| Objective:The second subclone of the vitro primary culture human umbilical vein endothelial cells(HUVEC) were used for experiment. The cells were dealed with 5.5mM and 20mM glucose respectively. observe the indicator of oxidative stress and the level of Cav-1, PKCαand SelS in each group to research whethere the damage effection of high glucose on HUVEC was through PKC pathway and the relationship among PKC, Cav-1 and SelS, in order to provide a new lab basis for researching endothelial protection.Method:1. A primary culture was set up and HUVEC were identify.2. The cells were treated with DMEM culture media containing 5.5 mM (Normal group),10 mM (HG10 group),20 mM (HG20 group),30 mM (HG30 group) glucose and both 5.5 mM glucose and 14.5 mM mannitol (M group) for 24 and 48 hours respectively. The effection of glucose on HUVEC proliferation by MTT was observed and the level of MDA and SOD in the supernatant of each group was measured.3. According to the result of MTT, the HUVEVC were devided into three groups:normal group (NG), high glucose group (HG) containing 20mM glucose and mannitol group (MG), after a cultured of 24 hours, the mRNA level of Cav-1,PKCa and SelS were detected in each group by real-time PCR, as well as the protein level in each group by Western blot.4. Also, after culture 7 days, the mRNA level of Cav-1,PKCαand SelS were detected in the NG, HG, MG and IG(intermittent group.which dealt with 5.5 and 20mM glucoserespectively on every day) by real-time PCR, as well as the protein level in each group by Western blot. Result:1. Primary HUVEC was successfully in vitro, of which 98.46% of the second generation cells were HUVEC.2. At 24 hours, the cells viabilities in HG20 and HG30 groups were decreased to 71±1.5 and 60±0.5 respectively, as Compared with NG (P<0.01), After dealing 48 hours, the survival rate of the in HG20 and HG30 groups were much lower than in NG (P<0.01), at 65±2.0 and 53±1.5 respectively. However, There were no differences among the HG10, NG and MG 24 hours(P>0.05) (89±1.5,91±1.2 and 93±1.5 respectively) as well as at 48 hours(P>0.05) (85±2.6,88±2.9 and 92±3.0 respectively)。3. The level of MDA (nmol/ml) in the culture media increased in a glucose concentration-dependent manner. After 24 hours culture, MDA level in HG20 and HG30 groups were much higher than in N group (P<0.01),they were 6.28±0.29,11.28±0.40 and 0.93±0.66 respectively. MDA level at 48 hours in HG20 and HG30 groups was also much higher (P<0.01),8.99±0.19 for HG20 group and 5.93±0.57 for HG30 group There were no differences among HG10 group, N group and M group at both 24 hours(P>0.05), (1.13±0.46,1.03±0.46 0.93±0.66 respctively)and 48 hours (1.22±0.33, 1.13±0.21. 0.97±0.36 respectively).4. SOD activity (U/ml) in the culture media decreased with the increase of glucose concentration. After a culture of 24 hours, SOD activities in HG20 and HG30 groups were lower than in N group (P<0.01).6.82±0.23 for HG20 group,5.70±0.22 for HG30 group and 8.62±0.09 for normal group. A more pronounced decline in SOD activities were also observed in the same group (P<0.01), they were 6.04±0.16,5.08±0.87 and 8.23±0.46 after culture48 hours. There were no differences among HG10 group, N group and M group in both 24 hours(P>0.05), SOD activities were 8.10±0.80,8.32±2.33 and 8.62±0.09 respectively, and 48 hours(P>0.05) SOD activities were 74±0.12, 7.80±1.58 and 8.23±0.46 respectively.5. When measured at 24 hours and 7 days, the expression of Cav-1 mRNA in HG was higher than in NG(P<0.05) (P<0.01),1.12±0.21,0.76±0.04 for 24 hours and 1.48±0.35,0.74±0.13 for 7 days. The expression of Cav-1 mRNA was much higher in IG, at 2.16±0.02. There were no differences between NG and MG (P>0.05). The same result was found in the level of Cav-1 protein expression. After dealing with 24 hours and 7 days, Cav-1 protein expression was higher in HG than in NG (P<0.01), the data were:1.62±0.07,1.21±0.01 and 0.69±0.02 0.32±0.01. No differences were found in NG and MG (P>0.05). The Cav-1 protein expression in IG was much higher than in HG and NG (P<0.01), it was 1.14±0.02. There was no differences between normal group and mannitol group(P>0.05) in both 24 hours and 7 days.6. Dealing with different concentration of glucose for 24 hours and 7 days, showd no differences in PKCa mRNA expression among HG, NG and MG (P>0.05), they were 1.38±0.14,1.36±0.33,1.40±0.06 for 24 hours, 1.38±0.28,1.34±0.03,1.35±0.46 for 7 days. The PKCa mRNA expression in IG was not different in NG(P>0.05), it was 1.42±0.01. The same result was found in the expression of PKCa protein level (P>0.05), the data of NG, MG, HG were 0.83±0.02,0.84±0.02,0.82±0.02 for 24 hours, and 0.99±0.02, 0.98±0.04,1.03±0.02 for 7 days. The expression of PKCa protein level in IG has no difference compared with NG(P>0.05).7. No differences in SelS mRNA expression were found between HG and NG after 24 hours and 7 days(P>0.05), they were 1.32±0.12,1.28±0.03 for 24 hours and 1.33±0.01,1.27±0.02, for 7 days. The SelS mRNA expression in IG has no difference with NG(P>0.05), it was 1.33±0.01. There was no difference in SelS mRNA expression after 24 hours and 7 days(P>0.05). There were also no differences in the level of SelS protein expression between HG and NG after 24 hours and 7 days(P>0.05), the data were 0.38±0.02,0.43±0.08 and 0.68±0.04,0.69±0.04 The SelS protein expression in IG has no difference with NG(P>0.05), it was 0.70±0.01. There were no difference in SelS protein expression after 24 hours and 7 days(P>0.05).Conclusion:1. High glucose can suppress the growth of endothelial cell and inhibit the SOD activity while the MDA content increased2. The role of constant and intermittent high glucose on endothelial cell damage may be associated with Cav-1, but there were no changes in the expression of PKCa and SelS from mRNA to protein at 24 hours and at 7 days in either group.
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