Vasostatin基因对胰腺癌细胞和血管内皮细胞增殖的影响

目的研究Vasostatin转基因对胰腺癌细胞、血管内皮细胞的作用，探讨其对胰腺癌生长抑制的作用机制。方法应用腺病毒载体将Vasostatin基因转入人胰腺癌细胞SW1990、人脐静脉血管内皮细胞ECV304，应用MTT方法检测转基因后细胞的生长活力。同时，应用小管形成实验研究Vasostatin对血管内皮细胞ECV304体外血管生成的影响。结果Vasostatin转基因对人胰腺癌SW1990细胞生长无显著影响。Vasostatin转基因（MOI分别为25和50）对人脐静脉血管内皮细胞ECV304作用72h后，Ad-Vasostatin组的ECV304细胞数目显著少于PBS组和Ad-lacZ组（P〈0．05）。小管形成实验结果显示，Ad-Vasostatin组内皮细胞数目较少，细胞排列连续性差，可见条索状的细胞链，但中空闭合的管状结构缺如。结论腺病毒介导的Vasostatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外细胞增殖，抑制其体外血管生成，而对人胰腺癌肿瘤细胞SW1990的体外细胞增殖无明显影响。     

Vasostatin基因对胰腺癌细胞和血管内皮细胞增殖的影响

目的研究Vasostatin转基因对胰腺癌细胞、血管内皮细胞的作用,探讨其对胰腺癌生长抑制的作用机制。方法应用腺病毒载体将Vasostatin基因转入人胰腺癌细胞SW1990、人脐静脉血管内皮细胞ECV304,应用MTT方法检测转基因后细胞的生长活力。同时,应用小管形成实验研究Vasostatin对血管内皮细胞ECV304体外血管生成的影响。结果Vasostatin转基因对人胰腺癌SW1990细胞生长无显著影响。Vasostatin转基因(MOI分别为25和50)对人脐静脉血管内皮细胞ECV304作用72h后,Ad-Vasostatin组的ECV304细胞数目显著少于PBS组和Ad-lacZ组(P<0.05)。小管形成实验结果显示,Ad-Vasostatin组内皮细胞数目较少,细胞排列连续性差,可见条索状的细胞链,但中空闭合的管状结构缺如。结论腺病毒介导的Vasostatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外细胞增殖,抑制其体外血管生成,而对人胰腺癌肿瘤细胞SW1990的体外细胞增殖无明显影响。     

Vasostatin基因对胰腺癌细胞和血管内皮细胞增殖的影响

目的研究Vasostatin转基因对胰腺癌细胞、血管内皮细胞的作用,探讨其对胰腺癌生长抑制的作用机制.方法应用腺病毒载体将Vasostatin基因转入人胰腺癌细胞SW1990、人脐静脉血管内皮细胞ECV304,应用MTT方法检测转基因后细胞的生长活力.同时,应用小管形成实验研究Vasostatin对血管内皮细胞ECV304体外血管生成的影响.结果 Vasostatin转基因对人胰腺癌SW1990细胞生长无显著影响.Vasostatin转基因(MOI分别为25和50)对人脐静脉血管内皮细胞ECV304作用72 h后,Ad-Vasostatin组的ECV304细胞数目显著少于PBS组和Ad-lacZ组(P < 0.05).小管形成实验结果显示,Ad-Vasostatin组内皮细胞数目较少,细胞排列连续性差,可见条索状的细胞链,但中空闭合的管状结构缺如.结论腺病毒介导的Vasostatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外细胞增殖,抑制其体外血管生成,而对人胰腺癌肿瘤细胞SW1990的体外细胞增殖无明显影响.     

内皮抑素对冠状动脉的影响

内皮抑素(Endostatin,ES)为胶原ⅩⅧ的-COOH末端片段,是一种内源性血管生成抑制因子,特异地抑制血管内皮细胞的增殖、迁移、促进血管内皮细胞的凋亡、抑制血管生成,从而抑制肿瘤的生长和转移。ES     

Canstatin基因转染对体外培养血管内皮细胞增殖与凋亡的影响

目的探讨电转染Canstatin基因对体外培养人脐静脉内皮细胞生长的影响。方法将Canstatin基因通过电穿孔法转染人脐静脉内皮细胞HUV-ECC,行G418筛选获得转基因细胞克隆。用SDS-PAGE电泳检测Canstatin蛋白在转基因细胞培养上清液中的表达,以流式细胞仪分析细胞周期,并比较转基因和未转基因细胞的生长特性。结果Canstatin在转染人脐静脉内皮细胞中表达并分泌至上清液中,Canstatin基因转染内皮细胞组的凋亡率（16.90%）高于空载体组（1.47%）和亲代细胞组（2.85%,P<0.01）,转染细胞生长明显受抑。结论Canstatin能特异地抑制血管内皮细胞增殖,并诱导细胞凋亡。     

内皮抑素对冠状动脉的影响

内皮抑素(Endostatin, ES)为胶原ⅩⅧ的-COOH末端片段,是一种内源性血管生成抑制因子,特异地抑制血管内皮细胞的增殖、迁移、促进血管内皮细胞的凋亡、抑制血管生成,从而抑制肿瘤的生长和转移.ES对胎儿肺的发育起着一定的作用,可能参与了出生后慢性肺损伤疾病的发生[1].     

持续释放人血管抑素的基因修饰化工程细胞hE/293细胞株的建立

目的:在人胚肾HEK293细胞中转染真核表达载体pSNA2/hEndostatin(hEndostatin,人血管抑素),建立能稳定分泌hES的基因工程细胞株.方法:将含有IL-2分泌肽的人endostatin(ES)全长cDNA插入真核表达载体pSNA2,产生重组质粒pSNA2/hEndostatin;利用阳离子脂质体介导将其转染入HEK293细胞中;用G418筛选出阳性克隆细胞,将其命名为hE/293细胞.用Western blot法检测hE/293细胞培养上清中分泌的hES蛋白.血管内皮细胞(ECV304)增殖抑制试验及鸡胚尿囊膜试验观察其分泌的hES蛋白的抗增殖活性.结果:经过双酶切和DNA测序证实构建出了含hES基因的真核表达载体.通过G418抗性筛选筛选出稳定表达hES的细胞株3株,将其命名为hE/293细胞;Western blot检测该细胞株培养上清中存在分子量为20 kDa的ES蛋白;ECV304增殖抑制试验显示,与HEK293细胞组相比,hE/293细胞组分泌的ES蛋白对bFGF刺激的血管内皮细胞增殖有明显的抑制作用(48h:0.125±0.007 vs 0.159±0.020,P<0.01;72 h:0.088±0.016 vs 0.249±0.070,P<0.01);鸡胚尿囊膜试验证实hE/293细胞分泌的ES蛋白可以抑制鸡胚尿囊膜血管生长.结论:所构建的hE/293细胞株可以稳定的分泌hES蛋白,并能抑制ECV304细胞生长及鸡胚尿囊膜血管生长.     

腺病毒介导的凋亡素、内皮抑素双基因对食管癌的实验研究

目的探讨携带有凋亡素(vp3、Apoptin)、内皮抑素(Endostatin)双基因的重组腺病毒载体在食管癌等多种肿瘤细胞内的表达情况及致凋亡作用,为进一步的食管癌治疗应用研究打下基础。方法将已纯化的携带凋亡素和内皮抑素基因的腺病毒Ad-vp3-IRES-sEndo-His感染食管癌细胞Eca-109、小鼠肝癌细胞Hepa1-6、结肠癌细胞LoVo提取各种细胞RNA,针对凋亡素、内皮抑素基因的表达进行RT-PCR检测,同时对感染腺病毒Ad-vp3-IRES-sEndo-His、Ad-vp3的食管癌细胞通过流式细胞仪、Hoechst33258染色等方式进行细胞凋亡率的检测。结果 RT-PCR检测显示被感染的多种肿瘤细胞均可表达凋亡素、内皮抑素基因的mRNA,Ho-echst33258染色显示被Ad-vp3-IRES-sEndo-His感染的食管癌细胞出现凋亡形态学改变,用流式细胞仪测定时段最高凋亡率达47.7%,与对照组相比具有统计学意义(P<0.05)。结论携带凋亡素及内皮抑素双基因的腺病毒载体能在多种肿瘤细胞中表达,感染食管癌细胞后,能有效诱导其凋亡且其致凋亡率高于仅携带凋亡素的单基因腺病毒载体。     

血管内皮生长因子反义寡核苷酸抑制甲状腺癌血管生成的效应

目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma

AIM: To investigate the effect of adeno-associated virus-mediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC). METHODS: HCC cell line Hep3B was infected with recombinant adeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2-hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined by ELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk. RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P<0.01). Intratumoral injection of rAAV2-hEndo (2×1010 v.g.) led to a sustained serum endostatin level of approximately (86.71±5.19) ng/mL. The tumor volume and microvessel density were less in rAAV2-hEndo group than in control groups(P<0.01). CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.     

人内皮抑素小环载体在真核细胞中的生物活性

目的通过对人内皮抑素小环载体在真核细胞中的表达和生物学效应的研究,探讨小环载体作为生物治疗载体的可行性。方法将mc-hES、pcDNA-hES分别转染人肝癌细胞HepG2后,通过ELISA、RT-PCR和Westernblot观察hES表达。MTT法检测其对人脐静脉内皮细胞（HUVEC）的增殖抑制作用。结果 mc-hES和pcDNA-hES均能表达hES,mc-hES能明显抑制HUVEC的生长,而对HepG2无明显作用（P〈0.05）。在相同条件下,小环较常规质粒在体外能快速介导转基因的表达,且较传统质粒高6～8.3倍。结论 mc-hES较普通质粒pcDNA-hES在肝癌细胞中能高效表达转基因,为小环载体进一步研究提供了很好的实验基础。     

一氧化氮/环鸟苷酸信号传导通路对高葡萄糖环境中人脐静脉内皮细胞内皮抑素表达的影响

目的 研究一氧化氮/环鸟苷酸信号传导通路对高葡萄糖时人脐静脉内皮细胞表达内皮抑素的调控.方法 采用5.6、11.2、22.4 mmol/L葡萄糖培养人脐静脉内皮细胞72 h,硝酸还原酶法测定培养上清液中一氧化氮含量,Western blot法检测内皮抑素蛋白表达水平.在11.2 mmol/L葡萄糖作用72 h(对照组)...     

The effect of endostatin mediated by human mesenchymal stem cells on ovarian cancer cells in vitro

Introduction  

Endostatin is the most potent inhibitor of tumor angiogenesis. However, endostatin protein has a short half-time and virus-mediated endostatin gene therapy has serious toxicity, which limits the application of endostatin in clinical therapy. Mesenchymal stem cells (MSCs) are considered to be able to accumulate at the site of cancers with high specificity and may be used as a new delivery of endostatin.     

Potent inhibition of angiogenesis and liver tumor growth by administration of an aerosol containing a transferrin-liposome-endostatin complex

AIM: To obtain an efficient delivery system for transporting endostatin gene to mouse liver tumor xenografts by administration of aerosol.METHODS: Recombinant plasmid pcDNA3.0/endostatin containing human endostatin gene together with signal peptide from alkaline phosphatase were transferred into human umbilical vein endothelial cell (HUVEC) by transferrin(TF)-Iiposome-endostatin complex. Western blot was used to detect the expression of human endostatin in transfected HUVEC cells and its medium. After the tumor-bearing mice were administrated with TF-liposome-endostatin complex,the lung tissue was analyzed by immunohistochemical method for expression of endostatin and the tumors were treated with CD-31 antibody to detect the density of microvesseles in tumor tissues. The inhibition of tumor growth was estimated by the weight of tumors from groups treated with different doses of TF-liposome-endostatin complex. DNA fragmentation assay was used to detect the apoptosis of the cells from primary liver tumor.RESULTS: Western blot analysis and immunohistochemical method confirmed the expression of endostatin protein in vitro and in vivo. After the tumor sections were treated with CD-31 antibody, the positive reaction cells appeared brown while the negative cells were colorless. The positively stained area of the TF-liposome-endostatin treated group was significantly smaller (P&lt;0.01, 645.8+55.2 μm^2) than that of the control group (1325.4&#177;198.5 μm^2). The data showed a significant inhibition of angiogenesis. After administration of TF-liposome-endostatin, comparing with the control group administrated with TF-liposome-pcDNA3.0, liver tumor growth in the mice treated with 50, 250 and 500 mg DNA/kg was inhibited by 36.6 %, 40.8%, and 72.8%, respectively(P&lt;0.01). And a typical DNA fragmentation of apoptosis was found in the cells from tumor tissues of the mice treated with TF-liposome-endostatin but none in the control group.CONCLUSION: Endostatin gene could be efficiently transported into the mice with TF-liposome-DNA delivery system by administration of aerosol. TF-liposome-mediated endostatin gene therapy strongly inhibited angiogenesis and the growth of mouse xenograft liver tumors. It also could promote the development of apoptosis of tumors without direct influence on tumor cells.     

人骨髓间充质干细胞上清液对体外肝星状细胞增殖周期及MMP-1表达的影响

目的 研究骨髓间充质干细胞(BMSC)对肝星状细胞(HSC)增殖周期及基质金属蛋白酶-1(MMP-1)活性的影响,以探讨其防治肝纤维化的作用机制.方法 采用密度梯度离心法分离鉴定人BMSC,收集原代培养7d的BMSC培养上清,加入HSC中培养24 h及48 h.通过流式细胞仪观察HSC增殖周期,ELISA方法检测MMP-1浓度.结果 与HSC单独培养组相比,共培养48 h组G1期细胞显著增加(P＜0.01),S期细胞显著减少(P＜0.01),并且共培养组MMP-1表达明显增加,与对照组相比差异均具有统计学意义(P＜0.05).结论 BMSC可抑制HSC的增殖,并且可能通过增加MMP-1的产生,从而起到抗纤维化的作用.     

Retrovirus—mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin. The inhibitory effect of endostatin expressed by transfected SMMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted. RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P<0.01). In vitro experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P<0.001). The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6+/-1.1, much fewer than that of 22.6+/-4.5 from SMMC-pLncx cells (P<0.01). CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.     

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P<0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.     

人肝细胞系5-羟色胺自分泌系统的研究

目的通过对人肝癌细胞系和人正常肝细胞系的研究,探讨肝细胞是否具有合成5-HT的功能。方法以人肝癌细胞系HepG2和人肝细胞系7702为研究对象,采用ELISA方法检测细胞培养上清液中的5-HT水平,采用荧光定量PCR法检测细胞表达5-HT合成的关键酶及5-HT受体。结果两种细胞系细胞上清液均可检测到5-HT;均可表达多种类型的5-HT受体,均表达合成5-HT的关键酶色氨酸羟化酶（TPH）及运载体（SERT）。结论人肝癌细胞系HepG2和人肝细胞系7702均表达合成5-HT的关键酶和受体,说明肝细胞具有5-HT自分泌系统,5-HT可能以自分泌或者旁分泌的形式发挥作用。