| PartⅠ Effects of 280 nm ultraviolet LED on murine leukemia cell L1210 and hematopoietic stem and progenitor cells in vitro and in vivoObjective Purification in vitro is critical for autologous hematopoietic stem cell transplantation.In the current study,we observed the impact of 280 nm ultraviolet LED(UV LED)on murine leukemia cell L1210,hematopoietic stem and progenitor cells(HSPCs)in vitro and in vivo.Methods 1.Hematopoietic stem cells(HSCs)sorting: mouse bone marrow cells were obtained,then we used the SCA1 antibody labeled with biotin or PE and purified HSCs by the immunomagnetic beads.The purity of HSCs was detected by flow cytometry.2.Effects of UV LED on colony formation: L1210 cells and HSCs were irradiated with UV LED at 0,2.4,4.8,7.2,9.6,12,14.4,16.8,19.2 J/m2.Cells were then inoculated in the Metho Cult? GF M3434 methyl cellulose culture medium,and cultured at 37℃,5% CO2 and saturated humidity for 8 and 12 days,respectively.The number of colonies was counted.3.Effects of UV LED on cell proliferation: L1210 cells and HSCs were irradiated with UV LED at 0,28,56,112,224 J/m2,then cells were cultured at 37℃,5% CO2 and saturated humidity for 3 h,6 h,12 h and 24 h,respectively.Cell proliferation was detected by CCK-8 assay.4.Effects of UV LED on cell apoptosis: L1210 cells and HSCs were irradiated with UV LED at 0,28,56,112,224 J/m2,then cells were cultured at 37℃,5% CO2 and saturated humidity for 3 h,6 h,12 h and 24 h(only incubation for 24 h when detecting apoptosis of HSCs),respectively.Cell apoptosis were detected by Annexin V-FITC/PI double staining with flow cytometry. 5.Effects of UV LED on peripheral blood HSCs transplantation: before transplantation,mice received myeloablative conditioning(MAC)of 9 Gy X-ray total body irradiation(TBI).2 x 107 bone marrow cells of DBA/2 male mice were irradiated with UV LED at 0 and 9.6J/m2 prior to being injected to the tail veins in female mice.The survival rate(90 days for observation),blood routine(d7、d14、d21、d42、d60)and sry gene in the genome DNA of the peripheral blood,bone marrow cells and spleen tissue of d60 transplanted mice in the two groups were then observed.6.Effects of UV LED on the tumorigenicity of L1210 cells in vivo: 2 x 105 L1210 cells were irradiated with UV LED at 0 and 224J/m2 and then injected to the DBA/2 female mice’ left forelimb armpit.The survival rate(90 days for observation),blood routine,blood smear,bone marrow cells smear and histological morphology were then observed.7.Effects of UV LED on the hematogenous metastasis of L1210 cells in vivo: before transplantation,mice received nonmyeloablative conditioning(NMC)of 2 Gy X-ray TBI and cyclophosphamide.2 x 105 L1210 cells were irradiated with UV LED at 0 and 9.6J/m2,mixed with 2 x 107 bone marrow cells of DBA/2 male mice,and finally injected to the female mice intravenously.The general condition,survival rate(90 days for observation),bone marrow cells smear and histological morphology were then observed.Results 1.Purity of HSCs: HSCs(PE positive)were obtained by immunomagnetic beads sorting using PE labeled SCA1 antibody,and the purity was up to 97.1%.2.Effects of UV LED on colony formation: UV LED significantly inhibited the CFU-L1210 and CFU-HPC(P<0.01)in the dose range of 0-19.2J/m2,with dose dependently.In the dose range of 2.4-14.4J/m2,the inhibitory effect of UV LED on CFU-L1210 was stronger than that of CFU-HPC(P<0.05),among which 9.6J/m2 was the most significant.3.Effects of UV LED on cell proliferation: UV LED significantly inhibited the proliferation of L1210 cells and HSCs(P<0.05)in the dose of 0-224J/m2 and incubation for 3-24 h,which was dose and time dependent.When cells were irradiated with 28J/m2 and incubated for 3h,there was no significant difference in their proliferation rate(t=1.28,P=0.27),while in other groups the inhibition of L1210 cell proliferation was stronger than that of HSCs(P<0.05).4.Effects of UV LED on cell apoptosis: during 0-224J/m2 irradiation dose and incubation for 3-6h,UV LED did not significantly increase the apoptosis rate of L1210 cells(P>0.05).When the incubation time prolonged to 12 h and 24 h,the apoptosis rate of L1210 cells was significantly increased(P<0.05),in a dose and time dependent manner,and the highest apoptosis rate of L1210 cells occurred when they were irradiated with 224J/m2 and incubated for 24 h.During 56-224J/m2 irradiation dose and incubation for 24 h,UV LED significantly led to apoptosis of HSCs(P<0.05),with no dose-dependent effect.When cells were incubated for 24 h,UV LED-induced apoptosis of L1210 cells was stronger than that of HSC in the dose of 0-224J/m2(P = 0.01),among which 224J/m2 was the most significant.5.Effects of UV LED on peripheral blood HSCs transplantation: mice of both groups survived more than 90 days and the survival rate had no significant difference(X2=1.00,P=0.32).There was no significant difference between the two groups in the number of WBC,RBC,Hb and PLT in d7,d14,d21,d42 and d60 after transplantation(P>0.05),and the hematopoietic function of mice in both groups had been normal since d14.Male sry gene could be detected in the genome DNA of the peripheral blood,bone marrow cells and spleen tissue of d60 transplanted mice in both groups,suggesting the stable donor cell chimerism.6.Effects of UV LED on the local tumorigenicity of L1210 cells in vivo: the tumor formation rate of mice in the control group was 100%,and mice were all dead by d21,whereas no tumors were formed in the experimental mice,who survived more than 90 days;indeed,their survival rate was significantly higher than mice in the control group(X2=11.76,P=0.00).The number of WBC,RBC,Hb and PLT in the dying mice of the control group was significantly lower than those of the experimental group(P<0.05),and mice in the experimental group had normal hematopoietic function.In the control group,the expanded central light staining area of red blood cells and leukemia cells infiltration in the spleen was observed,in contrast,no leukemia cells were found in the experimental mice.7.Effects of UV LED on the hematogenous metastasis of L1210 cells in vivo: mice in the control group had continued to decline in body weight since d21 after transplantation,and since d52 they had appeared mental malaise,poor diet,bow back,fur dull and hair removal,dyspnea,lower limb paralysis,and finally progressed to cachexia and death.Splenomegaly was found compared with their experimental counterparts anatomically and their median survival time was 56 days.In the experimental group,mice had continued to decline in body weight since d21 and had developed such symptoms since d80.Their median survival time was 84 days,which was significantly higher than that of the control group(X2=12.02,P=0.00).Leukemia cells infiltration was observed in the vertebra,spinal cord and the epidural space of the dying mice in both groups.Besides,leukemia cells were also found in the spleen and bone marrow of mice in the control group,which demonstrated no signs in the experimental group.Compared with the control group,mice in the experimental group had a late onset of leukemia,and the degree of leukemic cells infiltration was mild.Conclusion 1.280 nm UV LED can selectively kill murine L1210 leukemia cells in a dose and time dependent manner in vitro;its mechanism includes the inhibition of colony formation,inhibition of cell proliferation and induction of apoptosis,yet it exerts a relatively smaller amount of influence on HSC.2.280 nm UV LED is important for purification in vitro.After UV LED irradiation,the ability of local tumor formation and hematogenous dissemination of L1210 cells significantly decreases,while HSCs retain the in vivo hematopoietic reconstitution.Part Ⅱ The role of protein kinase in UV LED-induced apoptosis of L1210 cellsObjective UV-induced apoptosis is a highly complex biological process incorporating multiple signaling pathways,in which the protein kinase family plays an important role in the apoptotic signaling pathway.In this study,we observed the effects of 280 nm UV LED on PKA,PKCβ gene and protein expression to investigate the role of PKA and PKCβ in UV LED-induced apoptosis of L1210 cells.Methods L1210 cells were irradiated with UV LEDs at 0 and 224 J/m2,and incubated for 24 h at 37℃,5% CO2 and saturated humidity.The m RNA expression of prkacb and prkcb was detected by RT-q PCR.The protein expression of PKA and PKCβ was detected by Western blot.The DNA methylation levels of prkacb and prkcb were detected by pyrophosphoric acid sequencing.Then L1210 cells were incubated with 10 μM PKA inhibitor KT5710 and 5 μM PKCβ inhibitor Enzastaurin for 12 h,respectively,while their control counterparts were incubated with the same volume of DMSO for 12 h.Cell apoptosis was measured by Annexin V-FITC/PI double staining with flow cytometry.Results 1.The m RNA expression of prkacb and prkcb in both groups: The m RNA expression of prkacb and prkcb in the control group was referred as 1.The m RNA expression of prkacb in the experimental group was(0.22±0.03),which was significantly lower than that in the control group(t=51.06,P=0.00).The m RNA expression of prkcb in the experimental group was(0.39±0.04),which was significantly lower than that in the control group(t = 26.41,P = 0.00).2.The protein expression of PKA and PKCβ in both groups: the protein expression of PKA in the control and experimental groups was(1.04±0.15)and(0.37±0.06),respectively,and there was significant difference between the two groups(t=8.25,P=0.00).The protein expression of PKCβ in the control and experimental groups was(1.18±0.12)and(0.53±0.04),respectively,and there was significant difference between the two groups(t=10.62,P=0.00).3.The DNA methylation levels of prkacb and prkcb in both groups: The DNA methylation levels of the two positions of prkacb in the control and experimental groups were(85.33±1.51)% and(86.17±1.17)%,(84.33±2.58)% and(82.33±1.37)%,respectively,and there was no significant difference between the two groups(P>0.05).The DNA methylation levels of the nine positions of prkcb in the control and experimental groups were(97.00±0.63)% and(97.17±0.75)%,(95.67±1.37)% and(95.67±1.51)%,(92.83±0.41)% and(93.17±0.41)%,(90.00±0.41)% and(90.33±1.37)%,(95.67±3.93)% and(96.17±2.93)%,(85.33±0.52)% and(85.50±0.84)%,(93.17±1.72)% and(93.17±0.75)%,(96.17±0.75)% and(96.67±0.52)%,(96.50±0.55)% and(96.17±1.17)%,respectively,and there was no significant difference between the two groups(P>0.05).4.Effects of KT5720 on cell apoptosis: cell apoptosis rate of the control and experimental groups was(8.69±0.34)% and(39.66±8.56)% respectively,and cells in the experimental group had a higher apoptosis rate than their control counterparts(t=6.26,P=0.02).5.Effects of Enzastaurin on cell apoptosis: cell apoptosis rate of the control and experimental groups was(6.77±0.46)% and(17.11±1.29)% respectively,and cells in the experimental group had a higher apoptosis rate than their control counterparts(t=13.07,P=0.00).Conclusion 1.PKA and PKCβ play an anti-apoptotic role in L1210 cells.2.280 nm UV LED can reduce prkacb and prkcb m RNA expression and PKA and PKCβ protein expression in L1210 cells,and are involved in UV LED-induced apoptosis.3.The reduction of prkacb and prkcb m RNA expression induced by 280 nm UV LED is accomplished by non-DNA methylation modification. |
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