哺乳动物雷帕霉素靶蛋白信号通路相关基因对大鼠再生肝8种细胞生长过程的调节作用

个基因含于大鼠Genome 230 2.0芯片,82个基因与大鼠肝再生相关。其中,在启动阶段,mTOR信号通路主要参与激活、促进肝细胞、陷窝细胞、库普弗细胞、树突状细胞、肝星形细胞和窦内皮细胞的生长过程;在进展阶段,mTOR信号通路明显地促进肝细胞、胆管上皮细胞、卵圆细胞、肝星形细胞、库普弗细胞、陷窝细胞和树突状细胞的生长过程;在终止阶段,mTOR信号通路的大多数途径对肝细胞、库普弗细胞和树突状细胞的生长过程的促进作用减弱,而途径25却对肝细胞、窦内皮细胞、肝星形细胞和陷窝细胞的生长过程有明显的抑制作用。 结论 mTOR信号通路的25条途径和82个基因参与大鼠再生肝8种细胞的生长调控。     

JNK信号通路调控大鼠再生肝8种细胞的增殖和凋亡

目的 从基因转录水平了解JNK信号通路在大鼠再生肝8种细胞中的作用。方法 用密度梯度离心和免疫磁珠等方法分离肝细胞（HC）、胆管上皮细胞（BEC）、卵圆细胞（OC）、肝星形细胞（HSC）、窦内皮细胞（SEC）、库普弗细胞（KC）、陷窝细胞（PC）、树突状细胞（DC）等8种肝脏细胞,用大鼠Genome 230 2.0芯片检测大鼠再生肝8种细胞的基因表达谱,用生物信息学和系统生物学等方法分析基因表达变化预示的JNK信号通路在调控大鼠再生肝8种细胞增殖、凋亡中的作用。用实时荧光定量PCR方法验证了芯片结果的可靠性。结果 JNK信号通路涉及240个基因和42条途径,其中,225个基因与大鼠肝再生相关。基因协同作用分析显示,在大鼠肝再生启动阶段,JNK信号通路启动HC和KC增殖,促进OC凋亡,启动部分PC和SEC增殖和促进部分PC和SEC凋亡;在进展阶段,JNK信号通路促进HC、BEC、KC和DC增殖,促进部分PC增殖、部分PC凋亡。在终止阶段,JNK信号通路促进HC、OC和PC凋亡,促进部分KC增殖、部分KC凋亡。结论 大鼠肝再生中JNK信号通路的42条途径和225个基因参与调控大鼠再生肝8种细胞的增殖和凋亡。     

大鼠肝再生中肝脏细胞的基因转录谱预示EM>AI408225/EM>促进抗原肽与TCR结合

目的 了解新基因AI408225与大鼠肝再生涉及的体液免疫相关性。 方法 大鼠再生肝8种细胞的分离,采用Percoll密度梯度离心结合免疫磁珠分选方法进行,用Microsoft Excel、BLAST等软件分析基因的序列同源性及共表达关系,用生物信息学和系统生物学等方法分析新基因参与的生理活动。 结果 93个参与体液免疫的已知基因与肝再生相关,肝细胞、胆管上皮细胞、卵圆细胞、肝星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞的体液免疫相关基因数为33、46、17、59、48、35、53、68。其中,cd4与AI408225同源和共表达。 结论 大鼠肝再生与体液免疫相关,AI408225参与MHC II-抗原肽-CD4-TCR复合物形成。     

大鼠肝再生中8种肝脏细胞的细胞免疫相关基因转录谱预示的生理活动

目的 了解大鼠肝再生中8种肝脏细胞的细胞免疫相关基因转录谱,及其预示的细胞免疫活动。 方法 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、陷窝细胞和树突状细胞等8种细胞,用Rat Genome 230 2.0芯片等检测细胞免疫相关基因在上述细胞中表达变化,用Cluster等软件及生物信息学和系统学生物等方法分析它们的表达模式及预示的生理活动。 结果 大鼠肝再生中40个细胞免疫相关基因发生了表达变化,相应细胞的基因数为19、19、9、19、19、21、22、21。肝再生启动阶段和进展阶段抗原肽MHC复合物形成,NF-κB激酶活性和IL-2等细胞因子合成增加,终止阶段NF-κB促进细胞分化活动和caspase诱导T细胞凋亡活动增强。 结论 大鼠肝再生与细胞免疫相关。     

Gln和rhGH对门脉高压症术后肠粘膜屏障及增殖细胞核抗原表达的影响

目的：研究单独或联合应用谷氨酰胺（Gln）和重组人生长激素（rhGH）对门脉高压症术后肠粘膜屏障功能的保护作用及机制。方法:29例门静脉高压症术后患者随机分为4组：①Gln组（n=6），②rhGH组（n=8），③Gln+rhGH组（n=7）和④对照组（n=8）。术后3 d开始进行等氮、等热量的全胃肠外营养支持（TPN），持续7 d。对手术前、后的尿乳果糖排泄率/甘露醇排泄率（L/M）、十二指肠粘膜绒毛高度、陷窝深度及肠粘膜增殖细胞核抗原（PCNA）表达指数进行对比。结果:Gln+rhGH组术后L/M值升高小于对照组（P<0.05），肠粘膜绒毛高度和陷窝深度大于对照组（P<0.05）及术前（P<0.05），肠粘膜上皮PCNA表达指数大于术前及其它3组（P<0.05）；对照组术后绒毛高度小于术前（P<0.05），陷窝深度变化无显著（P＞0.05）。结论：联合应用Gln和rhGH能降低门脉高压症术后肠壁通透性并维护肠粘膜形态学完整性，单用Gln或rhGH无此作用。这种作用可能与增加肠黏膜PCNA表达而促进肠黏膜上皮细胞增殖有关。     

益骨胶囊含药血清对共育体系中破骨细胞活性和凋亡的影响

目的： 观察益骨胶囊含药血清在成骨-破骨细胞共育体系中对SD大鼠破骨细胞（OC）活性和凋亡的影响。 方法： (1)取1d龄SD大鼠颅骨分离培养成骨细胞（OB），取5d龄SD大鼠四肢股骨、胫骨分离培养OC，建立细胞上清相通但细胞间不相互混杂的平面式成骨-破骨细胞共育体系，实验分为含药血清组和对照组；（2）将10月龄SD雌性大鼠分为益骨胶囊灌胃组和生理盐水对照组，制备含药血清和对照血清；（3）重氮盐法检测抗酒石酸酸性磷酸酶（TRAP）和光镜观察骨陷窝数；（4）光镜和荧光显微镜下观察共育体系中OC凋亡情况。 结果： 含药血清组在48 h、72 h、96 h对成骨-破骨细胞共育体系中OC分泌TRACP的活性均明显降低于对照组，OC的存活数明显低于对照组，OC的凋亡率明显高于对照组且呈明显的时效关系；所形成骨吸收陷窝的数目明显低于对照组(P<0.01)。 结论： 益骨胶囊含药血清在共育体系中能够抑制OC活性，诱导破骨细胞的凋亡。     

应用破骨细胞-颅骨联合培养体系研究先天性成骨不全的骨再建过程

目的　采用先天性成骨不全(OI)小鼠,oim/oim为动物模型,应用破骨细胞-颅骨联合培养体系研究OB和OC两种细胞在OI骨再建过程中的功能改变和相互作用。 方法　实验采用小鼠颅骨（CAL）组织培养,实验设两组：WTCAL-WTOC组：联合培养对照组颅（WTCAL） 与对照破骨细胞（WTOC）;OICAL-OIOC组：联合培养OI颅骨（OICAL）与OI破骨细胞（OIOC）。以免疫组化染色方法　-TRAP识别破骨细胞,ALP免疫组化染色方法识别成骨细胞。破骨细胞骨吸收活性为骨吸收陷窝占颅骨表面百分比。单位OC吸收面积为总骨吸收陷窝除以破骨细胞数。 结果　于7d,OICAL-OIOC组破骨细胞数低于WTCAL-WTOC组;OICAL-OIOC组的OC/OB比例低WTCAL-WTOC组;OICAL-OIOC组单位破骨细胞吸收能力高于WTCAL-WTOC组。 结论　OI的小鼠模型骨再建中骨量丢失一方面由于其成骨细胞功能异常,另一方面也可能因为其破骨细胞的代偿性功能活跃。     

1, 25二羟基胆钙化醇诱导大鼠骨髓单核细胞向破骨细胞转化的机制

目的:探讨1,25二羟基胆钙化醇(1,25(OH)2D3)诱导大鼠骨髓单核细胞向破骨细胞转化时明胶酶表达及其参与骨陷窝形成机制。方法:分离乳鼠骨髓内细胞,诱导生成破骨样细胞。姬姆莎、抗酒石酸酸性磷酸酶(TRAP)染色鉴定。扫描电镜观察诱导出的细胞贴附于骨片上形成的骨陷窝,明胶酶谱检测细胞培养液中明胶酶表达水平。结果:单核细胞经1,25(OH)2D3诱导,第9日生成大量的破骨样细胞。姬姆莎染色显示出多核(≥3个),TRAP染色阳性,扫描电镜观察破骨细胞在骨片上培养时产生骨陷窝。1,25(OH)2D3组基质金属蛋白酶2(MMP-2)表达水平增加显著。结论:1,25(OH)2D3诱导单核细胞产生大量破骨细胞。参与骨陷窝形成的明胶酶是MMP-2。这可能是破骨细胞通过某些机制,促进其他非破骨细胞分泌有活性的MMP-2参与噬骨。     

阿仑膦酸钠对兔破骨细胞功能的影响

用骨陷窝形成分析法观察阿仑膦酸钠对体外培养破骨细胞功能的影响，探讨可能的作用机制。在建立兔破骨细胞培养方法的基础上，用不同浓度的阿仑膦酸钠分别与骨片或成骨细胞提前作用，然后再将骨片或成骨细胞分别与破骨细胞共同培养。结果发现当阿仑膦酸钠（10^-11,10^-9,10^-7mol/L)与骨片预处理后，破骨细胞在骨片上形成的骨吸收陷窝数目减少，其抑制率分别为19．5％（P＜0．05）、49．0％（P＜0．01）和74．5％（P＜0．01）。用阿仑膦酸钠（10^-9,10^-7,10^-5mol/L)预处理成骨细胞后，仅在高浓度（10^-5mol/L）时可见破骨细胞骨吸收陷窝的数目明显减少，其抑制率为62．8％（P＜0．01）。结果表明阿仑膦酸钠能直接或通过成骨细胞间接抑制破骨细胞的骨吸收活性。     

大鼠肝再生中8种肝脏细胞的凝血反应相关基因转录谱预示的生理活动

目的 了解大鼠肝再生中8种肝脏细胞的凝血反应相关基因转录谱及其预示的生理活动。 方法 大鼠再生肝8种细胞的分离,基因表达变化检测,预示的生理活动分析用Cluster等软件及生物信息学和系统生物等方法进行,用Microsoft Excel等软件分析基因的表达模式。 结果 40个参与凝血反应的基因与肝再生相关,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、树突状细胞的相应基因数为13、31、12、33、32、9、34、33。serpine1、a2m 在上述8种细胞,vwf、klkb1 在除库普弗细胞之外的7种细胞,其他基因在两种或两种以上细胞中发生有意义表达变化。上述基因转录谱预示,肝再生启动和进展阶段激肽释放酶和凝血酶原合成,凝血酶形成等活动增强。终止阶段纤维蛋白单体形成纤维蛋白聚合体等活动增强。 结论 大鼠肝再生与凝血反应密切相关。     

The roles of innate immune cells in liver injury and regeneration

For predominant abundance with liver-specific Kupffer cells, natural killer （NK） cells, and natural killer T （NKT） cdls and their rapid responses to several stimuli, the liver is considered as an organ with innate immune features. In contrast to their roles in the defense of many infectious agents like hepatitis viruses and parasites, hepatic innate immune cells are also involved in the immunopathogenesis of human clinical liver diseases and several murine hepatitis models such as concanavalin A （Con A）, lipopolysaccharide （LPS）, or polyinosinic-polycytidylic acid （Poly I：C）-induced liver injury. In this review, the destructive roles of NK cells, NKT cells and Kupffer cells in the processes of immune-mediated liver injury and regeneration will be discussed, and some putative mechanisms involving the impairment of liver regeneration caused by activated hepatic innate immune cells are also proposed.     

乙型肝炎病毒X蛋白对肝癌细胞生长的影响

目的探讨乙型肝炎病毒X蛋白（hepatitis Bvirus Xprotein，HBx）对肝癌细胞恶性程度的影响。方法构建携带HBV。基因真核表达载体pcDNA用Bx，以脂质体介导转染HepG2肝癌细胞，建立可稳定表达HBx的肝癌细胞系HepG2-HBx细胞，同时设空载体pcDNA，转染细胞HepG2-pcDNA，及未转染HepG2细胞为对照组。PCR法扩增Neo基因检测插入的质粒DNA片段．免疫荧光检测HBx的表达。通过生长曲线测定、平板克隆形成实验、MTr比色实验、Hoechst33342核形态学染色观察及流式细胞仪测定．了解稳定转染细胞的生物学行为变化。结果与对照组相比，转染pcDNA√IBx的HepG2-HBx细胞生长速度加快．其倍增时间缩短（28h对32．5h或34h，P〈0．05），克隆形成率增加[（10．12±0．23）％对（5．33±O．19）％或（5．19±0．28）％，P〈0．05]，细胞周期分析显示由GdG，期-S期的进程明显加快。细胞凋亡检测显示HepG2-HB。细胞可抵抗放线菌素D（ActD）诱导的凋亡作用。结论HBx可提高肝癌细胞的增殖活性，并增强肝癌细胞的抗凋亡能力．增加了肝癌细胞的恶性表型。     

Hepatic progenitor cells, stem cells, and AFP expression in models of liver injury

Adult hepatocytes and liver-cell progenitors play a role in restoring liver tissue after injury. For the study of progenitor cells in liver repair, experimental models included (a) surgical removal of liver tissue by partial hepatectomy; (b) acute injury by carbontetrachloride; (c) acute injury by d-galactosamine (GalN) and N-nitrosomorpholine (NNM); and (d) chemical hepatocarcinogenesis by feeding NNM in low and high doses. Serological and immunohistological detection of alpha-fetoprotein gene expression served to follow pathways of cellular differentiation. Stem cells were not required in models of surgical removal of parenchyma and in carbon tetrachloride intoxication of adult hepatocytes. In contrast, regeneration of liver occurred through biliary epithelial cells in injuries induced by GalN and NNM. These biliary epithelial cells, collectively called oval cells, are most probably derived from the canals of Hering. Proliferating bile duct cells reached a level of differentiation with reactivation of foetal genes and significant alpha-1-fetoprotein (AFP) synthesis signalling a certain degree of retrodifferentiation with potential stemness. Due to the same embryonic origin of bile ducts and hepatocytes, biliary epithelium and its proliferating progeny (oval cells) have a defined role in liver regeneration as a transit and amplification compartment. In their early proliferation stage, oval cells were heavily engaged in DNA synthesis ([3H]thymidine labelling). Pulse-chase experiments during experimental hepatocarcinogenesis exhibited their development into hepatocytes with high risk for transformation and leading to foci of altered hepatocytes. Hepatocellular carcinomas may arise either from proliferating/differentiating oval cells or from adult hepatocytes; both cell types have stem-like properties. AFP-positive and AFP-negative carcinomas occurred in the same liver. They may represent random clonal origin. The heterogeneity of phenotypic marker (AFP) correlated with a process of retrodifferentiation.     

The hepatic stellate (Ito) cell: its role in human liver disease

The hepatic stellate (Ito) cell lies within the space of Disse and has a variety of functions. Stellate cells store vitamin A in characteristic lipid droplets. In the normal human liver, the cells can be identified by the presence of these lipid droplets; in addition, many stellate cells in the normal liver express a-smooth muscle actin. In acute liver injury, there is an expansion of the stellate cell population with increased -smooth muscle actin expression; stellate cells appear to play a role in extracellular matrix remodelling after recovery from injury. In chronic liver injury, the stellate cell differentiates into a myofibroblast-like cell with marked expression of -smooth muscle actin and occasional expression of desmin. Myofibroblast-like cells have a high fibrogenic capacity in the chronically diseased liver and are also involved in matrix degradation. In vitamin A intoxication, hypertrophy and proliferation of the stellate and myofibroblast-like cells may lead to non-cirrhotic portal hypertension, fibrosis and cirrhosis. In liver tumours, myofibroblast-like cells are involved in the capsule formation around the tumour and in the production of extracellular matrix within it. The transition of stellate cells into myofibroblast-like cells is regulated by an intricate network of intercellular communication between stellate cells and activated Kupffer cells, damaged hepatocytes, platelets, endothelial and inflammatory cells, involving cytokines and nonpeptide mediators such as reactive oxygen species, eicosanoids and acetaldehyde. The findings suggest that the stellate cell plays an active role in a number of human liver diseases, with a particular reactivity pattern in fibrotic liver disorders.     

Nitric oxide in liver fibrosis: The role of inducible nitric oxide synthase

The inducible form of nitric oxide synthase (iNOS) is expressed in hepatic cells in pathological conditions. Its induction is involved in the development of liver fibrosis, and thus iNOS could be a therapeutic target for liver fibrosis. This review summarizes the role of iNOS in liver fibrosis, focusing on 1) iNOS biology, 2) iNOS-expressing liver cells, 3) iNOS-related therapeutic strategies, and 4) future directions.     

Hepatocyte progenitors in man and in rodents--multiple pathways, multiple candidates

In severe injury, liver-cell progenitors may play a role in recovery, proliferating, and subsequently differentiating into mature liver cells. Identifying these progenitors has major therapeutic potential for ex vivo pharmaceutical testing, bioartificial liver support, tissue engineering and gene therapy protocols. Potential liver-cell progenitors have been identified from bone marrow, peripheral blood, cord blood, foetal liver, adult liver and embryonic stem cells. Differences and similarities are found among cells isolated from rodents and humans. This review will discuss identifying markers and differentiation potential in in vitro and in vivo models of these putative progenitors in both humans and rodents.     

肝干细胞的研究进展

肝干细胞又称肝卵圆细胞 ,在一定病生理条件下可向肝细胞和胆管上皮细胞、胰腺外分泌上皮等有限的几种组织细胞分化 ,具有多向分化增殖潜力 ,体外培养肝干细胞将为生物型人工肝提供一种新的细胞来源 ,肝干细胞移植不但可以替代坏死的肝组织 ,还可以刺激受体组织再生以达自身修复 ,治疗急性或亚急性肝坏死 ;同时为基因治疗提供新的可能。本文将就肝干细胞分类、表面标志、来源、诱导分离方法及人类肝干细胞的研究综述如下     

肝卵圆细胞的研究进展

肝卵圆细胞（OCs）是肝脏的干细胞,能够分化为肝细胞、胆管上皮细胞及其他细胞,亦与肝再生和肝脏疾病密切相关。本文简要地总结了近几年有关肝卵圆细胞的来源、定位、增殖、分化、凋亡、移植及在肝再生和肝脏疾病中作用等研究进展。     

体外诱导骨髓间充质干细胞成肝(样)细胞的研究进展

肝病已成为世界上发病率和死亡率最高的疾病之一。目前有望治疗晚期重症肝病的方法有原位肝移植、肝细胞移植、生物人工肝等,但这些方法都面临着肝或肝细胞源紧缺的问题。如何能够获得体外增殖的、可用于移植疗法的肝细胞资源成为研究的要点。骨髓间充质干细胞（BMSCs）既具有自我更新的能力,也具有在合适的微环境里多系横向分化的潜能,在体外可以被诱导分化为肝(样)细胞,理论上可提供丰富的肝细胞,BMSCs成为了细胞治疗的研究热门。我们简要综述了体外诱导BMSCs向肝样细胞（HLCs）分化的研究成果和最新进展,总结了各种诱导方法的优势以及存在的问题,并对该研究领域进行了小结与展望。     

Differential motility stimulation but not growth stimulation or adhesion of metastatic human colorectal carcinoma cells by target organ-derived liver sinusoidal endothelial cells

Liver is the most common distant metastatic site for colorectal cancers and when blood-borne colorectal cancer cells reach the liver, they first encounter hepatic capillary and sinusoidal endothelial cells. Thus we studied differences between highly (HT-29LMM) and poorly (HT-29P) liver-metastatic sublines of human colorectal cancer cells by examining the interactions between tumor cells and liver microvessel endothelial cells. Using hepatic sinusoidal endothelial (HSE) and lung microvessel endothelial (MLE) cell-conditioned medium we measured the growth and motility stimulating activities released from these endothelial cells and adhesion of these cancer cells to the endothelial cells. Differences in the ability of HSE-conditioned medium (HSE-CM) or MLE-conditioned medium (MLE-CM) to stimulate HT-29 cell growth were not observed. There was a small but significant increase in the rate of adhesion of highly metastatic HT-29LMM cells to HSE cell monolayers than poorly metastatic HT-29P cells, but there was no difference in adhesion to MLE cell monolayers. HSE-CM stimulated the motility of highly metastatic colorectal cancer cells to a greater extent than the poorly metastatic cells. Motility-stimulating activity for the colorectal cancer cell lines was not detected in MLE-CM. The HSE-CM motility-stimulating activity for human HT-29 cells was not removed using antibodies against hepatocyte growth factor (HGF/SF), complement component C3 or laminin, indicating that it is not related to these known liver-derived motility factors. The results suggest that the ability of highly metastatic HT-29LMM colorectal cancer cells to colonize the liver is related to their ability to respond to liver sinusoidal endothelial cell-derived motility factors and to a lesser degree to adhere to liver sinusoidal endothelial cells.