Basic And Clinical Research Of Skin Repair And Regeneration After Burns (Regeneration Of Sweat Gland And Therapy Of Pyogenic Granuloma After Burns)

Objective:1.To investigate mechanism of autofluorescence of human eccrine sweat glands.2. To look for the promoters of key gene locis in the process of human bone marrow mesenchymal stem cells differentiating into sweat gland like-cell with methylation chips and to investigate the differentiation mechanism of mesenchymal stem cells.3. This retrospective study was to assess clinical results of cases with pyogenic granuloma after burns being healed with different therapy plans.Methods:1. Full-thinkness skin grafts were obtained from 5 volunteers. Autofluoresence of the eccrine sweat glands in the paraffin wax-embedded skin sections stained with hematoxylin-eosin (HE) were observed under the fluorescence microscope. It compared to that of indirectly immunofluorescence stained paraffin wax-embedded skin sections. Eccrine sweat glands were isolated and cultured. Intensity of autofluorensence of single eccrine sweat gland was measured with a laserconfocal scanning microscope system. 2. MSCs were induced with heat shock eccrine sweat gland in a transwell co-culture system for 10 days. Markers of KRT19 and the CEA were measured by immunofluoresence examination. Gene expression and proteins of CEA of MSCs were measured with RT-PCR and western blots at Od,5d and 10d. The methylation value of MSCs before and after being induced was measured with illumina 450K methylation chip. The methylation value of promoter of key gene locis were measured by flight mass spectrometry. Plasmids contained the key gene locis were constructed and transfected into MSCs. Gene expression and proteins of CEA were measured with RT-PCR and Western blots at 5d.3. This study included all cases with PGBs between 2003 and 2013 in our center. The diagnosis and treatments were based on clinical features, biological datas and pathological examination.Results:1. Autofluorescence of sweat gland in HE stained skin tissue sections was easily observed. It appeared green under GFP (470/40 nm), red under N2.1 (515-560nm) and blue under A(340-380nm). The single and living eccrine sweat gland showed various autofluorescence colours, including green under WB, red under WG and blue under UV. The spectrum excited at 488 nm exhibited two peaks located at approximately 530 nm (11.54±4.66) and 590 nm (10.38±4.33).2. MSCs induced by heat shock sweat glands differentiated into sweat gland like-cells, KRT19 and CEA were positive. DNA methylation value of total 76 gene locis changed significantly (0.169‰), DNA methylation value of 51 gene locis decreased significantly including SLC12A7, ENKUR, LRWD1, Pax6, HDAC4 and so on. DNA methylation value of 25 gene locis increased significantly including Hoxd11, ADARB2, ANGPT1, UNC84A, BMP6 and so on. Methylationvalue of Pax6 decreased with flight mass spectrometry, Plasmid contained Pax6 gene and 5-Azd10ng/μL enhance expression of CEA gene and protein of MSCs.3.15 cases included children and adults from 1 to 58 years old. The male-to-female ratio was 10:5.9 cases were scalded,4 cases were flamed and 2 were burned by electric spark. Pyogenic granulomas after burns were located in the face, trunk or limbs.10 cases were healed completely by conservative treatment. Full-thinkness excision of 3 cases included 2 grafted and 1 primary closure. Shave excision of 2 cases were healed completely. No recurrence of all cases was noted postoperatively except that 1 case recurred 7 days after shave excision.Conclusions:l.The autofluorescence of eccrine sweat gland sections is simple, helpful, and to observe the structure of eccrine sweat glands. The autofluorescence of the eccrine sweat gland may be due to the existence of flavin and lipopigment.2. DNA demethylation plays an important role in differentiation of MSCs. Pax6 gene and 5-Azd may influence CEA gene expression.3. Pyogenic granuloma after burns is a benign and different from classic pyogenic granulom. Infection agent is an important reason. Conservative treatments should be chosed firstly.