| Several researches have confirmed that CQHP was safety and good for MDS patients.In order to figure out the pharmacology and mechanism of CQHP,we not only focus on our clinical data but made full use of the transgenic MDS model to do relative experiments.Part Ⅰ Clinical Research of CQHP on MDS PatientsObject:To figure out the efficacy of CQHP on MDS patients after one and half years CQHP treatment.Methods:To arrange the clinical characters of MDS patients who treated in Xiyuan Hospital of China Academic Chinese Medicine from January 2015 to October 2016,finally 61 patients were analyzed in this study.Clinical response rates and relative factors associated with clinical response rates were mainly analysed here.We used descriptive method and mean ±standard deviation to calculate,also we use the chi-square sometimes.Results:(1)Efficacy of CQHP after 1.5 years treatment in 61 patients,91.8%of patients shown hematology improvement to the CQHP,among them,16 patients got improvement(16/61,26.2%),40 patients got stable status(40/61,65.6%),5 patients has no response(5/61,8.2%).(2)The hemoglobin level was significantly increased after 1.5 years treatment,before treatment is 79.23±21.28 g/L,after treatment is 85.59±26.6g/L,p=0.0162.(3)The netrophil count was increased after 1.5 years treatment,before treatment is 1.01±0.53×109/L,after treatment is 1.14±0.63×109/L,p=0.12.(4)The Platlet count was not change after 1.5 years treatment,before treatment is 44.35±38.23×109/L,after treatment is 43.67±37.79×109/L,p=0.93.(5)The transudion burden decreased obviously after 3 months(260ml),6 months(180ml)and 1.5 years(80ml),compared with before treatment(350ml)(p=0.0004).(6)The following factor have better relationship with drug efficacy:age<60 years old,good and medium karyocyte and low and medium risk of IPSS-R.(7)There was no evdience of adverse event in patients treated with CQHP.Conclusion:The hematology improvement in CQHP treated patients was about 91.8%in 1.5 years,which was quite useful for patients.Especially at improving the anemia,the drug efficacy was obviously.Part Ⅱ Pharmacology Effect of CQHP on MDS MiceObject:To figure out the pharmacology of CQHP,MDS transgenic model was established and used to give CQHP treatment.Methods:MllPTD/WT/Runx1Δ/Δ and TPM+bone marrow(BM)transplantation transgenic mice models were used in this study.When MllPTD/WT/Runx1Δ/Δ and TPM+ mice developed into disease,bone barrow transplantation was transfused to the normal mice.For TPM+BM transplantation mice,doxycycline was given for disease induction,while the MllPTD/WT/Runx1Δ/Δ BM transplantation mice need nothing but time.Chimerism was checked 1 months after transplantation.CQHP or PBS was given to 8 mice,five days a week,four weeks by oral gavage.Peripheral blood was checked to evaluate the drug effect.Survival time was recorded.Results:(1)Chimerism of BM transplantation mice shown the percentage of CD45.2+population in MllPTD/WT/Runx1Δ/Δ BM transplantation mice were 76.19±3.21%,while the percentage of GFP+ population in TPM+ BM transplantation mice was 74.59±7.22%.(2)The baseline of CQHP and PBS treated mice.There was no significant difference between CQHP and PBS treated mice of hemoglobin(HB),red blood cell(RBC)and platelet(Pit)counts(p=0.32,0.19,0.55).(3)Compared to PBS treated mice,HB and RBC in CQHP treated mice obviously increased at 8,1 5,21 days of treatment(p=0.0096,0.026,0.015 for HB;p=0.005,0.012,0.004 for RBC).Compared to PBS treated mice,Pit in CQHP treated mice obviously increased at 21 days of treatment(p=0.029).(4)At the day 128,the median overall survival(OS)for PBS treated mice was 47.5 days while CQHP treated mice did not got median OS yet.CQHP treated mice had longer survival time than PBS treated mice(p=0.04).Conclusion:We established the MDS transgenic mice model and finish the pharmacology study of CQHP successfully.It turned out that CQHP could improve the anemia and thrombocytopenia of MDS mice,and prolonged the overall survival.Part Ⅲ Possible Mechanism of CQHP on AnemiaObject:To clarify the possible mechanism of CQHP on anemia,we did in vitro and in vivo experiments with MDS and normal mice and human cells with molecular technology.Methods:(1)To validate the drugs effect,CCK-8 assay was used to test drug vailbility on human and mouse normal cells and MDS cells in this research.(2)To figure out the drug mechanism on erythropoiesis,flow cytometry was used to check the MDS models and normal mice reaction.(3)To figure out the CQHP drug effect on erythropoiesis,BFU-E and CFU-E assays were used on TPM+ BM transplantation,TPM+,TPM-and wild type mice bone marrow(4)For molecular mechanism,we used West-bolt and quantitative polymerase chain reaction to figure out the possible reasons for both MDS and normal clones.Results:(1)CCK-8 assay shown CQHP has was no cellular toxicity on MDS and normal mice and human cells but promoted MDSL cells differentiation.(2)Compared to PBS group,the erythropoiesis population as Ⅰ and Ⅱ increased(p=0.014,p=0.031)while mature red blood cell population as IV decreased(p=0.039)obviously in CQHP treated mice.Besides,the GFP-normal hematopoiesis stem cells increased obviously(p=0.006)while the spleen size decreased(p=0.032)in CQHP treated mice compared to PBS treated mice.(3)BFU-E and CFU-E assays with BM from TPM+ BM transplantation,TPM+,TPM-and normal mice shown that colony number and cell number increased obviously in CQHP treated group compared with PBS treated mice(p>0.05).(4)In normal mice,CQHP treated mice have more LSK,MPP2,MPP3,MPP4,Pre-CFU-E and Pro-E+CFU-E population compared with PBS treated mice(p>0.05).However,there was no significant difference of spleen size between CQHP and PBS treated mice(p>0.05).(5)The expression of HIF1A protein and downstream gene-Binp3,Binp3l,Ldha,Hk2,Ddit4 was decreased while the expression of P53、MDM2、VHL protein increased obviously in CQHP treated MllPTD/WT/Runx1Δ/Δ cells.(6)In normal cells,the expression of GATA1/GATA2/GATA3 increased obviously in CQHP treated group.Conclusion:CQHP could promote erythropoiesis both on MDS clones and normal clones.Moreover,we showed the possible molecular mechanism of the drug effect on erythropoiesis,by downregulating HIF1A to inhibit MDS colony and upregulating the expression of GATA factors to promote erythropoiesis.We also believe that CQHP could be broadly used for MDS patients and may even apply to other hematopoietic or non-hematopoietic conditions,which are needed to improve patients’anemia. |
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