The Expression And Significant Of Thrombospondin 1 In Human Tubular Epithelial-Myofibroblast Transdifferentiation

Objective: To investigate the expression of thrombospondin 1 (TSP-1) and its influence on the expression of vascular endothelial growth factor (VEGF) during the process of human tubular epithelial-myofibroblast transdifferentiation (TEMT) induced by transforming growth factor (TGF).Methods: Human tubular epithelial cell (HKCs) line were cultured in vitro with DMEM/F12 1:1 medium. HKCs were divided into four groups: (1) Normal control groups (C groups): HKCs were cultured with free serum medium (FSM) and were redivided into three subgroups C1, C2 and C3. (2) TGF groups (T groups): HKCs were treated with FSM contained TGF and were redivided into three subgroups T1, T2 and T3. (3) Antisense oligonucleotide groups (TA groups): HKCs were treated with FSM contained TGF and TSP-1 Antisense oligonucleotide (AS). (4) Scrambled oligonucleotide groups (TS groups): HKCs were treated with FSM contained TGF and TSP-1 Scrambled oligonucleotide (SC). Cells of C groups and T groups were cultured according to time point and cells of TA groups and TS groups for were cultured 72 hours in the conditional media. Morphological change was observed with microscope. The expression of cell keratin (CK), alpha-smooth muscle actin (SMA), TSP-1 and VEGF were detected with Immunohistochemistry. The expression of TSP-1 mRNA and VEGF mRNA were analyzed by RT-PCR. The corelation between the relative expression of VEGF mRNA and TSP-1 mRNA were analyzed.Results: 1. Cell morphology observation: (1) C groups: The shape of HKCs showed classic cobblestone morphology. (2) T groups: No HKCs in Tl were changed in shade, and some HKCs in T2 became elongated in shape and most of HKCs in T3 became elongated in shape. It suggested TGF induced HKCs morphology transforming. (3)TA groups: The proportion of elongated cells was less than that of T groups. It suggested AS inhibited HKCs morphology transforming induced by TGF .(4) TS groups: The proportion of elongated cells was similar to that of T2 groups. 2. Immunohistochemistry assays: (1)VEGF: VEGF were positive expression in C groups and were strong strained in T1 groups. However, there was weak positive expression in TA groups and T2 groups, and were negative expression in T3 groups and TS groups. AODVEGF were decreased significantly in 12, T3 groups and TS groups (P<0.01), and were decrease in TA groups (P<0.05) compared with C groups. AODvEGF were decreased in TA groups (P<0.01) and were unchanged in TS groups (P>0.05) compare with T2 groups. (2)TSP-1:TSP-1 were negative expression in C groups and Tl groups. However, there were strong positive expression in T2, T3 groups and TS groups and were weak positive expression in TA groups. AODTsp-1 were increased significantly in T groups and TS groups (P<0.01) and were increased in TA groups (P<0.05) compared with C groups. AODTsp-1 were decreased significantly in TA groups (P<0.01) and were unchanged in TS groups (P>0.05) compared with T groups. (3)CK: CK were positive expression in C groups and were weak positive expression in TA groups and were negative expression in T groups and TS groups. AODcK were decreased significantly in T groups and TS groups and in TA groups (P0.01) compared with C groups. AODcK were decreased in TA groups (P<0.05) and were unchanged in TS groups (P>0.05) compared with T2 groups. (4)SMA: a-SMA were negative expression in C groups and were strong positive expression in T groups and TS groups and were weak positive expression in TA groups. AOD-SMA were increased significantly in T groups and TS groups and in TA groups (P<0.01) compared with C groups. AOD-sMA were decreased in TA groups (P<0.05) and were unchanged in TS groups (P>0.05) compared with T2 groups. The expression of a-SMA induced by TGF indicates TEMT. The expression of VEGF and CK declined after TEMT. 3. RT-PCR assays: (1)C groups: VEGF mRNA expression were positive and TSP-1 mRNA expression were negative. (2)T groups: Expression of VEGF mRNA were declined significantly (P0.01) and TSP-1 mRNA expression were increased significantly (P<0.01) compared with that in C groups.