The Effects Of TGF-β₁ On Apoptosis Of Bovine Mammary Cell And Its Regulation Mechanism

The aim of this paper is to reveal the mechanism of bovine mammary cells response to TGF-β₁.Firstly,a bovine mammary tissue culture system was established,a TGF-β₁ treatment was determined,and the effects of TGF-β₁ on apoptosis of bovine mammary cell and its regulation mechanism were studied.1 Establishment of a bovine mammary tissue culture systemBovine mammary tissue was cultured in superfusion system or stationary system,and the influence of these two methods in the activity and ultrastructure of tissue was investigated according to LDH vigor,trypan blue dying,agrose gel electrophoresis, transmission microscope observation.The results showed that the mammary tissue cultured in superfusion system could keep normal tissue activity and ultrastructure within 24-72 h in DMEM plus 10%calf serum,while mammary tissue stationary culture could keep normal tissue activity and ultrastructure within 48-108 h.Both culture systems had some advantages and disadvantages.2 Effects of TGF-β₁ on activity,apoptosis and HSP70 expression of bovine mammary tissueThe effects of TGF-β₁ on activity,apoptosis and HSP70 expression of the mammary cells of dairy cow were investigated through multiple methods,including trypan blue staining,activity test of LDH,agarose gel electrophoresis,electron microscopy analysis, Immunohistochemistry analysis and activity analysis of Caspase-3.Method:Mammary was treated with TGF-β₁ of 10 ng/ml for 48h,then the mammary and its cultures were collected at different time for testing the related parameters.The group without TGF-β₁ treatment at 0h was defined to be control group.Results:The results show that the survival rate of mammary epithelial cells were decreasing gradually with the increasing of the time of TGF-β₁ treatment,and the survival rates of 24 h and 48 h groups were significantly lower than that of control group(p＜0.05);the LDH activity of 6 h,12 h,24 h and 48 h groups were significantly higher than that of control group(p＜0.05);the DNA degradation arisesed in 12 h,24 h,and 48 h groups' mammary epithelial cells;the activity of Caspase-3 reach to a peak at 24 h,which was significantly higher than that of control group(p＜0.05);the acinus of epithelial cells appears to break off and collapse with the increasing of treatment time,but the leydig structure keeps integrate;it was showed that there were obvious apoptosis characteristics in epithelial cells treated for 48 h,such as chromatin condensation, but the fibroblast cells keeps their compact structure and abundant mitochondrium;HSP70 was overexpressed when treated with TGF-β₁ for 3-12 h,which were significantly higher than that of control group(p＜0.05).Conclusion:The mammary epithelial cells were induced to apoptosis and overexpress the HSP70 by TGF-β₁,which indicates that TGF-β₁ accelerates mammary's involution in the way of promoting the apoptosis of epithelial cells. 3 Effects of TGF-β₁ on Proliferation,Apoptosis and HSP70 Expression of bovine Mammary Epithelial CellsBovine mammary epithelial cells were cultured in DMEM/F12+10%calf serum medium with TGF-β₁ and cells and supernatant were collected.The cell proliferation and apoptosis,activity of LDH,integrity of DNA,and expression of heat shock protein 70 (HSP70) were detected by MTT,Flow cytometry,lactate dehydrogenase(LDH) kit,agarose gel electrophoresis and Western bolt,respectively.The results showed that cells proliferations were inhibited after treated with TGF-β₁ in dose dependent way.The rate of apoptosis cells significantly increased after treated with TGF-β₁ for 6,12,24 hours (P＜0.01).The activity of LDH was significantly higher in the treated groups than that in the control group(P＜0.05).The nucleotide of DNA was degradated gradually as treatment time increased.The expression of HSP70 increased after TGF-β₁ treatment for more than 2 hours and it reached the highest level when treated for 6h,then decreased.Conculsion: TGF-β₁ could inhibit the proliferation of bovine mammary epithelial cells in a dose-dependent way.At a dose of 10ng/mL,TGF-β₁ could significantly stimulate cell apoptosis and increase LDH activity,which were resulted in degradation of DNA. Long-term culture of bovine mammary epithelial cells with TGF-β₁ could lead to the up-regulation of the expression of HSP70. 4 Signal mechanism of Bovine Mammary Epithelial Cell Induced by TGF-β₁Bovine mammary epithelial cells were cultured in DMEM/F12+10%calf serum medium with TGF-β₁ or SB-431542,and cells were collected.The cell proliferation and apoptosis,integrity of DNA,and expression of Smad2/3 were detected by MTT,Flow cytometry,agarose gel electrophoresis and Western bolt,respectively.The results showed that TGF-β₁-induced apoptosis of bovine mammary epithelial cell,which was observed with the phosphoration of Smad2/3 in 0.5-2 hours,and then the signal transferring into the nucleus.This process was further observed with a significant elevation of intracellular free Ca²⁺ concentration,an activation of Caspase-3 and DNA lysis,and thereby the programmed cell death was induced.This signaling pathway of TGF-β₁ was blocked by 10^-2μM of SB-431542 via inhibiting the activity of ALK-5 kinase,which thus reversed the anti-proliferation and apoptosis effect of TGF-β₁ in mammary epithelial cells.These results indicated that TGF-β₁ induced apoptosis of bovine mammary epithelial cells through ALK-5-Smad2/3 pathway,which plays an important role in inhibiting the survival of mammary epithelial cells.TGF-β₁ induced the epithelial cell apoptosis in a Ca²⁺- and Caspase-3-dependent manner.In conclusion,we revealed the mechanism of bovine mammary cells response to TGF-β₁.The results accomplished in this work show that TGF-β₁ accelerates mammary's involution in the way of promoting the apoptosis of epithelial cells,and TGF-β₁ induced apoptosis of bovine mammary epithelial cells through ALK-5-Smad2/3 pathway,which plays an important role in inhibiting the survival of mammary epithelial cells.TGF-β₁ induced the epithelial cell apoptosis in a Ca²⁺- and Caspase-3-dependent manner.It is proved that the apoptosis of epithelial cells is an important reason causing mammary gland involution.