The Research Of Hair Follicle Developmental Related Signal Pathways Of Thymosin Beta 4 In Arbas Cashmere Goat

Thymosin beta 4 is a 43 amino acid peptide which molecular weight is 4963 Da. It is highly conservative in mammals, which function is to promote hair follicle development and growth. The earliest thymosin beta 4 was isolated from the bovine thymosin. As the regulation material of cell migration and differentiation, is widely distributed in all kinds of tissues, organs and cells. Thymosin beta 4 complete the function through combined with other proteins in the cell, it has many biological activities, including promote angiogenesis by accelerate endothelial cell migration and differentiation, wound healing, anti-inflammatory and so on. Compared with non-transgenic cashmere goat, the cashmere production of thymosin beta 4 transgenic cashmere goats had been obviously improved. But the mechanism of thymosin beta 4 genes influence cashmere producuction is not clear, still need further research. Based on the above experiment purpose, thymosin beta 4 transgenic cashmere goat’s tissues and cells were used as experiment material to explore its function in MAPK-ERK/p38 and PI3K-AKT signaling pathways, and the proteins interacted with thymosin beta 4 were screened and identified as well, so that to provide experimental basis for understanding the mechanism of the hair follicle growth and development.1. Western Blot detection of related proteins expression levels in thymosin beta 4 transgenic cashmere goat skin samples.The skin samples of transgenic cashmere goats which cashmere production was improved and unimproved were used for Western Blot detection, and somatic cell cloning cashmere goat skin tissues from same cell line were used as control, gene expression and phosphorylation levels of AKT, ERK and p38 were detected. The results showed that compared with the control group, the ERK and AKT protein expression and phosphorylation levels in improved cashmere production goat were up-regulated obviously, but not in unimproved cashmere production goat.2. Screening of proteins interacted with thymosin beta 4 by Co-Immunoprecipitation (Co-IP) combined Mass Spectrometry.The fibroblasts were isolated from thymosin beta 4 transgenic cashmere goat skin tissues and somatic cell cloning cashmere goat skin tissues from the same cell line, the proteins interacted with thymosin beta 4 were enriched through Co-Immunoprecipitation, were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), after stained by Coomassie brilliant blue, then different bands were cut off to identify the composition by Mass Spectrometry.The results of the Mass Spectrometry showed that the three proteins might interact with thymosin beta 4: beta-actin (ACTB), calponinl (CNN1) and vasohibinl (VASH1). Those candidate proteins could be used for the next experiment.3. Identification the interaction between thymosin beta 4 and ACTB, CNN1, VASH1 by mammalian two-hybrid system.Thymosin beta 4 fragment with double enzymes sites was connected to the pACT plasmid, ACTB, CNNland VASH1 fragments were connected to the pBIND plasmid respectively. Those two plasmids and pG51uc plasmid were transfected together into the Hela cells, the activity of luciferase was measured after 24-48h cultivation. It is concluded that three kinds of protein all interacted with thymosin beta 4, and thymosin beta 4 and VASH1 was significantly related.Through this research, it was preliminarily determined that two signal pathways and three proteins might mediate hair follicle growth by thymosin beta 4. The interaction between those signal pathways and proteins might lead to the phenotype change in hair follicle growth, which will benefit to improve the cashmere production.