21 5 2011 9 Journal of Jiangsu University Medicine Edition Vol. 21 No. 5 Sept. 2011 TSP-1 C5b-9 210029-1 thrombospondin-1 TSP-1 sublytic C5b-9 glomerular mesangial cell GMC TSP-1 sir- NA small hair RNA shrna TSP-1 shrna shtsp-1 GMC C5b-9 PCR GMC TSP-1 D2 cyclin D2 proliferative cell nuclear antigen PCNA mrna 3 H- 3 H-thymidine incorporation 3 H-TdR CCK-8 cell counting kit-8 GMC DNA GMC α- α-smooth muscle actin α-sma shtsp-1 TSP-1 C5b-9 GMC 18 h cyclin D2 PCNA DNA shtsp-1 C5b-9 GMC 54 h C5b-9 GMC α-sma shtsp-1 GMC TSP-1 C5b-9 GMC α-sma shtsp-1 GMC α-sma C5b-9-1 α- R392. 31 A 1671-7783 2011 05-0369 - 06 Roles of thrombospondin-1 gene in glomerular mesangial cell proliferation induced by sublytic C5b-9 WU Yi-qin CHE Nan FENG Xue-feng QIU Wen WANG Ying-wei Department of Microbiology and Immunology Nanjing Medical University Nanjing Jiangsu 210029 China Abstract Objective To explore the effects of silencing thrombospondin-1 TSP-1 gene on sublytic C5b-9 complex-induced glomerular mesangial cell GMC proliferation. Methods Small hair RNA shr- NA against TSP-1 gene was designed and the corresponding expression plasmids were constructed. Cultured rat GMC was transfected with TSP-1 shrna shtsp-1 and then stimulated with sublytic C5b-9 complexes. The expression of TSP-1 cyclin D2 and proliferative cell nuclear antigen PCNA at mrna and protein levels in different groups of GMC was assessed by real-time PCR and Western blot respectively. In addition DNA synthesis and GMC number were detected by using 3 H-thymidine incorporation 3 H-TdR and cell counting kit-8 CCK-8. Finally the expression of α-smooth muscle actin α-sma in GMC was examined in different groups. Results TSP-1 gene knockdown by using shtsp-1 in rat GMC had no significant effect on the expression of cyclin D2 and PCNA and the synthesis of DNA in the GMC induced by sublytic C5b-9 for 18 h. But shtsp-1 could suppress the increase of GMC number due to sublytic C5b-9 stimulation for 54 h and production of α-sma at 18 h after sublytic C5b-9 stimulation. Conclusion Silencing TSP-1 gene in GMC with shtsp-1 can inhibit sublytic C5b-9-induced GMC proliferation and α-sma production. The proliferation of GMC treated with sublytic C5b-9 was possibly mediated in part by α-sma 31000396 81072402 10KJB310006 09NJMUM003 09JC007 1959 E-mail qiuwen@ njmu. edu. cn E-mail wangyw1508@ njmu. edu. cn
370 21 production induced by TSP-1 gene activation. Key words glomerular mesangial cell sublytic C5b-9 complex thrombospondin-1 proliferation α-smooth muscle actin mesangial proliferative glomerulonephritis MsPGN GMC 9 Congote 10 1 TSP-1 glomerular mesangial cell GMC extracellular matrix ECM C5b-9 GMC TSP-1 MsPGN GMC TSP-1 C5b-9 GMC MsPGN GMC MsPGN TSP-1 sirna small hair RNA shrna ECM GMC C5b-9 MsPGN Thy-1 Thy-1 nephritis Thy-1N anti-thymocyte serum nephritis ATSN MsPGN MsPGN 1 1 MPGN C5b-9 Thy-1 2 C5b-9 1. 1 GMC HBZY-1 C5b-9 3 C5b-9 ATS Thy-1 Ab 10 4 normal human serum NHS C8 5 ReverTra Ace-α- TM TOYOBA C9 C5b-9 TSP-1 D2 cyclin D2 Santa Cruz C5b-9 sub- proliferative cell nuclear antigen PCNA lytic C5b-9 6 C5b-9 α-smooth muscle actin α-sma CCK-8 cell counting kit-8 7 Thy-1 pgcsi. U6. neo. RFP GMC GMC C5b-9 1. 2 GMC 2 GMC 1. 2. 1 GMC 50 ml C5b-9 3 ml MEM 37 5% CO 2 C5b-9 GMC GMC 80% ~ 90% -1 thrombospondin-1 TSP-1 GMC TSP-1 C5b-9 GMC TSP-1 GMC Thy-1 C5b-9 GMC Thermo α- 5 min 1 000 r /min Hanks 2 8 1. 2. 2 TSP-1 shrna shtsp-1 Tuschl TSP-1 shrna
5. TSP-1 C5b-9 371 5'-CTCAATGAACGAGACAACT-3' DNA 5'-CTTGCATTGCATCCT- CATT-3' shrna shctr pgcsi. U6. neo. RFP DNA Xho I EcoR I ± s SPSS13. 0 Bonfferoni P < 0. 05 T 4 DNA 2 DH5α LB 37 2. 1 TSP-1 shrna shtsp-1 GMC TSP-1 1. 2. 3 shtsp-1 GMC 50% shtsp-1 shctr C5b-9 18 h TSP-1 GMC GenEscort TM III shtsp-1 48 h C5b-9 TSP-1 red fluorescence protein RFP shrna shctr TSP-1 GMC TSP-1 1 C5b-9 shtsp-1 + C5b-9 shctr + C5b-9 MEM shtsp-1 + C5b-9 shctr + C5b-9 shtsp-1 shctr GMC 48 h C5b-9 5% Thy-1 Ab 4% NHS C5b-9 18 h mrna 1. 2. 4 PCR mrna PCR GMC cyclin D2 PCNA α-sma mrna Trizol RNA 1 μg cdna PCR mrna 50 2 min 95 10 min 95 15 s 60 1 min 40 1. 2. 5 1 GMC TSP-1 Fig 1 Protein level of TSP-1 in GMC in various groups GMC TSP-1 cyclin D2 PCNA α-sma GMC 12 000 2. 2 TSP-1 GMC cyclin D2 PC- g 10 min SDS-PAGE NA PVDF 3% BSA 2. 2. 1 Cyclin D2 PCNA mrna 4 TBST 3 shtsp-1 GMC C5b-9 HRP 1 h ECL GMC cyclin D2 PCNA 1. 2. 6 GMC 96 18 h C5b-9 18 h 0. 8 10 4 24 h GMC 450 nm D 1. 2. 7 ± x TSP-1 shrna shtsp-1 GMC 48 h A 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 B * P < 0. 01 C5b-9 Δ P < 0. 01 shctr + C5b-9 PCR shtsp-1 C5b-9 cyclin D2 1. 2. 3 1 PCNA mrna 2 3 H-TdR 18 h 2. 2. 2 Cyclin D2 PCNA count per minute cpm mrna 3 2 18 36 54 h 16 34 52 h shtsp-1 GMC C5b-9 cyclin D2 PCNA CCK-8 2 h
372 21 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 * P < 0. 05 ** P < 0. 01 MEM 2 GMC D2 PCNA mrna Fig 2 The mrna level of cyclin D2 and PCNA in GMC in various groups A cyclin D2 a 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 b B PCNA ** P < 0. 01 MEM Fig 3 3 GMC D2 PCNA Levels of cyclin D2 and PCNA protein in GMC with different treatments 2. 3 TSP-1 C5b-9 GMC DNA 3 H-TdR GMC DNA cpm 18 h shtsp-1 + C5b-9 DNA shctr + C5b-9 C5b-9 cpm MEM shtsp-1 GMC C5b-9 DNA 4 2. 4 shtsp-1 GMC TSP-1 GMC CCK-8 GMC D 36 h 54 h 54 h 18 h 36 h shtsp-1 + 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 * P < 0. 05 * * P < 0. 01 MEM 4 Fig 4 GMC DNA Detection of DNA synthesis in GMC in varying groups C5b-9 C5b-9 shtsp-1 + C5b-9 GMC shctr + C5b-9 C5b-9 C5b-9 GMC shctr + shtsp-1 GMC
5. TSP-1 C5b-9 373 C5b-9 54 h GMC 5 sublytic 11 C5b-9 3 sublytic C5b-9 GMC TSP-1 12 * P < 0. 01 C5b-9 Δ P < 0. 05 shctr + C5b-9 P < 0. 01 MEM Fig 5 5 GMC The numbers of GMC in different groups 2. 5 TSP-1 α-sma 2. 5. 1 α-sma mrna PCR GMC α-sma shtsp-1 C5b-9 α-sma 6 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 * P < 0. 05 C5b-9 Δ P < 0. 01 shctr + C5b-9 6 Fig 6 GMC α-sma mrna The mrna levels of α-sma in GMC with different treatments 2. 5. 2 α-sma TSP-1 15 shtsp-1 GMC α-sma TSP-1 C5b-9 GMC mrna 7 18 h shtsp-1 + RNAi RNA dsrna C5b-9 α-sma shctr + mrna 16 RNAi C5b-9 C5b-9 RNAi 3 TSP-1 CDS sir- MsPGN GMC ECM A 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 4 MEM B * P < 0. 01 C5b-9 Δ P < 0. 01 shctr + C5b-9 pgcsi. U6. neo. RFP Thy-1 GMC TSP-1 TSP-1 MsPGN C5b-9 GMC 2 shtsp-1 GMC C5b-9 lytic TSP-1 C5b-9 18 h 7 Fig 7 GMC α-sma Levels of α-sma protein in GMC with different treatments TSP-1 TSPs TSP-1 13 TSP-1 14 NA TSP-1 shrna shtsp-1
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