血管内皮细胞生长因子反义核糖核酸对肝癌细胞生长的抑制作用

目的 探讨血管内皮细胞生长因子(VEGF)反义RNA转染人肝癌细胞后对细胞体内外生物学性状的影响。方法 将含正义、反义VEGFcDNA序列的质粒PCMV—VEGF、PCMV—FGEV及空载体质粒pcDNA3.1,在脂质体介导下导入SMMC—7721肝癌细胞,分别称为正义、反义及对照组,并通过G418筛选获得阳性克隆。细胞原位杂交和免疫组织化学方法检测转染后VEGF在肝癌细胞内的表达情况;MTT法和FCM检测转染后细胞在体外的增殖和凋亡情况;并制备裸鼠动物模型,观察转染后细胞的体内生长情况。结果 转染PCMV—FGEV后肝癌细胞内VEGF的转录及其蛋白的表达水平显著下降,但转染后体外细胞的增殖与凋亡情况均无明显变化。转染PCMV—FGEV后细胞在裸鼠体内的生长缓慢,反义组成瘤时间为(25.0±1.8)d,明显长于正义组(15.7±2.5)d和对照组(18.5±2.1)d,F=19.455,P<0.01;而平均瘤重以反义组最轻,为(0.96±0.28)g,F=21.501,P<0.01;同时反义组裸鼠肿瘤细胞发生明显的凋亡。结论 VEGF反义RNA转染人肝癌细胞可抑制肿瘤细胞VEGF的表达,在体外对细胞增殖和凋亡无影响,而体内可显著诱导细胞凋亡并抑制肿瘤生长。     

肝细胞癌中T淋巴瘤侵袭转移诱导基因1对肝细胞癌患者血管生成的作用

目的检测肝细胞癌中T淋巴瘤侵袭转移诱导基因(Tiam)1对血管生成的作用,探讨Tiam1、血管内皮生长因子(VEGF)及CD105表达的关系。方法以89例肝细胞癌患者术后存档蜡块为观察组,50例正常肝组织为对照组,采用免疫组织化学方法检测两组Tiam1、VEGF及CD105表达,探讨Tiam1、VEGF和MVD在不同临床病理特征的表达关系及相关性。结果观察组Tiam1、VEGF及MVD表达阳性率显著高于对照组;观察组Tiam1、VEGF及MVD均与肿瘤转移、体积及Ki67表达相关。肝癌Tiam1、VEGF及MVD表达呈正相关性。结论肝细胞癌中Tiam1、VEGF高表达,共同促进CD105阳性的血管形成,联合检测Tiam1、VEGF及CD105可能对判断肝细胞癌预后有重要意义。     

结肠癌血管内皮生长因子与微血管密度和p53的关系

目的探讨血管内皮生长因子(VEGF)在结肠癌中的表达及其与微血管密度(MVD)和p53之间的关系.方法用免疫组化SABC法检测68例结肠癌组织不同区域VEGF、MVD和p53的阳性表达.结果结肠癌区VEGF、MVD和p53的表达明显高于癌旁区和正常区.结肠癌组织VEGF及p53的表达与肿瘤浸润深度、淋巴结转移、远处转移、血管侵犯及Dukes分期密切相关,而与组织学分型无关.p53(+)或VEGF(+)组MVD(34.6±12.2;31.2±12.6)均显著高于p53(-)或VEGF(-)组(15.0±7.9;12.7±6.3,P＜0.01);VEGF和p53均为阳性时,MVD值最大(36.5±11.9,P＜0.01).MVD记数与VEGF表达明显相关(P＜0.01),p3表达与VEGF表达和MVD记数均显著相关(P＜0.01).结论在结肠癌血管生成过程中,可能存在p53-VEGF调节旁路,p53基因在调控肿瘤血管形成方面起重要作用.     

血管抑制素基因抑制结肠癌细胞增殖及肿瘤血管生成的研究

目的:研究血管抑制素基因体外对结肠癌细胞增殖的抑制及体内对肿瘤血管生成的抑制,探讨血管抑制素基因对结肠癌的抑制作用。方法:构建重组质粒pcDNA3.1( )／angio,用脂质体转染法将重组质粒、空白载体导入结肠癌细胞Colo205,培养细胞观察细胞生长,绘制细胞生长曲线,计算增殖抑制率,然后将pcDNA3.1( )／angio、pcDNA3.1( )／Colo205、Colo205分别接种至裸鼠右侧颈部皮下,观察肿瘤的生长,测量肿瘤体积,计算抑瘤率,免疫组化检测肿瘤组织中微血管密度(MVD)、血管内皮生长因子(VEGF)的表达。结果:体外pcDNA3.1( )／angio组细胞的生长速度略慢于pcDNA3.1( )／Colo205、Colo205组,但差异无统计学意义(P>0.05);体内pcDNA3.1( )／angio组细胞的致力显著降低,瘤体增长缓慢,抑瘤率较高(P<0.01),瘤体组织中MVD及VEGF的表达较低(P<0.05);pcDNA3.1( )／Colo205、Colo205两组之间的差异无统计学意义(P>0.05)。结论:在体外血管抑制素不直接影响结肠癌细胞Colo205的生物学特性,但在体内可强烈抑制肿瘤的生长,其机制可能是通过降低血管生成因子而抑制肿瘤的新生血管生成,发挥抑制肿瘤作用。     

药物和缺血预处理对供肝细胞增殖周期的影响

目的了解供肝复流后细胞增殖周期的变化,明确药物预处理和缺血预处理对其的影响.方法建立大鼠三袖套法原位肝移植模型,观察移植肝细胞复流前后不同时间增殖细胞核抗原的表达情况;对供肝分别作药物预处理和缺血预处理,比较正常肝、预处理供肝和未预处理供肝复流2 h后的增殖细胞核抗原、肝酶谱及肝细胞凋亡状况.结果移植肝细胞复流2 h后增殖细胞核抗原表达显著增高,12 h后恢复正常水平;正常肝、药物预处理供肝、缺血预处理供肝、未预处理供肝复流2 h后的增殖细胞核抗原表达和肝酶谱依次升高,PCNA(%):2.1±0.1,4.3±0.2,7.0±0.4,9.4±0.4,AST(nKat·L-1):3.5±1.6,28.1±11.9,53.8±12.3,75.2±24.1,ALT(nKat·L-1):2.8±2.7,46.1±17.6,61.7±22.9,90.5±30.1,LDH(nKat·L-1):49.6±7.6,61.4±15.3,95.4±23.2,148.3±30.1.结论供肝复流后肝细胞G1期缩短至2 h以内,整个细胞周期时间缩短至20 h左右.正常肝、药物预处理供肝、缺血预处理供肝、未预处理供肝肝损害程度依次加重,整个细胞周期时间依次缩短.     

心肌内注射腺病毒介导血管内皮生长因子基因的血管生成实验研究

目的探讨血管内皮生长因子165(VEGF165)基因的血管生成作用. 方法在猪冠状动脉回旋支起始处放置Ameroid闭合器,制备慢性心肌缺血模型,28 d时行选择性冠状动脉造影,观察回旋支狭窄程度.开胸,治疗组(6只)在缺血区心肌内多点注射含人VEGF165基因的重组腺病毒载体(Ad-VEGF165);对照组(6只)在缺血区心肌内多点注射含细菌半乳糖苷酶基因的重组腺病毒载体(Ad-β-gal).转染重组腺病毒载体28 d时重复行冠状动脉造影后,处死猪,取缺血区心肌,做组织学检查;应用酶标法检测血浆中VEGF浓度,采用Rentrop分级、缺血区心肌侧支血管积分和心肌碱性磷酸酶染色法评价血管生成作用. 结果与对照组比较,基因转染3 d时治疗组血浆VEGF增高[(114.6±5.4)pg/ml对(60.3±1.8)pg/ml,P<0.001],7 d时血浆VEGF为(128.4±3.0)pg/ml对(70.9±5.3)pg/ml,P<0.001,14 d后恢复到基线水平.与对照组比较,基因转染28 d时治疗组Rentrop分级增高(2.8±0.4对1.3±0.8,P<0.001),侧支血管积分增高(4.8±0.8对1.7±0.8,P<0.001),缺血区毛细血管密度增高(210±19对62±6,P<0.001). 结论心肌转染Ad-VEGF165后,缺血心肌表达VEGF,产生了血管生成作用.     

移植雄性骨髓的雌性小鼠肝组织肝再生相关基因的信号通路

目的:探讨骨髓形成肝细胞的分子机制.方法:采用移植♂骨髓的♀小鼠模型,♀Balb/c小鼠随机分为模型组和正常对照组,选用小鼠基因表达谱寡核苷酸芯片,利用反转录酶合成掺有荧光标记的cDNA探针,将探针与基因表达谱芯片杂交观察小鼠肝组织基因表达谱的变化,筛选样本之间杂交信号比值有差异表达的基因.采用生物信息学方法分析模型组中小鼠肝组织再生相关基因的信号通路变化规律.结果:♀小鼠在移植♂骨髓6 mo后肝组织的基因表达谱显著不同,对照组相对于正常组的差异表达基因有865条,已知功能基因有447条,其中上调基因92条,下调基因355条.与肝再生相关基因信号通路涉及HGF,TGF-β,Focal Adhesion,JAK-Stat,VEGF等信号通路,他们相关基因的上调表达促进肝细胞的增殖分化.TGF-β信号通路中相关基因下调,抑制信号通路的激活,减弱肝再生的负性作用,利于肝细胞的增殖分化.结论:♀小鼠移植♂骨髓后的肝组织基因表达谱显著变化,有可能通过其肝再生相关基因的信号通路激活或抑制调节肝再生.     

经肝动脉化疗栓塞术对肝细胞癌细胞增殖和细胞凋亡的影响

为研究经肝动脉化疗栓塞术 (TACE)对肝细胞癌细胞增殖和细胞凋亡的影响 ,分别采用免疫组织化学方法和 TUNEL方法检测 6 5例肝细胞癌患者术后存档石蜡切片组织中增殖细胞核抗原 (PCNA)与细胞凋亡的表达情况 ,并比较二者检测结果在术前行与未行 TACE两组间有无差异。结果显示 ,术前行 TACE组 (n=18例 )的 PCNA标记指数 (35 .83± 12 .2 3%)显著低于未行 TACE组 (n=47例 ,5 9.92± 2 3.5 7%) ,P<0 .0 5 ;前者的凋亡指数 (17.6 2± 6 .87%)显著高于后者 (9.76± 3.6 4%) ,P<0 .0 5。提示 TACE不仅能抑制肝细胞癌的细胞增殖 ,而且能够诱导细胞凋亡 ,这可能是其发挥作用的重要机制之一。     

VEGF基因转染同种异体骨髓间充质干细胞治疗心肌梗死的实验研究

目的探讨血管内皮生长因子（VEGF）基因转染同种异体骨髓间充质干细胞（MSCs）对猪急性心肌梗死（AMI）血管再生和心功能的影响。方法体外分离、纯化、培养猪骨髓MSCs，以PCNA染色标记细胞；制备、抽提、纯化质粒pd—VEGF。用球囊堵闭及拉伤冠状动脉法建立AMI模型1W后，随机将其分为4组（n=4），进行经冠状动脉途径移植MSCs和／或Ⅵ犯F转染。组Ⅰ：给予转染VEGF腺病毒的MSCs移植；组Ⅱ：单纯MSCs移植；组Ⅲ：VEGF转染；组Ⅳ：DMEM对照组。4W后行免疫组化和超声心动图检查。结果组Ⅰ及组Ⅱ在梗死区及缺血区均可见较大量的PCNA标记的移植细胞。Ⅶ因子染色阳性的新生血管密度：组Ⅰ〉组Ⅲ〉组Ⅱ〉组Ⅳ。4W后LVEF值：组Ⅰ〉组Ⅱ〉组Ⅲ〉组Ⅳ（均P〈0．01）。结论VEGF基因转染同种异体骨髓MSCs移植猪心肌梗死区及缺血区促进血管再生及心肌细胞的再生，改善心功能。     

心肌内控制释放碱性成纤维细胞生长因子血管再生机制的探讨

目的:评价心肌内控制释放碱性成纤维细胞生长因子(bFGF)对急性心肌梗死(AMI)犬局部相关血 管生长因子表达与细胞增殖水平的影响,探讨bFGF血管再生治疗的可能机制。方法:将12只成年健康杂种犬 结扎左前降支(LAD)制作AMI模型。动物随机分为两组:激光心肌血运重建(TMR)组于AMI30min后行透壁 心肌打孔;bFGF组则于AMI30min后行非透壁心肌打孔,并随后向孔道内注射含有bFGF的纤维蛋白胶(FG)。 术后第8周,采用免疫组织化学染色和计算机形态学分析评价缺血心肌局部血管内皮生长因子(VEGF)、转化生 长因子β1(TGF β1)和增殖细胞核抗原(PCNA)表达水平的变化。结果:免疫组织化学定量分析发现,bFGF组梗 死区心肌VEGF、TGF β1和PCNA染色的显色面积[(9.27±1.74),(9.82±0.99)与(32.11±5.78)μm3]均高于 TMR组[(5.65±0.99),(9.11±0.47)与(17.14±6.70)μm3](均P<0.05),bFGF组PCNA染色的平均吸收度 也高于TMR组(0.3696±0.0204与0.3043±0.0443,P<0.05)。结论:心肌内控制释放bFGF,能提高缺血心肌 局部相关血管生长因子(如VEGF、TGF β1等)的表达水平,增强细胞增殖,从而促进血管再生。     

血管内皮细胞生长因子真核表达质粒的构建及其在心肌细胞中的表达

目的 :构建血管内皮细胞生长因子 （ VEGF1 65）真核表达质粒 pc DNA3 .1（ -） /h VEGF1 65,并通过转染心肌细胞来验证其生物活性。方法 :应用 DNA重组方法 ,将编码 h VEGF1 65全长 c DNA克隆于真核表达载体 pc DNA3 .1（ -）中 ,构建 pc DNA3 .1（ -） /h VEGF1 65真核表达质粒 ;应用阳性脂质体介导的基因转染技术 ,将真核表达质粒 pc D-NA3 .1（ -） /h VEGF1 65瞬时转染培养的大鼠心肌细胞中 ;采用 RT-PCR,ELISA,Western blot及免疫组化染色等方法检测 VEGF1 65基因在心肌细胞中的表达情况 ;应用 MTT法检测转染后细胞上清液中 VEGF1 65的生物活性。结果 :将构建好的真核表达质粒 pc DNA3 .1（ -） /h VEGF1 65转染心肌细胞后 ,VEGF m RNA及蛋白表达水平明显增高 ,转染后的心肌细胞培养上清液具有促使内皮细胞增殖的生物活性。结论 :成功构建了真核表达质粒 pc DNA3 .1（ -） /h VEGF1 65,其转染心肌细胞后可获得较高水平 VEGF蛋白的表达 ,所表达出 VEGF蛋白具有生物学活性     

Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.     

导入外源肝细胞生长因子基因对肝细胞增殖的影响

目的 利用重组腺病毒载体将外源人肝细胞生长因子(HGF)基因导入原代培养的大鼠肝细胞, 观察HGF表达对肝细胞增殖特性的影响。方法 同源重组构建表达HGF的复制缺陷型重组腺病毒AdHGF,用 其感染原代培养的肝细胞。逆转录聚合酶链反应检测肝细胞HGF和c—met(HGF受体)mRNA的表达;酶联免 疫吸附实验测定培养上清液中HGF水平。MTS测定感染前后细胞增殖情况;流式细胞仪测定细胞周期的变化; 细胞免疫荧光法检测HGF基因导入后增殖细胞核抗原(PCNA)表达。结果 同源重组获得约4×10~(10)efu/ml 滴度的AdHGF。AdHGF感染原代培养肝细胞后,肝细胞HGF和c-met mRNA表达均明显上调;细胞上清液 中HGF分泌水平显著增加,达到(5 939.00±414.39)pg/ml(F=13.661,P<0.01)。细胞增殖能力增强(F ≥15.158,P<0.01),细胞周期由G_0/G_1期向S期转化(X~2=41.616,P<0.01);细胞免疫荧光法提示HGF 基因导入后PCNA指数显著提高(F=42.122,P<0.01)。结论 通过重组腺病毒载体将外源HGF基因 导入肝细胞后可维持HGF高效表达并能促进肝细胞增殖,是基因修饰供体肝细胞、增强肝细胞移植治疗效 果的有效方法。     

Inhibition mechanism of a newly cloned candidate tumor suppressor gene JST during hepatocarcinogenesis and its abnormal expression in human hepatocellular carcinoma from Qidong liver cancer risk area, China

BACKGROUND/AIMS: To study inhibition effect of a newly cloned candidate tumor suppressor gene (JST) during hepatocarcinogenesis and its normal expression in human hepatocellular carcinoma from Qidong liver cancer risk area, China. METHODOLOGY: By preparing rabbit anti-human JST polyclonal antibody, constructing of JST frameshift mutant and exploring RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot, cDNA expression microarray, co-immunoprecipitation and the tumorigenicity assay in vivo and in vitro, gene treatment, etc, JST gene expression and inhibition tumor growth effects were analyzed, including 150 pairs of HCC specimens and their adjacent para-cancerous tissues, 8 cases of normal liver tissues and QGY7701, HepG2, Hep3B cell line. Its relationship with the invasiveness of HCC from Qidong was also investigated. RESULTS: Our results showed that there is expression difference for JST between liver cancer and para-cancerous tissue and the results of RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot suggested that it is a down-regulation gene. The labeling index (LI) of cancer tissue and para-cancerous tissue was (70.2+/-8.7) and (9.4+/-2.8) respectively (p<0.01), lower LI was closely related with invasiveness of HCC, decreased expression of JST was also shown by Western blotting. Results of RT-PCR showed the JST gene expression index (EI) of HCCs was lower than that of para-cancerous tissue and normal liver tissue and there are some sequence differences between cancer and para-cancerous tissues. Northern blot showed JST having down-regulation expression among 92.20% (138/150) of patients. Using in situ hybridization, the signal corresponding to JST mRNA was particularly weak in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Less expression of JST was also found to be correlated with high metastasis potentiality of HCC. JST overexpression inhibits DNA synthesis and apoptosis in QGY7701 cells. QGY7701 cell transfected with JST is more inhibited in soft agar than that of vector transfected control cells (p<0.01) or QGY7701 cells stably transfected with the JST frameshift mutant. The tumor formation is more inhibited in the QGY7701-pcDNA3.1-JST group than that in the QGY7701-pcDNA3.1, QGY7701-pcDNA3.1-JST frameshift mutant group. cDNA expression microarray showed expression differences of 10% (20/200,18 up-regulation; 2 up-regulation) tumor genes were considered significant between QGY7701-pcDNA3.1-JST and QGY7701-pcDNA3.1. Using a co-immunoprecipitation approach, intracellular binding of JST and p53 was found. Higher levels of p53 were detected following infection with pcDNA3.1-JST when compared with pcDNA3.1. Induction of FasL could be demonstrated in Hep3B and in HepG2 cells following transfection pcDNA3.1-JST, but not following transfection pcDNA3.1. CONCLUSIONS: JST is a putative tumor suppressor gene. Overexpression of JST gene may contribute to inhibiting the occurrence, advancement and invasiveness of HCC from Qidong, a high risk area in China.     

VEGF真核表达载体构建及其在骨髓间充质干细胞中的表达

目的构建血管内皮生长因子（vascu lar endothelial growth factor,VEGF）真核表达载体,并研究其在骨髓间充质干细胞（m esenchym al stem cells,MSCs）中的表达情况。方法运用DNA重组技术,构建VEGF真核表达载体pcDNA3.1（-）/hVEGF165,将载体转染大鼠MSCs,RT-PCR,ELISA及免疫细胞化学等方法检测VEGF在大鼠MSCs中的表达情况,MTT法检测转染后细胞上清液中VEGF的生物活性。结果成功构建VEGF真核表达载体pcDNA3.1（-）/hVEGF165,其转染后的大鼠MSCs中VEGF表达水平明显增高,转染后细胞培养上清液具有促使内皮细胞增殖的生物活性。结论VEGF真核表达载体转染大鼠MSCs后能有效增加VEGF表达,并表现出较强的促内皮细胞增殖作用。     

Review: Regulation of liver regeneration by pro-inflammatory cytokines

The liver has tremendous regenerative capacity. This distinguishes it from other vital organs (e.g. the brain, heart and lungs) that cannot replace functional tissue once it has been destroyed. Although hepatocytes rarely proliferate in the healthy adult liver, virtually all surviving hepatocytes replicate at least once after 70% partial hepatectomy. Therefore, partial liver resection has been used to characterize mechanisms that regulate liver regeneration. Residual hepatocytes up-regulate both proliferative and liver-specific gene expression in order to preserve tissue specific function. In addition, hepatocyte proliferation is tightly co-ordinated to complement regenerative responses in hepatic nonparenchymal cells (e.g. endothelia, biliary epithelia, stellate and Kupffer cells), so that the entire organ can be reconstituted within days. Studies with neutralizing antibodies to tumour necrosis factor-α (TNF) clearly demonstrate that, after partial hepatectomy, TNF promotes liver cell proliferation. The present review focuses on the regulation of the hepatocyte proliferative response by pro-inflammatory cytokines.     

Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells

AIM: To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702. METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times. RESULTS: RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope, but not in HL-7702/pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression. CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.