| Differentiation therapy has become a new breakthrough among blood system neoplasms which makes tumor cells reversely differentiate into normal cells or nearly under the effect of the differentiation inducers.Retinoid is the representative of differentiation inducers.? We modified the cyclohexene ring and reformed the polar group at the end of the carbon chain with all-trans retinoic acid(ATRA) as the lead compound, synthesized a series of new retinoic acid derivatives which had been depurated and identified.4-amino-2-trifluoromethyl-phenyl retinate(ATPR) has powerful activity in inhibiting proliferation and inducing NB4 cell differentiation after screening of the derivatives with structure-activity,.In this paper,non-obese diabetic/severe combined immunodeficiency(NOD/SCID) is designed to investigate ATPR replication in vivo pharmacological effects of leukemia and we also disscuss how retinoic receptors develop role while ATPR induces NB4 cell differentiation in order to futher clarify its mechanisms.Objective: To investigate ATPR on human leukemia NOD/SCID mouse model and retinoid receptors how to develop role while ATPR induces NB4 cell differentiation.Methods:After adapt to a week in the SPF level Laboratory Animal Center of Anhui Province, NOD/SCID mice were received 2.5Gy X-ray irradiation, and they were inoculated i.p with 3×106 NB4 cells per animal within 24 hours after irradiation. After 24h inoculation ,the mices were randomly divided into 6 groups:control group; model group; ATRA group (10mg/kg); ATPR group (5mg/kg, 10mg/kg, 20mg/kg). With the first injection given 7 days after inoculation with leukemia cells,the control group and model group were injected with polyoxyethylene castor oil (solvent) and other groups were started to inject corresponding doses of drugs,once every other day,for four weeks.Observed animal time of death and recorded the survival days of Statistics;Observed and recorded body weight in each group;Collect ascites tumor cells in each group:①Nested RT-PCR detection of PML/RARαfusion mRNA in ascites tumor cells;②The chromosomes of ascites tumor cells and NB4 cell line were observed by the way of chromosome analysis;③To observe the morphology of ascites cells by microscope and electron microscope;④Ascites cells differentiation indexs were analyzed by NBT reduction test.Collect abdominal tumor cells in each group:①CD45,CD33 and CD11b of abdominal tumor cells were detected by flow cytometry(FCM);②The cell cycles of tumor was detected by flow cytometry(FCM);③The pathology of abdominal solid tumors were analyzed by haematoxylin-eosin. To inspect the infiltration of NB4 cells in mouse:①To odserve the body weight of each group;②The expression of the maturation specific cell surface markers CD45 and CD33 of liver, spleen, kidney and lung were detected by flow cytometry(FCM);③The cell cycles of liver and spleen were detected by flow cytometry(FCM);④The pathology of liver,spleen,kidhey,lung,myocardium,brain and bone marrow were analyzed by haematoxylin-eosin. Parts of mechanism study:①The expression of RARα,RARβ,RARγ,RXRα,RXRβ,RXRγmRNA and PML/RARα,RARβ,RARγproteins of NB4 cells were detected by RT-PCR and Western Blot respectively;②the expression of RARβ,RARγ,RXRαprotein of tumor tissue of leukemia model were detected by immunohistochemistry.Results:1 In the established animal model of leukemia, NB4 cells could infiltrate into the liver, spleen, kidney, lung and other organs, the model have been set up successfully.2 ATPR could induce differentiation on APL-NOD/SCID ascites tumor cells: ascites tumor cells could been induced differentiation and maturation of the granulocyte changes, and NBT-positive rate increased.3 ATPR could induce abdominal solid tumor cells differentiation on APL-NOD/SCID mouse: the expression level of the maturation specific cell surface marker CD11b increased, and the G0/G1 phase cells of solid tumor cells increased. Meanwhile,the pathological examination revealed that tumor cells had nulocyte differentiation and maturation changes.4 ATPR could reduce infiltration in vivo mouse model: The CD45+CD33+ positive rate of liver, spleen, kidney, lung and other major organizations reduced in different degrees,pathological examination was also aim to the key, and the G2-M+S phase cells of liver and spleen decreased.2 ATPR could prolong the survival of APL-NOD/SCID mouse.6 After treatmented with ATPR,the mRNA levels of the RARα,RARβ,RARγwere significantly increased in a time-dependent manner while RXRα,RXRβ,RXRγmRNA expression was no significantly reduced,PML/RARαfusion protein significantly reduced while RARβ,RARγprotein were significantly increased,and was time dependent while ATPR could increase retinoic acid receptor tumor expression of RARβand RARγ, but had no effect on RXRα.Conclusion:1 APL-NOD/SCID model can be successfully established.2 ATPR can significantly extend survival of leukemia mice, and has powerful activity in inducing leukemia cells differentiation.3 It is likely that ATPR binds RARs,and specific binds to the retinoic acid response element (RARE) in the target gene regulatory region to selective activation or inhibition of transcription in regulating NB4 cell proliferation and differentiation and on the other hand,ATPR can decompose the fusion protein of PML/RARαin NB4 cells,and stimulate the pathways of nuclear hormone differentiation depended by RXRα... |
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