Effect Of Danshen And Ligustrazine For The Expression Of Relaxin Recepter (LGR7) On Health People And Scleroderma Fibroblasts

ObjectiveBy observing the effect of Danshen and Ligustrazine,traditional Chinese medicine,on the fibroblast' s value appreciation of skin lesion of the normal people and the sick with scleroderma,together with relaxin receptor' s change in the fibroblast expression and the effect of Danshen and Ligustrazine on relaxin receptor,thus this paper probes into the inhibitory mechanism of traditional Chinese medicine against fibroblast' s value appriciation,and whether the relaxin receptor can serve as the target point of drug action in anti-fibrosis.Method1.Fibroblast culture In sterile surroundings,put plasma in DMEM'culture solution to rinse,snip it into plasma of 1 mm×1 mm, evenly coat it onto the culture bottle,pour in DEME culture solution with fetal calf serum of 20%and remove culture solution every 2-3days.Fibroblast will spring up around the plasma in 7-14 days.When nearly 80%of the culture bottle is covered by fibroblast after about 20days,continue transfer of culture in a 1/2 proportion, 'transfer the culture every 3-4 days,and select the fibroblast cultured in logarithmic phase to conduct experiment.2.MTT Value determination of fibroblast With 10%DMEM,adjust fibroblast cultured in logarithmic phase to 1×10⁴/mL,inoculate in the culture plate of 96 holes,each of which is filled with 180μL cell suspension,after 24hours,fibroblast will adherent to the bottle,then respectively add in drug and make a blank control group. After48h in the culture incubator of 5%CO₂,37℃,fill each hole with 5mg/mL MTT 20μL,continue culture for 4h,add in DMSO 150μL per hole,shake for 10min,exam the absorbance value at 492nm with ELISA.3.S-P of Fibroblast Adopt the S-P method,operate according to the workflow in the instruction of the S-P kit,and experiment with a negative control(PBS in the place of LGR7)4.Flow cytometer With 10%DMEM,adjust the fibroblast cultured in logarithmic phase to 1×104/mL,prepare 100ml culture bottle, and pour 10mL cell suspension in each bottle(1*105 fibroblast/bottle),preset culture for 48h.According to the experimental groups,after adding in pastille medium,continue culture fibroblast for 24h.prepare for the cell solution,add 4% paraform to fix,centrifugal,remove fixing solution,add 3mLPBS to wash for twice,add relaxin receptor antibody,incubate for 1h under 37℃,add 3mLPBS to wash for twice,add FITC antibody, incubate for 30min under 37℃,Keep in Dark Place,test by flow cytometer. 5.Adopt statistical package SPSS11.0,3,statistics are manifested as mean±standard deviation((?)±s),test counting data between groups with t.Result1.More than 10mg/mL Dans hen has inhibit function on the fibroblast value appreciation of the normal people and the sick with scleroderma;more than 0.5mg/mL Ligustrazine has inhibit function on the fibroblast value appreciation of the normal people and the sick with scleroderma,when reaching to 0.5mg/mL,it can completely inhibitate value appreciation of fibroblast.2.When normal people' s fibroblast plasmin and peplos are brown yellow,LGR7 is scored to be positive;when they in the sick with scleroderma show no brown yellow,LGR7 is scored negative.3.Traditional Chinese medicine Danshen and Ligustrazine both can depress the LGR7' s expression quantity and intensity on fibroblast of the health people and the sick with scleroderma.ConclusionConelusionBoth Danshen and Ligustrazine in some degree of concentration have inhibit function on the fibroblast value appreciation of normal people and the sick with scleroderma.Danshen and Ligustrazine both can depress the LGR 7' s expression quantity and intensity on fibroblast of the normal people and the sick with scleroderma.