| Occupational trichloroethylene(TCE)exposure could induce serious hypersensitivity dermatitis which named trichloroethylene induced hypersensitivity dermatitis(TIHD),the disease is a type of hypersensitivity syndrome characterized by a systemic skin lesions and accompanied with multiple organs damage.Human case-control study has found HLA-B*13:01 gene was closely associated with TIHD according to our previous study.In our current research,splenic lymphocyte-surface expression of HLA-B*13:01 molecular was detected in transgenic mice(TG mice),then TIHD disease model was established by using TG mice,and several indexes were involved to evaluate the mice disease model.The TG mice disease model were eatablished to provide a tool for researching the pathogenesis and the function of HLA-B*13:01 during the disease process.The results of our research as follows:1.Splenic lymphocyte-surface expression of HLA-B*13:01 molecular in TG miceSplenic lymphocytes cells were successively incubated with saturating concentrations of FITC-conjugated anti-human HLA-ABC and analyzed by flow cytometry.The result showed(59.08±16.45)%of positive cells in TG mice splenic lymphocytes while only(2.50±0.57)%of that in WT mice splenic lymphocytes,HLA-B*13:01 gene expressed stablely in the offspring and expressed well in both male and female.2.The establishment of the TG mice model of human TIHDAccording to the sacrificing time after the final challenge,the TCE treatment groups of TG and WT mice were subdivided into 24-hour(24h)group,48-hour(48h)group,72-hour(72h)group,and 7-day(7d)group(10 mice/group),both of the control groups(8 mice/group)of TG and WT mice were sacrificed at 24hs after the final challenge,adoptive transfer group of TG mice contained 14 mice,while that of WT mice contained 20 mice,sex in half in all group.The procedures are briefly described as follows:(1)induction period:treatment of exposure mice with TCE:100μl of 20%TCE and Freund complete adjuvant(1:1)mixed fluid was used for the first sensitization on first day by intradermal injection into the upper back,and 100μl of 50%TCE was then painted on the upper back skin on 5th,12th,and 19th day;(2)stimulation period:a week later,30%TCE was painted twice challenging the low back skin and left ear on 26th and 28th day.The solvent control group followed the same procedure without TCE during the induction period,while stimulation was the same.Cutaneous reaction scores of stimulating skin were judged by "Eczema Area and Severity Index(EASI)scoring system" after 24 hours.100μl EdU solution(5mg/ml,solvent:dimethylsulfoxide(DMSO))was intraperitoneal(IP)injection at 24hs before sacrificed to detect the lymphocyte proliferation.The result showed,TCE-treated TG mice groups contained 46 positive mice in 49 animals,incidence was93.88%,TCE-treated WT mice groups contained 22 positive mice in 59 animals and the sensitization rate was 37.32%,the difference was significant(P<0.05).The EASI Scores of TG positive mice(2.28±0.78)were significantly higher than that of WT mice(1.68±0.67)(P<0.05).3.The evaluation of the HLA-B*13:01 TG mice model of human TIHD1)Skin damage and draining lymph node swelling:skin and ear histopathology showed a large number of lymphocyte infiltration,and the lymphocyte infiltration increased when symptom aggravated.The results of immunohistochemical showed:compared with the degree of CD4+、CD8+T cell infiltration of control group ear[(5.40±1.67)%and(10.60±3.65)%],the TG mice TCE-treated positive group[(16.80±5.26)%and(19.20±5.07)%]increased significantly(P<0.05);compared with the degree of CD4+、CD8+T cell infiltration of control group skin[(17.20±4.15)%and(16.60±2.70)%],the TG mice TCE-treated positive group[(29.80±7.22)%and(21.00±2.92)%]increased significantly(P<0.05);the degree of CD4+T cell infiltration was more serious than that of CD8+(P<0.05).There were significant(P<0.05)increases in the relative ALN weight,which was observed in 72h TG mice sensitization-positive group(0.0300±0.0141)%compared with control(0.0094±0.013)%.The ear swelling of 48h(1.1357±0.1306)and 72h TG mice positivegroups(1.2678±0.1694)increased significantly(P<0.05)compared with TG mice control group(1.0467±0.0979).2)Liver damage:liver histopathology of positive TG and WT mice showed liver cell swelling,disordered arrangement,lymphocyte infiltration,the degree of TG mice liver damage was more serious than that of WT mice.The results of immunohistochemical showed:compared with the degree of CD4+、CD8+T cell infiltration of control group[(11.60±4.39)%and(15.60±7.23)%],the TCE-treated TG mice positive group[(28.80±5.36)%and(30.60±6.58)%]increased significantly(P<0.05);24h positive TG mice group had significantly higher levels of serum ALT(104.67±32.64)and AST(272.00±62.25)than those of control group[(49.33±6.77)and(133.14±34.59)](P<0.05).3)Passive hypersensitivity:separating positive mice(donor)serum and lymphocytes,serum or lymphocytes alone or combination both of them injected by tail vein into healthy mice(receptor),the incidence of receptor mice was observed at 24h after 2-time TCE inducing in vitro,the control was the immune adoption that the health mice played as donors.Accepting positive mice immune transfer was called positive receptor and accepting negative mice immune transfer was called control receptor.The incidence of control receptor mice was 0%,combination of serum and lymphocytes could transfer similar hypersensitivity skin reactions to recipient animals when they were challenged with TCE in both TG and WT mice;The results of adoptively transferred serum or lymphocytes alone showed that lymphocytes alone could make disease reappear on recipients(4 mice developed disease in 8 positive receptors,incidence was 50%)while serum alone failed(there was no positive mice in 7 positive receptors).The hypersensitivity incidence of combination of serum and lymphocytes adoptive transfer was higher than that of lymphocytes adoptive transfer,and the symptom of combination of serum and lymphocytes adoptive transfer was also more serious.4)T Lymphocyte Activation:In spleenThe percentage of CD62L+ T cells of TG control group was(60.85±5.56)%,while that of 48h positive TG mice was(43.87±11.52)%,the decreases were significant(P<0.05).In auricular lymph node(ALN)The percentage of CD44+ T cells of TG and WT control group were(71.33±2.51)%and(69.53±1.02)%,while 24h,48h positive TG mice and 72h positive WT mice were(76.67±0.74)%,(77.60±3.02)%and(76.03±0.45)%,all the increases were significant(P<0.05).The percentage of CD62L+ T cells of TG and WT control group were(67.87±0.85)%and(84.07±0.45)%,meanwhile 24h、48h positive TG mice and 48h,72h positive WT mice were(53.97±0.50)%,(54.04±6.82)%and(76.67±4.94)%,(54.22±15.18)%,respectively,all the decreases were significant(P<0.05).5)T Lymphocyte Proliferation:In spleen① The percentage of proliferation of lymphocyte of TG and WT control group were(3.59±0.50)%and(3.88±0.82)%,while 48h,72h positive TG mice and 72h positive WT mice were(6.54±3.18)%,(10.06±6.46)%and(6.59±2.51)%,the increases were significant(P<0.05).(2)The percentage of CD3+ proliferation of TG control group was(0.57±0.12)%,positive TG mice 48h,72h and 7d were(0.73±0.09)%,(1.24±0.43)%and(0.74±0.14)%,increases were significant(P<0.05).③The percentage of CD4+ proliferation of TG control group was(0.58±0.12)%,positive TG mice 48h and 72 h group were(0.78±0.15)%,(1.32±0.69)%,increases were significant(P<0.05);④The percentage of CD8+ proliferation of TG control group was(0.22±0.10)%,while that of 72h positive TG mice was(0.40±0.12)%,difference between them was significant(P<0.05).⑤The ratio of CD4+/CD8+ of TG control group was(1.36±0.05)%,while that of 24h positive TG mice was(1.58±0.11)%,the increase was significant(P<0.05).In inguinal lymph node(ILN)The proliferation of lymphocyte:compared with control group[(1.54±0.15)%],positive TG mice 48h group[(1.93±0.25)%]increased,difference between them was significant(P<0.05);②The percentage of CD4+ proliferation of TG and WT control group were(0.69±0.17)%and(0.90±0.09)%,positive TG mice 48 h,72 h and positive WT mice 24 h were(0.88±0.18)%,(0.88±0.10)%and(1.15±0.21)%,increases were significant(P<0.05);③the ratio of CD4+/CD8+:compared with control group[(0.79±0.05)%],positive TG mice 24 h and 48 h were(0.89±0.09)%and(0.95±0.08)%,increases were significant(P<0.05).In ALN① The percentage of proliferation lymphocyte of TG and WT control group were(1.53±0.06)%and(2.49±0.11)%,positive TG mice 24 h,48 h and positive WT mice 72h were(2.27±0.21)%,(2.25±0.21)%and(3.65±0.79)%,increases were significant(P<0.05);②the ratio of CD4+/CD8+ of TG and WT control group were(0.95±0.05)%and(0.99±0.10)%,positive TG mice 48 h and positive WT mice 24 h group were(1.09±0.05)%and(1.12±0.06)%,increases were significant(P<0.05).6)There was a rising trend in the Serum IgG level of positive TG mice from 24 h to 72 h,compared with control group[(4.42±1.07)μg/ml],positive TG mice 72 h group[(5.75±1.29)μg/ml]was higher,but there was no significant between them(P=0.056).In summary,1)the HLA-B*13:01 gene expressed well in TG mice;2)we had established TG and WT mice TIHD disease model successfully,TG mice were more sensitive than WT mice to TCE sensitization and obtained more serious symptoms;3)the symptoms of TG mice was more similar to human disease than WT mice model;The effect of cellular immune and humoral immunity of TG mice model were stronger than those of WT mice model;4)cellular immune played a important role while humoral immunity as an auxiliary role in disease;5)the immune reaction process of TG mice model was that:starting at 24h after stimulation,strengthing gradually from 24 to 72 h,reached cellular immune peak at 72h,7 d gradually declining;while the immune reaction progress in WT mice model was delayed. |
