Regulatory Role Of Lef1 And Msx2 On The Proliferation Of Arbas Cashmere Goat Derm Al Papilla Cells

Cashmere produces from cashmere goat hair follicles,which is regulated by dermal papilla cells through a series of signaling pathways.,and dermal papilla cell is considered the control center of the morphogenesis and cycling of hair follicle.Inner Mongolia cashmere goats have two types of hair follicles,which include the primary hair follicles and secondary hair follicles.Two types of hair follicles can produce two different types of hair fibers.The growth and development of these hair follicles are regulated by the interaction of multiple signal factors.In this study dermal papilla cells of the two types of hair follicles of Albas Cashmere Goats were used as cell models to understand the effects of LEF1 and Msx2 on the proliferation of dermal papilla cells.1.Construction of overexpression and interference vectorsThe LEF1 overexpression vector,LEF1 interference vector,Msx2 overexpression vector and Msx2 interference vector were successfully constructed by gene fragment clone,digestion and ligation.2.Culture and identification of the primary and secondary dermal papilla cells of Albas Cashmere GoatsThe primary and secondary dermal papilla cells of Albas Cashmere Goats were successfully cultured by common method.The specific markers a-SMA and CD 133 of dermal papilla cells were detected and confirmed by fluorescence immunoassay.3.Transfection and detection of overexpression and interference of LEF1 cell linesThe LEF1-overexpression and-interference vectors were respectively transfected into both of Arbas White Cashmere Goat primary and secondary dermal papilla cells by liposomes,and four types of LEF1 cell lines were obtained.The expression of LEF1,P-catenin,C-myc and cyclin D1 was assessed by quantitative real-time PCR and Western Blot.The cells transfected with no plasmid were used as a negative control.In primary dermal papilla cells,the LEF1 expression level of overexpression and interference group was 9.25 and 0.2 times than that of the control,respectively(P<0.001).Similar results were detected in the secondary dermal papilla cells,with a 10.53 and 0.21 times in the overexpression and interference group,respectively(P<0.001).These results confirmed that overexpression and interference of LEF1 in the dermal papilla cells were successfully achieved.It was also found that the expression of LEF1 in the two kinds of dermal papilla cells was consistent with that of cyclin D1,C-myc,and P-catenin.Therefore,the results showed that the expression levels of cyclin D1,C-myc,and ?-catenin were positively correlated with LEF1 expression.And these results indicated that overexpression of LEF1 promoted the proliferation of dermal papilla cells.Comparison of the gene expression levels between the primary and secondary dermal papilla cells of the control group showed that LEF1,?-catenin,and cyclin D1 mRNA expression levels in the secondary dermal papilla cells were 1.28,2.19,and 1.16 times higher than that of the primary dermal papilla cells,respectively.By contrast,the mRNA expression level of C-myc in the primary dermal papilla cells was 0.37 times than that of the primary dermal papilla cells.The result showed that the expression levels of LEF1,?-catenin,cyclin D1 and C-myc were different in the two hair papilla cells.4.Transfection and detection of overexpression and interference of Msx2 cell linesThe Msx2-overexpression and-interference vectors were respectively transfected into both of Arbas White Cashmere Goat primary and secondary dermal papilla cells by liposomes,and four types of Msx2 cell lines were obtained.The expression of Msx2,LEF1 ? BMP-2 was assessed by quantitative real-time PCR and Western Blot.The cells transfected with no plasmid were used as a negative control.In the primary dermal papilla cells,the Msx2 expression level of overexpression and interference group was 11.85 and 0.31 times than that of the control,respectively(P<0.001).Similar results were detected in the secondary dermal papilla cells,with a 10.59 and 0.45 times in the overexpression and interference group,respectively(P<0.001).These results confirmed that overexpression and interference of Msx2 in the dermal papilla cells were successfully achieved.It was also found that the expression of Msx2 in the two kinds of dermal papilla cells was consistent with that of LEF1 ?BMP-2.Therefore,the results showed that the expression levels of LEF1 and BMP-2 were positively correlated with Msx2 expression.And these resultes indicated that overexpression of Msx2 promoted the proliferation of dermal papilla cells.By observing the growth of each cell lines,it was found that the overexpression of LEF1 and Msx2 promoted the proliferation of dermal papilla cells,while down-regulation of Lefl and Msx2 decreased the proliferation of dermal papilla cells.The results of this study showed LEF1 was positively correlated with ?-catenin,C-myc and cyclin D1 in dermal papilla cells,and Msx2 was positively correlated with LEF1 and BMP-2.LEF1 and Msx2 have a positive effect on the proliferation of dermal papilla cells.