| The first part The effect of chemotherapeutic drugs on CD19-CAR-T cells in vitroBackground and purposeHematological malignancies has long been regarded as a problem that humans can not attack.From the initial radiation and chemotherapy to emerging hematopoietic stem cell translation,the progress of medical technology brings hope for cancer therapy.But even so,there still have a lot of ill-effected blood tumor therapy.Hematopoietic stem cell tram plantation still can't avoid the relapse and transplanting failure.Long-term side effects brought by the chemotherapy and radiotherapy greatly reduce the quality of life of patients.Relapse and complications after transplantation make the survival rate significantly reduced.Headed by CD19-CAR-T cells cellular immune therapy brings hope to patients with hematological malignancies.According to the tumor specific antigen,made the specifically CD 19-CAR transferred into T cells to have attack the tumor cells with the same surface marker of CD 19-CAR-T cells,as with biomarkers of CD 19-CAR-T cells in the treatment of acute B lymphocytic leukemia to has a very good therapeutic effect.Because of the limit of CD 19 antigen leading to emergence of some related complications,such as tow gamma globulin hematic disease,cytokine storm,off-target effects and so on caused by the lack of B cells.we need to explore some update and more effective way upon existing treatment in order to avoid or reduce the complications.Although there have researchers begain to let the suicide gene installed on the fourth generation of chimeric antigen receptor T cells to launch suicide program itself removed if necessary to mitigate complications,but is still in the research stage.The chemotherapy drugs because of its use of clinical experience,the exact effect,seems to be more likely to be used as a means of clearing the chimeric antigen receptor T cells,which is the starting point of our experiment.In clinical studies,B acute lymphoblastic leukemia is the most common and the most mature,also is maybe the best treatment in a class of diseases,so in this experiment we use CD19-CAR-T cells as the experimental cell in our study.If we can associate hematopoietic stem cell transplantation and CD19-CAR-T cells cellular immune therapy such as adopting CD19-CAR-T cell therapy before transplantation,in addition to avoiding the side effects of chemotherapy,it can also make full use of its advantages in CD19-CAR-T cell therapy targeting to improve the success rate of transplantation,or given chemotherapy drugs to decrease the survival time of cells in the body after CD19-CAR-T cells treatment and to reduce the side effect of CD19-CAR-T cells.What is the time point to give the chemotherapy drugs to kill tumor cells without creating influence on the subsequent infusion of CD19-CAR-T cells activity?Giving CD19-CAR-T cells after transplantation to remove residual tumor cells or relapse,and giving chemotherapy drugs to kill CD19-CAR-T cells to prevent corresponding side effects caused by CD19-CAR-T cell infusion is a good method.So what's the time on earth to give can achieve the purpose of killing tumor cells?This is the subject in which we need to clarify the problem of science,exploring the effect of chemotherapy drugs against CD 19-CAR-T cells.MethodsTo extract T cells from normal adult's peripheral blood,make it into T cells with CD 19 targets,and mechanically get it separated into single cell suspension,cultivating heavy suspension cells without serum,adjusting cell density,then inoculate in 96-well cultivation plate with each hole 90 ul,thus making living cells number 2*105/hole,and put them in the box for cultivation.Then add chemotherapeutic drugs 10 ul/hole,its density can be referenced by density index of peak plasma concentration(ppc)>add it in turn according to different concentration.Set five repeated holes for each concentration(A dosing group),and at the same time set a hole without medicine,only adding cell and medium(Ao dosing group),and the only medium of blank group(A blank)contract hole,continue to cultivate 24h,48h,72h and(or)after 96h,each hole should be added lOul CCK8 reagent,and cultivate for an appropriate time,use enzyme standard instrument in the 450 nm absorbance to detect absorbance.Calculate the inhibition rate=the OD value of AO dosing group-the OD value of A dosing group)*100%.The data uses SPSS 15.0 software to make a single factor analysis of variance and factorial design analysis of variance.If P<0.05,the difference is statistically significant.Result1.Fludarabine,as the extension of time and the increasing of the concentration,the inhibition rate of fludarabine to CD19-CAR-T cells is increased(P>0.05)2.Busulfan had no effects on CD19-CAR-T calls in vitro(P>0.05).3.Cyclophosphamide had no effects on CD 19-CAR-T cells in vitro(P>0.05).4.The inhibition rate of dexamethasone to CD19-CAR-T cells increased with prolonged time(P<0.05).And when the inhibition rate reached its peak at 48h,then it fells down.5.The inhibition rate of mafosfamide on CD19-CAR-T cells is increasing with the extension of time and the concentration of chemotherapeutic drugs(P<0.05).When the concentration is 10 kg/ml,the inhibition rate at 72h is over 90%,and then reduced from 96h.ConclusionMafosfamide is metabolic product of cyclophosphamide,has the inhibition effect to CD19-CAR-T cells in vitro.It is further verified that the cyclophosphamide metabolized in the liver at first when it comes into the body by metabolites.Fludarabine,dexamethasone were all have the inhibition on the CD19-CAR-T cells,and while extending overtime,the inhibition rate increased.In this study,the innovation is the first time indicate that fludarabine,mafosfamide and dexamethasone can inhibit the chimeric antigen receptor T cells in vitro by CCK8 test.The Second Part The signal pathway of apoptosis about CD19-CAR-T cells after induced by chemotherapy drugsBackground and purposeCell apoptosis is one of the basic characteristic of cells,referring to a high-level regulation course of cells removing their unwanted and losing function.According to different starting way,it's divided into death receptor pathway and mitochondrial pathway and endoplasmic reticulum pathway.It is different from cell-death,not a passive process,but an active process,and involving a series of gene expression,regulation and activation.It is body itself adapting to the environmental changes and voluntarily taking a death process.It can occur in cell growth,aging,and when the cells from being destroyed by the disease and toxic factors,such as buoyed by radioactive chemotherapy drugs or in the process of chemical stimulation.Fludarabine and mafosfamide have varying degrees of damage effect against CD 19-CAR-T cells in vitro,through the study of these two kinds of chemotherapy drugs acting on the CD19-CAR-T cells with Annexin V/PI current methods to detect the change of the cell surface membrane,with JC-1 as the probe to detect the change of mitochondrial membrane potential and cell of judging Caspase3/7 enzyme concentration change DC cell apoptosis,and discusses the mechanism of the chemotherapy drugs to kill your CD19-CAR-T cells,laying a theoretical foundation for further study.Methods1.CD 19-CAR-T cells were incubated in the presence of fludarabine,mafosfamide,dexamethasone for 24h(Fludarabine concentration:12.5?g/ml,mafosfamide concentration:10?g/ml and dexamethasone concentration:5?g/ml),respectively.Subsequently the early apoptotic CD 19-CAR-T cells was detected by Annexin V/PI.2.CD19-CAR-T cells were incubated in the presence of fludarabine for 24h and mafosfamide for 12h(Fludarabine concentration:12.5 ?g/ml,mafosfamide concentration:10 ?g/ml).Then the cell membrane potential was detected by Fluorescent probe JC-1.3.CD19-CAR-T cells were incubated in the presence of fludarabine for 24h and mafosfamide for 6h(Fludarabine concentration:12.5 ?g/ml,mafosfamide concentration:10 ?g/ml).The concentration of Caspase-3/7 was detected by flow cytometer.The factorial design analysis of variance,Paired-Sample T test and oneway anova of the results was analyzed by SPSS 15.0.It is statistically significant that P<0.05.Result1.The apoptotic rate of treated CD 19-CAR-T cells by fludarabine and mafosfamide was significantly increased than untreated.(P<0.05)2.The mitochondrial transmerabrane potential of treated CD19-CAR-T cells by fludarabine and mafosfamide was markly decreased than untreated.(P<0.05)3.The concentration of Caspase-3/7 of treated CD19-CAR-T cells by fludarabine and mafosfamide was obviously increased than untreated.(P<0.05).Conclusion We can estimate that fludarabine and mafosfamide can induce the early stage apoptosis through the change of mitochondrial transmerabrane potential and cell membrane.The increased concentration of Caspase-3/7 leads to the apoptosis.The innovation of this experiment 1.Fludarabine and mafosfamide can induce the apoptosis of chimeric antigen receptor T cellsThe third part The effect of Raji cell and Jurkat cell after treated with Chemotherapeutic drugs in vitroObjectiveThe date of 2015 shows that the solid tumor and hematological tumor cases in China and US are markedly increased.Therefore the clinical trial aiming at the different targets of dendrites' cell is steadily on the increase.In order to preferably research the effect of chemotherapeutic on different hematological tumor disease,we chose Raji cell and Jurkat cell as disease modes to discuss the best concentration of chemotherapeutic on different tumor modes.Methods1.We detected the OD of different concentration of Raji cell(1,2,3,4,8*104 cells per well)and Jurkat cell(1,2,3,4,5,6,7*104 cells per well)at 1h,2h,3h,4h and drawed standard curve.Then We chose the concentration of Raji cell and Jurkat cell(OD=1)to detect the OD at 24h,48h,72h,96h,respectively and then we selected the time points that OD is between 1 to 2 as the detected time of future experiment.2.According the peak plasma concentration of every drug,we added to each well successively according to different concentration and every concentration had five repeated wells.Meanwhile,we set up other groups including that cells with medium and medium alone followed by incubation for 24h,48h,72h,96h.At the designated time points,we added to each well with 10?l of CCK8,then detected the absorbance of the groups at 450nm by micro plate reader according to the time points was determined and repeated three times each group and calculated the inhibition ratio.Result1.The inhibition ratio of fludarabine on Raji cell and Jurkat cell was increased with time and increasing of concentration(P<0.05),and the inhibition ratios all began to drop at the concentration of 1.5625ug/ml after 72h.2.Busulfan had no inhibitory effect on Raji cell and Jurkat cell in vitro.3.Cyclophosphamide had no inhibitory effect on Raji cell and Jurkat cell in vitro.4.Dexamethasone had no inhibitory effect on Raji cell and Jurkat cell in vitro.5.The inhibition ratio of mafosfamide on Raji cell and Jurkat cell was increased with time and increasing of concentration(P<0.05).They began to drop at the concentration of 5ug/ml after 72h on Raji cell and 1.25ug/ml after 48h on Jurkat cell.ConclusionMafosfamide is a metabolite of cyclophosphamide and has inhibitory effect on Raji cell and Jurkat cell in vitro.That further verifies that cyclophosphamide has a role play in vivo through the metabolite in the hepatic and it has no effect in vitro.The inhibition ratio of fludarabine on Raji cell and Jurkat cell was increased with time.Busulfan and dexamethasonehad no killing effect on Raji cell and Jurkat cell in vitro. |
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