| Radiotherapy and chemotherapy are the main treatment methods for malignant tumors.However,radiotherapy and chemotherapy may cause bone marrow suppression,resulting in thrombocytopenia,often delaying the chemotherapy cycle and even affecting treatment.Severe thrombocytopenia may also cause spontaneous hemorrhage and endanger life.The effects of low-dose radiation and chemotherapy on the proliferation,development and platelet production of megakaryocytes and the specific mechanism are not yet fully understood.Therefore,it is of great significance to elucidate this problem and mechanism and to alleviate the side effect of radiotherapy and chemotherapy on thrombocytopenia.Objectives:To explore the effect and mechanism of gemcitabine on the proliferation,differentiation and platelet production of megakaryocytes,the effect of low-dose radiation on proliferation,differentiation and platelet production of megakaryocytes,and the protective effect of low-dose radiation on thrombocytopenia caused by gemcitabine.Methods:I.Effects and mechanisms of gemcitabine on proliferation,differentiation,and platelet production of megakaryocytes:(i)in vitro study:1.Changes in the proliferation inhibition rate of megakaryocytes after gemcitabine treatment were detected by WST-1 assay.2.Changes of megakaryocyte polyploid formation,megakaryocyte differentiation index(CD41+/CD61+)and apoptosis rate after gemcitabine were detected by cd41-fitc /PI double staining,CD41-FITC/CD61-PE double staining and Annexin V-APC /PI double staining flow cytometry.3.The effect of the process of producing preplatelet by megakaryocytes after gemcitabine was observed by the living cell differential intervention phase microscope of the living cell station,and video was made.Changes in the number of megakaryocytes producing preplatelet were observed and counted by optical microscopy.4.Real-time quantitative PCR(q PCR)was used to detect the expression changes of the transcription factors GATA-1,NF-E2,Fli-1and cyclin-related proteins after gemcitabine.5.The expressions of ERK1/2,p-ERK1/2,p-p38,P38,AKT,p-AKT and LC3 were detected by Western blot.(ii)in vivo studies:1.The method of blood collection from the orbital vein of mice was used to count the blood cells of the mice using the five-category hemocytometer.The changes of platelet count in the mice treated by gemcitabine were observed and the curve was drawn.2.Tail vein injection of NHS-biotin to mark platelets,Streptavidin-PE/CD41-FITC to mark platelets after blood collection from orbital vein,flow cytometry to detect the proportion of platelets marked by NHS-biotin each day after treatment by gemcitabine,and the platelet life curve was plotted for 5 consecutive days.3.Serum levels of TPO,SDF-1 and IL-1 were detected after blood was collected from the orbital vein of mice,and mice were sacrificed by cervical dislocation.A small piece of mouse liver was taken,total RNA was extracted,and TPO m RNA level was detected.II.Study on the protective effect of low-dose radiation on proliferation,differentiation,platelet production and thrombocytopenia induced by gemcitabine:1.Changes of megakaryocyte proliferation rate after X-ray irradiation in different doses were detected by WST-1 assay.2.Changes of megakaryocyte polyploid formation and megakaryocyte differentiation indexes(CD41+/CD61+)after LDR were detected by CD41-FITC /PI double staining and CD41-FITC/CD61-PE double staining flow cytometry.3.The effect of the process of the production of preplatelet by megakaryocytes after LDR was observed by the living cell differential intervention phase microscope of the living cell station,and video was made.Changes in the number of megakaryocytes producing preplatelet were observed and counted by optical microscopy.4.SPF grade male C57BL/6 mice at 6-8 weeks were divided into the control group,the low-dose radiation group(100 m Gy),the gemcitabine group(Gem 200 mg/kg),the low-dose radiation followed by the gemcitabine group(100 m Gy+ Gem 200 mg/kg),and the platelet count of each group was detected daily.To observe the changes of platelet count and draw a curve,the blood cells of mice were counted by the five-category hemocytometer.Results:1.Gemcitabine can inhibit the proliferation,differentiation and platelet release of megakaryocytes,but do not induce apoptosis;The proliferation activity of megakaryocytes was detected by WST-1 method.In the dose-effect study,it was found that,compared with the control group,the proliferation of megakaryocytes was significantly inhibited by gemcitabine in the 0.1-1 μM,showing a dose-dependent effect.In the time effect study,we found that compared with the control group,the proliferation inhibition rate of 24-96 h cells significantly increased after gemcitabine treatment,which was time-dependent,with the most obvious inhibition of 96 h cell proliferation.Flow cytometry was used to detect the polyploid formation of megakaryocytes,and the results showed that,compared with the control group,the polyploid formation of megakaryocytes could be inhibited by 0.1 μM and 0.5 μM of gemcitabine,which reduced the proportion of megakaryocytes in 16 N,32N and 64 N,while the proportion of megakaryocytes in 4N and 8N was increased.In addition,the mean ploidy of 0.1 μM and 0.5 μM of gemcitabine group was significantly reduced.Flow cytometry was used to detect the expression rate of CD41+/CD61+ in megakaryocytes,and it was found that the expressions of CD41+/CD61+ were both inhibited by 0.1 μM and 0.5 μM of gemcitabine,and the expression rate of CD41+/CD61+ in megakaryocytes of 0.5 μM was significantly reduced compared with the control group.Flow cytometry was used to detect the apoptosis rate of megakaryocytes,and it was found that apoptosis was associated with normal differentiation and maturation of megakaryocytes in the control group,while the apoptosis rate of 0.1 μM and 0.5 μM in the gemcitabine group was not significantly different from that in the control group.2.Gemcitabine can inhibit the expression of transcription factor GATA-1,NF-E2 and Fli-1,and promote the expression Cyclin D1,p21 and Cyclin E1.We divided gemcitabine group and solvent control group(DMSO),the results showed that compared with solvent control group(DMSO),can obviously inhibit transcription factor GATA-1,the expression of NF-E2,the Fli-1,promote the expression of Cyclin D1,p21 and Cyclin E1.3.The expression of LC3 and p-p38 was increased after gemcitabine treatment,and there was no significant change in p-ERK1/2 expression.We divided gemcitabine group and solvent control group(DMSO),the results showed that compared with solvent control group(DMSO),gemcitabine can promote LC3 and p-P38 expression,but ERK1/2,p-ERK1/2 expression has no obvious change.4.Gemcitabine induce thrombocytopenia in mice,the lowest platelet is about 7 days after the drug delivery,the platelet life is not affected.Mice were divided into control group and gemcitabine group,platelet daily continuous monitoring,and testing after life,the results show that compared with the control,gemcitabine can significantly induce mice platelet reduce gradually,about 7 days after the treatment,mice platelet life has no obvious change after the treatment.5.When the number of platelets was lowest after gemcitabine treatment,the level of TPO m RNA in the mouse liver decreased,while the level of TPO SDF-1 and IL-1 level in serum increased.C57BL/6 mice were divided into control group and gemcitabine group,daily continuous monitoring platelet,result show that compared with the control group,gemcitabine group of mice liver TPO m RNA level is reduced,and increased serum TPO level,serum SDF-1 and IL-1 levels.6.LDR promote the proliferation of megakaryocytesThe proliferation of megakaryocytes was detected by WST-1 method.In the study of the dose effect,the results showed that the X-ray of 25-100 m Gy could promote the proliferation of megakaryocytes compared with the control group,and the effect of 75 m Gy on proliferation was the most obvious.When the radiation dose was over 200 m Gy,the proliferation of megakaryocytes was significantly inhibited.In the time effect study,the results showed that compared with the control group,the proliferation of cells was significantly increased at 24-96 h after 75 m Gy radiation,with the most obvious proliferation at 24 h and no further proliferation at 48-96 h.7.LDR promote the polyploid formation and differentiation and maturation of megakaryocyte.Application of CD41-FITC/PI double flow cytometry detection dye LDR of megakaryocyte polyploid formation,the results showed that: compared with control group,75 m Gy can promote the proportion of 16 N,32N megakaryocyte,and 2N,4N and 8N megakaryocyte proportion reduced,the average number of ploidy of megakaryocyte increases.The expression rate of CD41+/CD61+ in megakaryocytes was detected by CD41-FITC/CD61-PE.Results: compared with the control group,75 m Gy X-ray promoted the expression of CD41+/CD61+ in megakaryocytes.8.LDR promote platelet production and platelet release in megakaryocytes.Workstation application living cells before the differential interference generated by phase contrast microscope observation of megakaryocyte platelet dynamic process and some application of ordinary optical microscopy before platelet megakaryocyte and statistical number,the results showed that: compared with control group,LDR2 group(75 m Gy 0 h + 75 m Gy 96 h)of megakaryocyte generated before the platelet and platelet release increased,while LDR1(75 m Gy 0 h)group of megakaryocyte thrombopoiesis before and had no effect on platelet release.9.LDR can reduce the degree of thrombocytopenia caused by gemcitabine.The mice were divided into control group,low-dose radiation group(100 m Gy),gemcitabine group(Gem 200mg/kg),followed by low-dose radiation followed by gemcitabine group(100 m Gy pre24 h + Gem 200mg/kg),and the platelet count of each group was detected daily.The results showed that compared with the control group,the platelet reduction was the most serious in the gemcitabine group,and the platelet reduction was reduced in the sequential gemcitabine group after low-dose irradiation,while there was no significant difference in platelet number between the low-dose irradiation group and the control group.Conclusions:1.Gemcitabine can inhibit the proliferation,differentiation and platelet release of megakaryocytes and induce apoptosis;2.Gemcitabine can inhibit the expression of transcription factor GATA-1,NF-E2 and Fli-1,and promote the expression of transcription factor Cyclin D1,p21 and Cyclin E1.3.The expression of LC3,p-p38 and P38 was increased after gemcitabine treatment,while the expression of ERK1/2 and p-ERK1/2 was not significantly changed.4.Gemcitabine can induce thrombocytopenia in mice,the lowest platelet is about 7 days after the drug delivery,the platelet life is not affected.5.When the platelet count was lowest after gemcitabine,the TPO m RNA level in the liver of mice was decreased,while the levels of TPO,SDF1 and IL-1 in the serum were increased,and the number of megakaryocytes in the bone marrow was decreased and the volume was decreased.6.LDR promote the proliferation,differentiation and maturity of megakaryocytes,polyploid formation,platelet generation and release.7.LDR play a protective role in reducing the degree of thrombocytopenia caused by gemcitabine. |
PDF Full Text Request