2009, 32 (2) : 1182123 Journal of N anjing A gricultural U niversity http: / / nauxb1njau1edu1cn,,,. 1 HSP 70 [ J ]., 2009, 32 (2) : 1182123 1 HSP 70 1, 1, 2, 1, 1, 1, 13 (1., 210095; 2., 225300) : (LDH ) Caspase23, 1 ) HSP 70 10 ng ml - 1 TGF2 1 48 h,, 0 h TGF2 1, TGF2 1, 24 h 48 h ( P < 0105), LDH, 6 12 24 48 h ( P < 0101) 12 24 48 h DNA ; Caspase23 24 h, ( P < 0101) ;,, ;, 48 h,, 3 12 h HSP 70, ( P < 0105) : TGF2 1 : 1 ) ; ; ; HSP 70 : S81313 : A : 100022030 (2009) 0220118206 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 210095, China; 2. J iangsu Animal Husbandry and Veterinary College, Taizhou 225300, 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 : 2008201217 : (2006BAD04A12, 2006BAD04A01) ; (BE200830622) :, 3 :,,, E2mail: glwang@njau1edu1cn
2, : 1 HSP 70 119 [ 1-4 ],, [ 1 ],, ( IGFBP) ( FasL) ( TGF2 ) [ 5-7 ] TGF2, ( DNA TdT ( TUNEL) ) [ 8-9 ], TGF2 ; TGF2 1,,,, [ 10 ] TGF2 TGF2 [ 11-12 ], TGF2, TGF2, TGF2 1 HSP 70, TGF2, 1 111 DMEM ( Gibco) ( Gibco) LDH ( ) ( ) Tris2 ( ) K (Calbiochem) TGF2 1 (Cy2 tolab) HSP 70 SABC ( ) Caspase23 ( Promega), 112 11211, 60 108 h, [ 13 ] 10% DMEM, CO 2 37, 60 h, 10 ng ml - 1 TGF2 1 48 h, 0 h TGF2 1,, 11212 TGF2 1 0 2 4 6 12 24 48 h, 215 g L - 1, 2 3 h, ; 300, 1 500 r m in - 1 10 m in;, 2, 1 500 r m in - 1 10 m in;, 5 10 6 L - 1 ; 20 g L - 1,, 4 ( ), : = / ( + ) 100% 11213 LDH TGF2 1 0 2 4 6 12 24 48 h, LDH (U L - 1 ) : LDH = ( D 440 - D 440 ) / ( D 440 - D 440 ) 01380 1 000 / 11214 DNA TGF2 1 0 6 12 24 48 h, 3 ml ( 20 L K), 50, 2 2 DNA, 1% 11215 TGF2 1 0 12 24 48 h, 10% 24 h,,, 5 m, 2, TGF2 1 48 h, 215% 4,, 1%,, 11216 HSP 70 TGF2 1 0 3 6 12 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 10 m in,,, - 70 Caspase23 Caspase23, Caspase23 Caspase23 (DEVD2pNA ) / ( mol m in - 1 mg - 1 ) 113 3 12, 3 x SD, SPSS1110 2 211 TGF2 1 LDH TGF2 1,, 12 24 48 h ( P < 0105) ( 12A ) ; LDH, 6 12 24 48 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 ) 3 3 3 (0 h) ( P < 0105) ( P < 0101) Compared with control group 3 P < 0105, 3 3 P < 0101. The same as fallows. 212 TGF2 1 TGF2 1 12 24 48 h DNA ( 22A ) ; Caspase23 12 h, 24 h, 12 24 48 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 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 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 (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 ) TGF2 1 3 6 h HSP 70 3 5 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,,
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