Empirical Study Of Dulcamara Effective Locus Cytostasis And Inducing Apoptosis To SGC-7901,Hela Tumor Cell

Apoptosis is the cell autonomic ordered decease by gene controlling in order to retain homeostasis. Apoptosis is the process by multiple gene strict controlling, these gene are extremely exactitude in the genus, exempli gratia Bcl-2 family,caspase family,anti-oncogene P53,oncogene C-myc and so on. From the new view angle about apoptosis to view the effect of Chinese crude drug opposing tumor, to reveal mechanism of action of Chinese crude drug, screening new anticancer Chinese crude drug possesses full crucial significance.Dulcamara is the plant of celastrus, distribute generally, its rattan,root,leafage,fruit are all put in drug. The modern chemical pharmacy has confirmed that some component(such as sesquiterpene esters) of dulcamara possess doughty antineoplasmic activity. at the present stage, there are no correlated report about dulcamara anticancer activity and mechanism. The topic is from the extraorgan to invest correlate study of dulcamara effective locus cytostasis and inducing apoptosis to tumor cell, to expand thinking on investigating the dulcamara further.1. purpose:To invest that acetoacetate extractive and n-butanol extractive of dulcamara inhibit tumor cell multiplication, ascertain that dulcamara effective locus possess the effectiveness of inducing apoptosis, reveal initially the route that these induce tumor cell apoptosis, all are established foundation to utilize in dulcamara exploitation.2. method:2.1 MTT methodApply MTT deoxidized method to survey the rate of acetoacetate extractive and n-butanol extractive of dulcamara inhibiting tumor cell in vitro. Gastric cancer-SGC 7901 and women carcinoma cervicis-Hela are effected by different density of the same extraction locus, it get dosage-effectiveness atlas by the inhibition ratio.2.2 Ambi-fluorescent labelling method-AO/EB Apply AO/EB ambi-fluorescent stain and observe apoptosis cell morphous in SGC-7901 and Hela cell effected by the acetoacetate extractive and n-butanol extractive of dulcamara through fluorescence microscope.2.3 FITC-Annexin V and PI ambi-staining methodApply flow cytometry to detect apoptosis cell population in SGC-7901 and Hela cell effected by the acetoacetate extractive and n-butanol extractive of dulcamara.2.4 Flow cytometry detect anti-oncogene-P53 proteinumApply flow cytometry to survey P53 proteinum transmutation in SGC-7901 and Hela cell, through indirect fluorescent labelling method. P53 proteinum Fluoresence Index(FI) express quantitative P53 proteinum. Green fluoresence LOG collect fluorescence intensity peak amplitude number, which convert linearity scale. It take peak amplitude number of contrast control tube as negative expression FI=1. FI calculated formula as follow: FI= mean fluorecence intensity of detector tube cell / mean fluorecence intensity of detector tube cell contrast control tube. For instance FI>1.0 for masculine expression, FI≤1.0 for negative expression2.5 Cell immunohistochemistryApply cell immunohistochemistry S-P staining method detect P53 proteinum masculine expressing rate in gastric cancer-SGC 7901 and women carcinoma cervicis-Hela.3. Result3.1 Four densities of the acetoacetate extractive and n-butanol extractive of dulcamara(15,30,60,120μg/ml) have determinate depressant effect on SGC-7901 and Hela cell, and to offer dose dependent. It has significance through statistics analysising discrepancy(P<0.01).3.2 Apoptosis cell definite marking is FITC+/PI- on flow cytometry atlas by the acetoacetate extractive and n-butanol extractive of dulcamara. The tumor cell is effected 24 hours by different densities of them, the rate of apoptosis cell step up obviously, and to offer dose dependent(P<0.01).3.3 Utilizing flow cytometry immunofluorescence technic detect P53 gene product, along with the accrescence of medicine density, P53 peak offset rightward overall. According to FI formula calculation, it could calculate FI numerical value. The tumor cell is effected 24 hours by the acetoacetate extractive and n-butanol extractive of dulcamara, P53 proteinum in them all show masccline expression, and the FI numerical value increase along with the accrescence of dosage.3.4 EB colorant is red on fluorescence microscope, it dye only cellular nucleus in decease cell. The cell membrane of normal living cell is integrity, EB could not percolate, and cell nucleus is unable to be dyed. AO colorant is green, could use for dyeing living cell. It could see the whole cell is green in control group, which is normal living cell. The tumor cell is effected by the acetoacetate extractive and n-butanol extractive of dulcamara, the whole cell background is green, the cell envelope is fairly integrity. The coincidence of red and green is yellow in cell nucleus, which is the morphology change on the apoptosising(apoptotic body).3.5 Utilizing cell immunohistochemistry detect P53 gene product, together with negative control group, P53 proteinum express all have different degree accrescence.4. Conclusion:The acetoacetate extractive and n-butanol extractive of dulcamara possess the fairly doughty effect of anti-tumor. Inducing cell apoptosis is one of the pathway that kill and wound the tumor cell, altering P53 expression is possible important mechanism that induce tumor cell apoptosis.