Construction Of Skin Tissue Culture System In Vitro And The Effect Of Serum From Diabetic Ulcer Patients On Skin Tissue Angiogenesis

Objective:To construct human skin tissue cultured in vitro and observe the effect of serum from patients with diabetic ulcer on tissue angiogenesis Methods:1.The culture of human prepuce: Collect skin tissues after clinical prepuce circumcision,Iodophor disinfection and sterile PBS rinsing and fix them on the stainless-steel stents.The tissue were,divided into two groups,one group was cultured by contacting the dermis side with the culture medium,and the epidermis side was kept dry in the gas phase,namely the gas-liquid junction group(G-L);the other group was cultured completely submerged in the culture medium,namely the immersion group(Im).Use two different concentrations of fetal bovine serum medium for culture(10%fetal bovine serum and 5% fetal bovine serum),and observe its status.2.The Histomorphological observation of human prepuce: Prepare paraffin sections for conventional HE stain and the morphology of the tissues was observed.3.Effects of serum from patients with diabetic foot ulcer on blood vessel formation of human prepuce: Prepare paraffin sections for immunohistochemistry and observe the number of blood vessels in the tissues(diabetic foot ulcer serum group and healthy control group).4.Effects of serum from patients with diabetic foot ulcer on related cytokines of human prepuce tissue.Extract the total RNA from the tissue,and analyze the m RNA expression of VEGF-192,TNF-α,TGF-β,IL-6 and IL-1βusing real-time quantitative PCR technology.Results:1.There is no significant difference between the tissue cultured with 10% fetal bovine serum medium and the tissue cultured with 5% fetal bovine serum medium,and the culture time can reach about 4 weeks.Therefore,the follow-up experiments are all cultured with 5% fetal bovine serum.2.Compared with the Im(immersion)group and the GL(gas-liquid junction)group at the same time,the GL(gas-liquid junction)group has a clearer and more complete epidermal structure,and the epidermal structure lasts longer;there is no significant difference in the dermal layer structure between the two groups.3.The degree of vascular damage and the cumulative number of blood vessels in the DFU group(diabetic foot ulcer group)were observed under the microscope more severe than those in the Ctrl group(healthy control group).Compared with the healthy control group,the number of blood vessels in the diabetic foot ulcer group decreased significantly at 3,7,and 10 days.4.The total RNA of the tissue was extracted,and the m RNA levels of VEGF-192,TNF-α,TGF-β,IL-6 and IL-1β were analyzed by real-time quantitative PCR technology: real-time fluorescent quantitative PCR detection and analysis showed that serum from diabetic foot ulcers can decrease the expression of VEGF-192 and increase the expression levels of TGF-β,IL-6 and IL-1β genes.Conclusion:It is feasible to construct the culture system of human skin tissue in vitro,and the culture time can reach about 4 weeks.There was no significant difference between the tissue cultured with 10% fetal bovine serum medium and the tissue cultured with 5%fetal bovine serum medium.The skin tissues cultured in the manner of either the gasliquid junction or immersion group can survive within 4 weeks,and the tissues kept integrate in 2 weeks,and was the best condition within 1 week.As for the organizational status,of G-L group was better than IM group.The serum from patients with diabetic foot ulcer reduced the angiogenesis of human prepuce tissue.At the same time,the serum of patients with diabetic foot ulcer can decrease the expression of VEGF-192 and increase the expression levels of TGF-β,IL-6 and IL-1β m RNA.