Establishment Of Animal Model Of Oral Lichen Planus And Quantitative Analysis Of Serum Proteins In Patients With OLP Before And After Treatment With Prescription Huashi Xingyu Qingre Decoction

Part one Establishment of animal model of oral lichen planusObjective: Established an animal model of oral lichen planus(OLP)by using OLP homogenate supplemented with Freund's adjuvant acupuncture in the buccal mucosa of NOD/SCID mice.And tried to establish an animal model of OLP based on the application of human PBL to establish an NOD/SCID mice model with human immunological properties.Methods:1 Establishment of OLP-NOD / SCID mice modelTake a little diseased tissue of OLP patients,and make it into tissue Homogenate.Twenty-four NOD/SCID mice were selected,aged 4 weeks,female,20~25 g,animal body fullness,and normal development.These mice which have a thick coat of shiny fur act quickly,react sensitively and have a good appetite.Feeding and experimental operations were conducted at the laboratory SPF laminar flow laboratory of the Animal Experimental Center.They were randomly divided into 2 groups,6 rats in each group.The first group was the blank control group,saline acupuncture buccal mucosa of NOD/SCID mice used for 4 weeks,and acupuncture once every two days.In the second group,OLP homogenate supplemented with Freund's adjuvant acupuncture in the buccal mucosa of NOD / SCID mice was used for 4 weeks,and acupuncture once every two days.The first and second groups were killed after 4 weeks,and specimen at the treated buccal mucosa was drawn.Make them into HE staining to observe whether the pathological changes under light microscope.2 Establishment and identification of Hu-PBL-NOD / SCID mice model2.1 18 NOD / SCID mice were randomly divided into 3 groups,6 in each group;the first group of mice were injected intraperitoneally with human peripheral blood lymphocytes of 2×107/0.5ml,as a low dose of human immune reconstitution group;the second group of mice were injected with human peripheral blood lymphocytes of 4×107/0.5ml,as a high dose of human immune reconstitution group;and the third group of mice were injected with0.5ml saline,as the blank control group.2.2 The mouse model was identified from the biological characteristics and immunological characteristics of two aspects: observe the biological signs of mice;pick the eyeball blood in the eighth week,detect Human CD3 +,CD4 + T lymphocytes in peripheral blood of mice by flow cytometry.Results:1 Establishment of OLP-NOD / SCID Mouse ModelThe macroscopic observation results of The experimental group treated with tissue homogenatel acupuncture scratches were mucosal congestion,and fragile texture.The pathological results of the experimental group showed that the mucosal epithelium parakeratosis,the prickle cell layer proliferated,the basal cells showed vacuolar degeneration,epithelial spikes extended irregularly,scattered lymphocytes infiltrated into lamina propria.However,there were no typical pathological features of proprialymphocyte infiltration zone in lamina propria.There was no significant change in the mucosa of the control group by macroscopic observation,and the pathological results of microscopic observation showed the mucosal epithelium hyperkeratosis,and that there was no infiltration of inflammatory cells in the lamina propria.2 Establishment and identification of Hu-PBL-NOD/SCID mouse modelThe First group of mice were fed to the 8th week and were in good nutritional state and mental state.The double positive expression rate of human CD3+ and CD4+ T lymphocytes in peripheral blood of mice was 3.03 ±0.47% by flow cytometry,lack of expression,failure of establishing Hu-PBL-NOD/SCID rat model.Second group of mice were fed to the 8thweek,graft versus host reaction(GVHR)occurred,and one died;The double positive expression rate of human CD3+and CD4+T lymphocytes in peripheral blood of mice was 36.13±4.79% by flow cytometry.Although the lymphocyte has reached a certain number,the mice appeared GVHR.The mice in third group were healthy,the nutritional and mental state were well.The expression of CD3+,CD4+ T lymphocytes in peripheral blood of mice was not detected by flow cytometry.Part two The quantitative analysis of serum proteins in patients with OLP before and after treatment with prescription Huashi Xingyu Qingre decoctionObjective: In the early stage of this study,we successfully identified the differential serum proteins of patients with oral lichen planus before and after the treatment of Huashi Xingyu Qingre Decoction(HXQD)by two-dimensional fluorescence difference gel electrophoresis(2-D DIGE)and mass spectrometry,found that there were some differences in the expression of human antithrombin III(AT-III)and vitamin D binding protein(Gc protein)in serum before and after treatment with OLP,and gave a preliminary semi qualitative and semi quantitative validation by Western-blot.In order to further explore the effect of HXQD on the OLP,this study intends to use ELISA technology to do a more accurate quantitative study on the protein level.This study provides a theoretical basis for the possible pathogenesis of OLP and the clinical application of the prescription of HXQD.Methods:A total of 20 cases of OLP patients were selected,and the venous blood of OLP patients before and after 8 weeks of HXQD treatment was collected.The differential serum proteins of patients with oral lichen planus before and after the treatment of HXQD by ELISA were identified.Results:The results of ELISA showed that the levels of human AT-III and Gc protein in oral lichen planus patients with after the treatment were lower than those before treatment(P<0.05).Conclusions:1 The diseased region after using OLP homogenate supplemented with Freund's adjuvant acupuncture occurred to the mucosal epithelium parakeratosis,the prickle cell layer proliferated,the basal cells showed vacuolar degeneration,epithelial spikes extended irregularly,scattered lymphocytes infiltrated into lamina propria.Compared with the previous experiments' pathological changes of the stratum epithelium of the mucosa appeared spinous proliferation,and inflammatory cell infiltration in the lamina propria,this experiment was closer to the pathological features of OLP.2 In this experiment,after injection of human PBL 2×107/0.5ml,the expression was not enough,the human PBL-NOD/SCID mouse model was not successfully established,and the GVHR reaction occurred after injection of human PBL 4×107/0.5ml.3 The expression of human AT-III and Gc protein in oral lichen planus patients was decreased after treatment,and the difference of expression of these two proteins was related to the mechanism of HXQD on oral lichen planus.