血管形成抑制因子HIAF-1在昆虫细胞中的高效表达和活性研究

背景与目的：大肠杆菌表达的人抑制血管生成因子－1（human inhibiting angiogenesis factor-1,HIAF-1）可抑制肿瘤血管的形成。本研究在昆虫细胞中表达HIAF－1,并研究其生物活性。方法：用DNA重组技术,构建了重组杆状病毒表达载体pAcuW51-HIAF-1,用Cellfectin将其与线性病毒DNA共转染昆虫细胞sf9,获得重组病毒。用SDS－PAGE电泳和Western blot鉴定重组病毒感染的昆虫细胞中重组HIAF－1重组蛋白的表达。重组蛋白经亲和层析纯化后,在体外用MTT法检测其对内皮细胞的作用,用食管癌移植瘤研究其体内抑瘤活性。结果：在昆虫细胞中高效表达了分子量为26kDa的HIAF－1重组蛋白,其表达量可达昆虫可溶性蛋白的5％－10％。重组HIAF－1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3．1μg/ml,而且显著抑制食管癌移植瘤的生长。结论：在昆虫细胞中高效表达了分子量为26kDa的HIAF－1重组蛋白,其表达量可达昆虫可溶性蛋白的5％－10％。重组HIAF－1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3．1μg/ml,而且显著抑制食管癌移植瘤的生长。结论：在昆虫细胞中高效表达了HIAF－1重组蛋白;HIAF－1重组蛋白的生物活性优于相应的原核重组蛋白。     

可溶性血管内皮生长因子受体2片段的克隆、表达及其在肿瘤血管形成中的作用

目的 对可溶性血管内皮生长因子受体2(VFGFR2)片段阻断血管内皮细胞生长因子(VEGF)与相应受体结合抑制血管形成的作用进行体内外实验研究。方法 应用RT-PCR技术,从胎鼠肝脏扩增Flk-1／KDR片段,重组于逆转录病毒载体PLXSN和表达载体pFT-28b( ),并行表达、纯化和鉴定。以原代培养的小鼠内皮细胞,观察可溶性受体蛋白对内皮细胞生长的影响。以脂质体法转染肿瘤细胞系S180和B16,观察基因转染后的体内生物学特点。结果 在受精后第9,11天的胎鼠肝组织中分离出1000bp大小的可溶性VEGFR2片段,连接TA克隆载体,经测序此片段为VEGFR2胞外段部分序列。将可溶性VEGFR2片段克隆入表达载体pET-28b( ),体外实验显示,可溶性受体蛋白能有效抑制内皮细胞的生长和增殖。将可溶性VEGFR2片段克隆入逆转录病毒载体PLXSN并成功转染肿瘤细胞系S180和B16,体内实验显示,转基因细胞系的瘤重减轻,体积明显缩小,且其血管密度明显降低,而Flkl蛋白表达明显增高。结论 可溶性VEGFR2片段是一种有效的抑制血管形成的生物工程产品,有望作为抗血管形成基因治疗的靶点。     

重组血管抑制剂CHM-Ⅰ对骨肉瘤的治疗作用

目的观察重组人软骨调节素(chondromodulin,CHM)蛋白对骨肿瘤的治疗作用.方法利用RT-PCR技术从人软骨组织中扩增出CHM-Ⅰ基因片段,并将其克隆到原核表达载体pET28a,在大肠杆菌中诱导表达和纯化重组人软骨调节素蛋白,将重组蛋白与人脐静脉上皮细胞或MG-63骨肉瘤细胞共培养,观察其对内皮细胞形成管样结构和MG-63骨肉瘤细胞生长的影响;将重组蛋白局部注射裸鼠皮下肿瘤,观察其对肿瘤形成的影响.结果获得纯化的重组人软骨调节素蛋白,该蛋白在体外可以抑制血管内皮细胞管样结构的形成而对肿瘤细胞的生长无影响;重组蛋白瘤内注射可以抑制肿瘤在裸鼠体内的生长.结论重组人软骨调节素蛋白通过抑制血管形成而抑制肿瘤生长,具有良好的临床应用价值.     

重组血管抑制剂CHM-I对骨肉瘤的治疗作用

目的：观察重组人软骨调节素（chondromodulin，CHM）蛋白对骨肿瘤的治疗作用。方法：利用RT-PCR技术从人软骨组织中扩增出CHM—I基因片段，并将其克隆到原核表达载体pET28a，在大肠杆菌中诱导表达和纯化重组人软骨调节素蛋白，将重组蛋白与人脐静脉上皮细胞或MG-63骨肉瘤细胞共培养，观察其对内皮细胞形成管样结构和MG-63骨肉瘤细胞生长的影响；将重组蛋白局部注射裸鼠皮下肿瘤，观察其对肿瘤形成的影响。结果：获得纯化的重组人软骨调节素蛋白，该蛋白在体外可以抑制血管内皮细胞管样结构的形成而对肿瘤细胞的生长无影响；重组蛋白瘤内注射可以抑制肿瘤在裸鼠体内的生长。结论：重组人软骨调节索蛋白通过抑制血管形成而抑制肿瘤生长，具有良好的临床应用价值。     

血管基膜衍生多功能肽的表达及其生物活性的鉴定

目的: 在毕赤酵母中表达血管基膜衍生多功能肽(vascular basement membrane-derived mulifunctional peptide,VBMDMP),并鉴定其生物学活性.方法: 利用PCR技术获得融合了谷胱甘肽转移酶(GST)的肿瘤抑素(tumstatin)活性成分的GST-VBMDMP基因,克隆到pPIC9K载体;然后原生质体转化法转化毕赤酵母细胞,行多克隆筛选和表型鉴定.获得His Muts表型的转化子,在MGY培养液中30 ℃摇床培养,每24 h从培养液中转移1 ml培养液上清于-70 ℃保存,并保持培养瓶中培养液的量和培养液中甲醇0.5%的浓度不变,第9天行SDS-PAGE检测表达的蛋白并纯化,然后进行细胞和动物生物活性检测.结果: SDS-PAGE发现阳性重组子在MGY培养液中表达的蛋白量在1～7 d逐渐增加,到第8天后开始减少,用Glutathione Sepharose 4B亲和层析柱纯化获得GST-VBMDMP融合蛋白; GST-VBMDMP能抑制C57BL/6鼠动脉内皮细胞管状结构的形成;同时GST-VBMDMP( 2 、6 、10 mg/kg)对小鼠Lewis肺癌原发瘤(瘤重抑制率分别为96.6%、82.1%、61.2%)和自发性肺转移瘤(瘤结节抑制率分别为96.8 %、87.9%、75.3%)均具有明显的抑制作用(P＜0.01).结论:VBMDMP能够抑制鼠动脉内皮细胞管状结构的形成,并对小鼠Lewis肺癌原发瘤和肺转移具有明显的抑制作用.     

IL—1和肿瘤转移

IL-l在不同的细胞中有多种不同的生物学作用,在肿瘤转移中.IL-l通过促进内皮细胞表达与粘附有关的因子如细胞间粘附分子1(ICAM-1)、E-选择素、血小板激活因子(PAF)、整合蛋白VLA-4、甘露糖受体等特异性受体的表达,促进肿瘤细胞与内皮细胞粘附,从而促进肿瘤转移;同时IL-1通过调节肿瘤生长,影响瘤细胞在继发部位的滞留和最初从血管移出而促进肿瘤转移,IL-1受体拮拉剂(IL-1ra)通过拮抗IL-1,抑制瘤细胞与内皮细胞的粘附而抑制IL-1引起的肿瘤转移.当IL-1与IL-1ra失衡时可促使肿瘤转移.     

视黄酸对肿瘤血管形成影响的研究进展

血管形成是肿瘤增殖及转移所依赖的基础。多种因素影响肿瘤血管形成，视黄酸在此方面有其独特的作用及机制，在多种实验模型中，视典酸均表明有明显的抑制肿瘤血管形成的作用，其作用机制可能与影响细胞因子活性、抑制内皮细胞增殖及迁移、调节细胞外基质成分及诱导相应抑制因子有关。     

视黄酸对肿瘤血管形成影响的研究进展

血管形成是肿瘤增殖及转移所依赖的基础.多种因素影响肿瘤血管形成,视黄酸在此方面有其独特的作用及机制,在多种实验模型中,视黄酸均表现有明显的抑制肿瘤血管形成的作用,其作用机制可能与影响细胞因子活性、抑制内皮细胞增殖及迁移、调节细胞外基质成分及诱导相应抑制因子有关.     

突变型flk-1基因转染抑制肝癌在裸鼠体内血管形成、生长和转移

为了研究血管内皮细胞生长因子(VEGF)及其受体(flk-1)系统在人肝癌血管形成、生长和转移过程中的作用,探讨预测和防治肝癌转移和复发的新途径,我们采用了仅表达flk-1蛋白的膜外和跨膜部分的突变型基因片段,与逆转录病毒载体pLXSN构建重组体,经过病毒包装,体内原位转染荷人肝癌转移瘤株LCI D-20裸鼠.结果显示,在被突变型flk-1转染的皮下肿瘤生长缓慢,21天时的平均体积为102.2mm~3,对照组为1374.5mm~3,两组相比P<0.01;肿瘤组织中新生血管极少,团状排列的肿瘤细胞被大量的纤维组织包裹,对照组新生血管丰富.细胞增殖活跃;肝内接种肿瘤转染后肺转移灶亦明显少于对照组,P<0.05.结果提示,VEGF/flk-1系统在肝癌血管形成、生长和转移过程中发挥重要作用,是预测和防治肝癌转移和复发较为理想的靶分子.     

可溶性VEGF受体基因sflt-1与反义VEGF核苷酸对新生血管形成的影响

背景与目的:肿瘤组织中新生血管提供的大量营养物质和生长因子是肿瘤快速生长的关键,因此如何抑制肿瘤组织中新生血管的形成、促使肿瘤组织坏死也是肿瘤治疗中的一条值得探讨的途径。本文拟研究和观察可溶性血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)受体基因sflt-1与反义VEGF对新生血管形成的影响。方法:用Ad-反义VEGF感染肝癌细胞株MM45T.Li后,观察反义VEGF对MM45T.Li分泌VEGF的影响;将人工重组的3'ΔFlt-1蛋白(C末端缺失的Flt-1蛋白)加入条件培养液中,观察3'ΔFlt-1对VEGF刺激的脐静脉血管内皮细胞(humanumbilicalvascularendotheliumcell,HUVEC)增殖的影响;并将Ad-反义VEGF、Ad-sflt-1注射于鸡胚尿囊绒毛膜中,观察sflt-1、反义VEGF对鸡胚新生血管形成的影响。结果:反义VEGF重组腺病毒感染MM45T.Li细胞后,细胞培养上清中VEGF浓度仅为对照组中VEGF浓度的15%(P<0.01);在含有3'ΔFlt-1蛋白的调价培养液中,HUVEC的增殖明显减慢,在一定的范围内与剂量呈负相关;两者都能有效地抑制鸡胚新生血管的形成。结论:sflt-1与反义VEGF均能有效抑制新生血管的形成,两者联合能增强抑制效果,但其作用机制不同。     

血管内皮抑素在非小细胞肺癌治疗中的研究进展

肿瘤血管生成及其生长、侵袭和转移是一个复杂的多因素多步骤的调控过程。肿瘤生长及转移依赖于新生血管形成，抑制血管形成是抗肿瘤治疗的新方法。血管内皮抑素是一种新型内源性血管生成抑制剂，体内外试验证明其能特异性抑制血管内皮细胞的增殖与迁移，诱导其凋亡，从而抑制肿瘤新生血管的形成，达到抑制肿瘤生长的目的。有关血管内皮抑素的具体作用机制及信号传导通路尚不十分明确。同时其表达水平与肿瘤具有明显的相关性．可作为肿瘤的临床预后指标。血管内皮抑素的基因治疗与化疗、放疗、免疫治疗联合应用不仅显著增强其他治疗抗肿瘤效应，而且可减少其他治疗的毒副反应，显示出良好的临床应用前景。其抗血管生成作用具有广谱、低毒的特点，更重要的是克服了肿瘤的耐药性，这对于人类最终克服肿瘤是非常重要的。多年来国内外对其用于非小细胞肺癌治疗的报道较多。本文就血管内皮抑素的结构、作用机制、在非小细胞肺癌中的临床前及临床试验研究做一综述。     

Recombinant adenovirus-mediated overexpression of TIMP-1 effectively suppresses hepatocellular carcinoma cell line HepG2 in vitro and in vivo

OBJECTIVE: To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation, invasion, metastasis, angiogenesis, and apoptosis in human hepatocellular carcinoma (HCC) cells in vitro and in vivo. METHODS: Recombinant adenoviral vector containing hTIMP-1 (AdhTIMP-1) was constructed previously. HepG2 cells were infected by AdhTIMP-1 and the changes of cell proliferation and invasion were detected in vitro. The anticancer activity of AdhTIMP-1 was evaluated in BAL B/c mice bearing HCC. Tumor volume and pulmonary metastases were observed. The mechanisms underlying the antitumor effect in vivo were investigated based on detection of microvessel density and apoptosis in tumor tissues. RESULTS: The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. AdTIMP-1 could effectively infect HepG2 cells and significantly inhibit the proliferative activity and invasive ability of the tumor cells. Compared with the controls, pre-infection of HepG2 cells by AdhTIMP-1 resulted in a significant inhibition of tumor formation by 75. 8%. A single local injection of AdhTIMP-1 into pre-established tumors significantly reduced the tumor growth rate by 45.4%, tumor-associated angiogenesis index by 47.8%, lung metastases by 70.4%, and showed a 3-fold increase of apoptotic tumor cells. CONCLUSION: Our data indicated that AdhTIMP-1 can significantly attenuate tumor proliferation and invasion, reduce metastasis, inhibit angiogenesis, and induce apoptosis in HCC-bearing mice and may pave the way for further liver cancer gene therapy.     

Tumor suppression through angiogenesis inhibition by SUIT-2 pancreatic cancer cells genetically engineered to secrete NK4.

NK4, composed of the N-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts not only as a competitive antagonist of HGF but also as an inhibitor of angiogenesis. By studying the antitumor effect of NK4, we evaluated the potential of gene therapy with NK4 as a treatment for pancreatic cancer. Expression vector pcDNA3-NK4 containing NK4 cDNA was used to transfect human pancreatic cancer cell line SUIT-2. Although the established NK4 transfectant continuously expressed NK4 protein, the expression was shown by migration assay to be insufficient to antagonize HGF in vitro. Proliferation of the NK4 transfectant did not differ significantly from that of a mock transfectant. In vivo, we used models of orthotopic implantation and liver metastasis to transplant NK4-transfected clone or mock-transfected clone into nude mice. Cell proliferation in vivo, evaluated by immunohistochemical staining of proliferating cell nuclear antigen, did not differ between NK4 and mock transfectants, and this was also the finding in the in vitro assay. However, the NK4-transfected clone showed significant inhibition of tumor progression in both the orthotopic implantation and liver metastasis models. The number of vessels within tumors was significantly decreased, and the apoptotic tumor cells were increased in number. The results of these experiments show that genetic modification of tumor cells with NK4 cDNA yields a significant antitumor effect and that this effect is mainly obtained by NK4's function as an angiogenesis inhibitor rather than as an HGF antagonist. We conclude that the potent angiogenesis inhibitor NK4 may be a promising molecule for gene therapy of pancreatic cancer.     

Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2-mediated JAK/STAT3 signaling in endothelial cell

Tumor angiogenesis is one of the hallmarks of the development in malignant neoplasias and metastasis. Many angiogenesis inhibitors are small molecules from natural products. Indirubin, the active component of a traditional Chinese herbal medicine, Banlangen, has been shown to exhibit antitumor and anti-inflammation effects. But its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is unknown. Here, we identified that indirubin inhibited prostate tumor growth through inhibiting tumor angiogenesis. Using chick chorioallantoic membrane (CAM) assay and mouse corneal model, we found that indirubin inhibited angiogenesis in vivo. We also showed the inhibition activity of indirubin in endothelial cell migration, tube formation and cell survival in vitro. Furthermore, indirubin suppressed vascular endothelial growth factor receptor 2-mediated Janus kinase (JAK)/STAT3 signaling pathway but had little effects on the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in endothelial cell. Our study provided the first evidence for antitumor angiogenesis activity of indirubin and the related molecular mechanism. Our investigations suggested that indirubin was a potential drug candidate for angiogenesis related diseases.     

Combined anti-fetal liver kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits angiogenesis and growth of human soft tissue sarcoma xenografts by induction of endothelial cell apoptosis

Vascular endothelial growth factor (VEGF) and VEGF receptor 2 [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] have been shown to play a major role in tumor angiogenesis. In this study, we investigated whether anti-Flk-1 monoclonal antibody DC101 could therapeutically inhibit growth and angiogenesis of human soft tissue sarcoma, and we explored its capacity to enhance the tumoricidal effects of doxorubicin. Treatment of well-established leiomyosarcoma SKLMS-1 and rhabdomyosarcoma RD xenografts in severe combined immunodeficient mice with DC101 resulted in significant antitumor activity. In a parallel study, we compared tumor inhibition with continuous low-dose "antiangiogenic" schedule versus once-every-2-weeks high-dose standard schedule of doxorubicin. We found that continuous low-dose treatment inhibited the tumor growth of RD xenografts about 46.5% of that with standard-schedule treatment, but that continuous low-dose treatment did not inhibit the tumor growth of SKLMS-1 xenografts. Notably, combined DC101 and continuous low-dose doxorubicin resulted in more effective growth inhibition of SKLMS-1 and RD xenografts than has been observed with any agent alone in a long-term s.c. tumor xenograft model. The combination therapy was associated with no additional toxicity to the host animal compared with low-dose doxorubicin alone. Histological examination of xenografts showed significantly reduced microvessel counts in the tumors given combined therapy compared with the tumors given either agent alone. These results are consistent with an enhanced inhibition of angiogenesis in vivo by combined DC101 and doxorubicin using Matrigel plug assay. Additionally, DC101 plus doxorubicin directly exerted enhanced inhibitory effects on endothelial cell migration, proliferation, and tube-like formation in vitro. Furthermore, the combination induced an enhanced apoptosis of endothelial cells that was associated with an increase of capase-3 activity. Thus, the inhibition of angiogenesis and induction of endothelial cell apoptosis are likely important mechanisms for the antitumor activity of combined DC101 and doxorubicin. Collectively, our data suggested that anti-VEGF receptor 2 in combination with continuous low-dose doxorubicin may provide a new therapeutic approach for human soft tissue sarcoma in the clinic.     

重组腺病毒介导基质金属蛋白酶组织抑制因子-1对人肝癌HepG2细胞的影响与作用机制

目的探讨携带人基质金属蛋白酶组织抑制因子-1(hTIMP-1)的重组腺病毒(AdhTIMP-1)对人肝癌HepG2细胞增殖、侵袭、转移、凋亡以及瘤内血管生成的作用。方法构建AdhTIMP-1并感染人肝癌HepG2细胞,采用Boyden chamber检测细胞体外侵袭能力;感染AdhTIMP-1的HepG2细胞接种于裸鼠皮下观察其成瘤情况;建立荷瘤鼠模型,瘤内注射AdhTIMP-1,观察其对肿瘤生长的作用;处死荷瘤鼠,计数肺转移结节;CD34免疫组化观察肿瘤血管生成;原位末端标记(TUNEL)法检测肝癌细胞凋亡。结果AdhTIMP-1感染的HepG2细胞体外侵袭力下降91.4%,成瘤量下降75.8%,荷瘤鼠瘤体内注射AdhTIMP-1使肿瘤生长减少45.4%,组织血管密度减少47.8%,肺转移结节减少70.4%,肿瘤组织中细胞凋亡则增加了3倍。结论AdhTIMP-1能抑制肝癌HepG2细胞的侵袭、转移及瘤内血管形成,诱导肝癌细胞凋亡,可用于肝癌基因治疗的研究。     

Vascular endothelial growth factor in human colon cancer: biology and therapeutic implications

Tumor growth and metastasis are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in the angiogenesis of numerous solid malignancies including colon cancer. Evidence from preclinical and clinical studies indicates VEGF is the predominant angiogenic factor in human colon cancer and is associated with formation of metastases and poor prognosis. Based on these results, it was hypothesized that inhibition of VEGF receptor activity could inhibit colon cancer liver metastasis. To test this hypothesis, the authors evaluated the ability of a small molecule inhibitor specific for the tyrosine kinase VEGF receptor Flk-1/KDR (SU5416) or multiple tyrosine kinase receptors (SU6668) to inhibit tumor angiogenesis and metastasis in a model of colon cancer hepatic metastasis. Both SU5416 and SU6668 inhibited metastases, microvessel formation, and cell proliferation while increasing tumor cell and endothelial cell apoptosis. These results showed that targeting the VEGF receptor/ligand system is a rational approach to inhibiting tumor growth and prolonging survival.