Expression And Function Of SERPINB1 In Psoriasis

As a kind of common, chronic, and recurrent inflammatory skin disease,psoriasis has a significant negative impact on patients'quality of life and also has been the focus for a long period in the area of skin diseases. Characteristical hispathological changes of psoriasis are the excessive proliferation of keratinocytes and accumulation of intraepidermal neutrophil. Neutrophils count is also high in the peripheral blood of patients with psoriasis. Neutrophils are important components of innate immunity, which play an important role in the generation and progression of psoriasis through a variety of ways. As an important mediator, NE (neutrophil elastase) promote the roles of neutrophils.Studies have shown that NE regulates inflammatory response in a complicated process because it can not only promote inflammation, but also inhibit the inflammation.About the relationship between NE and psoriasis, some studies had been shown that NE expressed highly in lesions and serum in patients with psoriasis, which match the severity of the disease. And some research had shown that NE could stimulate proliferation of keratinocytes through the EGFR signaling pathway. Trappin-2 andα1-antitrypsin,the inhibitor of NE, were significantly abnormal in psoriasis. Trappin-2 could also inhibit the proliferation of keratinocytes and reduce the expression of IL-8, IL-6, ICAM-1 and other inflammatory enhancement factors. But no studies have directly observed that NE inhibitors can inhibit the proliferation of keratinocytes induced by NE. Some research concluded that low concentrations of NE significantly increased transcription and expression of elafin, which is one kind of NE inhibitors, while high concentrations had the opposite effect. And now no further study about the relationship between NE and its inhibitors is presented.Vitamin D3 analogues are the first choice of local therapy for mild and moderate psoriasis. Research on its therapeutic mechanism has also been a hot area of skin disease. In my master's research, it was found that SERPINB1 played a role in curing psoriasis by calcitriol because ovalbumin-like serine protease inhibitor 1 (serine proteinase inhibitor, clade B, member 1, SERPINB1) (monocyte neutrophil elastase inhibitor, MNEI) overexpressed after calcitriol was put in HaCaT. As a neutrophil protease inhibitor, SERPINB1 can inhibit NE and cathepsin G (cat G). Currently more studies had been done about curing PMNs inflammatory diseases, especially acute lung injury and pulmonary Pseudomonas aeruginosa infection by SERPINB1. It was confirmed that SERPINB1 played an important role in the regulation of immune responses. However, there is no research on the relationship between psoriasis and SERPINB1.Based on my master's research, the first aim of this study is to further verify the differences of SERPINB1 in gene and protein level after calcitriol is given to HaCaT. And the second aim is to do some research about the impact of SERPINB1 on keratinocyte proliferation and differentiation so as to find the role SERPINB1 played in the process of treating psoriasis. This study may be necessary to find new therapeutic targets of psoriasis.PARTⅠObjective To study the effect of calcitriol on immortalized human keratinocyte cell line (HaCaT) SERPINB1 at the levels of mRNA and protein.Methods HaCaT cells were cultured with usual method. When 80% of cells got fused, fresh calcitriol on the concentration of 10-8mol/L was added while the negative control group was added with the same amount of serum-free DMEM. Two complex holes were set for each condition and then total cellular RNA and the whole protein were extracted after dark culture. Contents of SERPINB1 on mRNA and protein levels were analyzed with the methods of RT-PCR and western-blot, separately. And set the different time points to observe the impact of drugs on SERPINB1 mRNA.Results SERPINB1 mRNA was upregulated by 1.99 times in 4h-group compared with control group, and the at 8h, 12h,24h,48h SERPINB1 mRNA was separately upregulated by 2.41,3.28,3.46 and 3.31 times.There were statistical significance between every time group and control group. But there was no statistical significance between them.At the protein level, 24h-group and 48h-group were both upregulated when compared with control group and there were statistical significance. There was statistical significance between 24h-group and 48h-group.Conclusion Calcitriol could regulate SERPINB1 in HaCaT both at the mRNA and the protein levels.PARTⅡEffect of SERPINB1 on HaCaT in proliferation and the regulation of SERPINB1 Objective Select siRNAs interfering with SERPINB1 effectively. Discuss the effect of SERPINB1 on HaCaT cell proliferation by genetic interference, and study the differences of SERPINB1 when added different concentrations of NE.Methods Using RNA interference technology, design and synthesis the siRNAs to aim at SERPINB1, a total of 3 groups. HaCaT cells were transfected by Lipodectamin2000 and were observed on transfection efficiency by labeled siRNA. Use RT-PCR to Verify the efficiency of transfection and interference and then select the most effective siRNA (SERPINB1-homo-93) to interfere with HaCaT. MTT was observed before and after interfering. Different concentrations of NE also were joined into HaCaT,and then the quantity of SERPINB1 was studied by western-blot. At last, the effect of different concentration of NE and calcitriol on HaCaT was observed.Results 1. SERPINB1-homo-93 could interfere SERPINB1 of HaCaT effectively by 83%. 2. NE could promte cell proliferation at lower concentration at 1U/L to 5U/L compared with control group. At the concentration of 15U/L, the effect of NE on proliferation had no difference with control group, and at the level of 20U/L to 50U/L,NE could inhibit cell proliferation.3.Calcitriol could inhibit proliferation of HaCaT at the concentration of 10-9-10-6mol/L, but there were no statistical significance between different concentration groups.4. Cell proliferation was promoted when SERPINB1 was interfered. Calcitriol could not inhibit proliferation after SERPINB1 was interfered. 5. NE upregulated SERPINB1/GAPDH at lower concentration at 1,5,10,15U/L. There was on statistical significance compared with the control group with the concentration of 20U/L. And then, NE depressed the expression of SERPINB1.Conclusion SERPINB1 could inhibit the proliferation of HaCaT. NE could promote proliferation at lower concentration,but the effect was inhibition when the concentration was at a higher level. Lower concertration of NE could promote the expression of SERPINB1, higher concentration could inbibit its expression.PARTⅢThe expression of SERPINB1 in psoriatic lesionsObjective Study the difference of SERPINB1 between Lesions of psoriasis and normalMethods Collect severe skin lesions of psoriasis and normal skin to extract total protein from tissues and then analysis SERPINB1 differences using western-blot. Collect paraffins from previous biopsies of patients with psoriasis to investigate differences of SERPINB1 in tissue slices by immunohistochemistry.Result Immunohistochemical staining displayed that in the basal layer of normal skin SERPINB1 was positive with weak to moderate. But, in psoriatic lesions SERPINB1 staining was moderate to strong positive in the basal layer and spinous layer.There was statistically significance in difference. Total protein was extracted and the western-blot results showed that SERPINB1/GAPDH was lower in psoriasis group than health control. Conclusion The expression of SERPINB1 in paraffin sections of psoriatic lesions was significantly stronger than that in normal skin. In fresh tissue, expression of SERPINB1 was lower in psoriasis group than that in the control group.