Effects of transform ing growth factor2 1 on apoptosis and HSP 70 expression of mammary tissue of da iry cow

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1 2009, 32 (2) : Journal of N anjing A gricultural U niversity http: / / nauxb1njau1edu1cn,,,. 1 HSP 70 [ J ]., 2009, 32 (2) : HSP 70 1, 1, 2, 1, 1, 1, 13 (1., ; 2., ) : (LDH ) Caspase23, 1 ) HSP ng ml - 1 TGF h,, 0 h TGF2 1, TGF2 1, 24 h 48 h ( P < 0105), LDH, h ( P < 0101) h DNA ; Caspase23 24 h, ( P < 0101) ;,, ;, 48 h,, 3 12 h HSP 70, ( P < 0105) : TGF2 1 : 1 ) ; ; ; HSP 70 : S81313 : A : (2009) Ab stra c t: Effects of transform ing growth factor2 1 on apoptosis and HSP 70 expression of mammary tissue of da iry cow D I He2shuang 1, YANG Yuan2yuan 1, WANG L i2gang 2, CU IQun2wei 1, CHENG Kai2ning 1, HUANG J ing2yan 1, WANG Gen2lin 13 ( 1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing , China; 2. J iangsu Animal Husbandry and Veterinary College, Taizhou , China) The effects of transform ing growth factor2 1 ) on activity, apop tosis and HSP 70 exp ression of the mammary cells of dairy cow were investigated through multip le methods, including activity test of lactate dehydrogenase (LDH ), electron m icroscopy analysis, immunohistochem istry analysis and activity analysis of Caspase23. Mammary tissue was treated with TGF2 1 of 10 ng ml - 1 for 48 h, then the tissue and its cultureswere collected at different time for further analysis. The group without TGF2 1 treatment at 0 h was defined to be control group. The results showed that the survival rate of mammary ep ithelial cells decreased gradually with the increasing of the time of TGF2 1 treatment, and the survival rates of 24 h and 48 h group swere significantly lower than that of control group ( P < 0105). The LDH activities of 6 h, 12 h, 24 h and 48 h group s were significantly higher than that of control group ( P < 0101) ; the DNA of mammary ep ithelial cells was degradated in 12 h, 24 h, and 48 h group s; the activity of Caspase23 reached to a peak, which was significantly higher than that of control group ( P < 0101). The acinus of ep ithelial cells appeared to break off and collap se with the increasing of treatment time, but the leydig structure keeped integrity. U ltrostruc2 ture showed that there were obvious apop tosis characteristics in ep ithelial cells treated for 48 h, such as chromatin condensation and m itochondrial swelling, but the fibroblast cells keeped their compact structure and abundant m itochondrium. HSP 70 was overex2 p ressed when treated with TGF2 1 for 3212 h, which were significantly higher than that of control group ( P < 0105). Conclusion: TGF2 1 could induce the apop tosis of the mammary ep ithelial cells, which m ight result in the acceleration of mammary involution in dairy cow. Key wo rd s: transform ing growth factor2 1 ) ; mammary tissue; cell apop tosis; HSP 70 : : (2006BAD04A12, 2006BAD04A01) ; (BE ) :, 3 :,,, E2mail: glwang@njau1edu1cn

2 2, : 1 HSP [ 1-4 ],, [ 1 ],, ( IGFBP) ( FasL) ( TGF2 ) [ 5-7 ] TGF2, ( DNA TdT ( TUNEL) ) [ 8-9 ], TGF2 ; TGF2 1,,,, [ 10 ] TGF2 TGF2 [ ], TGF2, TGF2, TGF2 1 HSP 70, TGF2, DMEM ( Gibco) ( Gibco) LDH ( ) ( ) Tris2 ( ) K (Calbiochem) TGF2 1 (Cy2 tolab) HSP 70 SABC ( ) Caspase23 ( Promega), , h, [ 13 ] 10% DMEM, CO 2 37, 60 h, 10 ng ml - 1 TGF h, 0 h TGF2 1,, TGF h, 215 g L - 1, 2 3 h, ; 300, r m in m in;, 2, r m in m in;, L - 1 ; 20 g L - 1,, 4 ( ), : = / ( + ) 100% LDH TGF h, LDH (U L - 1 ) : LDH = ( D D 440 ) / ( D D 440 ) / DNA TGF h, 3 ml ( 20 L K), 50, 2 2 DNA, 1% TGF h, 10% 24 h,,, 5 m, 2, TGF h, 215% 4,, 1%,, HSP 70 TGF h, 2 ( 1 1) 10 m in, 2 ( 50 1) 30 m in, SABC HSP 70 (1 500), 37

120 32 1 h; PBS ; IgG, 37 20 m in, ABC ( avidin2biotin2peroxidase comp lox, 2 2 ) 37 20 m in, DAB,,,,, 5, 100, HSP 70 11217 C a sp a se 23 TGF2 1 0 6 12 24 48 h, 20 m in, 115 ml, 4 10 000 r m in - 1

3 h; PBS ; IgG, m in, ABC ( avidin2biotin2peroxidase comp lox, 2 2 ) m in, DAB,,,,, 5, 100, HSP C a sp a se 23 TGF h, 20 m in, 115 ml, r m in m in,,, - 70 Caspase23 Caspase23, Caspase23 Caspase23 (DEVD2pNA ) / ( mol m in - 1 mg - 1 ) , 3 x SD, SPSS TGF2 1 LDH TGF2 1,, h ( P < 0105) ( 12A ) ; LDH, h ( P < 0101) ( 12B ) TGF2 1 LDH, 1 1 ) (A) (LDH) (B ) F ig11 Effe c t o f tran sfo rm ing g row th fa c to r2 1 ) o n ce ll su rviva l ra te s (A) and the ac tivity o f lac ta te de hyd ro ge na se (LDH) (B ) (0 h) ( P < 0105) ( P < 0101) Compared with control group 3 P < 0105, 3 3 P < The same as fallows. 212 TGF2 1 TGF h DNA ( 22A ) ; Caspase23 12 h, 24 h, h ( P < 0101) ( 22B ), TGF 2 1,,, 2 TGF2 1 DNA (A) C a sp a se 23 (B ) F ig12 Effec t o f TGF2 1 o n ce lldna in teg rity (A) a nd the ac tivity o f C a sp a se 23 (B )

2, : 1 HSP 70 121 ( 3) TGF2 1 48 h, ( 42A),, ( 42B ) TGF2 1 Caspase23, DNA, 3 TGF2 1 F ig13 Effec t o f TGF2 1 o n m o rp ho

ρ The acinus of ep ithelial cells 4 TGF2 1 48 h (A) (B ) F ig14 The u ltra struc tu re o f m amm a ry ep ithe lia l ce lls

Hc: Heterochromatin TGF2 1 3 h HSP 70, 6 h, 12 h, ( 52A B ) TGF2 1 3 6 h HSP 70 3 5 TGF2 1 HSP 70 (A) (B ) F ig15 Effe c t o

4 2, : 1 HSP ( 3) TGF h, ( 42A),, ( 42B ) TGF2 1 Caspase23, DNA, 3 TGF2 1 F ig13 Effec t o f TGF2 1 o n m o rp ho lo gy and struc tu re o f m amm a ry tissue g The normal mammary ep ithelial cell; appears to break off. ρ The acinus of ep ithelial cells 4 TGF h (A) (B ) F ig14 The u ltra struc tu re o f m amm a ry ep ithe lia l ce lls (A) a nd fib ro b la st (B ) trea ted w ith TGF2 1 fo r 48 h Nu: Nucleus; M i: M icrovilli; 213 TGF2 1 HSP 70, ( P < 0105) Hc: Heterochromatin TGF2 1 3 h HSP 70, 6 h, 12 h, ( 52A B ) TGF h HSP TGF2 1 HSP 70 (A) (B ) F ig15 Effe c t o f TGF2 1 o n exp re ssio n (A) and e xp re ssio n ra te (B ) o f HSP 70 po sitive sta ine d ce lls 311 TGF2 1 TGF2 1 [ 14 ],, TGF2 1, TGF2 1, TGF2 1 LDH,,

5 122 32,,, [ 15 ] TGF2 1 LDH, LDH TGF2 1, TGF2 1,, LDH TGF2 1,, TGF2 1 TGF2 1, Bax/Bcl22, (A IF) C Ca 2 +, Caspases Caspases ( PARP), DNA, [ 2, ], TGF2 1 DNA,, Caspase23, TGF2 1,, TGF2 1 [ 18 ], TGF2 1, Caspases, DNA,, TGF2 1 DNA, TGF TGF2 1 HSP 70, (HSP), HSP,,, [ 19 ], 3 6 h HSP 70 [ 20 ] ; HeLa HSP 70 [ 21 ] HSP 70 [ 22 ], TGF2 1 HSP 70 TGF2 1 HSP 70, 3 6 h ; TGF h 12 h, TGF2 1, HSP h TGF2 1, HSP 70 HSP 70, : [ 1 ] Capuco A V, Akers R M. Mammary involution in dairy animals [ J ]. J Mammary Gland B iol Neop lasia, 1999, 4 (2) : [ 2 ] Motyl T, Gajkowska B, Wojew dzka U, et al. Expression of apoptosis2related p roteins in involuting mammary gland of sow [ J ]. Compara2 tive B iochem istry and Physiology Part B, 2001, 128: [ 3 ] W areski P, Motyl T, Ryniewicz Z, et al. Exp ression of apop tosis related p roteins in mammary gland of goat [ J ]. Small Rum inant Research, 2001, 40: [ 4 ] W ilde C J, Addey C V P, L i P, et al. Programmed cell death in bovine mammary tissue during lactation and involution [ J ]. Experimental Physiology, 1997, 82: [ 5 ] Tonner E, BarberM C, A llan G J, et al. Insulin2like growth factor binding p rotein25 ( IGFBP25) induces p remature cell death in the mam2 mary glands of transgenic m ice [ J ]. Development, 2002, 129: [ 6 ] Song J, Sap i E, B rown W, et al. Roles of Fas and Fas ligand during mammary gland remodelling [ J ]. J Clin Invest, 2000, 106 (10) : [ 7 ] Kolek O, Gajkowska B, GodlewskiM M, et al. Co2localization of apop tosis2regulating p roteins in mouse mammary ep ithelial HC11 cells ex2 posed to TGF2 1 [ J ]. Journal of Cell B iology, 2003, 82: [ 8 ] Faure E, Heisterkamp N, Groffen J, et al. D ifferential exp ression of TGF2 isoform s during postlactational mammary gland involution [ J ]. Cell Tissue Research, 2000, 300: [ 9 ] Plath A, Einspanier R, Peters F, et al. Exp ression of transform ing growth factors alpha and beta21 messenger RNA in the bovine mammary gland during different stages of development and lactation [ J ]. J Endocrinol, 1997, 155: [ 10 ] Kordon E C, McKnight R A, Jhappan C, et al. Ectop ic TGF 1 exp ression in the secretory mammary ep ithelium induces early senescence of the ep ithelial stem cell population [ J ]. Developmental B iology, 1995, 168: [ 11 ] Xie L, Law B K, Aakre M E, et al. Transforming growth factor beta2regulated gene expression in a mouse mammary gland epithelial cell line [ J ]. B reast Cancer Research, 2003, 5: [ 12 ] Ewan K B, Henshall2Powell R L, Ravani S A, et al. Transform ing growth factor2 1 mediates cellular response to DNA damage in situ [ J ].

6 2, : 1 HSP Cancer Res, 2002, 62: [ 13 ],,,. [ J ]., 2006, 22 ( 6) : [ 14 ] Savilahti E, Saarinen K M. Colostrum TGF2 21 associates with the duration of breast2feeding [ J ]. European Journal of Nutrition, 2007, 46 (4) : [ 15 ],,,. [ J ]., 2005, 26 (5) : [ 16 ] Kolek O, Gajkowska B, GodlewskiM M, et al, Molecularmechanism of TGF2 2beta12induced apop tosis in HC11 mouse mammary ep itheli2 al cells(mec) [ J ]. CellMol B io, 2001, 47: [ 17 ] Motyl T, Gajkowska B, Ploszaj T, et al. Exp ression and subcellular redistribution of Bax during TGF2beta12induced p rogrammed cell death of HC11 mouse mammary ep ithelial cells [ J ]. CellMolecular B iology, 2000, 46 (1) : [ 18 ] Musters S, Coughlan K, McFadden T, et al. Exogenous TGF2 1 p romotes stromal development in the heifer mammary gland [ J ]. Journal of Dairy Science, 2004, 87: [ 19 ] Hartl F U. Molecular chaperones in cellular p rotein folding [ J ]. Nature, 1996, 381: [ 20 ],,,. [ J ]., 2007, 30 ( 3) : [ 21 ],,,. HeLa 70 [ J ]., 2005, 17 (5) : [ 22 ] Dokladny K, W harton W, Lobb R, et al. Induction of physiological thermotolerance in MDCK monolayers: contribution of heat shock p ro2 tein 70 [ J ]. Cell Stress Chaperones, 2006, 11 (3) : :

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