| Background and objectivePure red cell aplasia(PRC A)is defined as reticulocytopenia(<1%)that persists for more than 60 days after hematopoietic stem cell transplantation.While the granulocytes,lymphoids,and megakaryocytes populations are implantated in the bone marrow,no erythroid precursors were found in the bone marrow.Almost all the the delayed erythropoiesis and PRCA occur in type O recipients who have underwent type A hematopoietic stem cell transplantation.The mechanism of the occurrence of PRCA is still unclear.The aim of this study is to construct ABO*A1.01 allele recombinant lentivirus,which was used to transfect K562 cells to obtain K562-A cells expressing ABO*A1.01 allele stably.The K562-A cells can be used as a cell model for simulating the erythroid precursors in hematopoietic stem cell transplantation.The characteristics of differentiation,proliferation and cell cycle of K562-A cells were analyzed,and the effect of human anti-A antibody on the mRNA expression level of ABO gene in stable K562-A cells was investigated.The study will lay a foundation for further research on the mechanism of graft accommodation in ABO-incompatible hematopoietic stem cell transplantion.Methods1.Acquisition of ABO*A1.01 allel and construction of recombinant vectorsThe GV492 vector was digested by two restriction endonucleases,and the fragment containing ABO*Al.01 allel was amplified from the plasmid library by PCR.The fragment containing ABO*A1.01 allel was inserted into the GV492 vector.After transformation,culture and colony identification,the positive clone transformants were cultured overnight,and the ABO allel in the bacteria was sequenced.The bacteria that have correct ABO allel were cultured overnight,and the plasmids were extracted from the bacteria and identified by enzyme digestion.2.Virus packaging,concentration and titer detectionThe recombinant vector and the empty vector were separately mixed with the two virus packaging plasmids in a certain ratio,and transfected into 293T cells.The cell culture supernatant was collected and ultracentrifuged to obtain a viral concentrate.The virus was serially diluted and transfected into 293T cells.The number of fluorescent cells after transfection was counted,and the virus titer was calculated.3.Establishment and detection of stable cell linesK562 cells were transfected with recombinant vector lentivirus and empty vector lentivirus respectively.After selecting with puromycin,the stable K562-A and K562-GFP cell lines were obtained.The cell images were captured using a fluorescence microscope.Sanger sequencing was used to ascertain the sequence of the exon 6 of the ABO allele in the cells.The cellular mRNA expression of ABO gene was assessed by quantitative PCR.Flow cytometry was used to detect the blood group A antigen on the cell surface.4.Expression of CD71 and CD235a on cell surfaceThe K562,K562-GFP and K562-A cells were incubated with anti-CD71 antibody and anti-CD235a antibody,washed and resuspended with PBS.The expression of CD71 and-CD235a on the cell surface was detected by flow cytometry.5.Analysis of cell cycle using propidium iodide(PI)stainingThe K562,K562-GFP and K562-A cells were fixed with ethanol.After fixing,the ethanol was removed.Then the cells were incubated with PI/RNase A staining solution.After incubation,the cells were washed and resuspended with PBS,and the PI fluorescence intensity on the cell surface was measured by flow cytometry.6.CCK8 assay for detecting the cell proliferation abilityThe cells in the logarithmic growth phase were seeded into a 96-well plate,and 5 replicate wells were set up.The cells were cultured for 0,24,48,72 h separately,and then the CCK8 reagent was added into each well.After incubation for 3 h,the absorbance value was detected at the wavelength of 450 nm using a microplate reader.7.To explore the effect of anti-A antibody on the mRNA expression of ABO genePlasma from healthy type O volunteers,as well as red blood cells suspensions from healthy type A and O volunteers were collected.Control group plasma that has no anti-A antibody:The anti-A antibody in type O plasma was absorbed and removed by type A red blood cells.Experimental group plasma containing anti-A antibody:The type O plasma was mixed with type O red blood cells for the same absorption treatment as the control group plasma,and anti-A antibody was not removed.Adding equal volume of experimental group plasma and control group plasma into the same number of cells(K562 cells or K562-A stable cells),adjusting the total volume of the culture medium to 2 mL,and incubating for 24 h.Then the RNA was extracted from the cells,reverse transcribed into cDNA,and the cellular mRNA expression of ABO gene in the experimental group and the control group was assessed by quantitative PCR.Results1.Acquisition of target genes and construction of recombinant vectorsThe target gene ABO*A1.01 allele was successfully obtained and the recombinant vector GV492-A was successfully constructed.2.Virus packaging,concentration and titer detectionThe packaged virus was obtained.The titer of the recombinant lentivirus was 1.5×109 TU/mL,and the titer of the GV492 lentivirus was 5..×108 TU/mL.3.Establishment and detection of stable cell linesThe rate of green fluorescent in K562-A cells achieved 98%.Sequencing results confirmed that the ABO*A1.01 allele was expressed in K562-A cells.Quantity PCR results showed that the mRNA expression levels of ABO gene was significantly increased in K562-A cells(P<0.01).Flow cytometry results showed that the blood group A antigen was significantly increased on the surface of K562-A cells.(P<0.05)4.Expression of CD71 and CD235a on cell surfaceCompared with K562 cells that were not transfected with lentivirus,the expression of CD235a on K562-GFP and K562-A cells increased(P<0.001),while the expression of CD71 decreased(P<0.01).Compared with K562-GFP cells,K562-A cells showed increased expression of CD235a(P<0.001),while the expression of CD71 decreased(P<0.05).5.Results of cell cycle analysisThe results of cell cycle analysis showed that compared with K562 cells,the percentage of cells in G1 phase was decreased(P<0.01)and the percentage of cells in G2 phase was increased(P<0.01)in K562-GFP and K562-A cells.Lentiviral transfection led to cell cycle arrest at G2/M phase.The percentage of cells in S phase was significantly increased in K562-A cells compared with K562 and K562-GFP cells(P<0.01).6.Results of CCK8 assayThe results of CCK8 assay showed that the proliferation rate of K562-GFP and K562-A cells was slower than K562 cells,especially at 72 h(P<0.001);and the proliferation rate of K562-GFP and K562-A cells had no difference.7.The effect of anti-A antibody on the mRNA expression of ABO gene in cellsThe mRNA expression levels of ABO gene in the experimental group cells were compared with those in the control group,and the results showed no statistical difference.It was indicated that the addition of anti-A antibody to the cell culture medium had no effect on the mRNA expression of ABO gene in K562 and K562-A cells.ConclusionsIn this study,a K562 cell line(K562-A)stably expressing the ABO*A1.01 allele was established,which expressed blood group A antigens on its surface.Incubating with anti-A antibody had no effect on the mRNA expression of ABO gene in K562 and K562-A cells.Lentivirus transfection promoted the differentiation of K562 cells.The expression of ABO*A1.01 allele promoted the differentiation of K562-A cells.Lentiviral transfection led to cell cycle arrest at G2/M phase.The expression of the ABO*A1.01 allele in K562-A cells resulted in a significant cell increase in S phase.Lentiviral transfection slow down the rate of cell proliferation. |
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