Résumé
Objective To construct the eukaryotic expression vector of hypoxia inducible vascular endothelial growth factor (VEGF), and establish its in vitro delivery method. Methods Erythropoietin (EPO) enhancer was inserted into eukaryotic expression vector pGL4.73 [hRluc/SV40] (pSV) promoter by gene recombination technique to construct hypoxia inducible expression system (pEPO-SV). Renilla luciferase (Rluc) was used as downstream reporter gene. Then the VEGF165 gene was inserted into the pEPO-SV plasmid instead of Rluc, and the pEPO-SV-VEGF and pSV-VEGF expression vectors were obtained by inserting the pEPO-SV-VEGF gene into pSV as control. The pSV plasmid expressing Rluc or VEGF165 and pEPO-SV plasmid were transfected in vitro into human embryonic kidney 293T cells. The expression of Rluc or VEGF165 was used to identify the hypoxia induction function of the constructed vector after being treated under normal and hypoxic conditions for 24h and 48h. The intracellular delivery method of plasmids was then established based on poly (lactic acid-glycolic acid) copolymer (PLGA) nanoparticles as carrier, and the efficiency of the eukaryotic expression plasmids induced by hypoxia was evaluated under the in vitro hypoxia model. Results In the construction of plasmid, the successful insertion and correctness of EPO enhancer and VEGF165 gene were confirmed by restriction endonuclease digestion, PCR amplification and DNA sequencing. The plasmid expressing Rluc or VEGF165 was transfected into 293T cells respectively. There was no significant difference in the expression of reporter gene Rluc (one, plasmid pSV and pEPO-SV fluorescence expression values were 2448.24±158.51 and 3173.97±379.92, the second, plasmid pSV and pEPO-SV fluorescence expression values were 55 500.00±3237.05 and 51 193.18±866.32, respectively) or target gene VEGF165 in normal culture (P>0.05). But the expression of Rluc (In the cobalt chloride of hypoxia, the fluorescence expression values of pSV and pEPO-SV were 4857.70±1223.28 and 16 432.64±1618.73, respectively. In the hypoxia incubator, the fluorescence expression values of pSV and pEPO-SV were 2504.45±213.20 and 17 274.35±685.60, respectively) or VEGF165 in hypoxia was significantly higher than that in control group (P<0.01). The results showed that the constructed pEPO-SV and pEPO-SV-VEGF plasmids had typical hypoxia inducible expression activity. PLGA nanoparticles were used to in vitro deliver pEPO-SV and pSV in 293T cells. The results of detecting the reporter gene Rluc in normal culture and hypoxic conditions were consistent with those mentioned above, that is, under normal conditions, the 24h and 48h fluorescence expression values of plasmids pSV and pEPO-SV were 149.44±4.01 and 127.09±15.05, 1074.91±114.78 and 1064.56±137.48, respectively; under hypoxic conditions, the 24h and 48h fluorescence expression values of pSV and pEPO-SV were 3265.34±440.00 and 8828.87±637.03, 3202.06±33.43 and 9114.75±292.06, respectively. Conclusion A typical hypoxia inducible VEGF eukaryotic expression system has been successfully established, and an in vitro effective delivery method is also established, which may have an important application prospect in ischemia, hypoxia and other tissue injury diseases.
Résumé
<p><b>OBJECTIVE</b>To understand the occurrence and development of adolescent students' type 2 diabetes mellitus (T2DM) by researching the characteristics of the adolescent students' impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) effected by overweight or obesity.</p><p><b>METHODS</b>From May to November 2007, 3856 middle school students aged 11 to 18 years old in Dongguan city were enrolled in the study. Overweight or obesity (b/Ob) depended on three indexes: the national unified school-age children and adolescent students' body mass index (BMI) and the temporary screening classification standard II established by the Working Group on Obesity in China, BP > or = 140/90 mm Hg (1mm Hg = 0.133 kPa) and fasting capillary whole glucose which was greater than or equal to 5.6 mmol/L. The fasting capillary whole glucose was screened by blood glucose meter from fingertips. Students who had any abnormal indexes were brought into this study. On basis of voluntary principle, blood lipid, fasting blood glucose (FPG) and 2-hour postprandial blood glucose (2 h PG), fasting insulin (FIns) of 368 male and 326 female students who conformed to these conditions were measured using their venous blood. By temporary BMI standard II, they were divided into overweight group (b) and obesity group (Ob). Data of different age groups (11 to 14; 15 to 18 years old) was analyzed.</p><p><b>RESULTS</b>The BMI, low density lipoprotein cholesterol (LDL-C), insulin resistance index (IR), IFG and IGT of the same age stage in two groups were compared. The BMI value was (22.1 +/- 2.4) kg/m2, LDL-C was (2.38 +/- 0.65) mmol/L, IR was 1.15 +/- 0.58 and the detection rates of IFG and IGT were 3.5% and 1.4% respectively in female students aged 11 to 14 years old in b group. In Ob group, BMI value was (24.4 +/- 3.9) kg/m2, LDL-C was (2.70 +/- 0.73) mmol/L, IR was 1.36 +/- 0.67 and the detection rates of IFG and IGT were 14.6% and 6.3% respectively. t or chi2 values of two groups which were compared were 4.83, 2.45, 2.10, 7.41 and 7.99 (P < 0.01 or P < 0.05). BMI value was (25.8 +/- 3.1) kg/m2, LDL-C was (2.35 +/- 0.62) mmol/L, IR was 1.14 +/- 0.64 and the detection rates of IFG and IGT were 3.1% and 4.1% respectively in 15 to 18 years old in b group. In Ob group, BMI value was (28.0 +/- 4.3) kg/m2, LDL-C was (2.69 +/- 0.69) mmol/L, IR was 1.43 +/- 0.84 and the detection rates of IFG and IGT were 12.8% and 15.4% respectively. t or chi2 values of two groups which were compared were 3.33, 2.79, 1.87, 4.75 and 5.17 (P < 0.01 or P < 0.05). BMI value was (22.4 +/- 2.3) kg/m2, LDL-C was (2.36 +/- 0.67) mmol/L, IR was 1.19 +/- 0.65 and the detection rates of IFG and IGT were 3.6% and 1.8% respectively in male students of 11 to 14 years old in b group. In Ob group, BMI value was (24.6 +/- 4.2) kg/m2, LDL-C was (2.68 +/- 0.71) mmol/L, IR was 1.44 +/- 0.89 and the detection rates of IFG and IGT were 13.3% and 9.4% respectively. t or chi2 values of two groups which were compared were 4.85, 2.72, 2.19, 6.75 and 6.76 (P < 0.01 or P < 0.05). BMI value was (26.4 +/- 2.8) kg/m2, LDL-C was (2.35 +/- 0.70) mmol/L, IR was 1.24 +/- 0.68 and the detection rates of IFG and IGT were 4.7% and 5.6% respectively in 15 to 18 years old in b group. In Ob group, BMI value was (28.2 +/- 4.8) kg/m2, LDL-C was (2.71 +/- 0.73) mmol/L, IR was 1.50 +/- 0.95 and the detection rates of IFG and IGT were 17.9% and 17.9% respectively. t or chi2 values of two groups which were compared were 2.80, 2.69, 1.84, 6.68 and 6.27 (P < 0.01 or P < 0.05). The male students' FPG of 11 to 14 years old in b group was (4.88 +/- 0.76) mmol/L and FPG of Ob group was (5.09 +/- 0.80) mmol/L. Two groups were compared and t = 1.84 (P < 0.05). The statistical differences were all observed. We compared different age stages and found that the male students' 2-hour PG of 11 to 14 years old in Ob group was (5.13 +/- 1.18) mmol/L and the 2-hour PG of 15 to 18 years old was (5.36 +/- 1.24) mmol/L. Two groups were compared and t = 1.78 (P < 0.05) near the adults value. Male students' IGT of 11 to 14 years old (b/Ob) had 8 positive cases and the positive detection rate was 3.6%. IGT of 15 to 18 years old (b/Ob) had 13 positive cases and the positive detection rate was 8.9%. Two age stages were compared and chi2 = 6.86 (P < 0.01). Female students' IGT of 11 to 14 years old (b/Ob) had 5 positive cases and the positive detection rate was 2.6%. IGT of 15 to 18 years old (b/Ob) had 10 positive cases and the positive detection rate was 7.4%. Two age stages were compared and chi2 = 4.02 (P < 0.05). All had statistical significance. The high IGT incidence rate of b/Ob group's male and female students was in the stage of 15 - 18 years old. Male students were more obvious.</p><p><b>CONCLUSION</b>T2DM prevention among adolescent students should start with body overweight control. Meanwhile, the adolescent students with high risk factors should be screened regularly and early measures should be taken to prevent the impaired glucose regulation (IFG, IGT) transforming into T2DM.</p>