| Objective: To observe the role of different concentration of lipoprotein and free fat acid in the expression of hUAT mRNA in HK-2 cells.Methods: All experiments were performed with HK-2 cells cultured in vitro. According to the concentration of lipoprotein and free fatty acids in culture medium, growth-arrested cells were devided into 6 groups. On ethanol added or not, control guoups were devided into C1, C2 group. And according to the concentration of VLDL, LDL, HDL, cells treated with lipoprotein were devided into 1, 2, 3, 4 group. And based on the concentration of PA and OA, cells treated with free fat acid were devided into 1, 2, 3 group. ①Control group: C1: DMEM/F-12 C2: DMEM/F-12+1% (V/V) ethanol. ②LDL group: L1: DMEM/F-12+50ug/ml LDL. L2: DMEM/F-12+ 100ug/ml LDL. L3: DMEM/F-12+200ug/ml LDL. L4: DMEM/F-12+400ug/ml LDL. ③HDL group: H1: DMEM/F-12+50ug/ml HDL. H2: DMEM/F-12+100ug/ml HDL. H3: DMEM/F-12 +200ug/ml HDL. H4: DMEM/F-12+400ug/ml HDL. ④VLDL group: V1: DMEM/F-12+50ug/ml VLDL. V2: DMEM/F-12+100ug/ml VLDL. V3: DMEM/F -12+200ug/ml VLDL. V4: DMEM/F-12 +400ug/ml VLDL. ⑤OA group: A1: DMEM/F-12 +100 umol/l OA+1% (V/V) ethanol. A2: DMEM/F-12+200 umol/l OA+1%(V/V) ethanol. A3: DMEM/F-12+400 umol/l OA+1% (V/V) ethanol. ⑥PAgroup: P1: DMEM/F-12+100 umol/1 PA+1% (V/V) ethanol. P2: DMEM/F-12+200 umol/1 PA+1%(V/V) ethanol. P3: DMEM/F-12+400 umol/1 PA+1% (V/V) ethanol. In each culture medium, 6 bottle of cells were cultured for 48h to get the mean. Then all were collected, counted and total RNA was extracted. 1 ug mRNA was reverse transcripted to cDNA and hUAT and GAPDH were amplified by real-time fluorescent quantitative PCR. Then relative expression quantity of hUAT mRNA was analyzed. Repeat the whole experiment. The data was analyzed by t-test and one-factor ANOVA and P<0.05 was considered significant.Results:①hUAT mRNA could be detected in all the samples. ②hUAT mRNA expression decreased with the increase of LDL concentration after treated for 48h. hUAT mRNA level of control group was significantly higher than that of L1, L2, L3, L4 group (P=0.000). There was significant difference between each two groups (P<0.05) except that between L2, L3 groups(P=0.440) and L3, L4 groups(P=0.158). (3)In VLDL groups, the expression of hUAT mRNA decreased with the increase of VLDL concentration. And hUAT mRNA of control groups was significantly higher than that of V1, V2,V3, V4group(P=0.000) and there was significant difference between each two groups (P<0.05) except that between V3, V4 groups (P=0.056). ?With the increase of HDL concentration, hUAT mRNA expression decreased after treated for 48h. hUAT mRNA of control group was significantly higher than that of HI, H2, H3, H4 groups (P=0.000). There was significant difference between each two groups (P<0.05) except that between HI, H2 groups and H3, H4 group. ?There was no significant difference in hUAT mRNA level between Cl and C2group after treated for 48h (P=0.342) . And there was no significant difference in hUAT mRNA level with different concentration of OA and PA. There was no difference in hUAT mRNA level between control groups and OA.PA groups(P>0.05). Condusions.'Increased concentration of VLDL,LDL,HDL down-regulate the expression of hUAT mRNA, which may be an important pathogenesis in hyperuricemia induced by lipid metabolic disorder. Free fatty acid may influence urate metabolism through the way other than hUAT regulation.Postgraduate student: Chen Ying (Endocrinology) Directed by: Professor Miao Zhimin ,Yuan Ying... |
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