The Experimental Study Of Opsin On The Construction Of Tissue-engineered Skin By Human Amniotic Mesenchymal Stem Cells On De-epidermal Dermis

Objective: Regenerative medicine is a branch of translational medicine.Its basic principle is to restore the structure and function of tissue or organ damaged by aging,diseases or other trauma.Tissue engineering skin research is an important part of regenerative medicine,tissue-engineered skin is a more appropriate choice in the treatment of skin defects,especially in large-scale skin damage.In the past,most of MSCs were derived from adult bone marrow and needed bone marrow puncture.However,the content of MSCs in human bone marrow was very low,and the number of cells and the ability of expansion and differentiation decreased significantly with age,which restricted the clinical and scientific research application of MSCs.Human amniotic mesenchymal stem cells(hAMSCss)derived from human placental amniotic membrane have the ability of multidirectional differentiation.Amniotic membrane is a kind of postpartum waste,which is easy to get and has no ethical dispute.Therefore,human amniotic mesenchymal stem cells are the ideal source of seed cells.Opsins(OPNs)belongs to the G-protein-coupled transmembrane receptor superfamily(GPCRs)which is sensitive to light.It can be divided into photoreceptor(opn1,opn2)and non photoreceptor(opn3,opn4,opn5).In the past,it has been thought that opsin is mainly distributed in the retina and other visual tissues,but in recent years,researchers have found that opsin distribution exists in many different parts outside the retina,which plays an important role in regulating skin pigment synthesis,circadian rhythm and immune function of the body,so the research on the distribution and function of non visual opsin outside the retina has become the current research highlights.There are no related reports about the expression of OPNs in hAMSCs.Therefore,we explore whether opsin exists in hAMSCs,which opsin dominates it,what effect does it have,what changes after UVA intervention,and how does OPNs affect MSC stemness? whether OPNs affect the maintenance of MSC stemness and keratin differentiation,Finally,we used MSC to inoculate into DED,and observed tissueengineered skin could be formed.Methods: Low concentration trypsin was used for hAMSCs digestion and separation in this experiment.Cells of P3-5 generation were selected for testing.The cultured cells were identified by flow cytometry and osteogenic and adipogenic differentiation.At the same time,we used RT-PCR and Western blot to detect the presence of Opsins expression in MSCs at the m RNA and protein levels respectively,and also performed immunofluorescence expression for OPN3 localization in hAMSCs.The CCK-8 method was used to detect the optimal intensity of UVA intervention in MSC.The UVA irradiation intensity was 0,0.5 J / cm 2,5J / cm 2,10 J / cm 2,20J/ cm 2,cell collection time was 0,12 h,24h,48 h,72h.In order to further verify the effect of UVA on MSCs,we intervened MSCs with UVA intensity of 5J / cm 2 for 48 h and observed whether UVA can cause the change of MSC cell cycle,promote cell apoptosis and affect cell aging RT-PCR and Western blot were used to detect the expression of Sox2 and Oct4 in MSC after UVA intervention.Si RNA and lentivirus vector were used to down regulate and up regulate opn3.It was found that UVA activated Ca2 + / Ca MK ? / p38 signal pathway through opn3 and up regulated Sox2 and Oct4.In order to understand the effect of UVA on the differentiation of MSCs into epithelium and cytokeratin,we selected CK pan and CK14 as observation indexes.We inoculated MSCs on DED,and used fluoresce in diacetate to observe cell viability under a stereo fluorescence microscope.The hAMSCs were cultured for 3 days under normal medium solution,and replaced with conditioned medium after 3 days to form tissue-engineered skin.Results: Flow cytometry and osteogenesis and adipogenesis induction experiments verified that the cells we obtained were amniotic mesenchymal stem cells and could be used in subsequent experiments.Western blot analysis showed that OPN3 expression was higher in hAMSCs,while OPN5 expression was lower.CCK-8 method was used to detect the optimal irradiation intensity of UVA intervention in MSC.The optimal irradiation dose was 5J / cm 2.The cells were collected at 0,12 h,24h,48 h,and 72 h after the intervention,and the OPN5 expression was gradually increased with time-dependent enhancement,but OPN3 did not increase significantly.hAMSCs cell cycle,apoptosis and aging had no effect with 5J / cm 2 48 h intervention.Western blot detected hAMSCs stemness indicators Sox2,Oct4,Nanog,HIF-1? and keratin Pan-CK,Ck14,CK19 expression.It was found that Sox2,Oct4 had an increasing trend.Pan-CK at 24 h,Ck14 at 12 h increased expression.We inoculated hAMSCs in DED,which can differentiate into keratinocytes and form epithelial-like structures,and use fluorescein diacetate(FDA)to dynamically observe the activity of DED-inoculated MSCs on a stereo fluorescence microscope to understand the state of tissue-engineered skin culture.Conclusion: hAMSCs can express Opsins,with the highest expression of OPN3.RT-PCR and Western blot were used to detect the expression of Sox2 and Oct4 in MSC after UVA intervention.Si RNA and lentivirus vector were used to down regulate and up regulate opn3.It was found that UVA activated Ca2 + / Ca MK?/ p38 signal pathway through opn3 and up regulated Sox2 and Oct4.hAMSCs can differentiate into keratinocytes in DED and form epithelial-like structures.