Effects Of VEGF164 On In Vitro Interaction Of Dermal Papilla Cells And Vascular Endothelial Cells Of Arbas Cashmere Goat

Arbas Cashmere goat is one of the endemic species of Inner Mongolia cashmere goats, which lives in a special ecological environment in arid desert and the semi-desert grassland. Arbas cashmere is known as "soft gold", "fiber jewel". The research about the factors that influence the production of cashmere has great scientific significance and value. In our laboratory, we had bred vascular endothelial growth factor 164 (Vascular endothelial growth factor 164, VEGF164) transgenic goats, whose cashmere production was significantly higher than that of control group. So it is necessary to study the reasons for the increase.In vitro conditions, the effects of VEGF164 transgenic dermal papilla cells (Dermal papilla cells, DPCs) on vascular endothelial cells (Vascular endothelial cells, VECs), VEGF164 cytokines on VECs growth status and tube-like structure formation, and VEGF164 transgenic dermal papilla cells on self-growth, were observed in this research. The purpose is to preliminarily understand the mechanism of VEGF164 on improving cashmere production whether promoting the increase of blood vessel around the hair follicle or promoting the increase number of hair follicle. The research results would have important reference value to understand the influence factors and their action mechanism on cashmere production.1. Establishment of cell lines of PHF-DPCs, SHF-DPCs and VECs of Arbas Cashmere goat(1)The culture of PHF-DPCs and SHF-DPCsPrimary hair follicle dermal papilla cells (PHF-DPCs) and secondary hair follicle dermal papilla cells (SHF-DPCs) storaged in our laboratory were cultured and passaged. The results showed that the PHF-DPCs and SHF-DPCs could be normal passaged, cryopreserved and recovered.(2) VECs isolation, culture and identificationVECs were isolated by enzymatic digestion and then cultured VECs were identified by immunohistochemistry, and cell purity was tested by flow cytometry. The results showed that VECs could be subcultured to seventh generation and expressed von Willebrand factor (von Willebrand factor, vWF), and the purity was 99.6%.2. Transfection and identification of PHF-DPCs and SHF-DPCsPHF-DPCs and SHF-DPCs were transfected by hair follicles-specific expression vector pCDsRed2-KV with Lipofectamine(?) LTX and PLUSTM. After 48h, PHF-DPCs and SHF-DPCs were cultured and selected in 500ng/ml G418-containing medium for 7 days, then cultured in 300ng/ml G418-containing medium for 3 days, the stably transfected cell clones were obtained. DNA of the cell clones was extracted for PCR identification, and specific bands were obtained. Total RNA of the cell clones was extracted after reverse transcription for real-time PCR analysis.The results showed that the mRNA expression of VEGF164 transgenic PHF-DPCs is 1.7 times than that of control group, the mRNA expression of VEGF164 transgenic SHF-DPCs is 3.9 times than that of control group. The results suggested that exogenous VEGF164 gene had been integrated into PHF-DPCs and SHF-DPCs genome, and could be transcribed.3. Effects of VEGF164 on in vitro interaction of DPCs and VECs(1) Effects of VEGF164 transgenic DPCs on VECsThe VEGF164 transgenic PHF-DPCs and SHF-DPCs were co-cultured with VECs in Transwell respectively. After co-culturing 7 days, VECs were collected to detect the changes in VECs and the control group by real-time PCR. The results showed that, the relative expression of CD31 mRNA of VECs co-cultured with transgenic PHF-DPCs was 1.2 times than that of control group; the relative expression of CD31 mRNA of VECs co-cultured with transgenic SHF-DPCs was 0.69 times than that of control group; the relative expression of CD34 mRNA of VECs co-cultured with transgenic PHF-DPCs was 1.18 times than that of control group; the relative expression of CD34 mRNA of VECs co-cultured with transgenic SHF-DPCs was 0.9 times than that of control group. Experimental results indicated that VEGF164 secreted from transgenic DPCs has hardly any significant effect on proliferation of VECs in co-culture condition, but the co-culture of transgenic DPCs with VECs could be benefit to slow down aging of VECs.(2) Effects of cytokines onVECCytokines of VEGF164 in 10ng/ml,20ng/ml,30ng/ml and VEGFD in 10ng/ml, 20ng/ml,30ng/ml were supplemented in VECs culture media respectively.The effects of VEGF164 and VEGFD on growth and formation of tube-like structure of VECs were observed.The results showed that the growth of VECs could be most effectively affected by 10ng/ml VEGF164 or 10ng/ml VEGFD. When cultured with lOng/ml VEGF164, CD31 mRNA relative expression of VECs was 1.55 times than that of control group. CD34 mRNA relative expression of VECs was 1.68 times than that of control group. The results indicated that VEGF164 and VEGFD could promote the growth of VECs and could promote the formation of tube-like structure of VECs as well and VEGFD was in stronger efficiency.(3) Effects of VEGF164 transgenic PHF-DPCs and SHF-DPCs on self-growthVEGF164 transgenic PHF-DPCs and SHF-DPCs were counted, while PHF-DPCs and SHF-DPCs cultured with 10ng/ml VEGF164 were counted as well. The results showed that the transgenic DPCs could promote self-growth and proliferation, and the promotive effect was stronger than that of VEGF164 supplementation.In summary, VEGF164 not only could promote growth and proliferation of VECs, but also could promote the growth and proliferation of DPCs. The effects of VEGF164 transgenic DPCs on the promotion of self-growth were more effective than that of VECs growth. The results of this research could provide some new clues for further understanding of the relationship between DPCs and VECs.