| Objective : Diabetic has liability to colorectal cancer, and the epidemiological investigation show that diabetes increases the risk of colorectal cancer.As the most important regulatory factor for vascularization, VEGF has an intensive promotion for neogenesis of the tumor vessel and for the development of tumor.At the same time, VEGF is an immunosuppressive molecules,it plays an important role in immunosuppression for tumor and it plays an important role for the development of tumor too.The secretion of VEGF is higher in diabetes patients than the population of nondiabetes,especially in diabetes patients with complication of capillary and the secretion of VEGF is related to the concentration of insulin and glucose closely.The abnormity of glucose and insulin exsits in the whole course of diabetes mellitus and has the apparente stage. The experience is to examine whether glucose and insulin can affect the secretion of VEGF in Colon26 cells and the interaction between them and it provides the experiment and theoretical evidence in exploring the relationship between abnormity of glucose and insulin and the high risk of diabetes mellitus with colorectal cancer. Methods : All experiments were performed using Colon26 tumor cells that cultured in vitro. According to the concentration of glucose and insulin in culture medium,cells were devided into 12-groups:A(control):RPMI1640;B:RPMI1640+5mmol/Lglucose;C:RPMI1640+10mmol/Lglucose;D:RPMI1640+25mmol/Lglucose;E:RPMI1640+5uIU/mLinsulin;F:RPMI1640+25uIU/mLinsulin;G:RPMI1640+125uIU/mLinsulin;H:RPMI1640+5mmol/Lglucose+5uIU/mLinsulin;K:RPMI1640+5mmol/Lglucose+125uIU/mLinsulin.L:RPMI1640+25mmol/Lglucose+125uIU/mLinsulin.M:RPMI1640+25mmol/Lglucose+5uIU/mLinsulin.N:RPMI1640+25mmol/Lglucose+1.25uIU/mLinsulin.Every group has three holes.After added to different culture medium, cells were cultured for 48h, supernatant were collected and stored at -70℃before detected. In all experiments VEGF protein concentration in the supernatant was determined by specific enzyme-linked immunosorbent assay(ELISA). Subsequently, total RNA was isolated and then VEGF mRNA andβ-actin mRNA examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Both VEGF andβ-actin PCR product were separated by flat bed electrophoresis in 1.5% agarose gels, stained with Goldview and photographed. The negative were scanned using a densitometer and the density of the bands compared to those of the housekeeping geneβ-actin. The experimence was repeated three times. Statistical analysis was performed using the one-way ANOVA and t test, with a value of P<0.05 considered to represent a significant differernce. Results: 1 The effect of different concentration of glucose on secretion of VEGF in Colon26 tumor cells. In B,C,D groups(including 5mmo1/L,10mmo1/L,25 mmo1/L glucose in culture medium, respectively), compared with A, there was no statistical difference about the secretion of VEGF (9.4167±0.4646,9.4667±0.1528,9.6167±0.2887vs9.5583±0.261, P>0.05) .2 The effect of different concentration of insulin on secretion of VEGF in Colon26 tumor cells. In G groups(including 125uIU/mL insulin in culture medium),the level of VEGF was higher significantly than A,E and F groups(27.7667±0.2517 vs 9.5583±0.2691,9.5833±0.0764,9.6167±0.1258 P<0.01).However no differences between A(control) ,E(including 5uIU/mL insulin in culture medium) and F(including 25uIU/mL insulin in culture medium) groups were found.3 The effect of different concentration of insulin and glucose on secretion of VEGF in Colon26 tumor cells.. In K and L groups(all including 125uIU/mL insulin in culture medium),the levels of VEGF were higher significantly than A (27.8333±0.3512, 27.6000±0.6557 vs 9.5583±0.2691, respecti -vely, all p<0.01).However no differences between K and L groups were found.H,M,N groups (including 5 uIU/mL, 5uIU/mL,1.25 uIU/mL insulin in culture medium), compared with A , there was no statistical difference about the secretion of VEGF (9.7167±0.00289,9.8667±0.0577,9.90±0.1732vs 9.5583± 0.2691, P>0.05) .4 The expression of VEGF mRNA in Colon26 tumor cells is corresponding with the level of VEGF in supernatant. Both VEGF andβ-actin PCR product were separated by flat bed electrophoresis in 1.5% agarose gels, stained with Goldview and photographed and made half-quantitative analysis according to the picture by computer. In G,K,L groups(all including 125uIU/mL insulin in culture medium), electrophoresis strips are thicker and brighter than A group .The levels of VEGF mRNA of G,K,L groups (0.8802±0.19;0.8605±0.18;0.8569±0.22) were higher significantly than the level of VEGF mRNA of A group(0.4653±0.02) and the statistical difference was significant (P<0.05). The electrophoresis strips of other groups have no difference with the electrophoresis strip of the control group and there was no statistical difference with the levels of VEGF mRNA in these groups ( P>0.05 ) .It imply that high concentration of insulin increase secretion of VEGF on the transcription。Conclusions: 1 The concentration of glucose have no effect on secretion of VEGF in Colon26 tumor cells.2 High concentration of insulin may increase secretion of VEGF in Colon26 tumor cells.3 High concentration of insulin may increase expression of VEGF mRNA in Colon26 tumor cells and it imply that high concentration of insulin increase secretion of VEGF on the transcription。...
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