反义寡核苷酸对人淋巴瘤细胞系Namalwa细胞血管内皮生长因子表达影响的体外研究

1、反义寡核苷酸对人淋巴瘤细胞系Namalwa细胞血管内皮生长因子表达影响的体外研究            作者：黎纬明，张敏，邹菁，童允洁，邹萍【摘要】  为了研究硫代磷酸化修饰的血管内皮生长因子（vascular endothelial growth factor, VEGF）反义寡核苷酸(antisense oligodeoxynucleotide, ASODN)对人淋巴瘤细胞系Namalwa细胞VEGF表达的影响， 将终浓度分别为5、10、20 mol/L的VEGF ASODN和错

2、义序列与人淋巴瘤细胞系Namalwa细胞分别孵育24、48小时，采用RTPCR检测VEGF mRNA的表达，采用链酶菌抗生素蛋白过氧化酶免疫组织化学法（streptavidin/peroxidase, SP法）检测VEGF的表达。结果表明： VEGF ASODN 3个浓度组（5、10和20 mol/L）处理的Namalwa细胞VEGF mRNA的表达分别为1.38、0.96、0.57, 错义序列组和对照组分别为1.79、1.84。当加入20 mol/L VEGF ASODN作用48小时后，细胞内VEGF蛋白水平显着减少，而错义序列组Namalwa细胞VEGF蛋白水平未见明显改变。结论： VEG

3、F ASODN在体外能够抑制Namalwa细胞VEGF的表达。 【关键词】  淋巴瘤   The recent studies have shown that in nonHodgkin lymphoma patients with bone marrow involvement, the expression of  vascular endothelial growth factor (VEGF) was associated with higher grading of NHL and highgrade transformation of low

4、grade lymphoma1. Data from Potti  et al2 indicated that signal transduction inhibition therapy using VEGFinhibitors may have distinct effect for the patients with mantle cell lymphoma.   Bellamy et al3 reported that VEGF over expressed in human lymphoma cell line Namalwa cells. We use

5、d VEGF ASODN and scrambled sequence  transfecting Namalwa cell respectively,  and confirmed the significant inhibition of VEGF ASODN on the VEGF mRNA expression, thus  provided experimental and theoretical basis for  the antisense gene therapy of  lymphoma.   Mater

6、ials and Methods   Drugs and reagents   VEGF ASODN sequence was designed based on the   human VEGF mRNA +261 to +281 nucleotides (5 TGGCTTGAAGATGTACTCGAT3), and the scrambled (SCODN) sequence was 5TACGTAGTATGGTGTACGATC34.  All these were prepared in the suitable co

7、ncentration with RPMI1640 (purchased from Shanghai Biotechnology Ltd). The mouse antihuman VEGF monoclonal antibody was purchased from Santa Gruz Co. The SP kits were purchased from Beijing Zhongshan Biotechnology Ltd. The TRIZOL kits were purchased from Gibco, cDNA firststrand synthesis reaction ki

8、ts were the products of MBI Co (purchased from Jingmei Biotech company). The dNTP and DNA enzyme were the products of Huamei Company.   Effect of Antisense Oligodeoxynucleotides on the Expression of Vascular Endothelial Growth Factor in Namalwa cell Cell culture and treatment of ASODN 

9、;  Human lymphoma cell line Namalwa cells were obtained from Shanghai Biochemistry Cell Institute. Namalwa cells were cultured in RPMI1640 (purchased from Gibco company) supplemented with 10 fetal bovine serum ( products of Hangzhou Sijiqing Biological Engineering Material Co.),100 U/ml penicil

10、lin, 100 g/ml streptomycin. They were cultivated in incubator of 37,5% CO2 and  saturated humidity. Cells in log phase were inoculated in 6well plates. Each plate consisting of 2 ml (cell density was 3×105/ ml) was added with VEGF ASODN and scramble sequence, whose final concentration were

11、 5, 10 and 20 mol/L respectively. Blank were also designed, each concentration was repeated for 3 times, incubated for 24 or 48 hours in normal condition.   RTPCR for VEGF mRNA   Cells of each group incubating for 24 hours were collected and washed with PBS for 2 times. 

12、0; Abstraction of cells RNA  All procedures were based on the instruction of TRIZOL RNA abstraction kits.  5×106 cells were got and  add with 1 ml TRIZOL,  RNA was abstracted after chloroform delamination , avantin precipitation,  washing and drying with 70% alcohol, an

13、d was dissolved with sufficient nonRNAase water, then  the purity and quality of RNA were assessed using extreme ultraviolet spectroscopy and agarose gel electrophoresis, OD260/OD280=1.9, the RNA concentration was 0.5 g/l.   RNA reverse transcription  The reverse transcription re

14、actions of RNA were based on the instruction of cDNA firststrand synthesis reaction kit (MBI). 1 l  total RNA, 1 l random primer and 10 l  DEPC water were mixed at 70 for 5 minutes; 5×reaction buffer 4 l, RNase inhibitor 1 l, 10 mmol/L dNTP  2 l  were added, mixed and warm b

15、athed at 25 for 5 minutes; 1 l  MMulv reverse transcriptase was added and warm bathed at 25 for 10 minutes, 42 for 60 minutes, 70 for 10 minutes.   PCR reaction  To use sufficient cDNA for PCR reaction. The primer sequence of VEGF5： sense: 5TCGGGCCTCCGAAACCATGA3,   

16、;          antisense: 5CCTGGTGAGAGATCTGGTTC3; The primer sequence of MG: sense: 5ATCTTCAAACCTCCATGATG3,             antisense: 5ACCCCCACTGAAAAAGATGA3.  The reaction condition:  94 force

17、degeneration for 3 minutes; 94 degeneration for 1 minute, 55 renaturation for 50 seconds, 72 elongation for 40 seconds, these 3 steps circulating for 30 times, and then 72 elongation again for 5 minutes.   Relative quantitative analysis of PCR amplification products  10 l  PCR am

18、plification products were detected through 1.5% agarose gel electrophoresis. The spot density scanning value of destination electrophoresis strip was obtained through FR200 image analysis software. The ratio of VEGF121 to MG was considered as the relative expression of VEGF. All relative amounts of

19、antisense groups were compared with blank groups, the ratios were considered as parameters for the comparison of all groups. Analysis of interclass variance was used with SPSS 10.0.   SP immunohistochemistry for the expression of VEGF protein   Cells were cultured and manipulated

20、 as RTPCR. Gather cells of each group after incubating for 48 hours, remove supernatant using centrifugation, resuspense after PBS washing, adjust to proper density, and place Namalwa cells on glass slides (1 × polylisine), observe in microscope, fix with cold acetone for 15 minutes (4). Replac

21、e PBS with VEGF monoclonal antibody as blank control. Incubate with 3% hydrogen peroxide for 10 minutes, wash with distilled water, soak with PBS for 5 minutes, drop normal goat serum fluid 50 l to incubate for 15 minutes, then pour it, add 50 l mouseantihuman monoclonal antibody (1:50 dilution), st

22、ay overnight in 4, wash with PBS for 3 minutes, 3 times, add biotin labeling goatantimouse IgG 50 l, incubate for 10 minutes at room temperature, wash with PBS for 3 minutes, 3 times, add horseradish enzyme labeling antibiotin fluid, incubate for 10 minutes at room temperature, wash with PBS for

23、0; 3 minutes,  3 times, DAB coloration ,wash with tap water, afterstain with hematoxylin, routine dehydrate, clear with dimethyl benzene, mouting with neutronresin.            Positive standard of VEGF: Cell which cytoplasm or membrane stain wit

24、h buffy was positive, otherwise was negative. Select 10 fields in light microscope randomly, count 100 cells in each field. Count the number of positive cells. The exponent formula of positive cells is : (number of positive cells/1000) ×100%.   Result   Effect of VEGF ASODN

25、on VEGF mRNA expression in Namalwa cells   RTPCR showed that the Namalwa cells expressed VEGF121 mRNA (516 bp) and VEGF165 mRNA(648 bp). The endogenous reference MG was 120 bp. The VEGF mRNA was measured by RTPCR in cells treated with various concentrations of VEGF ASODN (5, 10, 20 mol/L).

26、 After 24 hours, the band of VEGF165 mRNA 648 bp disappeared. With scanning the grey level ratio between VEGF 516 bp band and MG band, we detected the expression of VEGF121 mRNA treated with three concentration levels (5, 10, 20 molL) of ASODN were 1.38±0.21, 0.96±0.15, 0.57±0.09 resp

27、ectively (F=12.38, p0.05). ASODN showed a marked dosedependent inhibition on the expression of VEGF. The expression of VEGF121 mRNA in PBStreated cells and scrambled sequence treated cells were 1.79±0.28 and 1.84±0.31 respectively. There were no significant difference between these two gro

28、ups (p0.05). But  difference was significant,  as compared  between the ASODN group and PBStreated group (p0.05)（Figure 1）.   Figure 1. Expression of vascular endothelial growth factor mRNA in Namalwa cells treated by antisense oligodeoxynucleotides.  M: marker. Lane

29、60; 1,2: normal control. Lane 3,4: treated with scrambled sequence for 24 hours. Lane  5,6: treated with ASODN for 24 hours.   Expression of VEGF protein in Namalwa cells   The Namalwa cells were treated with VEGF ASODN and SCODN (20 mol/L) for 48 hours. The PBStreated group

30、 was used as blank control. The result showed that the VEGF expressed at high levels in SCODN treated group and control group, with the percentage of positive cells was 92.6% and 95.2% respectively. The cells treated with ASODN also expressed VEGF but at lower levels, with the percentage of 13.9%. C

31、ompared with the SCODN treated group and control group, the difference was statistically significant (p0.05) (Figure 2). ASODN showed inhibitory activity, but SCODN had minimal inhibitory effect on the expression of VEGF in Namalwa cells, which in accordance with the result reported by RTPCR, and fu

32、rther illustrated that  the effect of ASODN was sequencespecific. Though the levels of VEGF expression were different in these three groups, the growth of Namalwa cells were roughly the same. The Namalwa cells were treated by ASODN (20 mol/L), incubated for 24, 48 or 72 hours respectively, and the result s