Cooling Blood To Eliminate Mange Soup Clinical Studies Of Psoriasis And Keratinocyte Cell Proliferation And Apoptosis

ObjectiveTo observe the clinical effect on Vulgaris Psoriasis of Liangxue Xiaopi Decoction,and study medicated serum of Liangxue Xiaopi Decoction's effect on Hacat through Cell Experiment.To provide scientific basis for Liangxue Xiaopi Decoction clinical application.Menthods一.clinical observation1.Selecting 46 example patients dialogued Vulgaris Psoriasis who come from psoriasis clinic in Wuhan First Hospital.They were divided into treatment group and comparison group by randomly.Treatment group includes 26 cases,comparsion group includes 20 cases.Two groups have no obviously difference at age,sex and course of disease(P＞0.05),has comparability.2.We give treatment group patiences Liangxue Xiaopi Decoction once a day,a half in the morning and another in the evening.At the same time,we give comparison group patiences Yujinyinxie Pill, three times one day,each time four pills.Two groups are all with external application coal tar.3.We set four weeks as a treatment course,a follow-up of 46 patients for two treatment courses.At the same time noting the ill reaction.4.Statistics:After observation,we analyzed the datas,the counting materials were examined with x~2,the measuring materials were examined with U-test.二.experiment1.To prepare medicated serum:Medicine was made into animal's equal dosage according to body surface area.The rats were given orally in the dose of 12.825 g/kg(equivalent to the crude herb).Rats received Liangxue Xiaopi Decoction at 0.5,1and2times of equivalent dose.They were the low,middle and high dose groups of Liangxue Xiaopi Decoction.We use saline solution instead of traditional chinese medicine to perfusion rats,blood sample was collected as negative control group.For 7 consecutive days,2 hours after the last perfusion,aspiration of aether solvent and obtaining blood from the abdominal aorta.2.Confecting culture medium:basic medium:DMEM medium with double distilled water and 2g/L NaHCO3,0.22μm microscope for filtration sterilization,preservation at 4℃.complete medium:90% basic medium and 10%Fetal bovine serum,100U/mL penicillin and Streptomycin.medium including medicine:80%DMEM,10%Fetal bovine serum and 10%medicated serum.3.HACAT cell culture:We culture Hacat with complete medium and 37℃5%CO2 incubator,subculture every 3 days or 4 days,choose cellls that in the logarithm phrase.4.Batching:Dividing the experiment rats into four groups:the low dose group,middle dose group,high dose groups and saline solution group by randomly,there are 6 rats in every group.5.Effect of medicated serum of Liangxue Xiaopi Decoction on Hacat cell proliferation:Hacats were inplanted in 96-well microwell with density of 2×10~4,200μL every well,removal of primary medium when cells adhensive.medium including medicine entered,setting 3 wells in each group.24 hours later,adding MTT(20μL every well), continuously culturing for 4h,the supematant was removed,adding 150μL DMSO every well,concussion for 10min,getting OD value with microplate of 492nm.6.Effect of medicated serum of Liangxue Xiaopi Decoction on Hacat cell apoptosis:Choosing Hacat cells at logarithmic growth phase, dilution into 1×10~4 cell suspensions.There are 10mL cell suspensions in every 100mL culture bottle,Pre-incubating for 24 hours.Adding medium including medicine according to Batching, culturing continuously for 24h.Hacat cells were made into cell suspensions,getting into 70%cold alcohol,Centrifugation and fixative solution was removed.Hacat cells were washed twice with 3mL phosphate -buff-ered saline(PBS),adding 1mLPI,incubating cells in the dark with 4℃refrigerator for 30 min,filtering.The sample was tested by flow cytometry.7.Statistical methods:Using SPSS13.0 software,all datas are expressed by the mean±standard deviation((?)±s),the measuring materials were examined with t-test.Result一.clinical observationTreatment group convalescing rate7.7%,showing results rate 57.7%,efficiency rate 27%,totle efficiency rate92.31%;Comparison group convalescing rate5%,showing results rate30%,efficiency rate 40%,totle efficiency rate75%,has obviously difference after statistics analysis(P＜0.05).二.experimentMTT experiment:the comparison group and the surplus four groups has significant difference,the high-dose group and the surplus four groups has significant difference.The low-dose group and middle-dose group has insignificant difference.The low-dose group and normal salint group has insignificant difference.the middle-dose group and normal salint group has insignificant.Flow cytometry:Compared with negative controls,pronounced difference(p＜0.05))was detected by flow cytometry in apoptotic rate.ConclusionThe Liangxue Xiaopi Decoction is effective for the treatment of vulgaris psoriasis,is better significantly than the comparison group. Cell experiment indicated that Liangxue Xiaopi Decoction can obviously inhibit proliferative HaCaT cells,and encourage apoptosis HaCaT cells.This process is in a dose-dependent manner,and compared with that in the control group,the difference was significant.