肿瘤抑素

摘要： 目的 研究肿瘤抑素Tum5对人脐静脉血管内皮细胞血管生成活性以及碱烧伤诱导的大鼠角膜新生血管的抑制作用.方法 取对数生长期人脐静脉血管内皮细胞分为4组:正常对照组:无任何处理的人脐静脉血管内皮细胞;rAd-GFP组:人脐静脉血管内皮细胞经rAd-GFP感染并培养;rAd-Tum5组:人脐静脉血管内皮细胞经rAd-Tum5感染并培养;rAd-Tum5+ VEGF组:人脐静脉血管内皮细胞经rAd-Tum5感染后经外源性VEGF处理.通过CCK-8活性检测试剂盒、Transwell实验、Matrigel实验分别检测细胞增殖率、迁移细胞数及管腔形成情况.取雄性健康清洁级SD大鼠64只采用随机数字表法将大鼠分为:正常对照组、碱烧伤组、碱烧伤+ rAd-GFP组、碱烧伤+rAd-Tum5组,每组16只,除正常对照组外其余各组建立角膜碱烧伤模型.其中正常对照组无任何处理,碱烧伤组、碱烧伤+ rAd-GFP组、碱烧伤+rAd-Tum5组于烧伤后分别经结膜下注射等量无菌生理盐水、rAd-GFP、rAd-Tum5病毒,记录碱烧伤后第1天、第7天、第14天角膜新生血管相对面积及浸润炎性细胞数.结果 CCK-8实验结果显示rAd-Tum5组较正常对照组、rAd-GFP组细胞增殖率降低(均为P＜0.01);而rAd-Tum5+ VEGF组细胞增殖率较rAd-Tum5组显著升高(P=0.004),rAd-Tum5+ VEGF组与正常对照组和rAd-GFP组相比差异均无统计学意义(均为P＞0.05).Transwell实验结果显示rAd-Tum5组相对于正常对照组、rAd-GFP组每个视野细胞迁移数显著降低(均为P＜0.01);而rAd-Tum5+ VEGF组相对于rAd-Tum5组显著升高(P=0.000),但其迁移能力尚未恢复到正常水平,与正常对照组和rAd-GFP组相比差异均有统计学意义(均为P ＜0.05).Matrigel实验结果显示rAd-Tum5组相对于正常对照组、rAd-GFP组每个视野细胞成管数显著降低(均为P＜0.01);而rAd-Tum5+ VEGF组相对于rAd-Tum5组显著升高(P=0.001).大鼠角膜碱烧伤后第1-14天,各组角膜新生血管密度及数量逐渐增高,但是碱烧伤+ rAd-Tum5组第7天、第14天角膜新生血管相对面积均显著低于碱烧伤组和碱烧伤+ rAd-GFP组,提示Tum5能降低角膜新生血管生长速率并减少新生血管密度,抑制大鼠碱烧伤诱导的新生血管的形成.碱烧伤后第7天、第14天,正常对照组角膜完整、细胞排列有序,无炎性细胞浸润;碱烧伤后第7天,碱烧伤组、碱烧伤+ rAd-GFP组上皮结构紊乱,角膜水肿,炎性细胞浸润明显,而碱烧伤+rAd-Tum5组每个视野炎性细胞浸润数较碱烧伤组、碱烧伤+ rAd-GFP组明显减少,差异均有统计学意义(均为P＜0.01).碱烧伤后第14天,碱烧伤组、碱烧伤+rAd-GFP组和碱烧伤+ rAd-Tum5组每个视野炎性细胞浸润数较第7天显著下降,同时碱烧伤+rAd-Tum5组角膜上皮结构整齐,水肿消退,每个视野炎性细胞浸润数显著低于碱烧伤组以及碱烧伤+ rAd-GFP组,差异均有统计学意义(均为P＜0.01).结论 Tum5可能经VEGF通路抑制人脐静脉血管内皮细胞血管生成活性;Tum5可显著抑制碱烧伤诱导的角膜新生血管生成及炎性细胞浸润.%Objective To investigate the inhibitory effects of Tum5 on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and alkali-induced corneal neovascularization.Methods HUVECs in logarithmic growth phase were divided into 4 groups,cells with untreated as normal control group,cells with the infection of rAdGFP virus as rAd-GFP group,cells with the infection of rAd-Tum5 virus as rAd-Tum5 group,and cells with the infection of rAd-Tum5 virus followed by VEGF treatment as rAd-Tum5 + VEGF group.Then cell proliferation,migration,and tube formation of HUVECs were examined by CCK-8,Transwell and Matrigel assays,respectively.Sixty-four healthy male SD rats were randomly divided into 4 groups (n =16) by using random number table,and they were normal control group,alkali-burn group,alkali-burn + rAd-GFP group,and alkali-burn + rAd-Tum5 group.The alkali-burn rat model was then established except normal control group,and the normal control group received no treatment,whereas the alkali-burn,alkali-burn + rAd-GFP,and alkali-burn + rAd-Tum5 groups received subconjunctival injection of equal volumes of sterilized saline,rAd-GFP virus,and rAd-Tum5 virus,respectively following the alkaline burn.The relative area of corneal neovascularization and the number of infiltrating inflammatory cells were recorded on day 1,7,and 14 after injection.Results The CCK-8 assay showed that the proliferative rate of rAd-Tum5 group was lower than that of the normal control group and rAd-GFP group (both P ＜0.01),while rAd-Tum5 + VEGF group exhibited a significantly greater cell proliferative capability than rAd-Tum5 group (P =0.004).There were no statistical differences between rAd-Tum5 + VEGF group,normal control group and rAdGFP group (all P ＞ 0.05).Transwell assay showed the significantly lower number of migrating cells in the rAd-Tum5 group than those in the normal control group and rAdGFP group (both P ＜ 0.01).The number of migrating cells in rAd-Tum5 + VEGF group was higher than those in rAd-Tum5 group (P =0.000);however,the migration capacity had not been restored to normal level,and rAd-Tum5 + VEGF group had significant difference with normal control group and rAd-Tum5 group (both P ＜0.05).Matrigel assay showed that the number of meshes in rAd-Tum5 group was lower than that in the normal control group and rAd-GFP group (both P ＜0.01);while the number of meshes in rAd-Tum5 + VEGF group was significantly increased compared with rAd-Tum5 group(P =0.001).The density and number of corneal neovascularization increased gradually from day 1 to day 14 after alkali burn,while the relative neovascularization area in the alkali-burn + rAd-Tum5 group was significantly reduced as compared to those in the alkali-burn group and alkali-burn + rAd-GFP group on day 7 and day 14,suggesting that Tum5 could reduce the growth rate and density of corneal neovascularization,so as to inhibit corneal neovascularization induced by alkali burn.On day 7 and 14 after alkali burn,in the normal control group,the corneas were intact,no infiltrating inflammatory cells and cells were arranged in an orderly manner.On day 7 after alkali burn,there were disordered epithelial structure,corneal edema and infiltrating inflammatory cells in alkali-burn group and alkali-burn + rAd-GFP group.The number of infiltrating inflammatory cells in alkaliburn + rAd-Tum5 group was significantly lower than that in alkali-burn group and alkali-burn + rAd-GFP group (both P ＜0.01).On day 14 after alkali burn,the number of infiltrating inflammatory cells in alkali-bum,alkali-burn + rAd-GFP and alkali-burn + rAd-Tum5 group were significantly lower than those on day 7,while the corneal epitheliums were intact and dropsy was alleviated,while the number of inflammatory cells was significantly lower than that in alkali burn group and alkali-burn + rAd-GFP group,with significant difference (both P ＜ 0.01).Conelusion Tum5 can inhibit the angiogenic capability of HUVECs by VEGF pathway,as well as suppress the alkali-burn-induced corneal neovascularization and inflammatory cell infiltration in rats.

摘要： 目的 研究肿瘤抑素对血管瘤内皮细胞的增殖与凋亡过程的调节作用,为临床婴幼儿血管瘤的治疗提供参考.方法 血管瘤内皮细胞培养后随机分为5组:对照组、PD98059组、肿瘤抑制素低剂量、中剂量和高剂量组.除对照组给予生理盐水外,其余各组给予PD98059(10 μM)处理2h,肿瘤抑制素低剂量、中剂量和高剂量组分别给予1 μM、10 μM、100 μM的肿瘤抑制素处理2h.免疫印迹法或荧光定量法分析各组细胞凋亡相关蛋白Caspase3/9、ERK信号通路关键蛋白ERK1/2和MEK及氧化应激蛋白NOX4的表达,DHE染色法分析各组细胞的氧化应激状态.结果 与对照组相比,PD98059组血管瘤内皮细胞内ROS水平明显降低、细胞凋亡蛋白Caspase3/9的表达明显升高;同时ERK信号通路关键蛋白的表达明显增强,而肿瘤抑制素低剂量、中剂量和高剂量组作用与PD98059作用一致,且具有剂量依从性.结论 肿瘤抑制素通过激活氧化应激状态、ERK信号及抑制细胞凋亡蛋白通过发挥对血管瘤内皮细胞增殖的抑制作用.%Objective The present research aimed to explore the effect mechanism of tumstatin on the apoptosis process in hemangioma vascular endothelial cell.Methods Hemangioma vascular endothelial cells were divided into 5 groups:control,PD95059 group and tumstatin group at low,medium and high dose.The cells were treated with PD98059 for 2 h at 10 μM concentration except control.The tumstatin group cells were treated with tumstatin at 1 μM,10 μM,100 pM concentration for 2 h.The expression of NOX4,Caspase3/9,ERK and MEK was detected by Western Blotting or Real-time PCR.The ROS content in each group was assayed by DHE staining.Results The content of ROS was decreased in PD98059 medication group.The expression of Caspase3/9 was increased while the NOX4 expression was decreased in PD98059 medication group.The results also showed that the ERK signaling pathway proteins was increased after medication of in PD98059 medication.These abnormalities were normalized greatly after the medication of tumstatin with a dose dependent manner.Conclusion Oxidative-stress and apoptosis are involved in the tumstatin induced proliferation inhibition of hemangioma vascular endothelial cell.

摘要： Objective:To explore the inhibitory effect of Tum-5 gene on the tumor growth and its influence in the angiogenesis of hepatocellular carcinoma (HCC),and to illustrate its mechanism involved in antitumorigenesis.Methods:The human HepG2 cells were selected in in vitro experiment and treated with different multiplicity of infection (MOI) (0,1,5,10,25 and 50) of pLXSN and pLXSN-Tum-5 virus particles for 72 h.The proliferation rates of cells in various groups were detected by MTT method.In vivo,the H22 tumor-bearing mouse models were established.The mice were divided into saline group,pLXSN group and pLXSN-Tum-5 group (n=5).The mice in saline group were intratumorally injected with normal saline,and the mice in pLXSN group and pLXSN-Tum-5 group were intratumorally injected with virus particles (MOI =5),5 times every other day.The volumes and weights of transplanted tumor and the weights of mice in various groups were measured.The CD31 expressions in transplanted tumor tissue were detected by immunohistochemical method and the microvessel density (MVD) was calculated.Results:Compared with pLXSN group,the proliferation rates of cells in pLXSN-Tum-5 group after infected with different MOI of virus particles were not significantly different (P＞ 0.05).The volumes and weights of transplanted tumor of the mice in pLXSN-Tum-5 group were significantly smaller than thosein pLXSN group and saline group after intratumoral injection (P＜ 0.05 or P＜ 0.01).The immunohistochemical results showed that there was irregular angiogenesis in each group.Compared with saline group and pLXSN group,the mean value of MVD of the transplanted tumor tissue of the mice in pLXSN-Tum-5 group was significantly decreased (P ＜ 0.05).Conclusion:Tum-5 can exert its antitumor activity by inhibiting the formation of neovascularization in HCC.%目的:探讨肿瘤抑素5 (Tum-5)基因对肝细胞癌的抑制作用及对血管生成的影响,阐明其参与抗肿瘤生成的作用机制.方法:体外实验选择人肝癌HepG-2细胞,采用不同感染复数(MOI)(0、1、5、10、25和50)的pLXSN和pLXSN-Tum-5病毒颗粒感染72 h,MTT法检测各组细胞增殖率.体内构建肝癌H22荷瘤小鼠模型,分为saline组、pLXSN组和pLXSN-Tum-5组,每组5只.saline组小鼠瘤内注射生理盐水,pLXSN组和pLXSN-Tum-5组小鼠瘤内分别注射相应的病毒颗粒(MOI=5),隔日注射,共注射5次.测定各组小鼠移植瘤体积、质量和小鼠体质量,免疫组织化学法检测CD31蛋白在各组小鼠移植瘤组织中的表达,计算微血管密度(MVD).结果:与pLXSN组比较,不同MOI pLXSN-Tum-5组的细胞增殖率差异无统计学意义(P＞0.05);荷瘤鼠瘤内注射结束后,pLXSN-Tum-5组小鼠移植瘤体积和质量均明显小于pLXSN组和saline组(P＜0.05或P＜0.01);免疫组织化学检测,各组小鼠移植瘤内均有形态不规则的新生血管生成,与saline组和pLXSN组比较,pLXSN-Tum-5组小鼠移植瘤组织MVD平均值明显降低(P＜0.05).结论:Tum-5可通过抑制肝癌组织内新生血管的生成发挥抑瘤作用.

摘要： Background Tumstatin is the most active endogenous angiogenesis inhibitor,which has a marked inhibitory effect on pathological neovascularization,and Tum5 is an angiogenesis inhibitors fragment of fulllength tumstatin.Objective This study was to investigate the effects of adenovirus-mediated overexpression of recombinant Tum5 gene on the proliferation,migration and tube formation of human umbilical vein endothelial cells (HUVECs) in physiological status.Methods The empty adenoviral vector expressing green fluorescent protein (rAd-GFP) and the viral vector expressing recombinant Tum5 gene were constructed.The HUVECs cultured in RPMI1640 medium were divided into normal control group,empty vector group (rAd-GFP group) and Tum5 gene infection group (rAd-GFP-Tum5 group).The rAd-GFP and rAd-GFP-Tum5 adenoviral particles at the density of 1 × 1010/ml were added into the medium to infect the cells for 48 hours.The proliferation of the cells was assayed at 24,48 and 72 hours by cell counting kit-8 (CCK-8) to evaluate the proliferative rate;the migration number of the cells was detected at 48 hours after infection by Transwell chamber;the tube formation number of the cells were detected by Matrigel method.The concentration of vascular endothelial growth factor (VEGF) in cell supernatants was assayed by ELISA at 24,48,and 72 hours following adenoviral infection.Results The cultured cells showed green fluorescence in the rAd-GFP group and rAd-GFP-Tum5 group under the inverted fluorescence microscope,and the infection efficiency of rAd-GFP and rAd-GFP-Tum5 was 55.13％ and 50.31％,respectively.No significant difference was found in cell proliferative rate among normal control group,rAd-GFP group and rAd-GFP-Tum5 group both at 24 and 48 hours after infection (both at P＞0.05),and the cell proliferative rate was significantly lower in the rAd-GFP-Tum5 group than that in the normal control group and rAd-GFP group at 72 hours after infection (both at P＜0.01).The migration number of the cells at 48 hours after infection was 2 260.25-±930.44,2 370.00±441.06 and 723.75± 363.80 in the normal control group,rAd-GFP group and rAd-GFP-Tum5 group,showing a significant difference among the groups (F =8.524,P =0.008),and the migrated cells were evidently decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group and the normal control group (both at P＜ 0.01).The tube number at 48 hours after infection was 95.67±5.86,88.00±4.58 and 20.67±3.51 in the normal control group,rAd-GFP group and rAdGFP-Tum5 group,showing a significant difference among the groups (F=226.498,P＜0.01),and the tube number in the rAd-GFP-Tum5 group was significantly reduced in comparison with the normal control group and rAd-GFP group (both at P＜ 0.01).The considerably differences in VEGF concentration in the cell supernatants were found in different groups and various time points (Fgroup =73.260,P＜0.01;Ftime =73.477,P＜0.01),and VEGF concentration in the cell supernatants was significantly decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group at both 48 hours and 72 hours (both at P＜0.01).Conclusions The overexpression of the recombinant Tum5 can inhibit the proliferation,migration and tube formation of the HUVECs in physiological status,which may be associated with Tum-5-mediated down-regulation of VEGF protein in the cell supernatant.%背景 肿瘤抑素是迄今发现活性最强的内源性血管生成抑制因子,对病理性新生血管形成有明显抑制作用.Tum5片段为肿瘤抑素的抗血管生成活性片断. 目的 研究腺病毒介导的Tum5重组基因过表达对生理状态下人脐静脉内皮细胞(HUVECs)增生、迁移及管腔形成的影响. 方法 构建表达绿色荧光蛋白的空载体腺病毒(rAd-GFP)和携带重组Tum5基因的腺病毒载体(rAd-Tum5).将HUVECs分为正常对照组、空载体组(rAd-GFP组)和Tum5基因组(rAd-GFP-Tum5组).将rAd-GFP和rAd-Tum5病毒颗粒(1×101o/ml)各20μl分别加入rAd-GFP组和rAd-GFP-Tum5组培养液以感染培养的细胞48 h,倒置荧光显微镜下观察各组细胞中GFP的表达,并计算病毒的感染效率;采用细胞计数试剂盒(CCK)-8检测各组细胞在波长为450 nm处的吸光度(A)值并计算细胞增生率;采用Transwell小室实验测定各组的迁移细胞数目;采用基质胶(Matrigel)实验检测各组细胞的管腔形成数;采用人血管内皮生长因子(VEGF) ELISA试剂盒检测细胞感染后24、48和72 h各组细胞培养上清液中VEGF质量浓度. 结果 倒置荧光显微镜下可见rAd-GFP组和rAd-GFP-Tum5组HUVECs中呈绿色荧光,rAd-GFP组和rAd-GFP-Tum5组的感染效率分别为55.13％和50.31％.细胞感染后24 h和48 h,正常对照组、rAd-GFP组和rAd-GFP-Tum5组细胞增生率比较差异均无统计学意义(均P＞0.05);细胞感染后72 h,rAd-GFP-Tum5组细胞增生率明显低于正常对照组和rAd-GFP组,差异均有统计学意义(均P＜0.01).细胞感染后48 h,正常对照组、rAd-GFP组和rAd-GFP-Tum5组迁移细胞数分别为(2 260.25±930.44)、(2 370.00±441.06)和(723.75±363.80)个,总体比较差异有统计学意义(F=8.524,P=0.008),rAd-GFP-Tum5组迁移细胞数量较正常对照组和rAd-GFP组均明显减少,差异均有统计学意义(均P＜0.01).正常对照组、rAd-GFP组和rAd-GFP-Tum5组平均每张图片中管腔形成数目分别为(95.67±5.86)、(88.00±4.58)和(20.67±3.51)个,总体比较差异有统计学意义(F=226.498,P＜0.01),其中rAd-GFP-Tum5组细胞管腔形成数量较正常对照组和rAd-GFP组明显减少,差异均有统计学意义(均P＜0.01).细胞感染后24、48和72 h,各组细胞培养上清液中VEGF蛋白质量浓度总体比较差异均有统计学意义(F分组=73.260,P＜0.01;F时间码 =73.477,P＜0.01);其中感染后48 h和72 h,rAd-GFP-Tum5组VEGF质量浓度均明显低于rAd-GFP组,差异均有统计学意义(均P＜0.01). 结论 腺病毒介导的重组Tum5基因的过表达可抑制生理状态下HUVECs的增生、迁移及管腔形成,这可能与Tum5下调VEGF在细胞上清液中的含量有关.

摘要： 目的研究肿瘤抑素改造7肽(T-7R肽)对人非小肺癌细胞(A549)和人脐静脉内皮细胞(ECV304)增殖抑制和促凋亡活性,并进行对比研究。方法采用生物合成技术合成了肿瘤抑素改造7肽(T-7R),高效液相和质谱显示纯度高,含量大于97%;选取人非小肺癌细胞(A549)和人脐静脉内皮细胞(ECV304),通过MTT法、生长曲线观察T-7R肽对肿瘤细胞和内皮细胞增殖抑制作用;通过TUNEL法、HE染色和透射电镜观察T-7R肽对肿瘤细胞和内皮细胞形态结构影响。结果给药48 h后T-7R对人非小肺癌细胞(A549)具有明显的抑制作用,但对人脐静脉内皮细胞(ECV304)抑制作用不明显,且具有药物剂量依赖性。对人非小肺癌细胞(A549)抑制增殖作用较强,在给药的第2天细胞浓度开始下降第四天开始无法计数;对ECV-304作用不明显,给药组与空白对照组细胞生长状态一致仅在细胞数量上有所减少。T-7R肽可引起人非小肺癌细胞(A549)细胞数量明显减少,细胞形态改变,出现大量细胞变圆、长触角消失、体积缩小、核质比例降低、细胞核深染、胞质凝集、颜色变深等现象。中早期凋亡较多,晚期凋亡较少,凋亡小体明显,凋亡率为50%。对人脐静脉内皮细胞(ECV304)作用较弱,凋亡细胞较少。结论肿瘤抑素改造T-7R肽可抑制人非小肺癌细胞(A549)增殖,促进其的凋亡,但对非肿瘤细胞-人脐静脉内皮细胞(ECV304)作用不明显,其可能对肿瘤细胞具有一定的特异性。

摘要： Objective To explore the expression of recombinant tumstatin 30 peptide and its antitumor activity. Methods The 30 peptide sequence was designed, ligated into the fusion protein expression vector PTYB21, and then transformed to E. Coli BL-21 (DE3) . The fusion protein was expressed after induction by IPTG. Tumstatin 30 peptide was purified by using chitin affinity chroma-tography, and verified by SDS-PAGE and Tricine-SDS-PAGE. The antitumor activity of the 30 pep-tide was investigated through MTT assay, AO/ EB fluorescent staining, and the suppression experi-ments in mouse models of H22 ascites liver cancer. Results Recombinant tumstatin 30 peptide was successfully expressed. In vitro experiments showed that the 30 peptide could inhibit the proliferation of gastric cancer cells HGC-27 and human umbilical vein cells (HUVEC) and promote their apopto-sis. The in vivo experiments showed that the inhibition rate of H22 ascites liver tumor by the 30 pep-tide was 43. 18 %. Conclusion Recombinant tumstatin 30 peptide has strong antitumor activity.%目的：重组表达肿瘤抑素30肽并研究其抗肿瘤活性。方法设计并合成30肽基因序列,将此序列与融合蛋白表达载体 PTYB21重组,转化到大肠埃希菌 BL-21(DE3)。 IPTG 诱导表达,几丁质柱纯化得到30肽,经 SDS-PAGE 及 Tricine-SDS-PAGE 对纯化产物进行鉴定。利用 MTT 法、吖啶橙/溴化乙锭(AO/ EB)荧光染色法、小鼠 H22腹水转移型肝癌实体瘤抑瘤实验,研究30肽的抗肿瘤活性。结果成功重组并表达肿瘤抑素30肽。体外实验显示,30肽具有抑制 HGC-27胃癌细胞、 HUVEC 人脐静脉细胞增殖和促进这两种细胞凋亡的作用。体内实验显示,30肽对小鼠 H22腹水型肝癌抑瘤率达43.18 ％ 。结论重组的肿瘤抑素30肽具有较强的抗肿瘤活性。

摘要： 目的：构建肿瘤抑素41肽重组质粒，使其高效表达并纯化得到41肽，研究其抗肿瘤活性。方法人工设计并合成肿瘤抑素41肽基因序列，克隆到表达载体 pTYB21上，转化到大肠埃希菌 BL?21（DE3）中， IPTG诱导融合蛋白高效表达，几丁质亲和层析柱纯化后经Tricine?SDS?PAGE鉴定41肽。利用MTT法、吖啶橙／溴化乙锭（ AO／EB）荧光染色、小鼠H22腹水型转移型肝癌实体瘤模型抑瘤实验并结合组织病理切片来研究肿瘤抑素41肽的生物学活性。结果构建肿瘤抑素41肽重组质粒，获得可溶性肿瘤抑素41肽。体外实验显示，41肽具有抑制人脐静脉内皮 HUVEC 细胞、人胃癌 HGC?27细胞和人肝癌HepG2细胞增殖和促进HUVEC、 HGC?27细胞凋亡的作用。体内实验显示，41肽对小鼠H22腹水型转移肝癌实体瘤生长具有明显的抑制作用，抑瘤率达到34?35％。结论成功构建了pTYB21?41肽重组质粒，肿瘤抑素41肽具有显著的抗肿瘤活性，为肿瘤抑素机制研究和临床应用研究奠定了基础。

摘要： Objective To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.Methods Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs.Transfection efficiency was observed by fluorescence microscopy.Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR.Proliferation and apoptosis were detected by MTT and flow cytometry, respectively.The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 × 107 HepG2 cells into 30 BALB/c nude mice.The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.Results The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin.Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P ＜ 0.05).None of the treatments affected proliferation or apoptosis of the L02 cells (P ＞ 0.05).The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P ＜ 0.05).Tumor growth was significantly inhibited by the combination (P ＜ 0.05).In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells.Cell morphology of major organs such as liver, spleen and kidney were similar to the control group.The combination also produced the most significant effect on tumor microvascular density (P ＜ 0.05) and the highest apoptosis index (P ＜ 0.05).Conclusion The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells.Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.%目的 观察重组慢病毒Lv-hTERTp-TK与Lv-hTERTp-tumstatin在肝癌HepG2细胞中的靶向表达,探讨二者联合在体内外对HepG2细胞的抑制作用. 方法 用不同MOI重组慢病毒Lv-hTERTp-TK、 Lv-hTERTp-tumstatin转染肝癌细胞HepG2、正常肝细胞L02,72 h后荧光显微镜观察转染情况;逆转录PCR检测TK、 tumstatin mRNA在HepG2、 L02细胞中的表达;四甲基偶氮唑蓝法观察两种细胞增殖情况;流式细胞术检测两种细胞凋亡情况;RT-PCR、 Western blot检测HepG2细胞bcl-2、血管内皮生长因子(VEGF) mRNA和蛋白的表达;将HepG2细胞接种于30只BALB/c裸鼠皮下,构建动物移植瘤模型,随机分为5组,空白对照组、空载体组、tumstatm组、TK组及联合组,每组6只,瘤内分别注射磷酸盐缓冲溶液、空载体慢病毒Lv-hTERTp-eGFP、重组慢病毒Lv-hTERTp-tumstatin、重组慢病毒Lv-hTERTp-TK和重组慢病毒Lv-hTERTp-tumstatin+Lv-hTERTp-TK;4周后处死裸鼠,观察各组移植瘤体积及质量的变化;行HE染色进行各组肿瘤组织及主要脏器的形态学观察;免疫组织化学法检测肿瘤内微血管密度;TUNEL法检测肿瘤细胞凋亡率.数据采用方差分析. 结果 转染后TK、 tumstatin基因在HepG2细胞中特异表达,在L02细胞中无表达.TK组、tumstatin组及联合组均对HepG2细胞生长有抑制作用,联合组抑制率明显高于单基因作用组(F=731.679,P＜0.05),各组L02细胞抑制率差异无统计学意义(F=0.774,P＞0.05);联合组HepG2细胞总凋亡率达72.61％±3.29％,明显高于TK组(53.63％±3.06％)、tumstatin组(45.13％±2.15％)、空白对照组(13.29％±1.15％)(F=365.022,P＜0.05),各组L02细胞凋亡率差异无统计学意义(F=0.391,P＞0.05);与对照组相比,实验组HepG2细胞bcl-2及VEGF表达水平均下降(bcl-2 mRNA:F=322.780,P＜0.05;VEGF mRNA:F=561.136,P＜0.05;bcl-2蛋白:F=34.203,P＜0.05;VEGF蛋白:F=32.988,P＜0.05),其中联合组bcl-2及VEGF表达水平均低于单基因组(P值均＜0.05).tumstatin组、TK组和联合组的抑瘤率分别为40.68％、 44.96％和72.09％,联合组抑瘤效果最显著(P＜0.05);HE染色显示联合组肿瘤细胞凋亡最明显.与空白对照组相比,其他各组裸鼠体内肝、脾和肾等主要脏器组织形态学均无显著变化;空白对照组、空载体组、TK组、tumstatin组和联合组的微血管密度分别为28.4±4.28、29.2±2.59、20.8±3.35、 14.2±4.87、5.8±2.77,tumstatin组抗肿瘤血管生成作用强于TK组(P＜0.05),且联合组作用最明显(P＜0.05).联合组肿瘤细胞凋亡率为70.11％±5.67％,明显高于tumstatin组(23.75％±5.21％)、TK组(48.30％±4.99％)、空白对照组(3.89％±1.13％)(P＜0.05),其中TK组的促凋亡作用较tumstatin组显著(P＜0.05). 结论 hTERT启动子驱动TK、 tumstatin在HepG2细胞中靶向表达,两种基因联合在体内外显著抑制HepG2细胞增殖和促进HepG2细胞凋亡,作用明显强于单基因,其机制可能与bcl-2及VEGF表达下调有关.

摘要： Growth of tumor need new blood vessel to meet the demand for adequate oxygen and nutri -ents.Inhibiting the formation of blood vessel can inhibit growth of tumor .In all of the endogenous angiogenesis in-hibitors deriving from the type IV collagen .Study of tumstatin is the most widely .Tumstatin can inhibit endothelial proliferation and induce the apoptosis of endothelial cell .This article reviews the research status of the tumstatin .%肿瘤的生长需要新生血管来为其提供充足的营养物质和氧气，因此通过抑制血管的生成能够抑制肿瘤的生长。在所有来源于Ⅳ型胶原的内源性血管生成抑制剂中，肿瘤抑素的研究最广泛。肿瘤抑素具有抑制血管内皮细胞增殖、诱导内皮细胞凋亡、直接抑制肿瘤细胞增殖，促进肿瘤细胞凋亡等多种功能。本文就肿瘤抑素的研究现状进行综述。

摘要： Objective To explore the inhibitiory effect and mechanism of tetramethylpyrazine in combination with cisplatin on Lewis lung carci-noma rats.Methods A transplanted tumor model of Lewis lung carcino-ma was establised.Then randomly divided into 4 groups: control group (0.9%NaCl,0.2 mL),cisplatin group (2 mg・ kg -1,0.2 mL),tetram-ethylpyrazine group (100 mg・ kg -1,0.2 mL), and combination group ( the doses as above ) , each 10 rats .Different treatments were served for 14 days,then all of rats were put to death.The tumor weight and in-hibitory rate were calculated .The tumor cells and vascular structure were observed under electron microscope .The expression of cascular endothe-lial growth factor(VEGF) and tumstatin were decteted by immunohisto-chemistry and Western blot .Results The weight was increased in all the groups.The control group was obviously fast than the other group . The combination group was obviously the slowest .The inhibition rate in combination group was significantly higher than the other groups .The tumstatin expression of combination group was obviously lower than the other three groups.The VEGF expression of combination group were sig-nificantly lower than the other three groups .The expression of VEGF had negative correlation with tumstatin ( P <0.05 ) .Conclusion Tetrame-thylpyrazine can effectively inhibit the growth of Lewis lung cancer trans-planted tumor , and has synergetic effect when combined with cisplatin . The mechanism might be that it can inhibit tumor angiogenesis by reducing VEGF expression , and promoting tumstatin expression.%目的：探讨川芎嗪联合顺铂对Lewis肺癌的抑制作用及机制。方法建立小鼠移植瘤模型，随机分为4组：空白组（0.9％氯化钠，0.2 mL ）、顺铂组（2 mg・ kg-1，0.2 mL）、川芎嗪组（100 mg・ kg-1，0.2 mL）、联合组（川芎嗪联合顺铂，剂量同上，0.2 mL），10只／组，连续给药14 d；计算肿瘤重量及抑瘤率；电镜观察肿瘤细胞及血管结构；用免疫组化和蛋白印迹法检测血管内皮生长因子（ VEGF）、肿瘤抑素表达情况。结果空白组，肿瘤生长最快，联合组最慢。联合组，抑瘤率（59.81％）明显高于其他3组。联合组，VEGF的表达显著低于其他3组（ P ＜0.01），肿瘤抑素表达显著高于其他3组（ P＜0.01）。 VEGF与肿瘤抑素的表达呈负相关性（ P＜0.05）。结论川芎嗪可抑制肺癌生长，与顺铂联用有协同作用；其作用机制可能与抑制VEGF、促进肿瘤抑素表达有关。

摘要： 本发明公开了一种抗肿瘤蛋白内皮抑素的衍生物及应用，所述内皮抑素的氨基酸序列用SEQ ID NO.4所示；所述衍生物在内皮抑素的N端前面、或N端前面和C端后面引入糖基化位点的氨基酸短肽。本发明的抗肿瘤蛋白内皮抑素的衍生物在体内稳定，半衰期长、抗肿瘤生物活性高。

摘要： 本发明公开了一种抗肿瘤蛋白内皮抑素的衍生物，所述内皮抑素的氨基酸序列用SEQ ID NO.4所示；所述衍生物氨基酸序列的Arg128Arg129其中一个Arg发生点突变。本发明的抗肿瘤蛋白内皮抑素的衍生物在体内稳定，半衰期长、抗肿瘤生物活性高。

摘要： 本发明公开了一种具有抗肿瘤活性的内皮抑素33肽及其应用。所述的内皮抑素33肽由构成内皮抑素抗血管生成活性的24肽、RGDRGD序列和QRD序列组成，其氨基酸序列如SEQ ID NO.1所示。实验证明，该内皮抑素33肽具有广谱抗肿瘤活性，特别是对高表达α6β1亚型的前列腺癌组织的特异性抗癌功能。本发明的提出解决了目前30肽内皮抑素治疗存在RGD序列无法识别的整合素亚型的肿瘤时疗效欠佳的问题，为前列腺癌的治疗提供了一种新的有效的技术手段。

摘要： 苔藓抑素‑1刺激血液系统恶性肿瘤中肿瘤相关抗原的表达，使肿瘤能够被免疫疗法识别，从而增加对各种免疫疗法的临床反应和那些反应的持续时间。相应地，本文提供了基于这一观察的治疗方法。

摘要： 本发明属于生物医药领域，具体涉及重组人内皮抑素腺病毒与抗PD‑1抗体或抗PD‑L1抗体在制备抗肿瘤药物中的用途。本发明要解决的技术问题是提供一种具有较好抗肿瘤效果的药物。本发明解决技术问题的技术方案是提供一种含有编码人源内皮抑素的腺病毒与抗PD‑1抗体或抗PD‑L1抗体为主要活性成分的抗肿瘤药物。编码人源内皮抑素的腺病毒与抗PD‑1抗体或抗PD‑L1抗体共同治疗能有效抑制肿瘤生长、出现肿瘤消退且未复发，且未见明显毒副作用，说明了含有编码人源内皮抑素的腺病毒的抗血管生成治疗与抗PD‑1抗体或抗PD‑L1抗体免疫治疗相结合是治疗肿瘤的有效方法，具有很好的应用前景。

摘要： 本发明涉及一种具有抗肿瘤作用的肿瘤抑素30肽及其制备方法和应用。本发明所述的肿瘤抑素30肽的氨基酸序列为SEQ？No.1，编码所述肿瘤抑素30肽的核苷酸序列为SEQ？No.2。本发明在保持肿瘤抑素41肽抗肿瘤活性的前提下删除19肽中的非活性片段，减少融合肽的氨基酸数目，以降低融合肽的生产成本，尤其是化学合成的生产成本，增加用药的安全性。

摘要： 本发明涉及一种具有抗肿瘤作用的肿瘤抑素融合肽（41肽）及其制备方法和应用。本发明所述的肿瘤抑素融合肽的氨基酸序列为SEQ No.1，编码所述肿瘤抑素融合肽的核苷酸序列为SEQ No.2。本发明在肿瘤抑素42肽的基础上，使得21肽和19肽的连接不再依赖柔性连接片段，改由RGD序列承担，本发明抗肿瘤活性比肿瘤抑素42肽更强。

摘要： 本发明涉及一种具有抗肿瘤作用的肿瘤抑素融合肽（41肽）及其制备方法和应用。本发明所述的肿瘤抑素融合肽的氨基酸序列为SEQ No.1，编码所述肿瘤抑素融合肽的核苷酸序列为SEQ No.2。本发明在肿瘤抑素42肽的基础上，使得21肽和19肽的连接不再依赖柔性连接片段，改由RGD序列承担，本发明抗肿瘤活性比肿瘤抑素42肽更强。

摘要： 本发明涉及一种具有抗肿瘤作用的肿瘤抑素30肽及其制备方法和应用。本发明所述的肿瘤抑素30肽的氨基酸序列为SEQ No.1，编码所述肿瘤抑素30肽的核苷酸序列为SEQ No.2。本发明在保持肿瘤抑素41肽抗肿瘤活性的前提下删除19肽中的非活性片段，减少融合肽的氨基酸数目，以降低融合肽的生产成本，尤其是化学合成的生产成本，增加用药的安全性。

摘要： 一种重组肿瘤抑素－肿瘤坏死因子分泌型真核表达载体及其制备方法和用途公开了肿瘤抑素(tumstatin)与人肿瘤坏死因子(TNF－α)分泌型真核表达载体及融合蛋白制备方法，tumstatin－TNF分别能与新生血管内皮细胞表面αvβ3受体和肿瘤细胞死亡受体高度特异的相互作用。采用分步克隆法构建分泌型肿瘤抑素与人肿瘤坏死因子融合基因载体并在真核细胞中表达。通过肿瘤抑素与肿瘤组织新生血管内皮细胞表面αvβ3受体结合，特异地杀伤肿瘤组织血管内皮细胞和肿瘤细胞，发挥抗肿瘤和抑制肿瘤血管的作用。