The Study On The Effect Of Phorbol Ester Combination With Granulocyte Colony Stimulating Factor On Hematopoiesis In Myelosuppressed Patients

BackgroundMyelosuppression is the most common adverse reactions of cancer patients who usechemotherapy drugs, severe bone marrow suppression often makes chemotherapy difficult to continue asplanned, may be bring out the complications, could be life-threatening. Recently, G-CSF、EPO are usedwidely to treat leukopenia or anemia which coursed by chemotherapy and radiotherapy.But only G-CSF,EPO can not recover medullary hematopoiesis in the short term for who were accepted high intensity ormany times chemotherapy. Because chemotherapy drugs can hurt the normal hematopoietic cells andmicroenvironment in the bone marrow, when they are killing cancer cells. Meanwhile G-CSF、EPO play arole in the downstream of hematopoietic precursor cells. Such as G-CSF effects on myeloid progenitorstage, and stimulate them proliferation, differentiation and promote mature neutrophils to release into theperipheral blood. EPO paly a role on erythroid progenitor cells stage, to stimulate erythropoiesis,increasing the number of red blood cells in peripheral blood. But they have no effects on bone marrowmicroenvironment all. Therefore it is important to find a way to quickly restore bone marrowhematopoietic. It is reported that12-O-tetradecanoylphorbol-13-acetate which is called phorbol ester(TPA),not only can induce a variety of leukemia cells to normal cells, but also TPA can promote bone marrowhematopoietic.ObjectiveTo explore the effect of TPA alone or combined rhG-CSF on bone marrow hematopoietic cellsproliferation and colony formation ability which from patients of acute myeloid leukemia(AML) in the period of bone marrow suppression in vitro，and to observe the effect of TPA on bone marrow stromalcells(BMSCs) proliferation or inhibition from patients of AML in the period of myelosuppression and thehealthy persons.Methods1.Cultured bone marrow mononuclear cells(MNC) from AML patients in the period ofmyelosuppression by methylcellulose semi-solid medium. Each experiments use the same bone marrowfrom the same patients. Groups: control group, TPA concentration of10ng/ml, G-CSF concentration of50ng/ml, TPA combined with G-CSF group (final concentration of10ng/ml+50ng/ml), different groupsof drugs were added to the two culture medium,one contains no any stimulating factors, one contains EPO,phytohemato-agglutinin leukocyte conditioned medium(PHA-LCM),to observe the formation ofCFU-GEMM, CFU-GM, BFU-E, CFU-E and otherclonies.2. The TPA of different concentrations (range of0.1~30ng/ml) were added to incompletemethylcellulose semi-solid medium, and set up blank control, to observe the formation of CFU-GM,CFU-GEMM, BFU-E, CFU-E and otherclonies. The MNC were from the same sample of AML patients inthe period of myelosuppression.3. TPA of different concentrations (range of0.1~30ng/ml) and G-CSF(50ng/ml) were addedto incomplete methylcellulose semi-solid medium, and set up blank control, to observe the formation ofCFU-GM, CFU-GEMM, BFU-E, CFU-E and otherclonies. The MNC were from the same sample of AMLpatients in the period of myelosuppression.4. Cultivation of BMSCs from healthy persons and patients of AML in the bone marrowsuppression phase in vitro, added to TPA of different concentrations,0.1ng/ml,1.0ng/ml,5ng/ml,10 ng/ml,20ng/ml,30ng/ml, and setted up a control to to detect cell proliferation or inhibition. with CCK8method.Result1. Compared the clonies of four groups in incomplete medium, in the control group a small cellclusters can be seen for24-72hours,but the cells were dead as time prolong. Cultivated14days, controlgroup have noclonies formation, CFU-GM are dominated for G-CSF group,TPA alone and combinationwith G-CSF group have myeloid colony formation, including CFU-GM、BFU-E and CFU-GEMM. ForG-CSF group only have CFU-GM formation, G-CSF alone have no effect on erythroid and early mixedcolony forming; TPA and G-CSF synergistically promote the formation of CFU-GM(P<0.05), but thecombination of both have no effect on the formation of CFU-GEMM and BFU-E(both P>0.05).2. Compared theclonies of different concentration of TPA stimulation in incomplete medium, Wecan see CFU-GM formed in the concentration range of1~30ng/ml, CFU-GEMM formed in theconcentration range1~20ng/ml,BFU-E formed in the concentration range5~20ng/ml. For TPA alone,thebest final concentration is the range5~10ng/ml to stimulate CFU-GM and CFU-GEMM formation,thereare statistical significance (P<0.05).While decreased or increased the concentration of TPA can notimprove the numbers of the colonies. The best final concentration of TPA is10ng/ml to stimulated BFU-Ecolony forming (P <0.05).3. Compared theclonies of different concentration of TPA and G-CSF(50ng/ml) stimulation inincomplete medium.The main clone types are myeloid colones, CFU-GM, also have CFU-GEMM andfewer BFU-E. Combination with G-CSF the better concentration of TPA is1~5ng/ml for CFU-GMforming (both P<0.05). Combination with G-CSF the best concentration of TPA is5ng/ml forCFU-GEMM forming.In5~20ng/ml groups the numbers of BFU-E are the highest. 4. Compared theclonies of four groups in complete medium,clonies can be seen in four groupsincluding of CFU-GM, CFU-GEMM, BFU-E, CFU-E. The CFU-GM of joint group are highest comparedwith TPA group, G-CSF group and control group, difference have statistical significance (both P<0.05).The number of CFU-GEMM of TPA group and joint group are higher than control group and G-CSFgroup(both P<0.05),but there are no statistical significance between TPA group and joint group(P=0.98).The number of BFU-E have no statistical significance between TPA group and jointgroup(P=0.38);The number of CFU-E also have no statistical significance for four groups(P=0.34).5. Experiments show that TPA promote bone marroe stromal cells from healthy personsproliferation of concentration5~20ng/ml (cell number2x105/ml). Decreasing or increasing theconcentration of TPA, it shows that BMSCs are inhibited. Meanwhile, TPA promote bone marroe stromalcells from the AML patients proliferation of concentration5~30ng/ml (cell number2x105/ml).5ng/ml ofTPA is the best concentration of promoting proliferation, and G-CSF had no effect on the growth ofBMSCs from healthy persons or AML patients.Conclusion1. TPA alone can promote hematopoietic cell CFU-GM、CFU-GEMM、BFU-E forming,mainlyof CFU-GM for bone marrow of AML patients in the period of bone marrow suppression in vitro,the bestconcentration range is5~10ng/ml.2. TPA combined with G-CSF (50ng/ml) have synergistic effect on CFU-GM formation, the bestconcentration of TPA is1~5ng/ml，but have no synergistic effect on CFU-GEMM and BFU-E formation.3.G-CSF alone can promote hematopoietic cell CFU-GM forming for bone marrow of AMLpatients in the period of bone marrow suppression in vitro,but have no effect on the CFU-GEMM andBFU-E forming. 4. TPA can promote the growth of BMSCs of healthy persons and patients of AML in the bonemarrow suppression phase in vitro, the best concentration of promoting proliferation is5and10ng/mlrespectively.