Research On The Effects, Mechanism And Clinical Siginificance Of ATRA Drug-resistance Related New Gene HA117

PART ONE EFFECTS AND MECHANISMS OF HA117INTREATING ACUTE MYELOIDLEUKEMIA CELL LINEHL-60AND NB4, AND THE RESEARCH ON IN VIRTOLABORATORY EXPERIMENTS OF MECHANISMSObjective: To evaluate the expression of HA117in human acutemyeloid leukemia cell line HL-60and NB4by using the recombinantadenovirus-mediated gene high expression and interfere technique.Research the functions of HA117through studying effects of HA117tothe two cell lines ATRA and drug-resistance; Demonstrate the possiblemechanisms in morphology through researching effects of HA117to theproliferation, cell cycle, apoptosis in both cell lines; Clarify the actionmechanisms on a molecular level through detecting the relationshipbetween HA117and the potential key gene proteins MDR1(P-gp)、MRP1、LRP、PML-RARα、RARα、Bcl-2and Caspase-3that may associate withthe acting path. Lay a foundation for furthermore research on drug-resistance mechanisms of HA117gene in SCID mouse leukemiamodel.Methods:(1) Recombinated adenoviral supernatants with greenfluorescent protein and HA117or not (Ad-GFP-HA117and Ad-GFP)were harvested and concentrated by infection each other like Ping-Pongwith package cell HEK293.(2) Concentrated virus transfection methodwas used to establish the HA117gene high expression group, usingFluorescence microscope to observe the transfected HL-60and NB4which express green fluorescence protein (GFP).(3)Flow cytometry (FCM)and trypan-blue-stain was used to detect the transduction efficiency andsurvival rates of Ad-GFP-HA117and Ad-GFP, screening optimummultiplicity of infection(MOI).(4) The transcription and proteinexpression of ectogenous HA117was detected in the two cell lines byReal time PCR and Western blot.(5) Observing effects on morphologicalchange of HA117to ATRA inducing HL-60、NB4towards normal maturecell by Wright’s staining;(6) Detecting effects of restore function ofHA117to ATRA inducing HL-60、NB4towards normal mature cell byNBT test;(7) FCM was used to detect apoptosis of the both cell lines;(8)Drug sensitive test were made to detect the effects of HA117gene-transfected leukemia to the drug-resistance capability of manycommon clinical chemotherapeutics.(9) Mthy1-Thiazoly1-Tetrazolium(MTT) assay was used to assess the cell proliferation of HA117to HL-60 and NB4;(10) FCM was used to detect the cell cycle of HL-60and NB4;(11) The effects of HA117to potential key gene proteins MDR1(P-gp)、MRP1、LRP、PML-RARα、RARα、Bcl-2and Caspase-3expression wasdetected by Real time PCR and Western Blot.Results:(1) The viral titers of Ad-GFP-HA117was3.5×109plaqueforming units (PFU)/ml.(2) Ad-GFP-HA117was successfully transfectedinto HL-60and NB4, after transduction for48h, detecting the transfectionrate and survival rate in different MOI. The optimal MOI was100,transfection rate was30%-40%, survival rate larger than80%. Thetransfection rate and survival rate was high and status well, could be usedto the continual experiments;(3) Real time PCR and Western Blotdemonstrate that after Ad-GFP-HA117infection, the HA117get a hightranscription and protein expression in both cell lines.(4) Wright’sstaining revealed HA117retardant the cell morphology change ofleukemia cell line inducing normal cell line.(5) NBT test clarify thatHA117prevented restore function of ATRA inducing HL-60and NB4towards normal leukocyte differentiation.(6) The results of FCM revealedthat HA117retardant apoptosis of HA117inducing HL-60and NB4.(7)Drug sensitive test clarify the sensitivity of two leukemia cell line afterinfection improved significantly;(8) MTT assay showed HA117had noeffects on the capability of proliferation;(9) The results of FCM revealedthat the transfection and expression of Ad-GFP-HA117did not influence the cell cycle;(10) HA117had no significantly effects on the gene andprotein expression of MDR1(P-gp), MRP1, LRP, PML-RARα, Caspase-3,and RARα(P>0.05), up-regulation of Bcl-2significantly(P<0.01).Conclusions: Recombinated adenoviral supernatants could transfectectogenic HA117into HL-60and NB4stablely and effectively. HA117was drug-resistance in HL-60and NB4. The drug-resistance of HA117was not effective by regulating the cell proliferation, apoptosis, cell cycle,expression of MDR1(P-gp), MRP1, LRP, PML-RARα, Caspase-3, andRARα. Starting anti-apoptosis path by regulating Bcl-2, then takedrug-resistance may be the mechanism of HA117. PART TWO THE IN VIVO STUDY ON EFFECTS ANDMECHANISMS OF HA117IN SCID LEUKEMIA ANIMALMODELObjective: To establish SCID mice model suffering from humanAML, promote the in vivo experiments, verify effects and mechanisms ofHA117.Methods:(1) Modeling:150mg/Kg cyclophosphamidepreconditioned SCID mice for4days, divide mice into three groupsrandomly: Group A (SCID): tail vein injection HL-60; Group B(SCID+AD): tail vein injection HL-60+AD; Group C (SCID+HA117): tailvein injection HL-60+HA117, establish the SCID mice model sufferingfrom human AML.(2) Determination: Detecting the proliferation ofperipheral blood by Wrights’ Staining in mice tail vein blood smear,positive expression of CD33in bone marrow mononuclear cells detectedby immunohistochemistry and the infiltrated organization observed by HEstaining were used to identify whether the model is successfullyestablished.(3) Experiments in vivo: Using therapeutic dose ATRA andAS2O3to induce mice in treatment group respectively, compared with themice in control group, observing the normal situation of mice, record miceweight and survival time, detecting cell percentage of peripheral blood byWrights’ Staining in mice tail vein blood smear, positive rate of CD33in bone marrow mononuclear cells detected by immunohistochemistry,analysis whether there was drug-resistance in AML mice;(4) Feeding theSCID mice suffering from human AML for ten days, extracting mice bonemarrow, separating mononuclear cells, extracting total RNA and makingbone marrow smear, protein expression change of HA117, MDR1(P-gp),MRP1, LRP, PML-RARα, RARα, Bcl-2and Caspase-3was detected byimmunohistochemistry and Real time PCR, analyse the effects of HA117to MDR1(P-gp), MRP1, LRP, PML-RARα, RARα, Bcl-2and Caspase-3by statistical analysis in SCID mice model suffering human AML.Results:(1) After cyclophosphamide preconditioned SCID mice, tailvain injected HL-60, HL-60/Ad, HL-60/HA117, leukemia cellproliferating increasingly by wrights’ Staining, extracted bone marrowmononuclear cells CD33was significantly positive, liver, spleen, kidney,bone marrow and metastatic tumors revealed a heavy AML inflammatoryinfiltrate by HE Staining. It was demonstrated that SCID mice modelsuffering from human AML was established successfully.(2) Observingmice induced by ATRA and AS2O3in treatment group, SCID mice inGroup A and B gave a good response, but the control group and Group Cbad significantly. After chemotherapy for ten days, compared with controlgroup and Group C, SCID mice in Group A and B had a higher weight,more survival time, but HL-60cell percentage in peripheral blooddecreasing significantly (P <0.05). Results of immunohistochemistry and Real time PCR revealed that Bcl-2up-regulate significantly by HA117up-regulation, MDR1(P-gp), MRP1, LRP, PML-RARα, RARαandCaspase-3had no marked difference.Conclusions: HA117could be ATRA and other drugs resistance inSCID mice model.The Bcl-2up-regulation followed HA117up-regulatingmay be one of molecular mechanism of HA117drug-resistance. PART THREE CORRELATIVE STUDY ON THEEXPRESSION OF HA117、Bcl-2、P-gp、MRP1AND LRPIN MASS OF AML CLINICAL SAMPLESObjective: To explore the clinical significance of HA117encodingprotein in child AML and relationship among HA117and Bcl-2、P-gp、MRP1、LRP by detecting expression of HA117、 Bcl-2、P-gp、MRP1and LRP in bone marrow mononuclear cells.Methods: Clinical AL patiens was collected and divided into threegroups: newly diagnosed group, complete remission group, and refractoryor relapsed group, immune thrombocytopenic purpura as control group.The expression of HA117、Bcl-2、P-gp、MRP1and LRP in BMMNCwas detected by immunohistochemistry.Results:(1) Eighty-eight AL child patiens was collected, includingpositive expression of HA117encoding protein samples thirty-six,positive rate40.91%. Number of positive expression samples in newlydiagnosed group was22, positive rate51.16%(22/43); Completeremission group7, positive rate18.42%(7/38); all samples in refractory orrelapsed group expressed positively, but negatively in control group.Positive rate of HA117encoding protein in complete remission group,newly diagnosed group and refractory or relapsed group increasedsequentially, and that was higher significantly refractory or relapsed group compared with complete remission group(P <0.05, P<0.01), newlydiagnosed group higher than complete remission group (χ~2=9.409， P <0.01).(2) In newly diagnosed group, number of positive expression ofHA117encoding protein was22,17under-treatment included, CR18;negative expression samples number was21, under-treatment16, CR114;CR1rate in positive group was lower significantly than that in negativelygroup (P <0.05).(3) Positive rate of HA117encoding protein in childAML and ALL was40.98%and40.74%, which had no marked difference(P>0.05).(4) immunohistochemistry results revealed expression ofHA117in AML was positively related to Bcl-2significantly (χ2=37.079，P=0.0001， r=0.649), and MRP1positively (χ~2=6.335， P=0.012，r=0.268), but had no relationship with P-gp and LRP.Conclusions: Clinical cases detection results revealed thatoverexpression of HA117and its protein-encoding was closely related toclinical drug-resistance, and it was a unfavorable factor for child ALprognosis. The mechanisms of drug-resistance and Bcl-2may havedependence. That could be one of important mechanisms of HA117drug-resistance, but not unique.