血管内皮生长因子(VEGF)与肝再生

血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)能特异地直接作用于血管内皮细胞,刺激血管内皮细胞的分裂、增殖并诱导血管的形成.它通过与血管内皮细胞的受体结合发挥作用.肝受到各种损伤包括部分肝切除后,肝再生就得以启动.在肝再生过程中,VEGF对内皮细胞的生长增殖起有效促进作用,并且能诱导组织胶原酶、血纤维蛋白酶原的激活,增加血管的通透性,这对血管再生起着极其重要的作用.而血管再生是肝再生的一个重要组成部分,它不仅能给肝细胞提供血液支持,而且能促进肝脏结构的重构.因此VEGF在肝再生过程中具有重要的作用.     

Molecular cloning and expression of human hepatocyte growth factor

Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.     

VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.     

大鼠再生肝8种细胞的一碳代谢相关基因转录谱研究

为了解大鼠肝再生中8种肝脏细胞的一碳代谢相关基因转录谱及其预示的代谢活动,分离大鼠再生肝的8种细胞,检测它们的一碳代谢相关基因表达变化,分析其表达模式及预示的生理活动.结果表明,肝星形细胞的甲硫氨酸转化为S-腺苷甲硫氨酸活动在肝再生启动阶段增强.胆管上皮细胞的S-腺苷甲硫氨酸转化为S-腺苷高半胱氨酸、N5-甲基四氢叶酸将甲基转移给甲硫氨酸活动在进展阶段增强.肝细胞、陷窝细胞、树突状细胞的聚谷氨酸水解、四氢叶酸与N5-甲酰基四氢叶酸相互转化在肝再生3个阶段增强.结论:大鼠肝再生与一碳代谢相关.     

大鼠再生肝8种细胞的嘌呤核苷酸代谢基因转录谱预示的代谢活动

为了解大鼠肝再生中肝细胞、胆管上皮细胞、卵圆细胞、星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞等8种肝脏细胞的嘌呤核苷酸代谢基因转录谱及预示的代谢活动,按张丽君[1]等方法分离大鼠部分肝切除后10个恢复时间点大鼠再生肝的上述8种细胞,用RatGenome2302.0芯片等检测嘌呤核苷酸代谢基因在上述细胞中表达变化,用Excel等软件及生物信息学和系统生物学等方法分析它们的表达模式、预示的生理活动等.结果表明,85个嘌呤核苷酸代谢基因在大鼠肝再生中发生了有意义表达变化,8种细胞的相应基因数为40,43,30,42,22,26,36和48.上调、下调、上/下调的基因个数为49、11、31,相应细胞的基因数为27、20和1,31、4和1,15、7和3,12、10和0,23、15和3,19、7和2,39、3和1,33、6和0.其中,催化DNA合成的DNA聚合酶基因和催化RNA合成的RNA聚合酶基因在肝再生的多个时间点和多种细胞中表达增强,胆管上皮细胞和星形细胞的腺苷酸合成相关基因表达增强.肝细胞、卵圆细胞和星形细胞的核苷酸分解相关基因、肝细胞、星形细胞和树突状细胞的嘌呤核苷分解相关基因表达减弱.预示大鼠肝再生与嘌呤核苷酸代谢密切相关.     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.     

肝再生过程中肾desmin和GFAP的表达以及MMPs的变化

采用免疫组织化学法和明胶酶谱电泳法研究大鼠肝部分切除(PH)后再生过程中肾结蛋白(desmin)和胶质纤维酸性蛋白(GFAP)的表达以及基质金属蛋白酶-2(MMP-2),-9(MMP-9)的活性变化.免疫组织化学结果显示,肝再生过程中肾小球系膜细胞、足细胞和间质细胞表达desmin和GFAP,而且二者的表达在PH后均经历了先减少后恢复的过程.明胶酶谱电泳结果显示,在对照组,检测到1条92 kD(pro MMP-9,MMP-9酶原形式)蛋白酶带,具有强的活性.在PH后8 d到14 d检测到了92 kD,86 kD(MMP-9),72 kD(pro MMP-2,MMP-2酶原形式)和66 kD(MMP-2)4条蛋白酶带.从第10 d到14 d,pro MMP-9和MMP-9活性逐渐增强,pro MMP-2和MMP-2活性逐渐降低.     

大鼠肝再生中肝细胞基因表达与DNA裸露的相关性研究

为了解大鼠肝再生中肝细胞的基因表达与DNA裸露的相关性,分离大鼠肝细胞,提取裸露DNA,用SOLEXA方法对DNA测序,用BWA软件拼装测序片段,用IPA软件分析拼装的基因种类和作用,用Rat Genome230 2.0芯片检测基因的转录情况.研究表明,大鼠肝再生的12h(PH后12h),肝细胞的裸露DNA涉及16 912个基因,1 701个基因发生了有意义裸露量变化.其中,25个基因的裸露量上调,1 676个基因的裸露量下调.与Rat Genome 230 2.0芯片的检测的基因转录比对表明,肝细胞的AKR7A3,BMYC,CDC25B等26个基因的裸露量和表达量均下调,表明大鼠肝再生12h时,这些基因的表达可能受染色体结构调控.     

肝大部切除前后热休克处理对热休克蛋白、酸性磷酸酶和碱性磷酸酶活性影响的分析

综合了磷酸酶的原位复性电泳、体视学分析、比色分析等方法分析①切除大白鼠大部分肝后的肝再生期间,②热休克处理大白鼠（４６℃、３０ｍｉｎ）恢复８ｈ,再切除大部分肝后的肝再生期间,③切除大部分肝恢复４ｈ,再热休克处理（４６℃、３０ｍｉｎ）后的肝再生期间热休克蛋白（ＨＳＣ７０／ＨＳＰ６８）、酸性磷酸酶（ＡＣＰ）和碱性磷酸酶（ＡＫＰ）动态变化的资料,从６个方面比较分析了ＨＳＣ７０／ＨＳＰ６８,ＡＣＰ和ＡＫＰ在肝再生中的相互关系及对肝再生的可能作用     

Toll样受体信号通路的3条途径在大鼠肝再生中抑制库普弗细胞免疫反应

为从基因转录水平了解大鼠肝再生中Toll样受体信号通路调节库普弗细胞免疫反应的途径和方式,首先建立大鼠2/3肝切除(partial hepatectomy,PH)模型,从中分离库普弗细胞进行基因微阵列分析.发现Toll样受体信号通路的23个基因及其调节免疫反应的62个基因与大鼠肝再生相关.基因协同作用(Ep(t)值)和同类提取法分析表明,大鼠肝再生进展阶段,Toll样受体信号通路通过TLR/NF-κB,TLR/MAPK,TLR/IRF等3条途径和TLR4,MAL,UNC93B1,AP1S2,IRF1等5个关键基因抑制库普弗细胞免疫反应.     

大鼠肝窦内皮细胞的分离、鉴定及纯度、活性分析

按Higgins等方法制作大鼠2／3肝切除（parital hepatectomy,PH）模型,用两步灌流法分散肝脏细胞,用60％ Percoll梯度离心和免疫磁珠分离肝窦内皮细胞（hepatic sinusoidal endothelial cell,SEC）,用分化抗原簇14（cluster of differentiation 14,CD14）和内皮素-1（endothelin-1,ET-1）的免疫组织化学定性、定位再生肝（regenerating liver,RL）、分散的肝脏细胞及分离的窦内皮细胞,用RT—PCR定量窦内皮细胞的CDl1和ET-1的mRNA,用蛋白免疫印迹方法定量窦内皮细胞的CD14和ET-1蛋白．初步证实,分离的窦内皮细胞中CD14和ET-1阳性细胞占95％以上,从PH后各时间点分离的窦内皮细胞的CD14和ET-1mRNA量稳定,相应的蛋白量亦稳定．表明改进的分离窦内皮细胞方法具有收率和纯度高、活性好等特点,值得采用．     

大鼠再生肝8种细胞的嘧啶核苷酸代谢相关基因转录谱预示的生理活动

为了解大鼠肝再生中8种肝脏细胞的嘧啶核苷酸代谢相关基因转录谱及其预示的代谢活动,分离大鼠再生肝的8种细胞,检测它们的嘧啶核苷酸代谢相关基因表达变化,分析其表达模式及预示的生理活动.结果表明,50个嘧啶核苷酸代谢相关基因在大鼠肝再生中发生了有意义表达变化,8种细胞的相应基因数为25、33、16、24、15、15、21和18.肝细胞、胆管上皮细胞、窦内皮细胞的通过嘧啶核苷酸前体合成嘧啶核苷酸活动增强,除树突状细胞外其他7种细胞的嘧啶核苷酸分解代谢减弱.结论:大鼠肝再生与嘧啶核苷酸代谢密切相关.     

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.     

AA818342等6个新基因参与大鼠再生肝8种细胞的PLC信号转导

为了解AA818342、BM389035、BF289002、BF403759、AI170687、AI715484等6个新基因在PLC信号通路中的作用及与大鼠肝再生的相关性,分离大鼠再生肝8种细胞,检测它们的基因表达变化,分析新基因与已知基因的序列同源性、共表达关系及参与的生理活动.结果表明,AA818342与col8a1同源,在30 h和72 h再生肝的卵圆细胞中表达下调,在各期再生肝的库普弗细胞中表达上调.BM389035与itga1同源,在168 h再生肝的树突状细胞中表达下调.BF289002与gnai1同源,在12 h和168 h再生肝的卵圆细胞表达上调.BF403759与cacna1d同源,在2 h再生肝的星形细胞表达上调.AI170687与mef2c同源,在36 h再生肝的树突状细胞中表达下调.AI715484与prkce同源,在2 h再生肝的星形细胞中表达上调.上述基因转录谱预示,AA818342等6个新基因属于PLC信号通路成分,参与大鼠再生肝8种细胞的PLC信号转导.     

Mesodermal Wnt2b signalling positively regulates liver specification

Endodermal organs such as the lung, liver and pancreas emerge at precise locations along the primitive gut tube. Although several signalling pathways have been implicated in liver formation, so far no single gene has been identified that exclusively regulates liver specification. In zebrafish, the onset of liver specification is marked by the localized endodermal expression of hhex and prox1 at 22 hours post fertilization. Here we used a screen for mutations affecting endodermal organ morphogenesis to identify a unique phenotype: prometheus (prt) mutants exhibit profound, though transient, defects in liver specification. Positional cloning reveals that prt encodes a previously unidentified Wnt2b homologue. prt/wnt2bb is expressed in restricted bilateral domains in the lateral plate mesoderm directly adjacent to the liver-forming endoderm. Mosaic analyses show the requirement for Prt/Wnt2bb in the lateral plate mesoderm, in agreement with the inductive properties of Wnt signalling. Taken together, these data reveal an unexpected positive role for Wnt signalling in liver specification, and indicate a possible common theme for the localized formation of endodermal organs along the gut tube.     

Bojungbangdocktang inhibits vascular endothelial growth factor induced angiogenesis via blocking the VEGF/VEGFR2 signaling pathway in human umbilical vein endothelial cells

Oriental herbal medicines have been widely used for the prevention or treatment of various diseases including cancer in Asia. However, to prove their chemo preventive efficacies in modern times, scientific evidence for those herbal medicines is required. Thus, in the present study, an effective herbal cocktail Bojungbangdocktang (BJBDT) was investigated to elucidate antiangiogenic mechanism in vitro and in vivo. BJBDT significantly inhibited vascular endothelial growth factor (VEGF) induced proliferation in HUVECs at nontoxic concentrations, despite weak cytotoxicity against human umbilical vein endothelial cells (HUVECs). BJBDT also significantly suppressed VEGF-induced migration and tube formation of HUVECs. Furthermore, BJBDT treatment resulted in pale color and low hemoglobin level in Matrigel plugs, as well as dark red color and high hemoglobin level in untreated control. Interestingly, BJBDT specifically inhibited the binding of VEGF to vascular endothelial growth factor receptor 2 (VEGFR2), but not VEGFR1. In addition, friedelin, formononetin, ginsenoside Rb1, naringin, atractyloside, diosgenin, and allantonin were identified from BJBDT by high-performance liquid chromatography (HPLC) analysis as a quality of control. Taken together, these results suggest that BJBDT is a potent angiogenesis inhibitor blocking the VEGF/VEGFR2 signaling pathway in HUVECs. Supported by the Korea Science and Engineering Foundation Grant from the Korean Government (Ministry of Science and Technology) (Grant No. R13-2007-019-00000-0)     

AW915115参与大鼠再生肝星形细胞的Notch信号转导

为了解新基因AW915115在Notch信号通路中的作用及与大鼠肝再生的相关性,用Percoll密度梯度离心结合免疫磁珠分选方法分离大鼠再生肝的8种细胞,用Rat Genome 230 2.0芯片等检测上述细胞的Notch信号通路基因在大鼠肝再生中的表达变化,用BLAST、Microsoft Excel等软件分别分析新基因与已知基因的序列同源性和共表达关系,用生物信息学和系统生物学等方法分析上述基因参与的生理活动.结果表明,AW915115与活化Notch受体的N-乙酰葡糖基转移酶基因lfng同源,并且在2 h和12 h再生肝星形细胞中表达下调.根据上述基因的同源性和共表达关系推测,新基因AW915115参与大鼠再生肝星形细胞的Notch信号转导.     

腹腔镜肝癌切除术16例临床分析

目的介绍采用HIFU及Endo-GIA成功行16例肝癌切除手术的经验与体会.方法选择左半肝及右肝浅表癌肿患者,肝功能Child分级A级15例,B级1例,采用超声刀(High Intensity Focused Ultrasound,HIFU)及腹腔镜切割吻合器Endo-GIA行16例肝恶性肿瘤切除手术.结果 15例患者顺利完成行腹腔镜肝癌切除,1例因出血改为开腹手术.左肝局部切除4例,左半肝切除1例,左外叶切除8例,肝右叶局部切除3例.手术时间80~220 min,平均105 min.术中出血量90~1 000 m L,平均300 m L.病灶直径2~6.5 cm,平均4.5 cm,术后无并发症发生.结论对肝脏浅表肿瘤或左半肝的恶性肿瘤患者行HIFU及Endo-GIA腹腔镜肝癌切除术是可行和安全的.