| BACKGROUND AND OBJECTIVEβ-thalassemia is a common monogenic disease also one of the hereditary diseases with highest morbidity.Its molecular pathogenesis has been found to be related with mutation or absence of beta globin gene.Maturing defect and ineffective production of erythrocytes may result from excessive production of alpha globin peptide chain, leading to consequent occurrence of hemolytic anemia.The human globin genes' expression has phase specificity as expressed or closed according the different period of ontogenic.Theα-globin gene cluster shows as the changes ofξtoαgenes,and theβ- globin gene cluster shows as the changes ofεtoγandγtoβgenes.Gene regulation therapy with drugs which started from last century 80s have entered the stage of clinical research and gained certain optimistic therapeutic effect now.Theγ- globin gene inductors can activate the expression of nearly closedγ-globin gene and produce fetal hemoglobin(HbF)(α2γ2) to replace adult hemoglobin(HbA)(α2β2),then ameliorate clinical symptoms of patients with thalassemia.Because of established therapeutic effect of these therapies,the exploration on this research direction is very important.The presentγ-globin gene inductors include hydroxyurea(HU),5-azaeytidine (5-AZAC),butyrate and so on.These drugs have disadvantages of bone marrow depression,potential carcinogenicity,short half life and expensive price.Thus their clinical application has been confined.So there is an urgent need of novelγ-globin gene inductors which have high efficiency,low toxicity,and low price to improve the therapeutic status.Compared to the Western medicine research,the traditional Chinese medicine research has shorter cycle time,not only can avoid the problems of artificial synthesis of Western medicine,but also have distinctive recognition of curative and side effect,so it has favorable research prospect.The domestic research has reported that Chinese medicinal formulae such as Yisui Shengxue Granule, Huanggen Jiawei Soup ext.can reactivate the expression ofγ-globin gene.However, because of the different gene phenotypes of thalassemia and complicate components of Chinese medicinal formulae,many scholars are trying to single out the very sapidity and active component of the traditional Chinese medicine,and they have made some achievement,components as meisoindigo and harringtonine were separated and extracted from some anti-tumor drugs in order to eveluate their therapeutic efficacies and the mechanism.Our previous study also found that APS seperated from the traditional Chinese medicine such as Yiqi Buxue,Bushen Shensui etc. had the ability to make the K562 cell line differentiate into the erythroid cells efficiently and improveγ-globin gene expression.At the same time of inducing the expression ofγ-globin gene,all kinds of drugs have different side effects of various degrees or limitation of clinical application.So combined administration is a therapy worth trying,and we hope that at the same time of reducing drug doses,it also can up-regulate the expression ofγ-globin gene by different mode of action or mechanism.Some scholars abroad have explored different patterns of combined administration,such as butyrate with erythropoietin(EPO) or 5-AZAC and so on,to induce the expression ofγ-globin gene on animals or patients. They found that combined administration of some drugs could up-regulate the expression ofγ-globin gene significantly than single drug administration.But some researcher suggested that combined administration can increase the toxicity to the cell and have the variable regulation to the HbF synthesis,so it need investigated.Western medicine delivers clinical effect fast,but it can not last long time and have severe adverse effect.The traditional Chinese medicine affects slowly,but it can last long time and has little adverse effect.Our previously study suggested that one of astragalus mongholicus's active components is astragalus polysaccharide(APS) which can make the K562 cell line differentiate into the erythroid cells efficiently, improveγ-globin gene expression and increase HbF synthesis.Compared with one of the western medicine—sodium butyrate(NAB),APS have the weak effect to inhibite the growing of the cells and its effect in inducing can last a long time.APS has extensive pharmacologic action.This valuable immune- enhancer could protect the target organ,and lessen the adverse effect such as the bone marrow depression after radiotherapy and chemotherapy.The effect of stimulating bone marrow heamatogenesis,strengthening immune function and resisting tumor has been acknowledged unanimously among academic circles.Combined administration of Western medicine and traditional Chinese medicine is certainly worth further exploring in order to get the complementary advantages and decrease the dose.Recently,one of the most popular methods to culture the human erythroid progenitor cells in vitro is using two- phase liquid culture system to culture the human erythroid progenitor cells,which could imitate the process of erythrocytopoiesis better than K562 cells,and it also could display the expression of globins' switching on the process of human ontogeny.Our goal was to determine the effects of combined administration of APS and NaB on proliferation and globin gene expression of human erythroid progenitor cells. We selected cord blood as the study object which is rich of haemopoietic stem or progenitor cells and use the two- phase liquid culture system to culture the human erythroid progenitor cells,and observate the expression rule of the globin genes of the normal erythroid cells.We observed and identified the cultured erythroid cells by trypan blue dye exclusion counting,morphology observation,reverse transcription-polymerase chain reaction(RT-PCR) et al.The results of our study will provide the theoretical foundation for combined administration of low dose drugs in clinical application. METHOD1.The resoure of the cord blood samples:From the fetuses'umbilical cord who were bomed from the healthy parturient in the department of gynaecology and obstetrics of the Nanfang Hospital which is the affiliated hospital of Southern Medical University,and the parturients didn't have the communicable disease,blood disease,cancer and the variable severse pregnancy complications.The samples were maintained in the sterile blood collection bag on sealing.2.Trial grouping:In this study,there were 6 experiment groups,which include APS+NaB(250μmol/L+2.5mg/mL)(combined administration in low dose),APS 2.5 mg/mL and NaB 250μmol/L(single administration in low does),APS 5.0mg/mL and NaB 500μmol/L(single administration in routine dose),while the blank control group weren't added any drugs.3.Using two-phase liquid culture system to culture human erythroid progenitor cells:Mononuclear cells were isolated from the cord blood samples,cultured in two different culture systems in vitro.At the 0 day,2 day,4 day,6 day,8 day,10 day,12 day and 14 day of the second culturing,the cells were collected to eveluate the expression rule of the globin genes during the culturing in the liquid culture system; At 6 day of the second culturing phase,the drugs were added to the medium as this study need.At 8 day,10 days,12 days and 14 days,culturing was stopped and cells were collected.4.Using trypan blue dye exclusion counting of living cells,human erythroid progenitor cells at different time points were observed to evaluate the cell proliferation and the effect of the drugs on the cell inhibition.5.Using RT-PCR analyze the level of the mRNA expression of the 7 globin genes(ξ,α,ε,Gγ,Aγ,δandβ) of human erythroid progenitor cells in different time points during cells culturing,also the expression situations affected by the drugs were evaluated.6.Statistical analysis: Statistical software(SPSS 13.0) was used for statistical analysis.Repetitive measurement and analysis of variance and One-way ANOVA were used to analyze the mean value of each group.When differences were statistically significant,the LSD method was applied for multiple comparisons,and the Dunnett's T3 testing was applied when variance was irregular.P＜0.05 means there exists a significant difference.RESULTS1.The proliferation of the erythroid progenitor cells in the liquid culture system and the change of the expression of the golbin genes1.1 The change of the number of the cell in the second culturing phaseIn the different time points,there is a significant difference in the number of the cells(F=655.682,P＜0.001).Comparing with dO,the number of the cells at d6 started to increase exponentially,and its proliferation rate began to slow down at d10 (plateau phase),and it touched the peak at d12[(4.01±0.25)x 106/ml](about 4 times of d0).1.2 The change of the globin genes' mRNA of the cells in the second culturing phaseα-globin gene cluster:With the differentiation and maturation of the erythroid progenitor cells,the level of the mRNA ofα-globin gene increased(F=19.845,P= 0.046) and touched the peak at d14(0.96±0.12);the level of the mRNA ofξ-globin gene was low,but was not correlated with the maturating of the erythroid progenitor cells.β-globin gene cluster:With the differentiation and maturation of the erythroid progenitor cells,β- globin gene increased(F=28.936,P=0.028) and touched the peak at d14(0.96±0.20);the level of the mRNA of Gγ- globin gene and Aγ- globin gene decreased(F=48.923,P=0.016;F=19.353,P=0.042) and touched the nadir at d14(0.45±0.05 and 0.37±0.08);ε- globin gene andδ- globin gene were low,but were not correlated with the maturating of the erythroid progenitor cells.The cross transformation of the expression of theγ- mRNA of globin gene andβ-globin gene appeared at the d8-d9.2.The effect of combined and single administrations(APS and NaB) on proliferation and the mRNA of the globin gene of the human erythroid progenitor cells.2.1 The effect of combined and single administrations(APS and NaB) on proliferation of the human erythroid progenitor cells.Treated with different concentration of APS,NaB or combined administration, the proliferation of the human erythroid progenitor cells started weakening from d8, and achieved a steady state at d10.The number of the cells treated with low does combined administration of APS+NaB[(3.13±0.24)(×106/mL)]were more than that of the cells treated with NaB single routine dose[(2.88±0.19)(×106/mL)]and less than that of cells treated with APS single routine dose[(3.52±0.36)(×106/mL)].Treated with different concentration of APS,NaB or combined administration, the human erythroid progenitor cells were inhibited at different degree(F=18.616, P=0.000).As time went by,the inhibition ratio of the cells in the group treated with low does combined administration of APS+NaB touched the peak[(36.84±1.27)%]at d10,and was lower than that in the group of single routine dose NaB[(44.55±2.83)%] (P＜0.05) and higher than that in the group of single routine dose APS[(19.00±7.76) %(p＜0.05)]2.2 The effect on the mRNA of the globin genes of the human erythroid progenitor cells treated with combined and single administration.Treated with low does combined administration of APS+NaB,the cells's mRNA of the Gγ-globin gene were up-regulated and touched the peak at d12,and was the 2.65±0.13 times of that in untreated cells group(F=23.850,P=0.000),much higher than single routine dose APS and single routine dose NAB[1.23±0.09 and 1.38±0.22 times that respectively in untreated cells group(P＜0.05)).Treated with low does combined administration of APS+NaB,the cells's mRNA of the Gγ,-globin gene gene was up-regulated and touched the peak at d12,and was the 1.53±0.23 times that in untreated cells group(F=53.714,P=0.015);much higher than single low dose APS and single low dose NAB[1.14±0.21 and 1.15±0.18 times that respectively in untreated cells group(P＜0.05)]and when it compared with the single routine dose APS and single routine dose NaB(1.48±0.07 and 1.35±0.12 times that respectively in untreated cells group),there have no the significant difference(P＜0.05).At every time spot,comparing with the control group,there is no significant difference(P＞0.05) in the level of the cells' mRNA ofβ-globin gene,ε-globin gene,δ-globin gene,α-globin gene,andξ-globin gene in every experiment group.CONCLUSION1.We successfully established the two-phase liquid culture which could express the process of human erythroid cells' ontogeny in vitro well.This cell model can be used to study the globin switching during the process of erythroid cells' differentiation and maturation.2.First time on human erythroid cells,we demonstrated that routine dose single administration of APS could up-regulate the expression of gamma globin,especially the transcription of Gγ-mRNA,and relieve the inhibition of cells.And it doesn't affect the expression ofβ-globin gene cluster such asβ-,ε- andδ-globin genes,andα-globin gene cluster such asα- andξ-globin genes.3.First time on human erythroid cells,we demonstrated that low dose combined administration of APS and NaB could up-regulate globin gene expression expecially increase the transcription of the Gγ-mRNA and decrease the inhibition to the cell growing,and have no effect to the expressiong of theβ-globin gene cluster(β-,ε-,andδ- globin genes) and the same to theα-globin gene cluster(α-,ξ- globin genes).4.First time on human erythroid cells,we demonstrated that compared with the single routine dose of APS or NaB,combined administration of APS and NaB can increase the expression of theγ-globin genes and decrease the inhibition to the cell growing and the toxicity to the cell more effectly.5.Our data provides the new scientific basis and the sutdy idea of the combined administration research to increase the expression of theγ-globin genes and the theoretical foundation for low dose combined administration of difference drugs on clinical application. |
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