LX-2 组和共培养组(co-culture组)中LX-2细胞培养48h和72h的细胞周期情况。 [0133] To study the effect MenSCs on LX-2 cell cycle, using a cell cycle kit (Sigma) detection of LX-2 group and the co-culture group (co-culture group) in LX-2 cell culture cycle 48h and 72h of Happening. 具体的细胞周期实验步骤如下: Cell cycle specific experimental procedure is as follows:

[0134] (I) LX-2组(n = 4)和共培养组(co-culture组)(n = 4)共培养48h和72h; [0134] (I) LX-2 group (n = 4) and co-culture group (co-culture group) (n = 4) were cultured 48h and 72h;

[0135] (2)将细胞消化于15 mL离心管中,用7 0 %预冷的乙醇固定,随后室温沉淀细胞(1000rpm,5min),去上清; [0135] (2) The cells were digested in 15 mL centrifuge tube, chilled in 7 0% ethanol-fixed, and then the cells were pelleted at room temperature (1000rpm, 5min), the supernatant was discarded;

[0136] (3)加ImL冷PBS重悬细胞,随后沉淀细胞(1000rpm,5min),倒掉PBS; [0136] (3) was added ImL cells were resuspended in cold PBS, then the cells were pelleted (1000rpm, 5min), drained PBS;

[0137] ⑷配制PI染色液,按照下列比例配制,包括缓冲液(475yL),20XPI染色液(25yL) 和50 XRNAase A(IOyL); [0137] ⑷ PI staining solution formulation, prepared according to the following proportions, including buffers (475yL), 20XPI staining solution (25yL) and 50 XRNAase A (IOyL);

[0138] ⑶每管细胞加入500yL的PI染色液,然后缓慢并充分地重悬细胞; [0138] ⑶ cells were added to each tube 500yL PI staining solution, and then slowly and sufficiently resuspended cells;

[0139] (6) 37°C避光孵育30min,然后用300目的细胞筛过滤细胞,利用流式细胞仪(Beckman)检测细胞周期。 [0139] (6) 37 ° C dark for 30min, and then a 300 mesh screen filter cell cells, the cell cycle by flow cytometry (Beckman).

[0140] 通过流式细胞术检测了LX-2组和共培养组(co-culture组)中LX-2细胞48h和72h 的细胞周期情况。 [0140] Detection of LX-2 group and the group co-cultivation (co-culture group) period in the case of LX-2 cells for 48h and 72h by flow cytometry. 共培养48h后,LX-2组和共培养组(co-culture组)中的LX-2细胞G0/G1期, S期和G2/M期分别是47±2% (G0/G1),35±3% (S),18±1% (G2/M)和62±3% (G0/G1),23土2%⑶,15±1% ®2/M)。 After co-culture 48h, LX-2 group and the co-culture group (co-culture group) in LX-2 cells in G0 / G1 phase, S phase and G2 / M phase were 47 ± 2% (G0 / G1), 35 ± 3% (S), 18 ± 1% (G2 / M), and 62 ± 3% (G0 / G1), 23 soil 2% ⑶, 15 ± 1% ®2 / M). 类似地,共培养72h后,LX-2组和共培养组(co-culture组)中的LX-2细胞G0/G1 期,S期,G2/M期分别是71±3% (G0/G1),15±2% ⑶,14±1% (G2/M)和83±3% (G0/G1),8±2% ⑶,9±1% (G2/M)。 Similarly, co-culture after 72h, LX-2 group and the co-culture group (co-culture group) in LX-2 cells in G0 / G1 phase, S phase, G2 / M phase were 71 ± 3% (G0 / G1 ), 15 ± 2% ⑶, 14 ± 1% (G2 / M), and 83 ± 3% (G0 / G1), 8 ± 2% ⑶, 9 ± 1% (G2 / M). 也就是说在48h和72h时,共培养组(co-culture组)中, 处于G0/G1期的LX-2细胞显著高于LX-2组。 LX-2 cells at 48h and 72h that is, co-culture group (co-culture group), in the G0 / G1 phase was significantly higher than group LX-2. 说明MenSCs能够将LX-2细胞阻滞于G0/G1期,并能抑制其增殖。 DESCRIPTION MenSCs possible to LX-2 cells were arrested in G0 / G1 phase, and can inhibit proliferation.

[0141] 实施例10:抗体芯片检测细胞上清液的蛋白表达 10 [0141] Example: Antibody microarray expression cell supernatants

[0142] 为了进一步探究MenSCs分泌哪些因子来对LX-2细胞增殖产生抑制效应,利用人细胞因子G1000芯片(AAH-CYT-G1000,RayBiotech)来检测LX-2组,MenSC组和共培养组(co-culture组)培养72h后120个细胞因子的上清。 [0142] To further explore which factors MenSCs secretion the proliferation of LX-2 cells inhibitory effect using human cytokine G1000 chip (AAH-CYT-G1000, RayBiotech) to detect the LX-2 group, MenSC group and the co-culture group ( co-culture group) 120 culture supernatant cytokine after 72h. 细胞上清采用上下层培养液混合后统一收集的方式。 By way of cell supernatant was mixed culture on the lower rear of uniform collection. 具体的实验步骤如下: Specific experimental procedure is as follows:

[0143] (1)将新的玻片芯片(保存于-20°C)从盒子中取出来,在室温下平衡30min,然后将芯片放置于干燥器中2h,以保证芯片完全干燥; [0143] (1) A new chip slides (stored at -20 ° C) taken out from the box, equilibrated at room temperature 30min, and then placing the chip in a desiccator 2h, to ensure that the chip is completely dry;

[0144] (2)每个芯片孔中加入IOOyL的IX封闭液,然后在室温摇床上孵育30min(主要是为了避免产生气泡而影响实验结果); [0144] (2) Each chip is added to the wells IOOyL IX blocking buffer, then incubated shaker for 30 min (mainly to avoid air bubbles affect the results) at room temperature;

[0145] (3)将封闭液倒掉,在每个孔中添加IOOyL细胞上清样品(LX-2组,MenSC组和共培养组(co-culture组),n = 4),一个阵列加入一个样品(n = 4),将芯片平稳放置并于4°C孵育过夜; [0145] (3) The blocking solution was discarded, and the supernatant sample was added IOOyL cells (LX-2 group, MenSC group and the group co-cultured (co-culture group), n = 4), an array was added to each well a sample (n = 4), the chip is placed smoothly and incubated overnight at 4 ° C;

[0146] ⑷使用Thermo Scientific Wellwash Versa芯片洗板机清洗玻片; [0146] ⑷ using Thermo Scientific Wellwash Versa chip cleaning slide washer;

[0147] (5)配制生物素标记的抗体,然后快速离心标记有生物素抗体的小管,每个小管中加入300yL的IX封闭液,混合均匀后再每孔加入70yL的生物素标记抗体; [0147] (5) Preparation of biotin-labeled antibody, then quickly centrifuged labeled with biotin antibody tubule, each vial was added 300yL IX of blocking solution, mixed well and then added to each well of biotinylated antibody biological 70yL;

[0148] (6)每孔加入等量的(70μ〇荧光剂-链霉亲和素(1500倍稀释比例),然后快速离心并加入1.5mL的封闭液到小管中,接着用密封条将玻璃芯片贴住,然后再用铝箱纸包住玻璃芯片并在避光条件下室温孵育2h; [0148] (6) per well was added an equal amount of (70μ〇 phosphor - streptavidin-biotin (1500 times dilution ratio), and then quickly centrifuged 1.5mL blocking buffer was added to the vial, followed by glass sealing strip chip stick, then use to wrap the glass chip Aluminum and incubated 2h at room temperature in the dark;

[0149] (7)焚光检测:米用激光扫描仪(GenePix 4000B Microarray Scanner)扫描信号, 采用数据分析软件对因子进行相关的数据分析(具体筛选因子的条件:共培养组(coculture 组) 与LX-2 组/MenSC组均有显著性差异,荧光强度值不低于300) 。 [0149] (7) burning photodetection: m using a laser scanner (GenePix 4000B Microarray Scanner) scanning signal using data analysis software of the factors related to data analysis (specific factions of conditions: co-culture group (coculture group) and LX-2 group / MenSC groups were significantly different, the fluorescence intensity values ​​of not less than 300).

[0150] 2.2.4.3抗体芯片检测差异表达的因子 [0150] 2.2.4.3 factor expression microarray differences antibody

[0151] 结果发现,MCP-I,IL-6,HGF,GR0, IL-8和OPG高表达于共培养组(co-culture组) (相比较LX-2组和MenSC组)。 [0151] It was found, MCP-I, IL-6, HGF, GR0, IL-8 and OPG expression in co-culture group (co-culture group) (compared MenSC LX-2 group, and groups). 此外,对所有差异表达的因子(详见表2)进行了热图分析。 In addition, differences in cytokine expression of all (see Table 2) was subjected to thermal analysis in FIG. 进一步确信MCP-I,IL-6,HGF,GR0, IL-8和OPG是这些差异因子中更为显著的。 Further believed that MCP-I, IL-6, HGF, GR0, IL-8 and OPG these differences are more significant factors. 这些因子中,MCP-I 和IL-6的上升倍数最显著,其次是HGF,GR0和IL-8。 These factors, MCP-I and IL-6 fold increase in the most significant, followed by HGF, GR0, and IL-8. 有趣的是,OPG因子在MenSC组和共培养组(co-culture组)均能高表达,而在LX-2组几乎不表达。 Interestingly, expression of OPG can factor in coculture group MenSC group and (co-culture group), but hardly expressed in the LX-2 group.

[0152] 表2差异表达的因子和编号 [0152] Table 2 cytokine expression, and differences in numbers

Figure CN105944086BD00151

[0154] 实施例11 :ELISA检测细胞上清液以及细胞裂解液的蛋白表达 [0154] Example 11: ELISA detection of expression of cell supernatant and cell lysate

[0155] 为了进一步验证这些抗体芯片中得到的差异较明显的因子(MCP-1,IL-6,HGF, GRO,IL-8和0PG),利用ELISA试剂盒(RayBiotech),定量检测LX-2组,MenSC组和共培养组(co-culture组)培养72h后细胞上清液以及细胞的蛋白表达量。 [0155] To further verify these antibodies chips obtained obvious difference factor (MCP-1, IL-6, HGF, GRO, IL-8 and 0PG), using an ELISA kit (RayBiotech), quantitative detection of LX-2 group, and the cell supernatant protein expression after 72h MenSC cells co-cultured group and the group (co-culture group) culture. 细胞的测定是通过细胞裂解液(CST)来收集并检测,其中的共培养-LX-2代表共培养组(co-culture组)中下层的LX-2 细胞;共培养-MenSC代表共培养组(co-culture组)中上层的MenSCs。 Assay cells are collected by cell lysates (CST) and detection, wherein the co-cultured -LX-2 Representative co-culture group (co-culture group) in the lower layer LX-2 cells; co-culture -MenSC Representative co-culture group upper MenSCs (co-culture group). 具体的ELISA实验步骤如下: ELISA assays specific steps are as follows:

[0156] ⑴将芯片放置在试剂盒以及样品平衡至室温(18-25Γ),所有标准品以及样品使用单孔检测; [0156] ⑴ placing the chip in the kit and samples to equilibrate to room temperature (18-25Γ), and a sample using all standard detection hole;

[0157] (2)将已经包被抗体的ELISA板放置于室温下平衡30min,随后在对应的孔中加入IOOyL配制好的标准品(Raybiotech)及样品(细胞上清或细胞裂解液),接着用封板膜封住板条并于4°C孵育过夜; [0157] (2) has the antibody-coated ELISA plates were left at room temperature equilibrium 30min, followed by addition of the prepared standard IOOyL (Raybiotech) and sample (cell lysate or cell supernatant) in the corresponding holes, followed by sealed strips with an adhesive strip and incubated overnight at 4 ° C;

[0158] (3)将配制好的IX洗液放置于洗板机上,用洗板机来清洗板条(清洗4次,每次IOmin),随后在每个芯片孔中加入300yL的洗液; [0158] (3) The prepared lotion IX placed on a plate washer with a washer to clean the slats (washed four times IOmin), followed by addition of the washings each chip 300yL hole;

[0159] (4)将洗板清洗干净后,再在每个芯片孔中加入IOOyL配制好的检测抗体(用生物素抗体进行标记)并于室温下孵育Ih; [0159] (4) After the plate was washed clean, formulated IOOyL added detection antibody (an antibody labeled with biotin) and incubated for Ih at room temperature the wells of each chip;

[0160] (5)每个芯片孔中加入IOOyL配制好的HRP-链霉亲和素并于室温下孵育45min,然后继续清洗板条步骤同(3); [0160] (5) added to each chip IOOyL the prepared hole HRP- streptavidin, avidin and incubated for 45min at room temperature, then continue with step cleaning strip (3);

[0161] (6)加入IOOyL TMB (3,3',5,5'-Tetramethylbenzidine)显色液到每个芯片孔中并于室温下避光孵育30min; [0161] (6) was added IOOyL TMB (3,3 ', 5,5'-Tetramethylbenzidine) color reagent to each of the chips well and incubated in the dark at room temperature for 30min;

[0162] (7)加入50yL终止液到每个芯片孔中,然后用多功能酶标仪(Molecular Devices) 测定在450nm处的OD值,采用Sigmaplot 12.0软件来计算样品中的蛋白浓度值。 [0162] (7) was added to the protein concentration in the sample 50yL stop solution to each chip in the hole, OD value at 450nm was measured using the multi-functional microplate reader (Molecular Devices), calculated using Sigmaplot 12.0 software.

[0163] 为了进一步验证这些抗体芯片中得到的差异较明显的因子(MCP-1,IL-6,HGF, GR0,IL-8和0PG),利用ELISA定量检测LX-2组,MenSC组和共培养组(co-culture组)培养72h 后细胞上清液以及细胞的蛋白表达量。 [0163] To further verify these antibodies chips obtained obvious difference factor (MCP-1, IL-6, HGF, GR0, IL-8 and 0PG), quantified by ELISA detection of LX-2 group, MenSC group and co cell supernatant and protein expression after 72h of cell culture group (co-culture group) culture. 在细胞上清的结果中,其表达量虽然和抗体芯片不一致,但是其趋势和抗体芯片一致,进一步确认MCP-I,IL-6,HGF,GRO,IL-8和OPG因子的重要作用(图1A)。 Results in cell supernatants, although the expression level, and antibody chips inconsistent, but consistent with the trend and antibody chips, further confirmed MCP-I, IL-6, an important role of HGF, GRO, IL-8 and OPG factor (FIG. 1A).

[0164] 此外,为了确认这些因子中的主要分泌者是MenSCs还是LX-2细胞,使用细胞ELISA 来进一步验证。 [0164] Further, in order to confirm these factors is primarily secreted by MenSCs or LX-2 cells were used to further validate the cell ELISA. 三个组别的细胞(LX-2组,LX-2细胞;共培养组(co-culture组),共培养-LX-2和共培养-MenSC; MenSC组,MenSCs)用细胞裂解液进行裂解,然后进行ELISA检测。 Three groups of cells (LX-2 group, LX-2 cells; co-culture group (co-culture group), co-culture and co-culture -LX-2 -MenSC; MenSC group, MenSCs) were lysed with a cell lysis buffer , followed by ELISA. 从结果可知(图1B),共培养-MenSC组相比于单一的MenSC组:MCP-I (361±33vs.29±5pg/mg; 12 倍),11-6 (967±10切8.90±23口8/11^;11倍),邢? From the results can be seen (FIG. 1B), as compared to a group of co-culture -MenSC single MenSC group: MCP-I (361 ± 33vs.29 ± 5pg / mg; 12-fold), 11-6 (967 ± 10 8.90 ± 23 cut port 8/11 ^; 11 times), Xing? (927±20扣8.176±48口8/11^;5倍),61?0 (432±96丫8.224±69口8/11^;2倍),11^-8 (1033±163¥8.403±75口8/11^;3倍)和0?6(1338± 27(^8.510±103?8/11^;3倍)。类似地,共培养-1^-2组相比于单一的1^-2组:61?0(94± 25vs · 12 ± 6pg/mg; 8倍)和IL-8 (165 ± 46vs · 24 ± 9pg/mg; 7倍),而剩下的MCP-I,IL-6,HGF和OPG无显著性差异。 (927 ± 20 8.176 ± 48 snap opening 8/11 ^; 5 fold), 61 0 (432 ± 96 8.224 ± 69 Ah opening 8/11 ^; 2-fold)?, 11 ^ -8 (1033 ± 163 ¥ 8.403 ± 8 75/11 ^; 3 times), and 0 6 (1338 ± 27 (^ 8.510 ± 103 8/11 ^;? 3 times)?. Similarly, co-cultivation -1 (-2) compared to a single group 1 ^ -2 group:? 61 0 (94 ± 25vs · 12 ± 6pg / mg; 8-fold) and IL-8 (165 ± 46vs · 24 ± 9pg / mg; 7-fold), while the remaining MCP-I, IL -6, no significant difference between HGF and OPG.

[0Ίό5]实施例12:白介素-8 (IL-8)改善小鼠的肝纤维化 [0Ίό5] Example 12: Interleukin -8 (IL-8) improvement in liver fibrosis in mice

[0166] 如实施例6.1和6.2所示,以1.5mg/Kg的剂量通过尾静脉注射重组人白介素-8 (IL-8) (R&amp;D Systems公司)到已经构建好的纤维化模型小鼠中(白介素-8 (IL-8)组),对照为只注射等量PBS的肝纤维化模型小鼠(PBS组)。 [0166] As shown in Example 6.1 and 6.2, at a dose of 1.5mg / Kg by tail vein injection of recombinant human interleukin -8 (IL-8) (R & amp; D Systems, Inc.) to prebuilt fibrosis model mice in (interleukin -8 (IL-8) group), control of hepatic fibrosis model mice injected with an equal amount of PBS (PBS group). 在注射后的第1周和第2周,处死小鼠并进行SR 染色,观察并检测胶原纤维面积。 At 1 week and 2 weeks after injection, mice were sacrificed and SR staining and detecting collagen area. 结果表明,尽管在第1周白介素-8 (IL-8)组(1.8%)的胶原积累面积低于PBS组(2.4%),但其在统计学上并没有显著性差异;相反,在第2周白介素-8 (IL-8)组(1.3%)的胶原纤维面积相比于PBS组(3.4%)有明显减少,说明肝纤维化在白介素-8 (IL-8)的作用下,其状况得到了显著改善。 The results showed that, although lower than the PBS group (2.4%) in (IL-8) group (1.8%) of collagen accumulation area of ​​the first week of interleukin-8, but which is not statistically significant differences; contrary, in the 2 weeks interleukin -8 (IL-8) (1.3%) of a collagen fiber area compared to the PBS group (3.4%) significantly decreased, indicating liver fibrosis in the role of interleukin -8 (IL-8), which situation has improved significantly.

[0167] 与此同时,发明人按照实施例6.4进行了免疫组织化学检测α-SM和TGF-βΙ的蛋白表达水平,结果发现,注射白介素-8 (IL-8)到纤维化小鼠1周和2周后,相对于PBS组而言白介素_8 (IL-8)组的α-SMA和TGF-βΙ的表达明显受到了抑制。 [0167] Meanwhile, the inventors performed immunohistochemical detection of α-SM and protein level TGF-βΙ according to Example 6.4, found that injection of interleukin -8 (IL-8) to fibrosis in mice one week after 2 weeks, and, with respect to the set of expression of interleukin PBS _8 (IL-8) α-SMA and TGF-βΙ group significantly suppressed. 这进一步验证了白介素-8 (IL-8)在改善或治疗肝纤维化方面的作用。 This further validated the role of interleukin -8 (IL-8) in improving or treating hepatic fibrosis aspect.

[0168] 实施例13:白介素-8 (IL-8)和骨保护素(OPG)的组合改善小鼠的肝纤维化 [0168] Example 13: combination Interleukin -8 (IL-8) and osteoprotegerin (OPG) improved mice with liver

[0169] 如实施例6.1和6.2所示,分别以1.51^/敁和511^/敁的剂量通过尾静脉注射重组人白介素-8 (IL-8) (R&amp;D Systems公司)和重组人骨保护素(R&amp;D Systems公司)到已经构建好的纤维化模型小鼠中(联合组),对照为注射等量重组人白介素-8 (IL-8)和与重组人骨保护素等量的PBS的肝纤维化模型小鼠(对照组)。 [0169] As shown in, respectively, 1.51 ^ / Dian and 511 ^ / dose Dian by tail vein injection of recombinant human interleukin -8 (IL-8) (R & amp; D Systems, Inc.) as described in Examples 6.1 and 6.2 and Bone Protection Su (R & amp; D Systems, Inc.) to prebuilt fibrosis model in mice (combined group), an equal amount of the control injection of recombinant human interleukin -8 (IL-8) and human recombinant OPG bone equivalent amount of PBS liver fibrosis model mice (control group). 在注射后的第1周和第2周,处死小鼠并进行SR 染色,观察并检测胶原纤维面积。 At 1 week and 2 weeks after injection, mice were sacrificed and SR staining and detecting collagen area. 结果表明,在第1周联合组(1.0%)的胶原积累面积显著低于对照组(2.0%),在第2周联合组(0.6%)的胶原纤维面积相比于对照组(1.3%)有明显减少,说明肝纤维化在白介素-8 (IL-8)和骨保护素(OPG)联合作用下肝纤维化状况得到了更显著改善。 The results show that the combination group at week 1 (1.0%) of the area of ​​collagen accumulation was significantly lower than the control group (2.0%), in the second week combination group (0.6%) compared to the area of ​​the collagen fibers in the control group (1.3%) significantly decreased, indicating liver fibrosis under the combined effects of interleukin -8 (IL-8) and osteoprotegerin (OPG) liver fibrosis situation has improved more remarkably.

[0170] 与此同时,发明人按照实施例6.4进行了免疫组织化学检测α-SM和TGF-βΙ的蛋白表达水平,结果发现,联合组相对于对照组而言a-SMA和TGF-βΙ的表达受到了更明显的抑制。 [0170] Meanwhile, the inventors performed immunohistochemical detection of α-SM and protein level TGF-βΙ according to Example 6.4, the results found in the combination group relative to the control group in terms of a-SMA and the TGF-βΙ expression was more significantly inhibited. 这进一步验证了白介素-8 (IL-8)和骨保护素(OPG)的组合在改善或治疗肝纤维化方面的更加优异的作用。 This further validated the role of interleukin more excellent composition (IL-8) and osteoprotegerin (OPG) is - 8 in improving or treating hepatic fibrosis aspect.

[0171] 实施例14:白介素-8 (IL-8)和单核细胞趋化蛋白I (MCP-I)的组合改善小鼠的肝纤维化 [0171] Example 14: Interleukin -8 (IL-8), and combinations chemoattractant protein-I (MCP-I) monocytes improve mice with liver

[0172] 如实施例6.1和6.2所示,分别以1.51^/敁和411^/敁的剂量通过尾静脉注射重组人白介素-8 (IL-8) (R&amp;D Systems公司)和重组人单核细胞趋化蛋白I(MCP-I) (R&amp;D Systems公司)到已经构建好的纤维化模型小鼠中(联合组),对照为注射等量重组人白介素-8 (IL-8) 和与重组人单核细胞趋化蛋白I (MCP-I)等量的PBS的肝纤维化模型小鼠(对照组)。 [0172] As shown in, respectively, 1.51 ^ / Dian and 411 ^ / dose Dian by tail vein injection of recombinant human interleukin -8 (IL-8) (R & amp; D Systems, Inc.) as described in Examples 6.1 and 6.2 and recombinant human single monocyte chemoattractant protein-I (MCP-I) (R & amp; D Systems, Inc.) to prebuilt fibrosis model in mice (combined group), an equal amount of the control injection of recombinant human interleukin -8 (IL-8) and recombinant human monocyte chemoattractant protein-I (MCP-I) liver fibrosis model mice in an equal amount of PBS (control group). 在注射后的第1周和第2周,处死小鼠并进行SR染色,观察并检测胶原纤维面积。 At 1 week and 2 weeks after injection, mice were sacrificed and SR staining and detecting collagen area. 结果表明,在第1周联合组(1.5%)的胶原积累面积显著低于对照组(2.1%),在第2周联合组(0.9%)的胶原纤维面积相比于对照组(1.4%)有明显减少,说明肝纤维化在白介素-8 (IL-8)和单核细胞趋化蛋白I (MCP-I)联合作用下肝纤维化状况得到了更显著改善。 The results show that the combination group at week 1 (1.5%) of the area of ​​collagen accumulation was significantly lower than the control group (2.1%), in the second week combination group (0.9%) compared to the area of ​​the collagen fibers in the control group (1.4%) significantly decreased, indicating liver fibrosis under the combined effects of interleukin -8 (IL-8) and monocyte chemoattractant protein-I (MCP-I) liver fibrosis situation has improved more remarkably.

[0173] 与此同时,发明人按照实施例6.4进行了免疫组织化学检测a-SMA和TGF-βΙ的蛋白表达水平,结果发现,联合组相对于对照组而言a-SMA和TGF-βΙ的表达受到了更明显的抑制。 [0173] Meanwhile, the inventors carried out as in Example 6.4 immunohistochemical detection of a-SMA and protein expression of TGF-βΙ found in the combination group relative to the control group in terms of a-SMA and the TGF-βΙ expression was more significantly inhibited. 这进一步验证了白介素-8 (IL-8)和单核细胞趋化蛋白I (MCP-I)的组合在改善或治疗肝纤维化方面的更加优异的作用。 This further validates the more excellent effect of interleukin -8 (IL-8) and monocyte chemotactic composition protein I (MCP-I) in improving or treating hepatic fibrosis aspect.

[0174] 实施例15:白介素-8 (IL-8)和肝细胞生长因子(HGF)的组合改善小鼠的肝纤维化 [0174] Example 15: combination Interleukin -8 (IL-8) and hepatocyte growth factor (HGF) improved mice with liver

[0175] 如实施例6.1和6.2所示,分别以1.51^/敁和111^/敁的剂量通过尾静脉注射重组人白介素_8 (IL-8) (R&amp;D Systems公司)和重组人HGF蛋白(R&amp;D Systems公司)到已经构建好的纤维化模型小鼠中(联合组),对照为注射等量重组人白介素-8 (IL-8)和与重组人HGF蛋白等量的PBS的肝纤维化模型小鼠(对照组)。 [0175] As in Example 6.1 and 6.2 show, respectively, at a dose of 1.51 ^ / Dian and 111 ^ / Dian by tail vein injection of recombinant human interleukin _8 (IL-8) (R & amp; D Systems, Inc.) and recombinant human HGF protein (R & amp; D Systems, Inc.) to prebuilt fibrosis model in mice (combined group), an equal amount of the control injection of recombinant human interleukin -8 (IL-8) protein and an equivalent amount of recombinant human HGF in PBS liver fibrosis model mice (control group). 在注射后的第1周和第2周,处死小鼠并进行SR染色,观察并检测胶原纤维面积。 At 1 week and 2 weeks after injection, mice were sacrificed and SR staining and detecting collagen area. 结果表明,在第1周联合组(0.9%)的胶原积累面积显著低于对照组(2.0%),在第2周联合组(0.5%)的胶原纤维面积相比于对照组(1.4%)有明显减少,说明肝纤维化在白介素-8 (IL-8)和HGF联合作用下肝纤维化状况得到了更显著改善。 The results show that the combination group at week 1 (0.9%) of the area of ​​collagen accumulation was significantly lower than the control group (2.0%), in the second week combination group (0.5%) compared to the area of ​​the collagen fibers in the control group (1.4%) significantly decreased, indicating liver fibrosis in interleukin -8 (IL-8) and the combined HGF hepatic fibrosis situation has improved more remarkably.

[0176] 与此同时,发明人按照实施例6.4进行了免疫组织化学检测a-SMA和TGF-βΙ的蛋白表达水平,结果发现,联合组相对于对照组而言a-SMA和TGF-βΙ的表达受到了更明显的抑制。 [0176] Meanwhile, the inventors carried out as in Example 6.4 immunohistochemical detection of a-SMA and protein expression of TGF-βΙ found in the combination group relative to the control group in terms of a-SMA and the TGF-βΙ expression was more significantly inhibited. 这进一步验证了白介素-8 (IL-8)和HGF的组合在改善或治疗肝纤维化方面的更加优异的作用。 This further validates the more excellent effect of the combination of interleukin -8 (IL-8) and HGF in improving or treating hepatic fibrosis aspect.

[0177] 实施例16:白介素-8 (IL-8)和肿瘤生长相关因子(GRO)的组合改善小鼠的肝纤维化 [0177] Example 16: Interleukin -8 (IL-8) and related compositions tumor growth factor (GRO) improved mice with liver

[0178] 如实施例6.1和6.2所示,分别以1.51^/敁和211^/敁的剂量通过尾静脉注射重组人白介素-8 (IL-8) (R&amp;D Systems公司)和重组人GROa蛋白(R&amp;D Systems公司)到已经构建好的纤维化模型小鼠中(联合组),对照为注射等量重组人白介素-8 (IL-8)和与重组人GRO蛋白等量的PBS的肝纤维化模型小鼠(对照组)。 [0178] As in Example 6.1 and 6.2 show, respectively, 1.51 ^ / Dian and 211 ^ / dose Dian by tail vein injection of recombinant human interleukin -8 (IL-8) (R & amp; D Systems Co.) and recombinant human GROa protein (R & amp; D Systems, Inc.) to prebuilt fibrosis model in mice (combined group), an equal amount of the control injection of recombinant human interleukin -8 (IL-8) protein and an equivalent amount of recombinant human GRO of PBS liver fibrosis model mice (control group). 在注射后的第1周和第2周,处死小鼠并进行SR 染色,观察并检测胶原纤维面积。 At 1 week and 2 weeks after injection, mice were sacrificed and SR staining and detecting collagen area. 结果表明,在第1周联合组(1.0%)的胶原积累面积显著低于对照组(2.1%),在第2周联合组(0.6%)的胶原纤维面积相比于对照组(1.3%)有明显减少,说明肝纤维化在白介素-8 (IL-8)和GRO联合作用下肝纤维化状况得到了更显著改善。 The results show that the combination group at week 1 (1.0%) of the area of ​​collagen accumulation was significantly lower than the control group (2.1%), in the second week combination group (0.6%) compared to the area of ​​the collagen fibers in the control group (1.3%) significantly decreased, indicating liver fibrosis in interleukin -8 (IL-8), and the combined effect of hepatic fibrosis GRO situation has improved more remarkably.

[0179] 与此同时,发明人按照实施例6.4进行了免疫组织化学检测a-SMA和TGF-βΙ的蛋白表达水平,结果发现,联合组相对于对照组而言a-SMA和TGF-βΙ的表达受到了更明显的抑制。 [0179] Meanwhile, the inventors carried out as in Example 6.4 immunohistochemical detection of a-SMA and protein expression of TGF-βΙ found in the combination group relative to the control group in terms of a-SMA and the TGF-βΙ expression was more significantly inhibited. 这进一步验证了白介素-8 (IL-8)和GRO的组合在改善或治疗肝纤维化方面的更加优异的作用。 This further validates the more excellent effect of the combination of interleukin -8 (IL-8) and GRO in improving or treating hepatic fibrosis aspect.

[0180] 用重组人GRO0蛋白(R&amp;D Systems公司)和重组人GROy蛋白(R&amp;D Systems公司)分别替换上述的重组人GROa蛋白(R&amp;D Systems公司),也均显示出肝纤维化在白介素-8 (IL-8)和GRO联合作用下肝纤维化状况得到了更显著改善(其中与重组人GR0i3蛋白的联合下效果最优),以及a-SMA和TGF-βΙ的表达受到了更明显的抑制。 [0180] Recombinant human GRO0 protein (R & amp; D Systems, Inc.) and recombinant human GROy protein (R & amp; D Systems, Inc.) was replaced with the above-described recombinant human GROa protein (R & amp; D Systems, Inc.), also exhibited liver fibrosis in interleukin -8 (IL-8) under the conditions and fibrosis combined action GRO been more improved (optimal effect jointly wherein GR0i3 recombinant human protein), and the expression of a-SMA and subjected to the TGF-βΙ more significantly inhibited.

[0181] 这说明了白介素-8 (IL-8)和不同形式的重组人GRO蛋白的组合在改善或治疗肝纤维化方面的作用。 [0181] This illustrates the effect of interleukin -8 (IL-8) and a combination of different forms of recombinant human protein in GRO improving or treating hepatic fibrosis aspect.

[0182] 实施例17:白介素-8 (IL-8)和白介素-6 (IL-6)的组合改善小鼠的肝纤维化 [0182] Example 17: combination Interleukin -8 (IL-8) and interleukin -6 (IL-6) improvement of mice with liver

[0183] 如实施例6.1和6.2所示,分别以1.51^/敁和1.511^/敁的剂量通过尾静脉注射重组人白介素_8(IL_8) (R&amp;D Systems公司)和重组人IL-6蛋白(R&amp;D Systems公司)到已经构建好的纤维化模型小鼠中(联合组),对照为注射等量重组人白介素-8(IL-8)和与重组人IL-6 蛋白等量的PBS的肝纤维化模型小鼠(对照组)。 [0183] As shown in Example 6.1 and 6.2, respectively, 1.51 ^ / Dian and 1.511 ^ / dose Dian by tail vein injection of recombinant human interleukin _8 (IL_8) (R & amp; D Systems Co.) and recombinant human IL-6 protein (R & amp; D Systems, Inc.) to prebuilt fibrosis model in mice (combined group), and a control for the recombinant human IL-6 recombinant protein in equal amounts of equal amounts of injected human interleukin -8 (IL-8) is PBS liver fibrosis model mice (control group). 在注射后的第1周和第2周,处死小鼠并进行SR染色,观察并检测胶原纤维面积。 At 1 week and 2 weeks after injection, mice were sacrificed and SR staining and detecting collagen area. 结果表明,在第1周联合组(1.0%)的胶原积累面积显著低于对照组(2.3%),在第2周联合组(0.3%)的胶原纤维面积相比于对照组(1.6%)有明显减少,说明肝纤维化在白介素-8 (IL-8)和IL-6联合作用下肝纤维化状况得到了更显著改善。 The results show that the combination group at week 1 (1.0%) of the area of ​​collagen accumulation was significantly lower than the control group (2.3%), the area of ​​collagen fibers in the second week combination group (0.3%) compared to the control group (1.6%) significantly decreased, indicating liver fibrosis in interleukin -8 (IL-8) IL-6 and fibrosis combined effect was more significant health improvement.

[0184] 与此同时,发明人按照实施例6.4进行了免疫组织化学检测a-SMA和TGF-βΙ的蛋白表达水平,结果发现,联合组相对于对照组而言a-SMA和TGF-βΙ的表达受到了更明显的抑制。 [0184] Meanwhile, the inventors carried out as in Example 6.4 immunohistochemical detection of a-SMA and protein expression of TGF-βΙ found in the combination group relative to the control group in terms of a-SMA and the TGF-βΙ expression was more significantly inhibited. 这进一步验证了白介素-8 (IL-8)和IL-6的组合在改善或治疗肝纤维化方面的更加优异的作用。 This further verifies the excellent effect of more pigment compositions interleukin -8 (IL-8) and IL-6 in improving or treating hepatic fibrosis aspect.

[0185] 虽然用上述实施方式描述了本发明,应当理解的是,在不背离本发明的精神的前提下,本发明可进行进一步的修饰和变动,且这些修饰和变动均属于本发明的保护范围之内。 [0185] Although the above embodiments describe the present invention, it should be understood that, without departing from the spirit of the invention under the premise, the present invention may be further modifications and variations, and these modifications and variations belong to the protection of the present invention. within range.

Claims (6)

1. 白介素-8和单核细胞趋化蛋白1在制备用于治疗受试者中肝纤维化的药物中的用途。 1. Interleukin-8 and monocyte chemoattractant protein-1 uses a medicament for treating liver fibrosis in.
2. 根据权利要求1的用途,其中所述药物为适于注射、输注或口服的剂型。 2. Use according to claim 1, wherein the medicament is a dosage form suitable for injection, infusion or orally requirements.
3. 根据权利要求1的用途,其中药物的包装形式适于白介素-8和单核细胞趋化蛋白1被同时施用或按序先后施用。 Use according to claim 1, wherein the medicament is adapted packaging interleukin-8 and monocyte chemoattractant protein-1 is administered simultaneously or sequentially administered successively.
4. 根据权利要求1所述的用途,其中所述的受试者为哺乳动物。 4. Use according to claim 1, wherein the subject is a mammal.
5. 根据权利要求1所述的用途,其中所述的受试者为人。 5. The use according to claim 1, wherein the subject is a human.
6. 根据权利要求1所述的用途,其中所述的白介素-8为天然的白介素-8或人重组白介素-8 〇 6. Use according to claim 1, wherein said interleukin-8 as a natural interleukin-8 or human recombinant interleukin-8 billion
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1225016A (en) * 1997-07-04 1999-08-04 维罗塞尔公司 Interleukin-8 as an antiviral and antitumor agent
WO2001010899A2 (en) * 1999-08-09 2001-02-15 The Regents Of The University Of Michigan Treatment of liver disease and injury with cxc chemokines
WO2009042798A1 (en) * 2007-09-26 2009-04-02 Cold Spring Harbor Laboratory Methods for treating fibrosis by modulating cellular senescence
CN101443357A (en) * 2005-08-12 2009-05-27 先灵公司 MCP1 fusions

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Publication number Priority date Publication date Assignee Title
SK283096B6 (en) * 1994-07-05 2003-02-04 Steeno Research Group A/S Immunomodulators

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1225016A (en) * 1997-07-04 1999-08-04 维罗塞尔公司 Interleukin-8 as an antiviral and antitumor agent
WO2001010899A2 (en) * 1999-08-09 2001-02-15 The Regents Of The University Of Michigan Treatment of liver disease and injury with cxc chemokines
CN101443357A (en) * 2005-08-12 2009-05-27 先灵公司 MCP1 fusions
WO2009042798A1 (en) * 2007-09-26 2009-04-02 Cold Spring Harbor Laboratory Methods for treating fibrosis by modulating cellular senescence

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