WO2008077311A1 - Hepatopoietin and use thereof - Google Patents

Hepatopoietin and use thereof Download PDF

Info

Publication number
WO2008077311A1
WO2008077311A1 PCT/CN2007/003722 CN2007003722W WO2008077311A1 WO 2008077311 A1 WO2008077311 A1 WO 2008077311A1 CN 2007003722 W CN2007003722 W CN 2007003722W WO 2008077311 A1 WO2008077311 A1 WO 2008077311A1
Authority
WO
WIPO (PCT)
Prior art keywords
hppcn
liver
protein
sequence
group
Prior art date
Application number
PCT/CN2007/003722
Other languages
English (en)
French (fr)
Inventor
Zuze Wu
Bingxing Shi
Chunping Cui
Shaojun Du
Danli Wu
Original Assignee
Institute Of Radiation Medicine, Academy Of Military Medical Sciences, Pla
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute Of Radiation Medicine, Academy Of Military Medical Sciences, Pla filed Critical Institute Of Radiation Medicine, Academy Of Military Medical Sciences, Pla
Priority to US12/521,135 priority Critical patent/US8361795B2/en
Priority to JP2009543328A priority patent/JP5390397B2/ja
Priority to EP07855737.8A priority patent/EP2110384B1/en
Publication of WO2008077311A1 publication Critical patent/WO2008077311A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of biological products and protein drugs, in particular to the proteins of hepa topo iet in PCn (HPPCn), which can promote hepatocyte proliferation and liver regeneration, and inhibit cell growth and promote tumor cells in cells. Apoptosis has potential clinical application value.
  • HPPCn hepa topo iet in PCn
  • liver is an organ with strong regenerative capacity.
  • the research on its regulation mechanism has been carried out for more than 100 years, but currently known liver growth-related growth factors such as hepatocyte growth factor (HGF) and transforming growth factor-o ( TGF- ⁇ ), etc., lack of liver specificity, and it is difficult to explain the organ-specific regulation mechanism of liver regeneration. Therefore, it is always a hot spot in this field to continue to search for new liver regeneration regulatory factors.
  • HGF hepatocyte growth factor
  • TGF- ⁇ transforming growth factor-o
  • liver regeneration enhancer can promote liver regeneration in rats with partial hepatectomy, but lacks the stimulating activity of primary cultured hepatocytes and hepatic cell lines in vitro [5] .
  • LANP protein is a kind of multifunctional acidic nuclear protein, which is involved in cell signal transduction, protein degradation, cytoskeletal dynamics and morphogenesis [ 6-13 ] , but promotes hepatocytes as a growth factor. There is currently no report on proliferation and liver regeneration.
  • liver disease China is a large country with liver disease.
  • the number of patients with viral hepatitis, cirrhosis and liver cancer is more than 120 million. Therefore, an active factor capable of specifically promoting hepatocyte proliferation and liver regeneration has been developed for the treatment of liver damage caused by various causes. Significance.
  • malignant tumors such as liver cancer has become one of the serious threats to human health. It is also important to inhibit tumor cell growth or tumorigenesis.
  • Hepa topo iet in PCn is a novel hepatocyte growth factor isolated from the liver of newborn calves, obtained from human cDNA library its human nucleotide sequence, and in Escherichia coli
  • HPPCn The recombinant human HPPCn protein has the following basic characteristics: 1.
  • the protein is a member of the leucine-rich acidic nuclear protein family, has a molecular weight of about 30KD, is sensitive to proteases, and is heat-resistant; 2. It can promote primary culture in vitro. DNA synthesis of rat hepatocytes and hepatic cell lines; 3. In vivo can promote liver DNA synthesis in partial hepatectomy mice; 4.
  • a first aspect of the invention relates to HPPCn comprising the amino acid sequence set forth in SEQ ID NO: 1.
  • the sequence of HPPCn of the invention is set forth in SEQ ID NO: 1.
  • the invention also relates to homologs of HPPCn which have at least 80% homology, preferably at least 85%, 90%, 95% homology to the HPPCn of the invention, while retaining the activity of the HPPCn of the invention.
  • a further aspect of the invention relates to a nucleic acid molecule encoding HPPCn or a homolog thereof.
  • a further aspect of the invention relates to a pharmaceutical composition comprising the HPPCn protein of the invention, a homolog thereof or a nucleic acid molecule encoding the same.
  • the pharmaceutical composition may also optionally contain a pharmaceutically acceptable carrier or other conventional adjuvant. These carriers and adjuvants are within the skill of one of ordinary skill in the art.
  • a further aspect of the invention relates to the use of a HPPCn protein, a homolog thereof or a nucleic acid molecule encoding the same for the preparation of a medicament for promoting hepatocyte proliferation and liver regeneration, for treating liver damage caused by various causes, It is used to treat liver fibrosis, to inhibit tumor cell growth in cells or to treat various tumors.
  • the medicament is for the treatment of liver fibrosis or acute or chronic liver injury.
  • hepatocyte proliferation refers to an increase in the cell division ability of primary cultured normal cells derived from the liver, normal liver cell lines, and liver cancer cell lines, and the proliferation ability of the cells is mainly detected by detecting DNA synthesis.
  • liver regeneration refers to the ability of a mammalian liver to recover spontaneously after being damaged.
  • the HPPCn of the present invention can be expressed in Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae or animal cells.
  • Figure 1 is the amino acid sequence of HPPCn of the present invention (SEQ ID NO: 1).
  • Figure 2 shows the results of SDS-PAGE expression and purification of HPPCn in E. coli BL21.
  • the top panel shows the induced expression of HPPCn in E. coli BL21.
  • the lower panel shows the purification of recombinant HPPCn.
  • 1 inclusion body protein
  • 2 penetrant
  • 3 eluent elution product
  • 4 low molecular weight standard.
  • HPPCn HepatopoietinPCn
  • the recombinant protein with a purity of more than 95% was obtained by ion exchange or gel filtration.
  • the amino acid sequence is shown in Figure 1, and the electrophoresis results are shown in Figure 2.
  • Example 2 Identification of recombinant protein HPPCn promoting hepatocyte proliferation activity
  • HPPCn The proliferative activity of HPPCn was detected by 3H-TdR DNA incorporation assay.
  • the primary cultured rat hepatocytes were used as detection target cells for in vitro biological activity. Take the cell suspension (5 ⁇ 107ml) ⁇ 1, inoculate in 96-well culture plate for 6 hours, add different concentrations of HPPCn, continue to culture for 24 hours, add 1.85 x 10 4 Bq 3 H-TdR per well, 3 hours later, Perform liquid flash counting. The results indicate that HPPCn can promote DNA assembly of hepatocytes. In a dose-dependent manner (Table 1), this property is not available in the Liver Regeneration Enhancer (ALR).
  • ALR Liver Regeneration Enhancer
  • Example 3 Protective effect of recombinant protein HPPCn on acute liver injury
  • the protective effect of HPPCn on liver DNA synthesis in 34% of partially hepatectomized mice was examined by detecting the effect of HPPCn on liver DNA synthesis in 34% of partially hepatectomized mice.
  • Male C57 mice in good condition were surgically resected from the mid-leaf liver, and injected with 2. 5 mg HPPCn/kg body weight or an equal volume of normal saline at different time points after operation.
  • intraperitoneal injection of 20 ⁇ ⁇ ⁇ 3 H-TdR animals were sacrificed 2 hours after incorporation, and liver genomic DNA was extracted, and 3 H-TdR incorporation was detected by liquid scintillation.
  • the results showed that the amount of 3 H-TdR in the administration group was higher than that in the saline control group at each time point (see Table 2 for the results), suggesting that HPPCn can improve the liver regeneration ability after hepatectomy.
  • the drug group especially the middle dose group, had significantly reduced fibrous tissue proliferation, hepatocytes. Decreased necrosis suggests that recombinant HPPCn administration can alleviate liver fibrosis caused by chronic injury.
  • Table 4 Protective effect of recombinant HPPCn protein on liver fibrosis in rats
  • HPPCn inhibits tumor cell growth in cells
  • HPPCn was constructed into the eukaryotic expression vector pEGFP-N1 plasmid and transfected into human hepatoma cell line SMMC7721. After 36 hours of culture, 4% paraformaldehyde was used respectively. After fixation with 70% ethanol, the cell cycle was detected by FACS after PI staining. The results showed that the transfected hepatoma cells showed obvious G0/G1 phase arrest, and the G2/M phase cell ratio was significantly higher than that of the empty plasmid transfection group. There was little pEGFP-N1 transfection group, suggesting that HPPCn overexpression in cells has obvious growth inhibition on liver cancer cells (see Table 5).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Description

肝再生因子及其应用 技术领域
本发明涉及生物制品和蛋白质药物领域 具体说涉及外源性 肝再生因子 （hepa topo iet in PCn, HPPCn ) 这些蛋白能促进肝 细胞增殖和肝脏再生， 在细胞内则能抑制肿 细胞生长促进肿瘤 细胞凋亡， 具有潜在的临床应用价值。 背景技术
肝脏是机体具有强大再生能力的脏器， 有关其调控机理的研 究国际上已进行百余年， 但目前已知的肝再生相关生长因子如肝 细胞生长因子 （HGF ) ， 转化生长因子 - o ( TGF- α ) 等， 其作用 均缺乏肝特异性， 难以解释具有器官特异性的肝再生调控机理， 因此继续寻找新的肝再生调控因子一直是该领域研究的热点。 早 在上世纪 50年代，人们就发现哺乳动物肝脏中存在能够调解自身 生长的物质 [1]。 1975 年， LaBrecque 等[2]首先报道大鼠再生肝组 织中含有一种具有热稳定性并能够特异促进肝细胞增殖的混合 物， 称为肝刺激物 ( hepat i c s t imula tor subs tance, HSS ) 。 上世纪 80年代中期，我们在人胎肝组织中也发现同类因子， 并对 其生物活性、 理化性质、 蛋白质纯化以及临床应用进行了系统研 究。 由于此类因子应用临床治疗严重肝损伤取得较好的疗效， 因 而备受重视， 并于 1995年获得美国专利[3]。 但由于当时蛋白质纯 化及鉴定技术的限制， 未能对其成分作进一步鉴定， 这也限制了 对这类物质的开发利用。 与此同时， 国外的多家实验室也一直致 力于此类因子的分子克隆， 1995年， ¾817&等【4]从大鼠再生肝组 织中分离到一种肝再生增强因子， 并对其进行克隆表达， 发现重 组肝再生增强因子能够促进肝部分切除大鼠的肝脏再生， 但缺乏 体外对原代培养肝细胞和肝细胞系的刺激活性 [5]。
近几年， 我们采用多种分离方法从新生小牛肝脏中分离到一 种新的肝细胞生长因子， 能够促进肝细胞的 DNA合成， 并对急慢 性肝损伤具有保护作用， 将其命名为 HPPCn, 序列分析发现它是 富含亮氨酸酸性核蛋白家族 ( Leuc ine-r ich ac idic nuc lear prote in, LANP ) 的成员之一。 LANP 蛋白是一类多功能的酸性核 蛋白， 在细胞的信号转导、 蛋白质降解、 细胞骨架动力学以及形 态发生等过程中都参与作用 [6— 13]， 但作为一种生长因子促进肝细 胞增殖和肝脏再生方面目前没有报道。
我国是一个肝病大国， 病毒性肝炎、 肝硬化与肝癌病人数在 1. 2 亿以上， 因此开发一种能够特异的促进肝细胞增殖和肝脏再 生的活性因子对于治疗各种原因引起的肝损伤具有重要意义。
除此以外， 肝癌等恶性肿瘤的发生也已成为严重威胁人类健 康的杀手之一， 抑制肿瘤细胞生长或肿瘤发生也同样具有重要的 社会意义。
发明内容
本发明现已发现， Hepa topo iet in PCn (HPPCn)是从新生小牛 肝脏中分离到的一种新的肝细胞生长因子，从人 cDNA文库获得其 人源核苷酸序列， 并在大肠杆菌中表达获得重组人 HPPCn蛋白， 具有以下基本特性： 1、该蛋白为富含亮氨酸酸性核蛋白家族成员 之一， 分子量约 30KD, 对蛋白酶敏感， 耐热； 2、 体外能够促进 原代培养的大鼠肝细胞和肝细胞系的 DNA合成； 3、体内能够促进 部分肝切除小鼠的肝脏 DNA合成； 4、对急性肝损伤和肝纤维化具 有保护作用； 5、 在肿瘤细胞内过表达能够抑制肿瘤细胞生长。 因此， 本发明第一方面涉及 HPPCn, 其含有 SEQ ID NO: 1所 示的氨基酸序列。 优选地， 本发明的 HPPCn的序列如 SEQ ID NO: 1 所示。 本发明也涉及 HPPCn的同源物， 该同源物与本发明 HPPCn 具有至少 80 %的同源性， 优选至少 85 %、 90 %、 95 %的同源性， 同时保留本发明 HPPCn的活性。
本发明再一方面涉及编码 HPPCn或其同源物的核酸分子。 本发明再一方面涉及药物组合物， 其含有本发明的 HPPCn蛋 白、 其同源物或编码其的核酸分子。 所述药物组合物还可以任选 地包含药物可接受的载体或其它常规辅剂。 这些载体和辅剂在本 领域普通技术人员的技术范围之内。
本发明再一方面涉及 HPPCn蛋白、其同源物或编码其的核酸分 子在制备药物中的应用， 所述药物用于促进肝细胞增殖和肝脏再 生、 用于治疗各种原因引起的肝损伤、 用于治疗肝纤维化、 用于 在细胞内抑制肿瘤细胞生长或用于治疗各种肿瘤。
优选地， 所述药物用于治疗肝纤维化或急性或慢性肝损伤。 根据本发明， 术语 "肝细胞增殖"是指来源于肝脏的原代培养 的实质细胞、正常肝细胞系以及肝癌细胞系细胞分裂能力的增强， 主要通过检测 DNA合成来检测细胞的增殖能力。
根据本发明， 术语 "肝再生" 是指哺乳动物肝脏受到损伤后 能够自发恢复的能力。
根据本发明， 本发明的 HPPCn可在大肠杆菌、 毕赤酵母、 酿 酒酵母或动物细胞中表达。 附图说明
结合附图举例说明本发明， 不构成对本发明的限制。 图 1为本发明 HPPCn的氨基酸序列 （ SEQ ID NO: 1 ) 。
图 2为 HPPCn在大肠杆菌 BL21中表达和纯化的 SDS-PAGE结果。 上图表示 HPPCn在大肠杆菌 BL21中的诱导表达。 1: 阴性对 照； 2: 包涵体蛋白； 3: 可溶性蛋白。
下图表示重组 HPPCn的纯化。 1: 包涵体蛋白； 2: 穿透液； 3: 洗脱液洗脱产物； 4: 低分子量标准。 下面的实施例用来进一步说明本发明， 但并不意味着对本发 明的任何限制。
实施例 1 HPPCn的生产制备
采用 DEAE-cellulose、 Sourcel5Q等层析方法、 SDS-PAGE分 段回收以及 MALDY-T0F和 Q-T0F质谱技术， 从新生小牛肝脏中纯 化并鉴定了一个能够特异促进肝细胞增殖和肝脏再生的蛋白因 子， 即 HepatopoietinPCn ( HPPCn ) 。 通过人胎肝 cDNA文库筛 选，获得人 HPPCn cDNA序列。在基因序列两端加上 BamH I和 Xhol I 酶切位点， 构建到原核表达载体 PET-24a 中 ， 得到 PET-24a-HPPCn质粒。 将其转化至大肠杆菌， IPTG诱导表达。 经 离子交换、 凝胶过滤得到纯度达 95%以上的重组蛋白， 其氨基酸 序列如图 1所示， 电泳结果如图 2所示。 实施例 2 重组蛋白 HPPCn促肝细胞增殖活性的鉴定
3H-TdR DNA掺入法检测 HPPCn的促增殖活性。 以原代培养的 大鼠肝细胞为体外生物活性的检测靶细胞。 取细胞悬液 （ 5 χ 107ml ) ΙΟΟμ 1, 接种于 96孔培养板中培养 6小时， 加入不同浓 度 HPPCn, 继续培养 24小时， 每孔加 1.85 x 104Bq 3H-TdR, 3小 时后， 进行液闪计数。 结果表明 HPPCn能够促进肝细胞的 DNA合 成， 且具有明显的剂量依赖性 (表 1)， 此特性为肝再生增强因子 ( ALR ) 所不具备的。
表 1 重组 HPPCn蛋白对原代培养肝细胞的促增殖活性检测
Figure imgf000007_0001
实施例 3 重组蛋白 HPPCn对急性肝损伤的保护作用 通过检测 HPPCn对 34%部分肝切除小鼠肝脏 DNA合成的影响 检测其对部分肝切除小鼠的保护作用。状态良好的雄性 C57小鼠， 手术切除其中叶肝脏， 并分别于术后不同时间点尾静脉注射 2. 5mg HPPCn/kg体重或等体积的生理盐水， 作用 1 8小时后， 腹 腔注射 20 μ θ ί 3H-TdR , 掺入 2小时后处死动物， 并提取肝脏基 因组 DNA， 液闪仪检测 3H-TdR掺入量。 结果显示， 各时间点给药 组的 3H- TdR掺入量均高于生理盐水对照组 （结果见表 2 ) ， 提示 HPPCn能够提高肝切除后肝脏的再生能力。
表 2 重组 HPPCn蛋白对 34%肝切除小鼠的肝脏 DNA合成的影响
3H掺入量 （ cpm/m| 1 DNA )
时间 （小时） 重组人 HPPCn 生理盐水
24 3244 ± 339. 4 1641. 3 土 944. 4
36 4216 ± 1512. 7 1582. 6 ± 179. 8
48 6128 ± 736. 1 4876 ± 867. 04
60 4749. 66 ± 359. 7 2775 土 0
72 3825. 333 ± 294. 2294. 66 ± 716. 5
84 2962. 667 ± 938. 2 2197. 3 ± 823. 7 30只状态良好的 Ba lbc小鼠， 注射 lml /kg体重的 CC14后， 随机分成 3组： I组静脉注射 lmg/kg体重的 HPPCn; Π组静脉注 射 2. 5mg/kg体重的 HPPCn; ΙΠ组静注 5mg/kg体重的 HPPCn; IV组 为生理盐水对照组。 每 12小时注射一次， 于 48小时后观察血液 中天冬氨酸氨基转移酶活性 （AST ) 和丙氨酸氨基转移酶（ALT ) 变化并作病理学检测。结果显示对照组小鼠血清中 AST, ALT值都 明显升高， 肝组织细胞大部分坏死， 细胞核消失， 呈空泡样， 但 HPPCn治疗组小鼠 AST, ALT值均有所恢复（结果见表 3 ) ， 坏死 肝细胞明显减少， 肝损伤程度明显减轻。 提示重组蛋白对 CCUl 起的急性肝损伤具有保护作用。
Figure imgf000008_0001
实施例 4 重组蛋白 HPPCn对肝纤维化的保护作用
40只雄性 Wi s tar 大鼠， 采用酒精、 CC14复合造模法制备肝 纤维化模型， 造模四周后开始分组， 随机分为 4组， 分别为对照 组、大剂量组（ 5mg/kg )、中剂量组（ 2. 5mg/kg )、小剂量组 ( lmg/kg )。 每日腹腔给药。 至第八周， 处死动物， 取血检测其 ALT、 AST等生 化指标活性， 取肝脏观察其表观变化， 检测羟脯氨酸、 丙二醛含 量并做组织病理检测。 结果显示给药组天冬氨酸氨基转移酶活性 ( AST ) 和丙氨酸氨基转移酶（ALT )较对照组有所降低， 羟脯氨 酸（Hyp )含量明显减少， 而丙二醛（MDA )变化不明显（见表 4 ) 对照组肝脏灰黄色， 且表面不光滑， 呈现颗粒状， 而给药组大鼠 肝脏则较为红润。肝组织 HE染色和 Mas son' s三色染色结果显示 对照组肝脏出现细胞坏死和脂质沉积， 纤维组织增生， 并形成假 小叶， 给药组特别是中剂量组纤维组织增生明显减少， 肝细胞坏 死减少，提示重组 HPPCn给药能够緩解慢性损伤引起的肝纤维化。 表 4 重组 HPPCn蛋白对肝纤维化大鼠的保护作用
Figure imgf000009_0001
实施例 5 HPPCn在细胞内抑制肿瘤细胞生长 将 HPPCn构建到真核表达载体 pEGFP-Nl质粒中，并将其转染 至人肝癌细胞系 SMMC7721中， 培养 36小时后， 分别用 4%多聚甲 醛和 70%乙醇固定， PI染色后， FACS检测其细胞周期的变化， 结 果表明转染后的肝癌细胞出现明显的 G0/G1期阻滞， G2/M期细胞 比例明显较空质粒转染组（pEGFP-Nl转染组）少， 提示 HPPCn在 细胞内过表达对肝癌细胞有明显的生长抑制 （见表 5 )
表 5 HPPCn在 SMMC7721细胞中过表达对细胞 期的影响
组别 G0-G1 G2-M S
pEGFP-Nl-HPPCn转染组 92. 59% 0. 12% 7. 28 % pEGFP-Nl转染组 45. 67% 14. 42 % 39. 91 % 参 考 文 献
1. Blomquist K, et al. Growth stimulation in the liver and tumour development following intraperitoneal inject ions of liver homogenates in the rat. Acta Pathol Microbiol Scand 1957; 121(Suppl): 375-382.
2. LaBrecque DR, Pesch LA. Preparation and partial characterization of hepatic regenerat ion stimulator substance from rat liver. J physiol, 1975. 248(3): 273-284.
3. Wu Ct, Tu Q, He FC, et al. Hepatokine and methods for its use. Unite States Patent, 1995. 5440022.
4. Hagiya M, Francavi 1 la A, Pol imeno L, et al. Cloning and sequence analysis of the rat augmentor of liver regenerat ion (ALR) gene: express ion of biological ly active recombinant ALR and demonstration of tissue distribution. PNAS, 1995, 92 (7): 3076-3080.
5. Francavi 1 la A, Hagiya M， Starzl E. Mama li an ALR: human and rat. United States Patent, 1996. 1996-08-27: 5550037.
6. Matsuoka K， Taoka M， Satozawa N, et al. A nuclear factor containing the leucine— rich repeats expressed in murine cerebellar neuron. Proc Natl Acad Sci USA 1994; 91 (21): 9670-9674.
7. Malek SN， Katumuluwa Al, and Pasternack GR. Ident if icat ion and prel iminary characterizat ion of two related pro 1 if era t ion-associated nuclear phosphoproteins. J. Biol. Chem 1990; 265 (22): 13400-13409.
8. Brody JR, Kadkol SS， Mahmoud MA, et al. Identification of sequences required for inhibit ion of oncogene- media ted transformation by pp32. J Biol Chem 1999; 274(29): 20053-20055.
9. Chen TH, Brody JR， Romantsev FE, et al. Structure of pp32, an acidic nuclear protein which inhibits oncogene- induced formation of transformed foci. Mol Biol Cell 1996; 7(12): 2045-2056.
10. Li M， Makkinje A, Damuni Z. Molecular identification of I1PP2A, a novel potent heat-stable inhibitor protein of protein phosphatase 2A. Biochemistry 1996; 35 (22)： 6998-7002.
11. Brennan CM, Gal louzi IE, Steitz JA. Protein 1 igands to HuR modulate its interact ion with target mRNAs in vivo. J Cell Biol 2000; 151(1): 1-14.
12. Seo SB, McNamara P, Heo S, et al. Regulation of histone acetylat ion and transcription by INHAT, a human cellular complex containing the set oncoprotein. Cell 2001; 104(1): 119-130.
13. 13. Opal P, Garcia JJ, McCall AE et al, Generation and Characterization of LANP/pp32 Null Mice, Mole Cell Biol; 24 (8): 3140-9.

Claims

权利要求
1. HPPCn蛋白， 其含有 SEQ ID NO: 1 所示序列， 或者含有与 SEQ ID N0: 1具有至少 80 %、 优选 85 %、 90 %或 95 %同源性的序 列。
2. 权利要求 1的 HPPCn蛋白， 其序列为 SEQ ID NO: 1所示。
3. 一种核酸分子， 其编码权利要求 1或 2的 HPPCn蛋白。
4. 一种药物组合物， 其含有权利要求 1或 2的 HPPCn蛋白或 权利要求 3的核酸分子。
5. 权利要求 1或 2的 HPPCn蛋白或权利要求 3的核酸分子在 制备药物中的应用， 其中所述药物用于促进肝细胞增殖和肝脏再 生、 用于治疗各种原因引起的肝损伤、 用于抑制肿瘤细胞生长、 用于治疗各种肿瘤或用于治疗肝纤维化。
6. 权利要求 5 的应用， 其中所述药物用于治疗急性肝损伤或 慢性肝损伤。
PCT/CN2007/003722 2006-12-26 2007-12-21 Hepatopoietin and use thereof WO2008077311A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US12/521,135 US8361795B2 (en) 2006-12-26 2007-12-21 Hepatopoietin and use thereof
JP2009543328A JP5390397B2 (ja) 2006-12-26 2007-12-21 ヘパトポイエチン及びその使用
EP07855737.8A EP2110384B1 (en) 2006-12-26 2007-12-21 Hepatopoietin and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200610156298 2006-12-26
CN200610156298.0 2006-12-26

Publications (1)

Publication Number Publication Date
WO2008077311A1 true WO2008077311A1 (en) 2008-07-03

Family

ID=39562092

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2007/003722 WO2008077311A1 (en) 2006-12-26 2007-12-21 Hepatopoietin and use thereof

Country Status (5)

Country Link
US (1) US8361795B2 (zh)
EP (1) EP2110384B1 (zh)
JP (1) JP5390397B2 (zh)
CN (1) CN101600733A (zh)
WO (1) WO2008077311A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5440022A (en) 1992-10-14 1995-08-08 Panorama Research, Inc. Hepatokine and methods for its use
US5550037A (en) 1994-02-16 1996-08-27 University Of Pittsburgh Mammalian augmenter of liver regeneration (ALR): human and rat
CN1896100A (zh) * 2005-06-01 2007-01-17 中国人民解放军军事医学科学院放射与辐射医学研究所 肝再生因子及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1146909A4 (en) * 1999-02-03 2004-12-29 Univ Johns Hopkins METHOD FOR TREATING CANCER BY RECOVERING THE PP32 FUNCTION

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5440022A (en) 1992-10-14 1995-08-08 Panorama Research, Inc. Hepatokine and methods for its use
US5550037A (en) 1994-02-16 1996-08-27 University Of Pittsburgh Mammalian augmenter of liver regeneration (ALR): human and rat
CN1896100A (zh) * 2005-06-01 2007-01-17 中国人民解放军军事医学科学院放射与辐射医学研究所 肝再生因子及其应用

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
BLOMQUIST K ET AL.: "Growth stimulation in the liver and tumour development following intraperitoneal injections of liver homogenates in the rat.", ACTA PATHOL MICROBIOL SCAND, vol. 121, 1957, pages 375 - 382
BRENNAN CM; GALLOUZI IE; STEITZ JA.: "Protein ligands to HuR modulate its interaction with target mRNAs in vivo", J CELL BIOL, vol. 151, no. 1, 2000, pages 1 - 14, XP002230286, DOI: doi:10.1083/jcb.151.1.1
BRODY JR; KADKOL SS; MAHMOUD MA ET AL.: "Identification of sequences required for inhibition of oncogene-mediated transformation by pp32", J BIOL CHEM, vol. 274, no. 29, 1999, pages 20053 - 20055, XP002303700, DOI: doi:10.1074/jbc.274.29.20053
CHEN T.H. ET AL.: "Structure of pp32, an Acidic Nuclear Protein Which Inhibits Oncogene-induced Formation of Transformed Foci", MOLECULAR BIOLOGY OF THE CELL, vol. 7, no. 12, December 1996 (1996-12-01), pages 2045 - 2056, XP000916325 *
CHEN TH; BRODY JR; ROMANTSEV FE ET AL.: "Structure of pp32, an acidic nuclear protein which inhibits oncogene-induced formation of transformed foci", MOL BIOL CELL, vol. 7, no. 12, 1996, pages 2045 - 2056
DATABASE GENBANK [online] 7 June 2006 (2006-06-07), CUI C.P. ET AL., XP008111109, Database accession no. (DQ083510) *
DATABASE GENBANK [online] 7 June 2006 (2006-06-07), CUI C.P. ET AL., XP008111110, Database accession no. (AAZ31261) *
DU S.J. ET AL.: "Progress in Research on the Family of Leucine-Rich Acidic Nuclear Protein", BULL. ACAD. MIL. MED. SCI., vol. 30, no. 4, August 2006 (2006-08-01), pages 369 - 372, XP008125285 *
HAGIYA M; FRANCAVILLA A; POLIMENO L ET AL.: "Cloning and sequence analysis of the rat augmentor of liver regeneration(ALR)gene: expression of biologically active recombinant ALR and demonstration of tissue distribution", PNAS, vol. 92, no. 7, 1995, pages 3076 - 3080
LABRECQUE DR; PESCH LA.: "Preparation and partial characterization of hepatic regeneration stimulator substance from rat liver", J PHYSIOL, vol. 248, no. 3, 1975, pages 273 - 284
LI M; MAKKINJE A; DAMUNI Z.: "Molecular identification of I1PP2A, a novel potent heat-stable inhibitor protein of protein phosphatase 2A", BIOCHEMISTRY, vol. 35, no. 22, 1996, pages 6998 - 7002, XP002929316, DOI: doi:10.1021/bi960581y
MALEK SN; KATUMULUWA AI; PASTERNACK GR: "Identification and preliminary characterization of two related proliferation-associated nuclear phosphoproteins", J. BIOL. CHEM, vol. 265, no. 22, 1990, pages 13400 - 13409, XP002299272
MATSUOKA K; TAOKA M; SATOZAWA N ET AL.: "A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neuron", PROC NATL ACAD SCI USA, vol. 91, no. 21, 1994, pages 9670 - 9674
OPAL P; GARCIA JJ; MCCALL AE ET AL.: "Generation and Characterization of LANP/pp32 Null Mice", MOLE CELL BIOL, vol. 24, no. 8, pages 3140 - 9
See also references of EP2110384A4 *
SEO SB; MCNAMARA P; HEO S ET AL.: "Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein", CELL, vol. 104, no. 1, 2001, pages 119 - 130

Also Published As

Publication number Publication date
US20100144628A1 (en) 2010-06-10
JP5390397B2 (ja) 2014-01-15
EP2110384A4 (en) 2010-10-13
EP2110384B1 (en) 2016-07-06
EP2110384A1 (en) 2009-10-21
US8361795B2 (en) 2013-01-29
JP2010514426A (ja) 2010-05-06
CN101600733A (zh) 2009-12-09

Similar Documents

Publication Publication Date Title
AU2003248419B2 (en) Recombinant super-compound interferon
US10259852B2 (en) Conjugate comprising P21 protein for the treatment of cancer
US20230288402A1 (en) Molecules targeting ribosomal protein rpl35/ul29 for use in the treatment of diseases, in particular epidermolysis bullosa (eb)
CN116836233B (zh) 抗炎活性多肽及其应用
BRPI0318594B1 (pt) Recombinant protein that has an antibody effect, its coding gene, its use, cancer drug, expression vector and transgenic microorganism
JP7514841B2 (ja) ４－アルキル－５－ヘテロアリール－３ｈ－１，２－ジチオール－３－チオンの回転異性体
Zhang et al. Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats
CN107629114B (zh) 多肽、其衍生物及其在制备抗肺纤维化的药物中的应用
WO2008077311A1 (en) Hepatopoietin and use thereof
CA3020339C (en) Compositions and methods for treating pancreatitis and pain with death receptor agonists
EP1397154B1 (en) Abin-mediated hepatitis protection
WO2020175935A1 (ko) 알부민이 결합된, slit3 단백질의 lrrd2를 포함하는 골 관련 질환 예방 또는 치료용 조성물
CN116789750B (zh) 预防和/或治疗宫颈癌的多肽及其应用
CN105985445B (zh) 一种肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途
CN111973743B (zh) Rna结合蛋白zcchc4的靶向药物的应用
CN116789751B (zh) 预防和/或治疗纤维化疾病的多肽及其应用
PT2325202E (pt) Utilizações de interferões com estrutura espacial alterada
CN111732631B (zh) 一种多肽及其在制备治疗和预防肿瘤的药物中的应用
WO2013166944A1 (zh) 一种rtn4b相关多肽及其制备与应用
EP4341280A1 (en) Polypeptides related to hmgb1 useful for promoting tissue regeneration, compositions comprising same, and uses thereof
TWI328011B (en) Recombinant super-compound interferon
JPH05271280A (ja) 新規ポリペプチド、その製造方法及び該ポリペプチドを 有効成分とする医薬
KR20120118978A (ko) ｐ53을 유효성분으로 포함하는 면역질환의 예방 또는 치료용 조성물
JP2015509949A (ja) Ｎｅｍｏ結合ドメイン融合タンパク質

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780048304.3

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07855737

Country of ref document: EP

Kind code of ref document: A1

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
ENP Entry into the national phase

Ref document number: 2009543328

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2007855737

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2007855737

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 12521135

Country of ref document: US