PP2A-B55α Calbiochem, PP2A-B56α B56γ PP2A-B56δ Santa Cruz, ( ) ; ECL Santa Cruz 1.2 HL % RPMI1640, 5%CO 2, 37,,, cell/ml, 24h, RPM

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1 35 2 Vol. 35, No ACTA HYDROBIOLOGICA SINICA Mar., DOI: /SP.J LR HL7702 2A 郑群梁婧徐立红 (, ) 摘要 : 2A, PP2A, MC-LR HL h PP2A mrna, h PP2A, B55α 1000 nmol/l, ; 15 PP2A mrna 100 nmol/l, nmol/l 7 6 mrna MC-LR 6h, PP2A,, h MC-LR 24h HL7702 PP2A, ; MC-LR PP2A : ; 2A; ; mrna; 中图分类号 : X503.1 文献标识码 : A 文章编号 : (2011) (Microcystins, MCs) [1], MC-LR MCs [2,3],, 2A [4] MCs 2A(Protein phosphatase 2A, PP2A) /, DNA PP2A, C(35 kd) A(65 kd) B A C α β [5], B 4 B( PR55 B55) B ( PR61 B56) B ( PR72) B PP2A C PP2A, MCs DNA [6 8] MCs, MC-LR PP2A A [9], PP2A,, MCs PP2A mrna PP2A MC-LR HL7702 PP2A HL-7702 MC-LR Alexis, 95%; RPMI1640 Gibco ; ; PP2A Promega ; Trizol Invitrogen ; SYBR PrimeScript RT-RCR kit TaKaRa ; Tag dntp ; ; PP2A-C Cell Signal, 收稿日期 : ; 修订日期 : 基金项目 : ( ); (2008ZX ); (Y206088) 作者简介 : (1984 ),, ; ; qunpurple@hotmail.com 通讯作者 :, xulihong@zju.edu.cn

00313 LR HL7702 2A 郑群梁婧徐立红 (, 310058) 摘要 : 2A, PP2A, MC-LR HL7702 24h PP2A mrna, 3 6 12 24h PP2A, B55α 1000 nmol/l, ; 15 PP2A mrna 100 nmol/l, 500 1000 nmol/l 7 6 mrna MC-LR 6h, PP2A,, 3 12

2 PP2A-B55α Calbiochem, PP2A-B56α B56γ PP2A-B56δ Santa Cruz, ( ) ; ECL Santa Cruz 1.2 HL % RPMI1640, 5%CO 2, 37,,, cell/ml, 24h, RPMI1640 MC-LR(DMSO ),, MC-LR nmol/l DMSO ( 0.1%), 24h 1.3 Western blot PP2A, 150 µl (50 mmol/l Tris C1, 150 mmol/l NaC1, 15 mmol/l EDTA, 0.1%Triton X-100, ph 8.0) 30min, 4, r/min 30min, Bradford, 60 µg/, 80 Western blot [10], ECL,, quantity one, 1.4 PCR PP2A mrna PP2A, nmol/l 24h, Trizol RNA, RNA SYBR PrimeScript RT-RCR kit cdna, 20 µl 1 µg RNA Genbank PP2A ( 1), PCR 20 µl cdna 1 µl, 2 SYBR Premix Ex Taq 10 µl, 10 µmol/l 0.4 µl, ABI 7500 PCR, 95 30s; 95 15s, 56 30s, 72 35s, 40 2 Ct 1.5 PP2A PP2A 24h PP2A mrna, 12h 24h MC-LR 2000 nmol/l, 3h 6h MC-LR nmol/l MC-LR 表 1 各亚基实时定量 PCR 扩增引物序列 Tab. 1 Primer sequences of subunits for realtime PCR Subunit Forward primers (5-3 ) Reverse primers (5-3 ) Accession number Aα GCATCAAGAAGCTGTCCACC CACCAGGGTAGTGAAGGTT NM_ Aβ TCGCGGTTTTAATCGACGAG CCTTTCTACTCCAAGTGCT NM_ Cα CATCACCATTCTTCGAGGG GGCAGTGAGAGGAAGATAG NM_ Cβ AGAGGAAATCACGAAAGCCG CAGAATATCTGTCCATCTAC NM_ B55α AGGAGCAGTAGATGATGATG TTGTTCTCCTGCTCCTGT NM_ B55β ATCCTGCCACCATCACAAC GTTCCATAGGTTAATCCTC NM_ B55γ GGAAGATTACCGAACGAGAT TGGCCATTGGCAAAGATCCT NM_ B55δ AGTAGCAAAGGGACCATC TTCACCGACAGGTAGTCTCTG NM_ B56α ACGGCTTCACCCGGAAAT GAAGTGGCATCTTTGAGCT NM_ B56β ACTTCTTGGACTGTGTGGCC CAAATTCAGGGTTCTCACTGG NM_ B56γ AGTTACGTCAGTGTTGCG TAGGATTGGAGGAAGGTGG NM_ B56δ ACTTCGTGTCAGACCCACTC ATTCGATGAAGGTGGCAGC NM_ B56ε ACTTCATGGACACGCTATC CTGGATCAAATTCATTGCTG NM_ PR72 AGGTGCAGAATGGCTCACAT ATACTCCTCAGCGGCAAACCG NM_ PR48 GCTGGCTAACGTCTTCTTCG GCCACCAGGATGTCGTACT NM_ GAPDH AGAAGGCTGGGGCTCATTTG AGGGGCCATCCACAGTCTTC BC004109

3 Western blot PP2A, 150 µl (50 mmol/l Tris C1, 150 mmol/l NaC1, 15 mmol/l EDTA, 0.1%Triton X-100, ph 8.0) 30min, 4, 15000 r/min 30min, Bradford, 60 µg/, 80 Western blot [10], ECL,, quantity one, 1.

3 2期郑群等: 微囊藻毒素 LR 对人 HL7702 细胞蛋白磷酸酶 2A 的影响染毒终止后, 用细胞刮收集所有细胞, 重悬于 μl 细胞裂解液 (50 mmol/l Tris C1, 0.1 mmol/l EDTA, 0.05%Triton X-100, 10%甘油, 15%蛋白酶抑 2.1 结果 MC-LR 对 HL7702 细胞内 PP2A 各亚基蛋白表达水平的影响分别用 nmol/l MC-LR 制剂混合物, 0.5 mmol/l PMSF, 0.05% β-巯基乙醇, ph 7.5), 所得蛋白液按照试剂盒说明书过柱纯化 315 处理 HL7702 细胞 24h 后, 检测 5 种 PP2A 相关蛋白定量, 然后以丝/苏氨酸磷酸肽为底物, 加入等量的表达量, 除了 B55α 有比较明显的下降趋势并在最蛋白液反应, 37, 20min 后加入染料终止反应并显高剂量组有显著性降低(P<0.05)外, 其他 4 种蛋白在色, 630nm 波长下检测各组的吸光度, 根据磷标准该染毒条件和作用时间下基本保持不变(图 1) 曲线将吸光度折算成磷浓度以计算酶活性, 最后结果用处理组酶活性和对照组酶活性的比值表示 1.6 统计分析每个实验重复三次, 每次处理组都分别与对照 2.2 MC-LR 对 HL7702 细胞内 PP2A 各亚基 mrna 转录水平的影响选 nmol/l MC-LR 处理 HL7702 细胞 24h 后, 细胞内 PP2A 相关蛋白的 mrna 水平组相比, 采用 Student s t-test 做统计检验 P<0.05, 发生了变化(图 2) 包括 Aα Aβ Cα Cβ B55α 认为有显著性差异 B55β B55γ B55δ B56α B56β B56γ B56δ 图1 Fig. 1 MC-LR 染毒 24h 后 HL7702 细胞内 PP2A 各亚基蛋白水平变化(n=3, *P<0.05, ** P<0.01) Protein levels of subunits of PP2A in HL7702 cells exposure to MC-LR for 24h(n=3, *P<0.05, ** P<0.01)

5), 所得蛋白液按照试剂盒说明书过柱纯化 315 处理 HL7702 细胞 24h 后, 检测 5 种 PP2A 相关蛋白定量, 然后以丝/苏氨酸磷酸肽为底物, 加入等量的表达量, 除了 B55α 有比较明显的下降趋势并在最蛋白液反应, 37, 20min 后加入染料终止反应并显高剂量组有显著性降低(P<0.

4 MC-LR 24h HL7702 PP2A mrna (n=3, *P<0.05, **P<0.01) Fig. 2 mrna levels of subunits of PP2A in HL7702 cells exposure to MC-LR for 24h (n=3, *P<0.05, **P<0.01) a. A C ; b. B55 ; c. B56 ; d. PR70 a. A and C family; b. B55 family; c. B56 family; d. PR70 family B56ε PR728 PR48 15 PP2A 100 nmol/l MC-LR, 24h mrna [11] (P<0.05), 500 nmol/l 1000 nmol/l, mrna, 500 nmol/l Aβ B55α B55β B56α B56δ B56ε PR728 mrna, 1000 nmol/l B55α B55β B56α B56δ B56ε PR728 mrna 2.3 MC-LR HL7702 PP2A MC-LR HL7702 6h, nmol/l PP2A (P<0.05),, ( 3) 57.32% h, PP2A MCs PP2A [4], 3 MC-LR 6h HL7702 PP2A (n=3, *P<0.05, **P<0.01) Fig. 3 PP2A activity in HL7702 cells after 6h of MC-LR exposure (n=3, *P<0.05, **P<0.01) Mdha(Methyldehydroalanine) PP2A C, PP2A [12], MCs PP2A, MC-RR PP2A A [13], MC-LR [9],,

05), 500 nmol/l 1000 nmol/l, mrna, 500 nmol/l Aβ B55α B55β B56α B56δ B56ε PR728 mrna, 1000 nmol/l B55α B55β B56α B56δ B56ε PR728 mrna 2.

5 2 : LR HL7702 2A 317 PP2A, mrna,, 6h MC-LR PP2A MC-LR, PP2A, 24h,, PP2A PP2A,, [14], MC-LR PP, PP1 PP2A mrna,, 40min, 60min [15], PP2A,, (Organic anion transporting polypeptide, OATP) 1B1 OATP1B3 [16],, MCs, [17],, PP2A,, [18], PP2A,,, PP2A, PP2A [1] Dawson R M. The toxicology of microcystins [J]. Foxicine, l998, 36(7): [2] Wu X Q, Gong Y, Wang Z, et al. Residue levels and distribution on features of microcystins in fish samples from lake Dianchi [J]. Acta Hydrobiologica Sinica, 2010, 34(2): [,,,.., 2010, 34(2): ] [3] Li X Y, Liang J B, Huang P P. Toxic effects of microcystis cell extracts containing microcystin-lr on the blood of mice [J]. Acta Hydrobiologica Sinica, 2008, 12(6): [,,.., 2008, 12(6): ] [4] Honkanen R E, Zwiller J, Moore R E, et al. Characterization of microcystin-lr, a potent inhibitor of type 1 and type 2A protein phosphatases [J]. The Journal of Biological Chemistry, 1990, 265(32): [5] Zhou J, Pham H T, Ruediger R, et al. Characterization of the A alpha and A beta subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution [J]. The Biochemical Journal, 2003, 369(2): [6] Arroyo J D, Hahn W C. Involvement of PP2A in viral and cellular transformation [J]. Oncogene, 2005, 24(52): [7] Sablina A A, Chen W, Arroyo J D, et al. The tumor suppressor PP2A abeta regulates the rala GTPase [J]. Cell, 2007, 129(5): [8] Eichhorn P J, Creyghton M P, Bernards R. Protein phosphatase 2A regulatory subunits and cancer [J]. Biochimica et biophysica acta, 2009, 1795(1): 1 15 [9] Xing M L, Wang X F, Xu L H. Alteration of proteins expression in apoptotic FL cells induced by MCLR [J]. Environmental Toxicology, 2008, 23(4): [10] Ausubel F M, Brent R, Kingston R E, et al. Short Protocols in Molecular Biology [M]. Beijing: Academic Press. 1998, [,,,.. :.1998, ] [11] Liang J, Huang P, Fu W Y, et al. Effect of microcystin LR on the transcription of subunits of PP2A of FL and HL7702 [J]. Carcinogenesis, Teratogenesis and Mutagenesis, 2009, 21(6): [,,,. LR FL HL7702 PP2A., 2009, 21(6): ] [12] Xing Y N, Xu Y H, Chen Y, et al. Yu Chen Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins [J]. Cell, 2006, 127: [13] Fu W Y, Yu Y N, Xu L H. Identification of temporal differentially expressed protein responses to microcystin in human amniotic epithelial cells [J]. Chemical Research in Toxicology, 2009, 22(1): [14] Anja P, Linda L, Stephan P. In vivo influence of cyanobacterial toxins on enzyme activity and gene expression of protein phosphatases in Alfalfa (Medicago sativa) [J]. Toxicon, 2008, 52: [15] Mezhoud K, Praseuth D, Francois J C, et al. Global quantitative analysis of protein phosphorylation status in fish exposed to microcystin [J]. Advances in Experimental Medicine

Residue levels and distribution on features of microcystins in fish samples from lake Dianchi [J]. Acta Hydrobiologica Sinica, 2010, 34(2): 388 393 [,,,.

6 and Biology, 2008, 617: [16] Masaharu K, Tatsuhiko F, Ryuji I. Involvement of mitogen-activated protein kinase signaling pathways in microcystin-lr induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3 [J]. Toxicological Sciences, 2007, 97(2): [17] Lars H, Frode S. Microcystin produces disparate effects on liver cells in a dose dependent manner [J]. Mini-Reviews in Medicinal Chemistry, 2006, 6(3): [18] Zora B, Axel H S. Autoregulation of protein phosphatase type 2A expression [J]. The Journal of Biological Chemistry, 1998, 273(30): EFFECT OF MICROCYSTIN LR ON PP2A IN HL7702 CELL ZHENG Qun, LIANG Jing and XU Li-Hong (Department of Biochemistry and Genetics, Medical School, Zhejiang University, Hangzhou ) Abstract: Microcystins (MCs) are hepatotoxins which inhibit the activity of protein phosphatase 2A (PP2A) notably. The cells were incubated with different concentrations of MC-LR to determine the effect of MCs on PP2A in HL7702 respectively for 24h. The mrna and protein levels of PP2A subunits were detected. Furthermore, the activity of PP2A was assayed after 3h, 6h, 12h and 24h exposure. The protein levels were not changed significantly except B55α which decreased concentration-dependently and significantly in 1000 nmol/l. For mrna levels, 15 subunits increased in 100 nmol/l group, whereas 7 subunits in 500 nmol/l group and 6 subunits in 1000 nmol/l group decreased. The activity of PP2A was inhibited significantly and concentration-dependently after 6h exposure while there were no changes in 3, 12 and 24h exposure. In conclusion, MC-LR affects the transcription of PP2A subunits after 24h exposure, without concomitant changes in the interrelated proteins. Exposure time might be a determinant when considering the effect of MC-LR on PP2A activity. Key words: Microcystin; Protein phosphatase 2A; Activity; mrna; Protein

Toxicological Sciences, 2007, 97(2): 407 416 [17] Lars H, Frode S. Microcystin produces disparate effects on liver cells in a dose dependent manner [J].

2004级研究生现代免疫学实验技术结业报告

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