作者：崔国惠， 陈卫华， 薛克营， 刘芳， 陈燕
【摘要】 为了探讨肿瘤坏死因子 （TNF-α ）和雷公藤内酯醇作用Raji细胞VEGF（血管内皮细胞生长因子）的表达及VEGF与ECV血管形成的关系， 采用MTT法检测雷公藤内酯醇抑制Raji细胞增殖的影响；用ELISA法对Raji细胞上清的VEGF定量；用基质胶上的内皮细胞网络形成检测ECV304（人类脐静脉内皮细胞起源的细胞系）血管生成；用RT-PCR检测VEGF mRNA含量。结果表明：雷公藤内酯醇对Raji细胞增殖抑制作用呈时间和剂量依赖方式，最适合作用时间为24小时，24小时半数抑制浓度IC50为25 nmol/L； Raji细胞上清VEGF的含量在TNF-α 组明显高于空白对照组，雷公藤内酯醇处理组则明显低于空白对照组，两者比较有极显著差异(P<0.01)； Raji细胞内VEGF mRNA主要为VEGF165和VEGF121，其含量在TNF-α 处理组与空白对照组比较有增加，而雷公藤内酯醇抑制VEGF mRNA表达，并呈剂量依赖方式； Raji细胞上清、VEGF(10 ng/ml)和 TNF-α (10 ng/ml)处理的Raji细胞上清在基质胶中作用ECV304细胞后可促进血管形成，而雷公藤内酯醇(25 nmol/L)处理的Raji细胞上清和1640培养基作为的对照组加入基质胶中ECV304细胞未见血管形成。结论： 在TNF-α 、雷公藤内酯醇作用Raji细胞过程中，TNF-α 促进VEGF的表达而雷公藤内酯醇则抑制其表达； TNF-α 处理的Raji细胞上清在基质胶中促进血管形成，而雷公藤内酯醇则抑制血管形成。
【关键词】 肿瘤坏死因子-α； 雷公藤内酯醇； VEGF； Raji细胞； ECV304细胞； 血管形成
Vascular endothelial growth factor (VEGF) promotes angiogenesis and is associated with the invasion and metastasis of malignant tumours［1］. VEGF promotes acute myelogenous leukemia (AML) cell growth and survival, and may contribute to drug resistance［2，3］. Previous studies showed that TNF-α increased VEGF mRNA expression［4］ . Triptolide, a traditional Chinese medicine, has been reported to be effective in the treatment of auto-immune diseases, and it can also induce anti-neoplastic activity on several human tumour cell lines. Triptolide can inhibit VEGF expression［5］. Therefore, to better understand the possibilities of antiangiogenic tumour therapy, we investigate the effect of TNF-α and triptolide on the expression of VEGF in human B lymphoma Raji cells lines, and their effect on angiogenesis in human umbilical vein endothelial cells (HUVECs)-derived cell line (ECV304) to elucidate the mechanisms controlling the process of tumour neovascularization.
Materials and Methods
Drugs and reagents
Recombinant human TNF-α was purchased from Peprotechec, USA (#0604CY25,≥2×107 units/mg). Triptolide (molecular weight 360， from Tripterygium Wilfordii 98% (HPLC) solid )； Matrigel was purchased from Becton Dickinson Labware (Bedford, MA, USA), the Quantikine human VEGF ELISA kit was purchased from R&D Systems (USA). RevertAidAM FirstStrand cDNA Synthesis kit was purchased from Fermentas. Primer pairs were synthesized by BioAsia Biotechnology Co.Ltd.
Cell lines and culture
Raji and ECV304 cells were obtained from China Center for Typical Culture Collection in Wuhan and were grown in RPMI 1640 (Gibco BRL, USA) containing 10% FCS, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin at 37℃ in a fully humidified atmosphere with 5% CO2. To test the VEGF production, Raji cells were plated at 105 cells/well in a 6-well plate for 24 hours before the experiment. U937 cells were used at 106 cells/ml.
MTT assay
The antiproliferative effects of triptolide against Raji cells were determined by the MTT dye uptake method as described earlier. Briefly, the cells (40 000 per well) were incubated in triplicate in 96-well plate. Different concentrations of triptolide were added, and the final concentrations were 3.13, 6.25, 12.50, 25.00 and 50.00 nmol/L, then were collected at different times incubated for 12, 24, 36, 48, 60 and 72 hours. MTT assay was performed to determine the proliferative inhibitory rate (PIR).
PIR=(1-average A value of experimental group/
average A value of control group)×100%
Quantification of VEGF Production
To test the VEGF production treated by TNF-α and triptolide, Raji cells were incubated in the presence of TNF-α (10 ng/ml) or triptolide (25 nmol/L). After 24 hours, cell culture supernatants were collected, and VEGF concentrations were determined by ELISA using the Quantikine human VEGF ELISA kit ( R&D Systems, USA). The VEGF protein concentration of each fraction was measured by ELISA (pg VEGF/ml supernatant). VEGF concentration in cell culture supernatant was measured which has been calibrated against a highly purified recombinant human VEGF165. In short, 1 ml of media was centrifuged at 10 000×g for 5 minutes at 4℃, and the supernatant was stored at -70℃ for up to 72 hours. Thawed supernatant (200 μl) was used in the ELISA according to manufacturer's instructions. The absorbance of the samples was determined by using an EXL-800 microplate reader. A serial dilution of human recombinant VEGF was included in each assay to obtain a standard curve from which sample VEGF concentrations were obtained. Computer software Curve Expert 1.3 was used to obtain a standard curve from which sample VEGF concentrations (pg/ml) were obtained.
Matrigel capillary tube formation
This assay was carried out by the method as previously described［6］. Matrigel was purchased from Becton Dickinson Labware (Bedford, MA, USA), diluted to 4 mg/ml with cold phosphate-buffered saline (PBS) and added to 24-well plates in a total volume of 400 μl in each well. Plates were placed at 37℃ for an hour to form a gel layer. After gel formation, a total of 2×105 /ml ECV304 cells were applied to each well and incubated with the supernatants of Raji culture for 24 hours. Experiments were divided into 5 group as follows: (A) untreated group; (B) VEGF (10n g/ml) group; (C) TNF-α (10 ng/ml) group; (D) triptolide(25 nmol/L)group, and (E) control (1640 medium instead of the supernatants of Raji culture ). Plates were incubated at 37℃ for 24 hours with 5% CO2, viewed (magnification ×40) and photographed using an Olympus microscope (Olympus, Japan). At least 5 fields were examined per well; each experimental condition was tested in triplicate.
Reverse transcription-PCR
Total RNA was isolated by TRIZOL reagent (Life Technologies, Inc., USA) according to the manufacturer's instructions. The reverse transcription reaction was performed using the RevertAidTM First-Strand cDNA Synthesis kit (Fermentas, Lithuania， USA). The newly synthesized cDNA was amplified by PCR. The 26.25 μl reaction mixture contained 10×buffer with MgCl2 2.5 μl， 10 mmol/L dNTP 0.5 μl， Taq polymerase (5 U/μl) 0.25 μl, cDNA 1.25 μl,1.25×2 μl of forward and reverse primers 1 (5 pmol/μl), 125×2 μl of forward and reverse primers 2（5 pmol/μl）and ddH2O 17 μl .The following primer pairs were designed from human cDNA sequences available in GenBank and synthesized by BioAsia Biotechnology Co., Ltd.; PCR primers were as follows: VEGF, 5'-T- CGGGCCTCCGAAACCATGA-3' and 5'-CCTGGTGA-GAGATCTGGTTC-3'; PCR was carried out at 94℃ for 5 minutes, followed by 34 cycles of 94℃ for 30 seconds, 55℃ for 30 seconds, and 72℃ for 1 minute, with a final extension at 72℃ for 10 minutes, which can amplify all included 516,588,648,720 bp fragment; and β-actin, 5′-TCTACAATGAGCTGCGTGTG-3′ and 5′-CAACTAAGTCATAGTCCGCC-3′, which amplify an 878 bp fragment; Notch1, 5′-GACATCACGGATCATATGGA-3′ and 5′-CTCGCATTGACCA-TTCAAAC-3′, which amplify a 666 bp fragment; PCR was carried out at 95℃ for 2 minutes, followed by 30 cycles of 95℃ for 1 minutes, 60℃ for 2 minutes, and 72℃ for 1.5 minutes, with a final extension at 72℃ for 7 minutes. After amplification, 8 μl aliquots of products were resolved on a 1.7% agarose gel. DNA bands were visualized by UV light and documented with Smart View 2000 software processor. The ratio between the target gene and β-actin gene band densities was used for quantitative evaluation. The data presented is representative of three experiments.
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