| Cell gap junction, composed by several connexins, plays an important role in cell signaling transduction during the hair follicle growth and development. Our previous researches showed that the transcriptional expression levels of connexin40(Cx40) and connexin43(Cx43) were significantly differently expressed between the anagen and telogen pelage skin of the cashmere goats by using high-throughput transcriptome sequencing. In this study, Cx40and Cx43genes were investigated to find their potential roles during the hair growth and development, and the results may provide experimental basis for further study on the gene regulatory system of the hair growth and development of the cashmere goat.In this study, three types of hair follicle-related cells (pHFDPCs, sHFDPCs, epidermis cells) had been successfully isolated from the cashmere goat, and the expression levels of Cx40and Cx43genes in the three types of cells were measured. Meanwhile it had been observed that the expression levels of both Cx40and Cx43genes in anagen pelage skin of the cashmere goat were higher than those in telogen.Based on the successfully cloned CDS sequences of Cx40and Cx43genes by RT-PCR, pIRES2-EGFP-Cx40and pIRES2-EGFP-Cx43plasmids were successfully constructed. Later six transgenic cell lines were obtained after transfecting the two plasmids respectively to pHFDPCs, sHFDPCs and epidermis cells by using liposome transfection method. The overexpression of Cx40and Cx43genes in the three types of follicle-related cells was achieved. The transcriptional levels of related genes involved in the hair follicle growth and development in the six types of transgenic cells were measured, and the results indicated that:1. VEGF gene transcriptional levels:The expression levels of VEGF in Cx40transgenic pHFDPCs, sHFDPCs and epidermis cells were0.84,0.14b and0.57a folds, respectively, compared with non-transgenic cells; The expression levels of VEGF in Cx43transgenic pHFDPCs, sHFDPCs and epidermis cells were0.81,0.18a and0.62a folds, respectively, compared with non-transgenic cells (a:P<0.05, b:P<0.01).2. IGF-1gene transcriptional levels:The expression levels of IGF-1in Cx40transgenic pHFDPCs, sHFDPCs and epidermis cells were4.02b,1.8a and1.91a folds, respectively, compared with non-transgenic cells; The expression levels of IGF-1in Cx43transgenic pHFDPCs, sHFDPCs and epidermis cells were4.27b,1.93a and1.42a folds, respectively, compared with non-transgenic cells (a:P<0.05, b:P<0.01).3. β-catenin gene transcriptional level:The expression levels of (3-catenin in Cx40transgenic pHFDPCs, sHFDPCs and epidermis cells were2.19a,1.13and5.05b folds, respectively, compared with non-transgenic cells; The expression levels of β-catenin in Cx43transgenic pHFDPCs, sHFDPCs and epidermis cells were1.32,1.12and2.46a folds, respectively, compared with non-transgenic cells (a:P<0.05, b:P<0.01).4. HIF-la gene transcriptional level:The expression levels of HIF-la in Cx40transgenic pHFDPCs, sHFDPCs and epidermis cells were0.51a,0.46a and0.72folds, respectively, compared with non-transgenic cells; The expression levels of HIF-1α in Cx43transgenic pHFDPCs, sHFDPCs and epidermis cells were0.52a,0.54a and0.56a folds, respectively, compared with non-transgenic cells (a:P<0.05).5. Thymosins-β4gene transcriptional levels:The expression levels of Thymosins-β4in Cx40transgenic pHFDPCs, sHFDPCs and epidermis cells were1.08,0.74and1.23folds, respectively, compared with non-transgenic cells; The expression levels of Thymosins-β4in Cx43transgenic pHFDPCs, sHFDPCs and epidermis cells were1.13,0.67a and0.87folds, respectively, compared with non-transgenic cells (a:P<0.05).The above results suggested that in pHFDPCs, sHFDPCs and epidermis cells, the overexpression of Cx40and Cx43genes have a trend of suppressing the transcriptional expression of VEGF and HIF-1α genes, while promoting the expression of IGF-1and β-catenin genes. Therefore the overexpression of Cx40and Cx43genes may play a negative regulation role in transcriptional expression of the VEGF and HIF-1α genes, and a positive regulation role in transcriptional expression of IGF-1and β-catenin genes. The overexpression of Cx40and Cx43genes may play a nonsignificant role in transcriptional expression of Thymosins-β4. However, the functions of Cx40and Cx43genes on the hair follicle growth and development of the cashmere goat gene regulation system still needs further research. |
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