肝细胞生长因子的分子生物学研究

肝细胞生长因子 (HGF)是一种多功能细胞因子 ,其分子为异二聚体糖蛋白 ,有NK1,NK2 ,NK4三个变种。HGF启动子的结构很复杂 ,其表达受多种因素和调控元件的调节。因HGF在体内有重要作用 ,HGF的基因工程表达和基因治疗正在研究中     

肝细胞生长因子基因转染对人淋巴瘤细胞凋亡的影响

探讨肝细胞生长因子（HGF）基因转染人淋巴瘤细胞系Raji细胞后,拮抗足叶乙甙（VP－16）诱导细胞凋亡的研究。将三种细胞：未转染Raji细胞、空载体pVITR02－mcs转染细胞和HGF基因转染细胞,分成正常对照组和经vP－16处理的药物组。采用Westernblot法验证HGF蛋白的表达：CCK－8法检测诱导Raji细胞凋亡的药物浓度;通过透射电镜、流式细胞术、吖啶橙（A0）染色、苏木精咿红（HE）染色等方法观察Raji细胞的凋亡情况,并进行相关分析。结果显示：Westernblot法验证了HGF蛋白质的表达;CCK．8法显示100μg／mL足叶乙甙可明显抑制Raii细胞增殖;透射电镜下可发现典型的凋亡细胞;流式检测结果表明：给药组与正常组相比,三组细胞的凋亡率明显升高（P〈0．01）,提示VP－16具有诱导细胞凋亡的作用：但给药组间：HGF基因转染组凋亡率明显低于未转染组（P＜0．05）和空载体pVITR02．mcs转染组（P〈0．05）,提示嬲F基因转染可明显抑制VP-16诱导的Raji细胞的凋亡,AO染色和HE染色结果也同样提示HGF具有拮抗VP－16诱导的细胞凋亡效应。     

重组人肝细胞生长因子真核表达的定性及定量检测

将人肝细胞生长因子（human hepatocyte growth factor hHGF)全长cDNA重组入pEE14真稳定表达质粒,用lipofectin脂质体将pEE14/rhHGF转染入CHO－K1细胞,蛋氨酸亚氨基代砜（methionine sulfoximine,MSX)筛选出阳性细胞克隆,利用RT－PCR检测rhHGF mRNA的表达通过ELISA法测定rhHGF的蛋白表达,3H掺入法检测培养上清液对大鼠原代培养肝细胞DNA合成的影响,结果表明转染pEE14/rhHGF的细胞可扩增出hHGF特异的396bp RT-PCR片段,培养上清液明显促进大鼠肝细胞DNA的合成,ELISA法测出上清液中,rhHGF的含量在8ug/L以上,显示rhHGF在CHO细胞中以活性形式得到表达。     

Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration

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Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

A complementary DNA (cDNA) encoding human hepatocyte growth factor was introduced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homologous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8% ±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was detected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0 x 10⁵ cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6 x 10⁹ pfu, 8.6 x 10⁸ pfu, 8.6 x 10⁷ pfu, 8.6 x 10⁶ pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of Hl (hypertrophie index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 10⁹ pfu and 10⁸ pfu. Whereas no therapeutic effects were seen at lower dose (10⁷ pfu and 10⁶ pfu of Ad-HGF) groups. In addition, clusters of hair were observed to different extent on healed wound treated with Ad-HGF. Histopathologic examination revealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amount of type I collagen in the Ad-HGF-treated scars was diminished markedly, compared to that in Ad-GFP group, in which a huge amount of type I collagen was still observed; (iv) immune response against HGF was absent. Antibody against HGF was not detectable by ELISA in serum from rabbit treated with Ad-HGF; (v) no local or systemic side-effects and toxicity associated with the gene transfer were found. These results demonstrated the potential use of treating pathologic scar by Ad-HGF, an alterative strategy of gene therapy for scar in clinical practice.     

Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

Pathologic scar, characterized by excessive dermal fibrosis and scarring, is a common im-portant clinical sequela after wound healing. It often appears during wound healing after deep burn, surgical cutting and other injured skin. Accumulation of extracellular matrix (ECM) proteins is a manifestation of increased collagen synthesis and/or reduced matrix degradation, resulting in excessive scarring with a deformed appearance and dysfunction[1]. To date, treatment modalities to scar include sur…     

肝细胞生长因子基因修饰的人脐带间充质干细胞对大鼠慢性肝损伤的治疗研究

目的：研究肝细胞生长因子（HGF）基因修饰的人脐带间充质干细胞（MSC）对Wistar大鼠慢性肝损伤的治疗作用。方法：利用携带人HGF基因的重组腺病毒（Ad～HGF）对MSC进行基因修饰;通过皮下注射CCl4-橄榄油溶液建立大鼠肝损伤模型;56只Wistar雄性大鼠随机分为正常对照组、模型组、MSC组、HGF组和HGF／MSC组,分别尾静脉注射生理盐水、MSC、Ad-HGF或HGF／MSC,通过血清肝功能检测及肝组织的生化指标、病理切片等评价治疗效果。结果：利用腺病毒将HGF基因转入MSC并确定了最佳感染条件。CCl4-橄榄油诱导4周后,动物肝脏外观出现明显的脂肪变性和血清转氨酶明显升高,表明成功诱导了大鼠慢性肝损伤模型。经过4周治疗,治疗组动物的体重明显升高、肝指数明显降低,动物存活率高于模型组（模型组：50％;MSC组：70％;Ad-HGF组：70％;HGF／MSC组：90％）。肝功能指标测定结果显示,与模型组比较,HGF／MSC组动物的天门冬氨酸氨基转移酶（406.75±35.98 vs.513.75±12.71U／L,P〈0.01）、丙氨酸氨基转移酶（124.6±10.6 vs.169.67＋15.38 U／L,P〈0.05）和总胆红素（14.6±2.08 vs.19.25±1.38g／dL,P〈0.01）均明显降低,白蛋白含量（29.1±1.3vs.22.05±2.61g／L,P〈0.05）明显升高;Ad-HGF组只有天门冬氨酸氨基转移酶（436.0±18.40vs.513.75±12.71U／L,P〈0.05）明显降低;而单纯MSC治疗对肝功能改善不明显。丙二醛测定结果显示,治疗后动物肝组织中的过氧化反应均较模型组显著减弱。HGF／MSC组和MSC组动物肝组织中的羟脯氨酸含量与模型组比较明显降低（69.27±14.58,63.23±13.23 vs.96.59±15.05mg／mL,P〈0.01）。上述实验结果提示3种治疗方式对CCl4-橄榄油引起的大鼠肝损伤均有一定的治疗效果,可减轻CCl。导致的肝损伤程度,增加动物体重,降低肝脏指数,减轻肝组织的过氧化反应,提高动物的存活率,其中HGF／MSC治疗效果最为明显,而且细胞注射没有引起动物的不良反应。结论：3种治疗方式均可改善CCl。导致的肝损伤,其中以HGF／MSC对肝损伤的治疗效果最好。本实验为HGF／MSC的临床研究提供了新的实验数据,为治疗肝损伤提供了新的治疗措施。     

人胰岛素生长因子Ⅰ基因的人工合成,克隆及其表达

采用固相亚磷酸三脂法,化学合成了人胰岛样生长因子Ｉ结构基因的两个１２９聚体长单链ＤＮＡ片段,通过其中的２３ｂｐ互补配对和Ｋｌｅｎｏｗ酶酶促补齐成为ＩＧＦ－Ｉ中进行ＤＮＡ全序列测定分析及寡核苷酸引导的定向点空变校正,获得了人工合成的ＩＧＦ－Ｉ结构基因。进一步分别重组构建了在Ｐｌａｃ和ＰＬＰｒｏｍｏｔｅｒ控制下的人工合成ＩＧＦ－Ｉ基因表达质粒ＰＨＭ５９０和ＰＢＬＥ０１１,在大肠杆菌中进行了表达研究。经     

Hepatocyte growth factor plays a dual role in tendon-derived stem cell proliferation,migration, and differentiation

Heterotopic ossification is common in tendon healing after trauma, but the detailed mechanisms remain unknown. Tendon-derived stem cells (TDSCs) are a type of progenitor cell found in the tendon niche, and their incorrect differentiation after trauma may lead to tendon calcification. The expression of hepatocyte growth factor (HGF) presents drastic fluctuations in serum/tissue after trauma and was found to activate quiescent stellate cells and contribute to wound healing; however, its potential role in TDSCs remains elusive. In this study, TDSCs isolated from rats were cultured in media containing HGF with or without a signaling inhibitor, and the proliferation, migration, and differentiation ability of TDSCs were measured to determine the role and mechanism of HGF in TDSCs. We showed that HGF promotes TDSC proliferation and migration but inhibits TDSC osteogenic differentiation ability. HGF activated-HGF/c-Met, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, which was positively correlated with TDSCs proliferation and migration but negatively related to TDSC osteogenic differentiation ability. The phosphorylation of Smad1/5/8 was also negatively related to HGF/c-Met, MAPK/ERK1/2, and PI3K/AKT signaling, which demonstrated that the inhibition of osteogenic differentiation was dependent on BMP/Smad1/5/8 signaling. Overall, we showed that HGF could promote TDSCs proliferation and migration and inhibit osteogenic differentiation in vitro, suggesting a potential role for HGF as a cytokine treatment of tendon trauma.     

大鼠肝脏F抗原基因的克隆、表达及产物纯化

肝脏F抗原是一种新型的能直接反映肝损伤程度的血清学标志,它是1968年由美国学者Fravi从小鼠肝中分离出来的[1].F抗原是存在于哺乳动物肝细胞质中的一种水溶性不稳定的单链蛋白质,相对分子量为43kD[2],正常肝组织中的F抗原含量是血清中的1370倍,只有肝细胞被破坏时,F抗原才释放     

血管内皮生长因子基因治疗大鼠脑缺血的实验研究

为探讨血管内皮生长因子(VEGF)基因治疗大鼠脑缺血的可行性,构建了pcD2/hVEGF121真核表达质粒,建立持续性大脑中动脉堵塞(MCAO)的局灶性脑梗塞模型,大鼠脑皮质直接注射法转移pcD2/hVEGF121真核表达质粒。应用逆转录聚合酶链反应(RT-PCR)、VEGF免疫组织化学法、脑血管计数及梗塞面积测定等方法检测转移pcD2/hVEGF121真核表达质粒后大鼠脑中VEGF基因表达及生物学效应。结果发现,与转移空载质粒的对照组相比,转移VEGF基因后7d的大鼠脑组织中有VEGFmRNA高表达,VEGF免疫组化染色可见VEGF蛋白表达水平增高,脑血管数增多,梗塞体积缩小。因此,直接注射法转移VEGF基因能够在缺血脑组织中表达,表达产物能够发挥生物学效应,进而起到保护脑组织作用。     

重组人角质细胞生长因子-2基因克隆、表达、纯化与活性分析

 从人胚肺二倍体细胞KMB17中抽提总RNA ,经RT PCR扩增获得编码人角质细胞生长因子 2 (keratinocytegrowthfactor 2 ,KGF 2 )的cDNA .克隆于硫氧环蛋白表达载体pThioHisA ,序列分析表明与文献报道一致 .经IPTG诱导 ,在大肠杆菌BL2 1中实现高效表达 ,表达量可达菌体总蛋白 10 %～15 % .菌体超声破碎 ,上清经CM SepharoseFF阳离子交换 ,Heparin Sepharose亲和层析 ,Superdex 75凝胶过滤层析纯化得到重组人KGF 2 ,纯度高于 95 % .生物活性分析表明 ,它能够促进成纤维细胞NIH 3T3的增殖 ,诱导鸡胚背根神经结神经轴突的生长 ,促进鸡胚尿囊膜血管生成 .研究结果表明 ,获得了 95 %纯度的具有生物学活性的重组人KGF 2 ,为进一步的基础与应用研究提供了基础     

Gene Expression and Polymorphism of Myostatin Gene and its Association with Growth Traits in Chicken

Myostatin is a member of TGF-β super family and is directly involved in regulation of body growth through limiting muscular growth. A study was carried out in three chicken lines to identify the polymorphism in the coding region of the myostatin gene through SSCP and DNA sequencing. A total of 12 haplotypes were observed in myostatin coding region of chicken. Significant associations between haplogroups with body weight at day 1, 14, 28, and 42 days, and carcass traits at 42 days were observed across the lines. It is concluded that the coding region of myostatin gene was polymorphic, with varied levels of expression among lines and had significant effects on growth traits. The expression of MSTN gene varied during embryonic and post hatch development stage.     

突变型人HGF(tvNK1)对CCl_4诱导的大鼠肝纤维化的影响

目的:探讨突变型人肝细胞生长因子(HGF~(K132E,R134E),tvNK1)对四氯化碳(CCl_4)诱导的SD大鼠肝纤维化的影响。方法:生物发酵大量制备tvNK1,并经腹腔注射于CCl_4诱导的纤维化SD大鼠体内,6周后取肝脏组织,通过real-time PCR和Western blot检测tvNK1对纤维化SD大鼠肝脏中I型胶原蛋白(Collagen type I,Col1A1)、IV型胶原蛋白(Collagen type IV,Col4A1)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)在mRNA和蛋白水平表达的影响,并进一步通过HE和Masson染色观察tvNK1对纤维化SD大鼠肝脏形态和肝组织胶原纤维的影响。结果:SDSPAGE检测结果显示,获得纯度≥95%的tvNK1。Real-time PCR和Western blot结果显示,tvNK1降低纤维化大鼠肝脏中Col1A1、Col4A1和α-SMA在mRNA和蛋白水平上的表达。HE和Masson染色结果显示,tvNK1减缓纤维化大鼠肝结构的病理性改变,并降低肝脏中胶原纤维含量。结论:tvNK1能抑制CCl_4诱导的大鼠肝纤维化,为预防肝纤维化和其他器官纤维化疾病提供实验依据。     

重组腺病毒Ad-HGF体外感染成纤维细胞后转染效率及表达的检测

押检测携带人肝细胞生长因子基因的重组腺病毒Ad-HGF在体外对成纤维细胞的感染效率以及感染细胞对目的蛋白的表达。以不同感染复数(m.o.i.)(25,50,100,200)的Ad-GFP感染NIH3T3细胞,48h时用流式细胞仪检测转染效率;以50m.o.i.感染NIH3T3细胞后48h,用ELISA和Western印迹杂交法分别检测感染上清中HGF的表达。分别以50m.o.i.的Ad-GFP和Ad-HGF感染原代培养人瘢痕成纤维细胞,以检测重组腺病毒对原代培养人瘢痕成纤维细胞的转染效率和其对HGF的表达。结果表明,当m.o.i.为50时,重组腺病毒对NIH3T3细胞的转染效率已达95%以上;HGF的表达量可达每2×106细胞249ng;并可检测到HGF蛋白的一特异杂交带。以50m.o.i.的Ad-GFP感染原代培养人瘢痕成纤维细胞,72h时GFP表达达高峰,此时转染效率可高达36.75%。Ad-HGF感染原代培养人瘢痕成纤维细胞后HGF的表达在72h时达高峰,表达量可达每3.3×105细胞66ng。初步认为重组腺病毒可有效地介导HGF基因转染正常或瘢痕成纤维细胞,且感染细胞可有效表达目的蛋白。     

Elf Electromagnetic Fields Affect Gene Expression of Hegenerating Rat Liver Following Partial Hepatectomy

Pulsed extremely-low-frequency magnetic fields (ELF-PEMFs) influence the expression of oncogenes c-myc and c-ras and of ornithine decarboxylase (ODC) in the regenerating rat liver following partial hepatectomy. In fact, while the mRNA's encoding both oncogenes are present in very low amounts in the normal liver, their concentration is dramatically increased during regeneration. Ornithine decarboxylase and c-myc mRNA's reach a maximum during the early phases of regeneration (3 hours after surgery) and decrease thereafter. c-ras mRNA reaches a maximum 40 hours after the operation. Treatment with ELF-PEMFs delivered to the animals immediately after the operation and every 12 hours thereafter increases The concentration of both oncogenes and of ornithine decarboxylase mRNA' s at 3 hours (c-myc and ODC) and at 40 hours (c-ras) respective-ty.     

Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B

Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.