A Study Of The Treatment Effect Of Tanshinone ⅡA Combinated With Arsenic Trioxide On Xenograft Of NB4 Cell In Nude Mice

ObjectiveTo observe the therapeautic effect of Tanshinone ⅡA Combinated with Arsenic Trioxide by building a model of Xenograft of human leukemia cell line NB4 in nude mice,in order to provide a reliable basis for clinical treatment or auxiliary treatment for acute promyelocytic leukemia(APL) of Tanshinone ⅡA.Methods1. NB4 cells were cultured in RPMI1640 medium which containing 1% penicillin and streptomycin,also containing 10% fetal bovine serum.the temperature of the incubator was 37 ℃ and the carbon dioxide saturation was 5%,the medium was changed every three days.the logarithmic growth phase cells were used by subsequent.2. Xenograft tumor model of human acute promyelocytic leukemia cell line NB4 cell in nude mice was built by ways of inoculating subcutaneously, All the tumor bearing mice were randomly divided into four groups:the control group,30mg/kg.d TanⅡA group,5mg/kg.d ATO group,combination group (30mg/kg.d tan ⅡA +5mg/kg.d ATO).the mice were given drugs for 14 days continuously.3. Observing the time of forming xenograft in nude mice,growth characteristic of the xenograft during the administration time.measuring the maximum diameter and the shortest diameter of transplanted tumor,according to the volume of the tumor drawing a growth curve of the transplanted tumor.the quality of the tumor were measured after the mice killed.the inhabition rate of tumor were calculated accrding to the calculation formula.4. The organization of tumor,liver, spleen,kidney and lymph node were stained by HE after the bearing tumor mice sacrificed.5. RT-PCR was used to detect the mRNA level of mTOR and AKT of the tumor.immunohistochemical staining was used to detect the expression level of the protein of caspase-3 and bax.the professional image system was used to manage the picture.6. The data of experiment were analyzed and processed by SPSS 17.0 for windows.Results1. Macroscopic tumors were observed in all of the mice from the fifth day to the ninth day after inoculation,the transplanted tumor grown rapidly.2. The qualityof the tumor in control group was the largest,followed by Tan ⅡA group and ATO group.And the combination group was the smallest.he inhibition rate of the tumor in tan ⅡA group was the lowest,followed by ATO group.And the rate in combination group was the highest.3. All the organizational structure were intact,There were no pathologic change and metastasis in the liver, spleen,kidney and lymph node of all the mice,4. The relative expression level of the mRNA of mTOR in control group, tan ⅡA group,ATO group and combination group is 0.907±0.059,0.883±0.070, 0.821±0.039,0.643±0.103.and the AKT is 0.872±0.075,0.839±0.071,0.776±0.054, 0.705±0.060.5. The positive expression of the protein of Caspase-3 in control group, tan ⅡA group ATO group and combination group is 3(16.7%),4(22.2%),9(50.0%) and 13(72.2%).and the Bax is 4(22.2%),7(38.9%),12(66.7%) and 16(88.9%).ConclusionATO alone and Tan ⅡA in combination with ATO can inhibit the growth of the xenogrfts.Tan ⅡA in combination with ATO can inhibit the relative expression level of the mRNA of mTOR and AKT ,TanⅡA in combination with ATO can induce apoptosis through upregulation the expression of Caspase-3 and Bax.