| Objective: Cancer is the most hazardous disease to human health.There are many ways to treat cancer including application of Chinese herbal medicines.Based on Shenqi tablet,Shenqi compound (SQ)is made with addition of mongolian milkvetch root,chinese angelica,zedoray,spreading hedyotis herb and etc.It is reported that SQ can suppress cancer in vivo and in vitro of mouse.However,it is still unknown that whether SQ has the same effect on human cancer.For providing theoretic foundation for developing anti-tumor drugs,this experiment will find out how SQ work on human cancer and its correlate mechanisms by studies on SQ's effects on human high-metastatic lung cancer(PG)and its effects on the expression of PG's surface adhesion molecules(CD44,CD54).1.Methods:1.1 Applicating MTT assay and flow cytometer and study SQ's suppression effect on PG cells in vitro.PG cells cultivated in vitro are made to cell suspension.Adjust cell concentration to 1.0×106/ml,add cells to 96 pores plate,then add 100μl SQ of different concentration,make it as experimental group.Add NS 100μl as cntrol group.5 parallel pores in each group,and put them all in incubated box and cultivate.Centrifugate and reject superior clear fluid on each 12,24,48,72 hours later.Then add 20μl MTT application fluid ,4 hours later,add each pore DMSO resoluting fluid 100μl,agitate it fully and make particles resolute wholly,estimate OD value of every pore by enzyme-labelled meter and get the inhibition rate of PG cell.After transfer of culture of PG cells,adjust cell concentration to 5.0×105/ml,then add it to 24 pores plate,about 1ml per pore.Then add different concentration of SQ,about 1ml per pore.Add 1ml NS to every pore in control group.4 parallel pores in every group.Put them in 37℃,5%CO2 incubated box for 48 hours.After PI staining according to literature,detect PG cell's proliferation cycle after the effect of SQ by flow cytometer and get the proliferation index(PI).1.2 Applicating fluorescence microscope and flow cytometer fluorescence labeling assay,study adhesion between PG cell and endothelial cell,expression of adhesion molecules and anti-metastasis effect of SQ in vitro.After transfer of culture of PG cells,divide into 3 groups basing the concentration of SQ(25μg/ml,50μg/ml,100μg/ml)and control group (the same volume of NS added to culture medium ).After 72 hours,CD44-FITC and CD54-FITC are added to SQ groups,isotype contrast IgG-FITC is added to control group,after incubating for 30 minutes at 4℃,detect the expression of CD44 and CD54 on the surface of PG cells by flow cytometer.After the fetal umbilical vein endothelial cells are collected,inoculate them on 96 pores plate.Then add PG cells marked with 5,6-CFDA,and add SQ of different concentration to SQ groups.After 60 minutes incubating in CO2 cultivated box,add CD34-PE for continuous incubating with adhesive cells for more 30 minutes,detect the adhesion rate of PG and endothelial cells by fluorescence microscope. Collect PG cells,then add them to 96 pores plate which is already covered with endothelial cells.Add SQ of different concentration,cultivating for 60 minutes in CO2 box,then elute adherent PG cells by pancreatin.Label these PG cells with CD44-FITC,CD54-FITC,then detect the expression of CD44 and CD54 on the surface of PG cells by flow cytometer.Put lipopolysaccharide and endothelial cells together and incubate them for 6 hours at room temperature so as to make endothelial cells activated sufficiently.Then perform adhesion examination between PG cells and endothelial cells and detect effect of SQ on the adhesion of them as the methods which have been mentioned.2.Results:2.1 The results of the effect of SQ on PG cell in vitro:All 3 different concentrations of SQ has clear direct kill effect to PG cells,and among the concentrations 50μg/ml SQ can show us strong kill vitality.The kill intensity of SQ shows dependence on concentrations and time in a certain range.As the prolongation of time and the increase of the concentration,the kill e...
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