Experimental Study On Reversal Of P-gp Expression In P388 / VCR Nude Mice Model By Compound Jiedu Huayu Chinese Medicine

Objective:This study is to establish P388/VCR multi-drug resistant leukemia cells in animal models, it compared with the normal control group by extracting ascites, peripheral blood and bone marrow smearing, establish an animal model of success is identified by the number of leukemia cells; This experiment to determine medicine Jiedu huayu large, medium and small-dose group compound with vincristine compatibility reversing multidrug resistant leukemia effect, which through the basic situation after treatment of multi-drug resistant leukemia nude model and quality of life in animal models. The experiment explore the molecular mechanisms of compound Jiedu huayu Chinese medicine to reverse multidrug resistance of leukemia by detecting multidrug resistance protein (P-gp).Methods:(1)Cell culture:the leukemia cell lines P388and the drug resistance of leukemia cell lines P388/VCR were placed in RPMI1640containing10%fetal bovine serum medium, cultured in the condition of37℃, saturation humidity,5%C02conditions. Each nude mice was inoculated directly to0.2ml (the concentration of1X107个/ml) of cell suspension under sterile conditions using the logarithmic growth of leukemia cells. The mice were randomly divided into normal-control group, model group, vincristine treatment group、Experimental group1(large-dose combination group Jiedu Huayu medicine+vincristine), experimental group2(medium dose combination medicine Jiedu Huayu+vincristine group), group3(small dose combination medicine+vincristine Jiedu Huayu alkali group)6groups after ascites tumor touch-type establishment, Mice were given distilled water, saline, intraperitoneal injection of vincristine, large, medium, small doses of medicine administered intraperitoneal injection of vincristine, are0.1ml/10g volume weight. Among them, the mice were injected with vincristine once every two days, five consecutive times; Compound Jiedu huayu medicine administered once daily, continuous medication for10days. Peripheral blood was detected during the experiment once every two days. Dynamic observation of the animals spirit, diet and weight change before making the model and after administration, we should pay attention to this situation of ascites, tumor mass and survival of mice.(2)Multiple drug resistance was detected by MTT assay of P388/VCR resistant cells in vitro and in vivo.(3)Leukemia cells:Peripheral blood of mice, and ascites (ascites cells were mixed with25μl fetal bovine serum after centrifugation) and bone marrow (marrow mixed with25μl fetal bovine serum) is extracted for smear, Giemsa staining, cell morphology was observed in oil microscope, Percentage of leukemia cells (original+promyelocytic) calculated by pressing the cell morphology,which determine the animal model is successful.(4)Nude mouse model of leukemia MDR cell remission is observed, according to the spirit of nude mice, dietary conditions and extraction nude ascites, peripheral blood or bone marrow smears sternum observe ascites, peripheral blood and bone marrow blast percentage+promyelocytic cells, P-gp expression, and to measure the volume of tumor mass nude before the end of the experiment.(5) Expression of multidrug resistance protein P-gp were observed in the multidrug resistance of leukemia cell line P388/VCR, which detected by flow cytometry on nude mice bearing human peripheral blood, to stop drug, feed the extraction of nude mice peripheral blood. Results:(1) Nude mice by intraperitoneal injection of0.2ml/只cell volume of1X107cells/ml, the average time of ascites formation was5.38±0.84days, the average number of days to form the tumor masses was8.63±0.67days, The weight of nude mice with the extension of time, the body weight increased significantly before nude mice were inoculated with leukemia cells throughout the course of the experiment; The weight of nude mice was significantly increased when ascites, tumor formation at approximately5-8days after inoculation of leukemia cells, the spirit of the nude mice and diet deteriorate with the extension of time in model group, but the spirit of nude mice and diet significantly improved in drug group compared with the model group, nude body weight decreased slowly.especially large、medium-dose medicine group.(2) P388/VCR cell resistance multiples in vitro is5.72times by MTT assay;The resistance multiples is4.8times by measure when leukemic cells inoculated in nude mice. Compared to the in vivo and in vitro P388/VCR multiple drug resistance,Multiple drug resistance in vivo has been reduced, but remained resistant.(3) Nude mice will form ascites、tumor mass after leukemia cells were inoculated in it, and peripheral blood、ascites and sternal bone marrow were drawn、smears, they were infiltrated leukemia cells,And nucleated cells per smear were more than200/片.(4) The relief of nude mouse model of leukemia multidrug resistance: Vincristine treatment remission is1/6, the experimental group is4/6, the second group is3/6, the third group is1/6, leukemia relief situation is better in the experimental group1, experimental group2, But the leukemia is poor in vincristine treatment group and experimental group3.(5)Nude mice peripheral blood were detected by flow cytometry, p-gp almostly no express in normal control group, p-gp is high expression in model control group, the vincristine treatment group and small doses of Chinese medicine Combination with vincristine group can reduce the p-gp experssion, But the comparison was not statistically significant (P>0.05),the small dose of Chinese medicine group compared with high, middle dose of Chinese medicine group combined with vincristine group have significant difference (P<0.01), the expression of P-gp was significantly decreased. Large, middle dose of compound Jiedu Huayu Chinese Medicine group combined with vincristine groups can better suppress the expression of P-gp. there were significant differences between the two phases (P <0.05).Conclusion:P388/VCR resistant leukemia cell lines still keep the cell resistance in vivo after the cells were seeded in nude mice. The spirit of nude mice, diet and activities significantly improved in drug group compared with the model group, nude body weight decreased slowly, especially large、medium-dose medicine group. Medium-dose Practitioners staggering.The compound Jiedu Huayu Chinese medicine can reverse P-gp expression in P388/VCR cells in the model nude mice; The compound Jiedu Huayu Chinese medicine can reverse the mechanism of leukemia cell lines resistant P388/VCR may be related to the reduced expression of the multi-resistant genes P-gp. Compound jiedu Huayu Chinese medicine can reduce the expression of P-gp of P388/VCR resistant cells in nude mice model, which decreased in a dose-dependent manner.