苦参碱诱导K562细胞γ珠蛋白基因表达和向红系分化的研究
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摘要
研究背景及目的
β珠蛋白生成障碍性贫血(βthalassemia)是世界上最常见的常染色体单基因缺陷所致的遗传性疾病，也是对人类健康影响最大的慢性溶血性贫血病，其分子病理基础是由于β珠蛋白基因的突变或缺失，造成α珠蛋白肽链与非α珠蛋白肽链之间的不平衡，过剩的α肽链造成红细胞成熟缺陷和无效生成，最终使患者出现溶血性贫血。本病在世界范围内分布广，发病率高，危害大，长期以来珠蛋白一直是生物医学领域研究的热点之一，常被作为遗传病的重要范例。目前除通过社区筛查、遗传咨询和产前诊断等预防手段控制患者出生外，至今对重型和部分中间型β珠蛋白生成障碍性贫血患者的治疗仍有较大难度：规律输血并配合除铁剂，平均寿命仅达30岁左右；脾切除及脾动脉栓塞只对部分中间型β珠蛋白生成障碍性贫血患者有一定的疗效；造血干细胞移植能够根治，但相合配型的供体难寻、移植难度大而又费用高昂，限制了其广泛应用；目前最有前景的治疗方法是基因治疗(gene therapy)，广义的基因治疗包括基因矫正治疗和基因调控治疗，基因矫正治疗是指运用DNA重组技术修复患者细胞中有缺陷的基因，使细胞恢复功能而达到治疗遗传病的目的；理论上β珠蛋白生成障碍性贫血此类单基因遗传病是基因矫正治疗最理想的模型，人们一直期望其能成为第一个通过基因矫正而治愈的疾病，但因诸多技术难题至今尚未解决，目前仍停留于基础研究阶段。基因调控治疗是广义的基因治疗，是指运用药物来调控基因的表达而达到治疗疾病的目的。在临床上可见重型β珠蛋白生成障碍性贫血患者可以被表达增高的γ珠蛋白改善临床症状，受此启发进行的如何利用药物调控基因以阻止或延迟γ珠蛋白基因向β珠蛋白基因表达的切换及如何重新激活出生后已趋于关闭的γ珠蛋白基因表达的研究已被进行了20余年，由于此类治疗方法疗效确切，是目前唯一进入临床研究的基因治疗方法，给β珠蛋白生成障碍性贫血患者带来了希望。
迄今所报道的已进行了比较深入研究的化学药如羟基脲(hydraxyurea)、5—氮胞核苷(5-azacytidine)、重组人类促红细胞生成素(recombinant human EPO)及丁酸盐类及其衍生物(butyric acid and derivatives)等存在不同程度的骨髓抑制、潜在的致癌性及费用高昂、用药不便等缺点；另一些处于基础实验研究的化学药离临床应用尚远，且远期对人体的毒副作用还无法明了，因此目前迫切需要发掘出一些新的更有效且安全实用的激活γ珠蛋白基因的药物。中药以其副作用小，资源丰富等优点引起人们的注意，因此许多国内外学者致力于在中药中筛选出更为有效、低毒和低成本的γ珠蛋白基因诱导剂。
大多数γ珠蛋白基因诱导剂是从抗肿瘤药中发现的，化学药如5—氮胞核苷、羟基脲、阿糖胞苷、白消安、顺铂及阿霉素衍生物等，中药如三尖杉酯碱、甲异靛、长春新碱(三者是白血病化疗药)等。在本研究中我们选取11种在体外或体内研究中具有抗白血病功能的中药有效部位及活性成分作用于K562细胞，通过诱导K562细胞后联苯胺染色的血红蛋白定性实验，筛选出苦参碱具有诱导K562细胞向红系分化(erythroid differentiation)的作用，应用台盼蓝拒染活细胞计数和XTT法检测苦参碱对K562细胞增殖抑制(proliferative inhibition)的影响，通过联苯胺染色、western blotting及实时荧光定量RT-PCR分别从细胞水平、蛋白质水平和mRNA水平三个不同层次探讨苦参碱调控γ珠蛋白基因表达的作用，旨在从天然植物中筛选出新的γ珠蛋白基因诱导剂，为β珠蛋白生成障碍性贫血的治疗开辟新的途径。
研究方法
一、苦参碱对K562细胞增殖抑制与诱导向红系分化的研究方法
1．苦参碱对K562细胞增殖抑制的影响：K562细胞培养于RPMI-1640完全培养基中，在37℃、5％CO_2条件下培养。①台盼蓝拒染活细胞计数：取指数生长期K562细胞，以5×10~4 cells／mL密度接种于培养瓶中。分设苦参碱浓度为0.05g／L、0.10g／L和0.20g／L的3个实验组，只加等量培养液的阴性对照组和丁酸钠浓度为0.5mmol／L的阳性对照组。从接种当时起每隔24 h取部分细胞进行台盼蓝拒染活细胞计数，连续计数6 d。②XTT法：取指数生长期K562细胞，制成1×10~4 cells／mL的细胞悬液，设培养液组(只加培养液-本底背景)、苦参碱组(浓度分别为0.05g／L、0.10g／L和0.20g／L)、阴性对照组(未加任何药处理)和阳性对照组(丁酸钠浓度为0.5mmol／L)，于d 3检测苦参碱对K562细胞增殖抑制的剂量效应。
2．苦参碱诱导K562细胞向红系分化的影响：①联苯胺染色检测苦参碱诱导K562细胞血红蛋白的表达：细胞培养及实验分组见台盼蓝拒染活细胞计数，从接种当时起每隔24 h先进行台盼蓝拒染活细胞计数，细胞活力在95％以上时，再取部分细胞进行联苯胺染色，计算联苯胺染色阳性细胞率(BZ％)，连续计数6 d。②Wright-Gimesa染色检测苦参碱诱导K562细胞的形态学变化：将阴性对照组及0.10g／L苦参碱诱导d 4的K562细胞，分别离心制作细胞涂片，观察细胞形态学上的改变。
二、苦参碱诱导K562细胞γ珠蛋白基因表达的研究方法
细胞培养及实验分组方法同台盼蓝拒染活细胞计数。
1．Western blotting检测苦参碱诱导K562细胞胎儿血红蛋白(HbF)的合成：做剂量效应时，收集0.05g／L、0.10g／L和0.20g／L浓度苦参碱和0.5mmol／L丁酸钠诱导d 4及阴性对照组K562细胞；做时间效应时，分别在d 3、d 4、d 5收集0.10g／L苦参碱诱导后及阳性对照组、阴性对照组细胞。对上述收集的细胞提取总蛋白，先做蛋白定量，再以β-actin作为看家基因，应用Western blotting获得各组HbF及相对应的β-actin的蛋白印迹结果，通过凝胶分析软件进行蛋白条带的灰度测定，用各组HbF条带的灰度值除以各自相对应的内参β-actin的灰度值，进行蛋白上样量的校正，再以校正后的阴性对照组的表达量作为“1”，计算出各组相对于阴性对照组的倍数，对所得倍数进行统计学处理。
2．实时荧光定量RT-PCR检测苦参碱诱导K562细胞γ珠蛋白基因mRNA的表达：采用相对定量解析方法进行mRNA表达量的分析。应用标准曲线进行样品间扩增效率校正；选择β-actin作为参比基因对所有样品进行归一化处理(RNA量校正)。制作标准曲线时以高浓度总RNA反转录后的cDNA作为样品，用EASY Dilution对样品进行10倍梯度稀释后，构建相对定量标准曲线。做剂量效应时，收集0.05g／L、0.10g／L和0.20g／L浓度苦参碱和0.5mmol／L丁酸钠诱导d3及阴性对照组细胞；做时间效应时，分别在d2、d3、d4时收集0.10g／L苦参碱诱导后及阳性对照组、阴性对照组细胞。对所收集的细胞提取总RNA，对其进行质量鉴定后，行实时荧光定量RT-PCR反应，调整待测样品浓度，使所得Ct值落在标准曲线上，然后与标准样品同时分管进行扩增，得到的各个目的基因及看家基因mRNA的Ct值分别代入各自对应的标准曲线。以看家基因的定量结果作为“1”，计算出各组相对于看家基因的倍数，即为误差校正后的总RNA量。再以校正后的阴性对照组的表达量作为“1”，计算出各组相对于阴性对照组的倍数，对所得倍数进行统计学处理。
结果
一、苦参碱对K562细胞增殖抑制与诱导向红系分化的结果
1．苦参碱对K562细胞增殖抑制的影响：通过台盼蓝拒染活细胞计数和XTT法检测，苦参碱诱导K562细胞后不同时间之间有显著差异(P=0.000)；不同浓度苦参碱诱导后的细胞数之间有显著差异(P=0.000)。各浓度苦参碱对K562细胞增殖均有抑制作用，增殖抑制程度随苦参碱浓度的增大抑制作用增加。苦参碱能够以剂量依赖的方式抑制K562细胞的增殖。
2．苦参碱诱导K562细胞向红系分化的影响：①联苯胺染色检测苦参碱诱导K562细胞血红蛋白(Hb)合成：不同浓度苦参碱诱导不同时间联苯胺染色阳性细胞率(BZ％)有显著差异(P=0.000)。苦参碱能以剂量依赖和时间依赖的方式诱导K562细胞Hb合成，在0.10g／L浓度苦参碱诱导K562细胞d 4 BZ％达到高峰15.7％。②Wright-Gimesa染色检测苦参碱诱导K562细胞形态学变化：未诱导的K562细胞表现为未分化的祖细胞形态；诱导后的K562细胞在形态学上出现向红系分化的特征。
二、苦参碱诱导K562细胞γ珠蛋白基因表达的结果
1．Western blotting检测苦参碱诱导K562细胞胎儿血红蛋白(HbF)的合成：0.05g／L、0.10g／L和0.20g／L浓度的苦参碱及丁酸钠诱导K562细胞d4 HbF合成增加倍数分别为1.08、1.53、1.36和1.52。0.10g／L、0.20g／L浓度的苦参碱与阴性对照间有显著差异(P＜0.01)；0.10g／L浓度苦参碱诱导K562细胞d3、d4、d5时及丁酸钠诱导d3时HbF合成增加倍数分别为1.38、1.81、1.54、1.92。苦参碱诱导K562细胞d3、d4、d5合成的HbF均与阴性对照组间有显著差异(P＜0.01)。苦参碱能以剂量依赖和时间依赖的方式诱导K562细胞合成HbF。在0.10g／L浓度苦参碱诱导K562细胞d4达到最佳剂量和时间效应。
2．实时荧光定量RT-PCR检测苦参碱诱导K562细胞γ珠蛋白基因mRNA的表达：①~Aγ珠蛋白基因mRNA的表达：各浓度苦参碱诱导K562细胞d3及0.10g／L浓度苦参碱诱导K562细胞d2、d3和d4时，~Aγ珠蛋白基因mRNA的表达均与阴性对照组间无显著差异(P＞0.05)。②~Gγ珠蛋白基因mRNA的表达：0.05g／L、0.10g／L和0.20g／L浓度苦参碱及丁酸钠诱导K562细胞d3 ~Gγ珠蛋白基因mRNA表达增加倍数分别为：1.40、2.72、2.20和3.39。0.10g／L、0.20g／L苦参碱与阴性对照间有显著差异(P＜0.05)；苦参碱诱导d2、d3和d4及丁酸钠诱导d3时K562细胞~Gγ珠蛋白基因mRNA表达增加倍数分别为1.57、3.08、2.54和3.45。3个不同天数的苦参碱诱导后的~Gγ珠蛋白基因mRNA的表达均与阴性对照组间有显著差异(P＜0.01)。故苦参碱能以剂量依赖及时间依赖的方式诱导K562细胞~Gγ珠蛋白基因mRNA表达。在0.10g／L浓度苦参碱诱导K562细胞d3达到最佳剂量和时间效应；苦参碱诱导K562细胞γ珠蛋白基因mRNA表达增加，主要是通过诱导~Gγ珠蛋白基因mRNA表达增加实现的。
结论
1．联苯胺染色显示：苦参碱能够以剂量依赖及时间依赖的方式诱导K562细胞血红蛋白的合成。
2．Western blotting检测结果显示：苦参碱能够以剂量依赖及时间依赖的方式诱导K562细胞血红蛋白F的合成，这表明苦参碱通过诱导K562细胞血红蛋白F的表达而促使血红蛋白的合成。
3．实时荧光定量RT-PCR检测结果显示：苦参碱能够以剂量依赖及时间依赖的方式上调K562细胞~Gγ，珠蛋白mRNA表达，而对~Aγ珠蛋白mRNA表达无明显作用。这表明苦参碱是通过上调~Gγ珠蛋白mRNA的表达诱导K562细胞血红蛋白F的合成。
4．苦参碱能够诱导K562细胞向红系分化，具有与丁酸钠相同的诱导γ珠蛋白基因的表达，是一种低毒、价廉的γ珠蛋白基因诱导剂。
5．本研究为药物诱导调控治疗β珠蛋白生成障碍性贫血提供基础实验依据。
主要创新点
1．通过对K562细胞被诱导后联苯胺染色的血红蛋白定性实验，从11种在体外或体内研究中具有抗白血病功能的中药有效部位及单体中筛选出苦参碱具有诱导K562细胞向红系分化的作用。
2．0.05g／L、0.10g／L及0.20g／L苦参碱对K562细胞增殖有抑制作用，随剂量加大细胞增殖抑制加著，苦参碱在抑制细胞增殖的同时伴有向红系分化的作用。首次证实苦参碱具有诱导胎儿血红蛋白合成增加的作用。
3．首次证实苦参碱具有诱导K562细胞γ珠蛋白基因表达的作用，且主要是通过诱导~Gγ珠蛋白基因的表达实现的。
4．苦参碱具有与丁酸钠相似的诱导γ，珠蛋白基因表达的作用，是一种低毒、价廉的γ珠蛋白基因诱导剂。
Background and Objectives
Beta-thalassemia is one of the most common somatic chromosome monogenicdiseases and a chronic hemolytic anemia that imposes greatest impact on human health,its molecular pathogenesis has been found to be related to mutation or absence of betaglobin gene that leads to the imbalance between alpha globin peptide chain andnon-alpha globin peptide chain. Maturation defect and ineffective production oferythrocytes may result from excessive alpha globin peptide chain, leading toconsequent occurrence of hemolytic anemia. Due to its worldwide distribution, highincidence and severe consequences, globin has been being one of the hottest topics inthe field of biomedicine as an important paradigm for the treatment of geneticdisorders. So far, it still remains difficult in treating beta-thalassemia major and partialbeta-thalassemia intermedia, though such preventive measures as community screening,genetic counseling and prenatal diagnosis have been taken to control the birth ofperilous fetuses. Presently, therapies for beta-thalassemia include regular bloodtransfusion with iron chelating agents, patients receiving which have a mean life-spanof about 30 years; splenectomy and partial splenic embolization, which is effective for only partial beta-thalassemia intermedia; and hematopoietic stem cells (HSCs)transplantation, a radical cure for the disease, but the widespread application of whichis restricted due to the difficulty in tissue matching, complicated transplantationprocedures and high cost. At present, the most promising treatment forbeta-thalassemia is the gene therapy, including gene correction therapy and generegulation therapy in general. Gene correction therapy is to achieve functional recoveryof the cells by repairing the defective gene with DNA recombinant techniques fortreatment of genetic disorders. Theoretically, beta-thalassemia, for example, themonogenic disease, is the most ideal model for gene correction therapy, so it has beenexpected to be the first curable monogenic disease through correction of the defectivegene at the molecular level. However, it is still in its initial stage due to sometechnological puzzles. Gene regulation therapy, in a broad sense, refers to themedication therapy for genetic disorders by regulating target gene expression. It is wellknow that an increase in gamma-globin production could ameliorate the clinicalsymptoms of patients with beta-thalassemia major, which sheds lights on studies of thepast two decades to find how to prevent or postpone the expression switch fromgamma-globin gene to beta-globin gene and how to reactivate the expression ofgamma-globin gene that tends to postnatally terminate, It is the unique gene therapythat has entered into the stage of clinical research and brings about hope for patientswith beta-thalassemia.
So far, the reported pharmaceutical chemicals with high therapeutic efficacy, forexample, hydraxyurea, 5-azacytidine, recombinant human EPO and butyric acid andderivatives, have such disadvantages to certain extents as marrow inhibition, potentialcarcinogenesis, high cost and inconvenient intake, while other compounds that arebeing undergone basic experimental studies have been far from clinical application and their long-term toxicological effects are still unknown. So, it is badly needed todevelop new agents that might be able to more effectively and safely stimulategamma-globin gene. Traditional Chinese herbal remedies have gained widespreadattention from both domestic and overseas scholars due to their minor side effects andabundant resources. Therefore, many scientists are attempting to screen out moreeffective, less toxic and cheaper agents as the inductor of gamma-globin gene.
Many gamma-globin gene inductors were found from antitumor agents, includingchemical compounds (e.g. 5-azacytidine, arabino-furanosyl-cytosine, hydraxyurea,tallimustine, cisplatin analogs and doxorubicin derivatives), herbs (harringtonine,meisoindigo and vinblastine) for chemotherapy of leukemia. In our study, 11 effectiveparts or simple substances of Chinese medicines that are anti-leukemic confirmed by invitro and in vivo studies are used to induce hemoglobin production of human chronicmyelogenous leukemia (CML) cell line K562, by benzidine staining. Matrine wasfirstly screened out as an inductor for erythroid differentiation of K562 cells.Meanwhile, effects of matrine on K562 cells proliferative inhibition were determinedby trypan-blue dye exclusion test and the colorimetric (XTT) assay, followed bywestern blotting and real time fluorescence quantitative RT-PCR to investigate theexpression of gamma-globin gene at protein and mRNA level, respectively. We attemptto screen out a new gamma-globin inductor for the treatment of beta-thalassemia.
Methods
1. Methods for proliferation inhibition and erythroid differentiation of K562 cellsby matrine
1.1 Methods for proliferation inhibition of K562 cells by matrine: K562 cells werecultured in RPMI-1640 containing 10% fetal bovine serum, 100U/mL penicillin,100U/mL streptomycin and 2 mmol/L glutamine in a 5% CO_2 incubator at 37℃.
1.1.1 Trypan-blue dye exclusion test: K562 cells at the exponential growth phase wereharvested to be inoculated into the culture flask of 25 cm~2 at the density of 5×10~4 cells/mL, 10mL per culture flask. According to the final mass concentration of matrine,three experimental groups were established at the matrine final mass concentrations of0.05g/L, 0.10g/L and 0.20g/L. Instead of matrine, partes aequales of RPMI-1640culture fluid and 0.5mmol/L sodium butyrate were taken as the negative and thepositive controls, respectively. Trypan-blue dye exclusion test was performed atintervals of 24 h from the time of inoculation and aliquots were removed daily for up to6 days.
1.1.2 Colorimetric (XTT) assay: K562 cells at exponential growth phase wereharvested and re-suspended at a density of 1×10~4 cells/mL. Six group including threecontrols (RPMI-1640 culture medium only for a blank, a negative and a positive withaddition of 0.5mmol/L sodium butyrate) and three experimental group (with finalmatrine mass concentrations of 0.05g/L, 0.10g/L and 0.20g/L, respectively) wereinvolved in this study. At day 3, the dosage effect of matrine on K562 cellsproliferative inhibition was recorded.
1.2 Methods for erythroid differentiation of K562 cells by matrine:
1.2.1 Benzidine staining: K562 cells culture procedures and experimental design werethe same as those in Trypan blue exclusion assay. Trypan blue exclusion assay wasperformed at the intervals of 24 h from the time of inoculation. Benzidine staining wascarried out only in case of the cell viability greater than 95%. The percentage ofbenzidine-positive cells (visualized as cells with blue crystals) was calculated for up to6 days.
1.2.2 Wright-Gimesa staining: K562 cells of negative control group and the matrinegroup at the final mass concentration of 0.10g/L at day 4 was centrifuged respectively for the preparation of cell smears and then stained with Wright-Gimesa for subsequentmorphological observations.
2. Methods for matrine-induced gamma-globin gene expression in K562
K562 cells culture procedures and experimental design were performed asdescribed in the section of Trypan blue exclusion assay. Western blotting techniquesand real time fluorescence quantitative RT-PCR was used to detect levels of proteinsynthesis and mRNA expression, respectively.
2.1 Methods for matrine-induced fetal hemoglobin (α_2γ_2) synthesis in K562 cells
For dose-dependent effects of matrine, K562 cells were collected fromexperimental group at the final matrine concentrations of 0.05g/L, 0.10g/L and 0.20g/L,from the positive control group at day 4 and from the negative control group,respectively. For time-dependent effects ofmatrine, K562 cells were collected from the0.10g/L matrine group at day 3, 4 and 5, from the positive and the negative controls.The total protein was extracted from the collected K562 cells and firstly was performedprotein quantitation, taking beta-actin as the house-keeping gene. These proteinblotting results of Fetal hemoglobin (α_2γ_2) and the corresponding of beta-actin fromeach group were separted by Western blotting techniques and analyzed by Gel analysissoftware package, Gel-pro analyzer 3.1, was used to determine the gray scale of theprotein bands. The ratio of each gray scale of fetal hemoglobin band to itscorresponding gray scale of beta-actin band was used for correction of protein samplequantities. The corrected expression of the negative control was taken as "1" forcalculating relative fold of each other groups, which were used for subsequentstatistical comparisons.
2.2 Methods for matrine-induced ~Gγglobin gene and ~Aγglobin gene mRNA expressionin K562 cells
Quantitative of mRNA expression was performed with relative quantitationanalysis. The standard curve was used for inter-sample amplifying efficiencycorrection. All these samples were performed normalization (quantitative correction ofRNA), taking beta-actin, as the house-keeping gene. When making the standard curve,cDNA sample, reverse transcription for total RNA at high concentration, was diluted10 times by specific agent, EASY Dilution. Five Ct values of amplified gradientconcentration samples were obtained through real time fluorescence quantitativeRT-PCR. Based on the 5 points, the linear relative quantitation standard curve wasfinally constructed with Ct values of each gradient samples as the y axis and with Logvalues of corresponding original template concentrations as the x axis (y=ax+b). Fordose-dependent effects, K562 cells from all groups at day 3 were collected. Fortime-dependent effects of matrine, K562 cells from the experimental group (0.10g/L ofmatrine) at day 2, 3 and 4, positive and negative controls were harvested, respectively.The extracted total RNA was firstly identified by agarose gel electrophoresis (AGE)and ultraviolet spectrophotometry, and then by real time fluorescent quantitativeRT-PCR to regulate the sample concentration for placement of Ct values on thestandard curve. The samples to be assayed and the standard sample were spontaneouslyamplified in different tubes for respective destination gene. The Ct values of eachdestination gene mRNA and house-keeping gene mRNA were then substituted into thecorresponding standard curve. The quantified house-keeping gene mRNA was taken as"1" to calculate the relative times of each destination gene mRNA as the errorcorrected total RNA. Subsequently, the corrected expression of negative control wastaken as "1" for calculating the relative expression of other group, which were used forstatistical comparisons.
Results
1 Effects of matrine on proliferation inhibition and erythroid differentiation inK562 cells
1.1 Results of matrine on proliferation inhibition in K562 cells by trypan-blue dyeexclusion test and XTT assay: After pretreatment with matrine, decreased K562 cellscounts have statistical significance not only at different time points (P=0.000), but atdifferent matrine concentrations (P=0.000). K562 cells were inhibited after treatedwith each matrine concentration, the more proliferation inhibit strong and the moreincreasing matrine concentrateion, matrine can inhibit proliferation of K562 cells bydose-dependent way.
1.2. Result of matrine on erythroid differentiation in K562 cells:
After pretreatment with different concentrations of matrine, the percentage ofbenzidine-positive cells (BZ%) of K562 cells increased statistically not only atdifferent time points (P=0.000) in a time dependent way, but at different matrineconcentrations (P=0.000) in a dose dependent way. The reault indicates that matrineinduce hematoglobin production of K562 cells by dose-dependent and time-dependentway. The peak value reached 15.7% on day 4 at 0.10g/L ofmatrine.
Morphological assessment of the K562 cells stained with Wright-Gimesa: theuntreated K562 cells showed features of undifferentiated progenitor in shape, whilematrine-induced K562 cells on day 4 were characterized of erythroid differentiation.
2 Matrine-inducedγ-globin gene expressions in K562 cells
2.1. Matrine-induced fetal hemoglobin (HbF) synthesis in K562 cells by Westernblotting: HbF production incresed were 1.08-, 1.53-, 1.36- and 1.52- fold of K562 cellsin the presence of 0.05g/L, 0.10g/L and 0.20g/L of matrine and sodium butyrate,respectively. K562 cells which induced by 0.10g/L and 0.20g/L matrine have statistical significance relative to negative control (P＜0.01). HbF production incresed were 1.38-,1.81-, 1.54- and 1.92- fold on day 3, 4 and 5 after matrine treatment and on day 3 afterincubation with sodium butyrate, respectively, as compared to negative control (P＜0.01). Matrine-induced fetal hematoglobin synthesis of K562 cells by dose-dependentand time-dependent way. The action reached its peak of K562 cells on day 4 at 0.10g/Lof matrine.
2.2 Matrine-induced accumulation ofγ-globin gene mRNA in K562 cells by real timeFQ-RT-PCR.
No statistical significance of ~Aγ-globin gene mRNA expression with differentconcentration matrine and on different time points among all experimental group ascompared to negative control (P＞0.05).
Accumulation of ~Gγ-globin gene mRNA expression in K562 cells were 1.40-,2.72-, 2.20- and 3.39- fold on day 3 in the presence of 0.05g/L, 0.10g/L and 0.20g/L ofmatrine and sodium butyrate, respectively. ~Gγ-globin gene mRNA expression inducedby 0.10g/L and 0.20g/L of matrine have statistical significance relative to negativecontrol (P＜0.05); Accumulation of ~Gγ-globin mRNA were 1.57-, 3.08-, 2.54- and3.45-fold on day 2, 3 and 4 in the presence of matrine and on day 3 after incubationwith sodium butyrate, respectively. ~Gγ-globin gene mRNA expression induced ondifferent time have statistical significance relative to negative control (P＜0.05);Matrine-induce K562 cells accumulate ~Gγ-globin gene mRNA in a dose-dependent andtime-dependent way. The effect reached its peak on day 3 at 0.10g/L of matrine. Theseresults suggested that matrine-induced accumulativeγ-globin gene mRNA expressionwas associated with accumulation ~Gγ-globin gene mRNA expression.
Conclusions:
1. Matrine (at 0.05g/L, 0.10g/L and 0.20g/L) can inhibit K562 cells proliferation and induce erythroid differentiation by increasing hematoglobin synthesis in adose-dependent and time-dependent way. The effect reached its peak on day 4 at0.10g/L of matrine.
2. Matrine can induce K562 cells to synthesis fetal hematoglobin in a dose-dependentand time-dependent way. The effect reached its peak on day 4 at matrine concentrationof 0.10g/L.
3. Matrine-induced accumulativeγ-globin gene mRNA expression was asscciated withaccumulation ~Gγ-globin gene mRNA expression. The effect reached its peak on day 3 atmatrine concentration of 0.10g/L.
4. The induction ofγ-globin genes expression by matrine is similar to that of sodiumbutyrate, which was used as positive control.
5. The study paves the experimentation base for pharmacologically mediatedupregulation expression of humanγ-globin genes and production of fetal hemoglobinas a potential therapeutic strategy in theβ-thalassaemia.
Main new ideas:
1. 11 kinds of effective parts and simple substances of Chinese medicines that areanti-leukemic confirmed by in vitro or in vivo studies are used to induce hemoglobinproduction of K562 cells by benzidine staining, and matrine was firstly screened out asan inducer for erythroid differentiation of K562 cells.
2. 0.05g/L, 0.10g/L and 0.20g/L of matrine can inhibit proliferation and erythroiddifferentiation in K562 cells and firstly verity that matrine can induce fetal hemoglobinsynthesis.
3. Firstly verity that accumulation ofγ-globin gene mRNA after treatment of humanK562 cells with matrine and was associated with the increase ~Gγ, globin gene mRNAexpression.
4. Matrine is a hypo-toxious and effective inducer ofγ-globin gene and similar tosodium butyrate.
β珠蛋白生成障碍性贫血(βthalassemia)是世界上最常见的常染色体单基因缺陷所致的遗传性疾病，也是对人类健康影响最大的慢性溶血性贫血病，其分子病理基础是由于β珠蛋白基因的突变或缺失，造成α珠蛋白肽链与非α珠蛋白肽链之间的不平衡，过剩的α肽链造成红细胞成熟缺陷和无效生成，最终使患者出现溶血性贫血。本病在世界范围内分布广，发病率高，危害大，长期以来珠蛋白一直是生物医学领域研究的热点之一，常被作为遗传病的重要范例。目前除通过社区筛查、遗传咨询和产前诊断等预防手段控制患者出生外，至今对重型和部分中间型β珠蛋白生成障碍性贫血患者的治疗仍有较大难度：规律输血并配合除铁剂，平均寿命仅达30岁左右；脾切除及脾动脉栓塞只对部分中间型β珠蛋白生成障碍性贫血患者有一定的疗效；造血干细胞移植能够根治，但相合配型的供体难寻、移植难度大而又费用高昂，限制了其广泛应用；目前最有前景的治疗方法是基因治疗(gene therapy)，广义的基因治疗包括基因矫正治疗和基因调控治疗，基因矫正治疗是指运用DNA重组技术修复患者细胞中有缺陷的基因，使细胞恢复功能而达到治疗遗传病的目的；理论上β珠蛋白生成障碍性贫血此类单基因遗传病是基因矫正治疗最理想的模型，人们一直期望其能成为第一个通过基因矫正而治愈的疾病，但因诸多技术难题至今尚未解决，目前仍停留于基础研究阶段。基因调控治疗是广义的基因治疗，是指运用药物来调控基因的表达而达到治疗疾病的目的。在临床上可见重型β珠蛋白生成障碍性贫血患者可以被表达增高的γ珠蛋白改善临床症状，受此启发进行的如何利用药物调控基因以阻止或延迟γ珠蛋白基因向β珠蛋白基因表达的切换及如何重新激活出生后已趋于关闭的γ珠蛋白基因表达的研究已被进行了20余年，由于此类治疗方法疗效确切，是目前唯一进入临床研究的基因治疗方法，给β珠蛋白生成障碍性贫血患者带来了希望。
迄今所报道的已进行了比较深入研究的化学药如羟基脲(hydraxyurea)、5—氮胞核苷(5-azacytidine)、重组人类促红细胞生成素(recombinant human EPO)及丁酸盐类及其衍生物(butyric acid and derivatives)等存在不同程度的骨髓抑制、潜在的致癌性及费用高昂、用药不便等缺点；另一些处于基础实验研究的化学药离临床应用尚远，且远期对人体的毒副作用还无法明了，因此目前迫切需要发掘出一些新的更有效且安全实用的激活γ珠蛋白基因的药物。中药以其副作用小，资源丰富等优点引起人们的注意，因此许多国内外学者致力于在中药中筛选出更为有效、低毒和低成本的γ珠蛋白基因诱导剂。
大多数γ珠蛋白基因诱导剂是从抗肿瘤药中发现的，化学药如5—氮胞核苷、羟基脲、阿糖胞苷、白消安、顺铂及阿霉素衍生物等，中药如三尖杉酯碱、甲异靛、长春新碱(三者是白血病化疗药)等。在本研究中我们选取11种在体外或体内研究中具有抗白血病功能的中药有效部位及活性成分作用于K562细胞，通过诱导K562细胞后联苯胺染色的血红蛋白定性实验，筛选出苦参碱具有诱导K562细胞向红系分化(erythroid differentiation)的作用，应用台盼蓝拒染活细胞计数和XTT法检测苦参碱对K562细胞增殖抑制(proliferative inhibition)的影响，通过联苯胺染色、western blotting及实时荧光定量RT-PCR分别从细胞水平、蛋白质水平和mRNA水平三个不同层次探讨苦参碱调控γ珠蛋白基因表达的作用，旨在从天然植物中筛选出新的γ珠蛋白基因诱导剂，为β珠蛋白生成障碍性贫血的治疗开辟新的途径。
研究方法
一、苦参碱对K562细胞增殖抑制与诱导向红系分化的研究方法
1．苦参碱对K562细胞增殖抑制的影响：K562细胞培养于RPMI-1640完全培养基中，在37℃、5％CO_2条件下培养。①台盼蓝拒染活细胞计数：取指数生长期K562细胞，以5×10~4 cells／mL密度接种于培养瓶中。分设苦参碱浓度为0.05g／L、0.10g／L和0.20g／L的3个实验组，只加等量培养液的阴性对照组和丁酸钠浓度为0.5mmol／L的阳性对照组。从接种当时起每隔24 h取部分细胞进行台盼蓝拒染活细胞计数，连续计数6 d。②XTT法：取指数生长期K562细胞，制成1×10~4 cells／mL的细胞悬液，设培养液组(只加培养液-本底背景)、苦参碱组(浓度分别为0.05g／L、0.10g／L和0.20g／L)、阴性对照组(未加任何药处理)和阳性对照组(丁酸钠浓度为0.5mmol／L)，于d 3检测苦参碱对K562细胞增殖抑制的剂量效应。
2．苦参碱诱导K562细胞向红系分化的影响：①联苯胺染色检测苦参碱诱导K562细胞血红蛋白的表达：细胞培养及实验分组见台盼蓝拒染活细胞计数，从接种当时起每隔24 h先进行台盼蓝拒染活细胞计数，细胞活力在95％以上时，再取部分细胞进行联苯胺染色，计算联苯胺染色阳性细胞率(BZ％)，连续计数6 d。②Wright-Gimesa染色检测苦参碱诱导K562细胞的形态学变化：将阴性对照组及0.10g／L苦参碱诱导d 4的K562细胞，分别离心制作细胞涂片，观察细胞形态学上的改变。
二、苦参碱诱导K562细胞γ珠蛋白基因表达的研究方法
细胞培养及实验分组方法同台盼蓝拒染活细胞计数。
1．Western blotting检测苦参碱诱导K562细胞胎儿血红蛋白(HbF)的合成：做剂量效应时，收集0.05g／L、0.10g／L和0.20g／L浓度苦参碱和0.5mmol／L丁酸钠诱导d 4及阴性对照组K562细胞；做时间效应时，分别在d 3、d 4、d 5收集0.10g／L苦参碱诱导后及阳性对照组、阴性对照组细胞。对上述收集的细胞提取总蛋白，先做蛋白定量，再以β-actin作为看家基因，应用Western blotting获得各组HbF及相对应的β-actin的蛋白印迹结果，通过凝胶分析软件进行蛋白条带的灰度测定，用各组HbF条带的灰度值除以各自相对应的内参β-actin的灰度值，进行蛋白上样量的校正，再以校正后的阴性对照组的表达量作为“1”，计算出各组相对于阴性对照组的倍数，对所得倍数进行统计学处理。
2．实时荧光定量RT-PCR检测苦参碱诱导K562细胞γ珠蛋白基因mRNA的表达：采用相对定量解析方法进行mRNA表达量的分析。应用标准曲线进行样品间扩增效率校正；选择β-actin作为参比基因对所有样品进行归一化处理(RNA量校正)。制作标准曲线时以高浓度总RNA反转录后的cDNA作为样品，用EASY Dilution对样品进行10倍梯度稀释后，构建相对定量标准曲线。做剂量效应时，收集0.05g／L、0.10g／L和0.20g／L浓度苦参碱和0.5mmol／L丁酸钠诱导d3及阴性对照组细胞；做时间效应时，分别在d2、d3、d4时收集0.10g／L苦参碱诱导后及阳性对照组、阴性对照组细胞。对所收集的细胞提取总RNA，对其进行质量鉴定后，行实时荧光定量RT-PCR反应，调整待测样品浓度，使所得Ct值落在标准曲线上，然后与标准样品同时分管进行扩增，得到的各个目的基因及看家基因mRNA的Ct值分别代入各自对应的标准曲线。以看家基因的定量结果作为“1”，计算出各组相对于看家基因的倍数，即为误差校正后的总RNA量。再以校正后的阴性对照组的表达量作为“1”，计算出各组相对于阴性对照组的倍数，对所得倍数进行统计学处理。
结果
一、苦参碱对K562细胞增殖抑制与诱导向红系分化的结果
1．苦参碱对K562细胞增殖抑制的影响：通过台盼蓝拒染活细胞计数和XTT法检测，苦参碱诱导K562细胞后不同时间之间有显著差异(P=0.000)；不同浓度苦参碱诱导后的细胞数之间有显著差异(P=0.000)。各浓度苦参碱对K562细胞增殖均有抑制作用，增殖抑制程度随苦参碱浓度的增大抑制作用增加。苦参碱能够以剂量依赖的方式抑制K562细胞的增殖。
2．苦参碱诱导K562细胞向红系分化的影响：①联苯胺染色检测苦参碱诱导K562细胞血红蛋白(Hb)合成：不同浓度苦参碱诱导不同时间联苯胺染色阳性细胞率(BZ％)有显著差异(P=0.000)。苦参碱能以剂量依赖和时间依赖的方式诱导K562细胞Hb合成，在0.10g／L浓度苦参碱诱导K562细胞d 4 BZ％达到高峰15.7％。②Wright-Gimesa染色检测苦参碱诱导K562细胞形态学变化：未诱导的K562细胞表现为未分化的祖细胞形态；诱导后的K562细胞在形态学上出现向红系分化的特征。
二、苦参碱诱导K562细胞γ珠蛋白基因表达的结果
1．Western blotting检测苦参碱诱导K562细胞胎儿血红蛋白(HbF)的合成：0.05g／L、0.10g／L和0.20g／L浓度的苦参碱及丁酸钠诱导K562细胞d4 HbF合成增加倍数分别为1.08、1.53、1.36和1.52。0.10g／L、0.20g／L浓度的苦参碱与阴性对照间有显著差异(P＜0.01)；0.10g／L浓度苦参碱诱导K562细胞d3、d4、d5时及丁酸钠诱导d3时HbF合成增加倍数分别为1.38、1.81、1.54、1.92。苦参碱诱导K562细胞d3、d4、d5合成的HbF均与阴性对照组间有显著差异(P＜0.01)。苦参碱能以剂量依赖和时间依赖的方式诱导K562细胞合成HbF。在0.10g／L浓度苦参碱诱导K562细胞d4达到最佳剂量和时间效应。
2．实时荧光定量RT-PCR检测苦参碱诱导K562细胞γ珠蛋白基因mRNA的表达：①~Aγ珠蛋白基因mRNA的表达：各浓度苦参碱诱导K562细胞d3及0.10g／L浓度苦参碱诱导K562细胞d2、d3和d4时，~Aγ珠蛋白基因mRNA的表达均与阴性对照组间无显著差异(P＞0.05)。②~Gγ珠蛋白基因mRNA的表达：0.05g／L、0.10g／L和0.20g／L浓度苦参碱及丁酸钠诱导K562细胞d3 ~Gγ珠蛋白基因mRNA表达增加倍数分别为：1.40、2.72、2.20和3.39。0.10g／L、0.20g／L苦参碱与阴性对照间有显著差异(P＜0.05)；苦参碱诱导d2、d3和d4及丁酸钠诱导d3时K562细胞~Gγ珠蛋白基因mRNA表达增加倍数分别为1.57、3.08、2.54和3.45。3个不同天数的苦参碱诱导后的~Gγ珠蛋白基因mRNA的表达均与阴性对照组间有显著差异(P＜0.01)。故苦参碱能以剂量依赖及时间依赖的方式诱导K562细胞~Gγ珠蛋白基因mRNA表达。在0.10g／L浓度苦参碱诱导K562细胞d3达到最佳剂量和时间效应；苦参碱诱导K562细胞γ珠蛋白基因mRNA表达增加，主要是通过诱导~Gγ珠蛋白基因mRNA表达增加实现的。
结论
1．联苯胺染色显示：苦参碱能够以剂量依赖及时间依赖的方式诱导K562细胞血红蛋白的合成。
2．Western blotting检测结果显示：苦参碱能够以剂量依赖及时间依赖的方式诱导K562细胞血红蛋白F的合成，这表明苦参碱通过诱导K562细胞血红蛋白F的表达而促使血红蛋白的合成。
3．实时荧光定量RT-PCR检测结果显示：苦参碱能够以剂量依赖及时间依赖的方式上调K562细胞~Gγ，珠蛋白mRNA表达，而对~Aγ珠蛋白mRNA表达无明显作用。这表明苦参碱是通过上调~Gγ珠蛋白mRNA的表达诱导K562细胞血红蛋白F的合成。
4．苦参碱能够诱导K562细胞向红系分化，具有与丁酸钠相同的诱导γ珠蛋白基因的表达，是一种低毒、价廉的γ珠蛋白基因诱导剂。
5．本研究为药物诱导调控治疗β珠蛋白生成障碍性贫血提供基础实验依据。
主要创新点
1．通过对K562细胞被诱导后联苯胺染色的血红蛋白定性实验，从11种在体外或体内研究中具有抗白血病功能的中药有效部位及单体中筛选出苦参碱具有诱导K562细胞向红系分化的作用。
2．0.05g／L、0.10g／L及0.20g／L苦参碱对K562细胞增殖有抑制作用，随剂量加大细胞增殖抑制加著，苦参碱在抑制细胞增殖的同时伴有向红系分化的作用。首次证实苦参碱具有诱导胎儿血红蛋白合成增加的作用。
3．首次证实苦参碱具有诱导K562细胞γ珠蛋白基因表达的作用，且主要是通过诱导~Gγ珠蛋白基因的表达实现的。
4．苦参碱具有与丁酸钠相似的诱导γ，珠蛋白基因表达的作用，是一种低毒、价廉的γ珠蛋白基因诱导剂。
Background and Objectives
Beta-thalassemia is one of the most common somatic chromosome monogenicdiseases and a chronic hemolytic anemia that imposes greatest impact on human health,its molecular pathogenesis has been found to be related to mutation or absence of betaglobin gene that leads to the imbalance between alpha globin peptide chain andnon-alpha globin peptide chain. Maturation defect and ineffective production oferythrocytes may result from excessive alpha globin peptide chain, leading toconsequent occurrence of hemolytic anemia. Due to its worldwide distribution, highincidence and severe consequences, globin has been being one of the hottest topics inthe field of biomedicine as an important paradigm for the treatment of geneticdisorders. So far, it still remains difficult in treating beta-thalassemia major and partialbeta-thalassemia intermedia, though such preventive measures as community screening,genetic counseling and prenatal diagnosis have been taken to control the birth ofperilous fetuses. Presently, therapies for beta-thalassemia include regular bloodtransfusion with iron chelating agents, patients receiving which have a mean life-spanof about 30 years; splenectomy and partial splenic embolization, which is effective for only partial beta-thalassemia intermedia; and hematopoietic stem cells (HSCs)transplantation, a radical cure for the disease, but the widespread application of whichis restricted due to the difficulty in tissue matching, complicated transplantationprocedures and high cost. At present, the most promising treatment forbeta-thalassemia is the gene therapy, including gene correction therapy and generegulation therapy in general. Gene correction therapy is to achieve functional recoveryof the cells by repairing the defective gene with DNA recombinant techniques fortreatment of genetic disorders. Theoretically, beta-thalassemia, for example, themonogenic disease, is the most ideal model for gene correction therapy, so it has beenexpected to be the first curable monogenic disease through correction of the defectivegene at the molecular level. However, it is still in its initial stage due to sometechnological puzzles. Gene regulation therapy, in a broad sense, refers to themedication therapy for genetic disorders by regulating target gene expression. It is wellknow that an increase in gamma-globin production could ameliorate the clinicalsymptoms of patients with beta-thalassemia major, which sheds lights on studies of thepast two decades to find how to prevent or postpone the expression switch fromgamma-globin gene to beta-globin gene and how to reactivate the expression ofgamma-globin gene that tends to postnatally terminate, It is the unique gene therapythat has entered into the stage of clinical research and brings about hope for patientswith beta-thalassemia.
So far, the reported pharmaceutical chemicals with high therapeutic efficacy, forexample, hydraxyurea, 5-azacytidine, recombinant human EPO and butyric acid andderivatives, have such disadvantages to certain extents as marrow inhibition, potentialcarcinogenesis, high cost and inconvenient intake, while other compounds that arebeing undergone basic experimental studies have been far from clinical application and their long-term toxicological effects are still unknown. So, it is badly needed todevelop new agents that might be able to more effectively and safely stimulategamma-globin gene. Traditional Chinese herbal remedies have gained widespreadattention from both domestic and overseas scholars due to their minor side effects andabundant resources. Therefore, many scientists are attempting to screen out moreeffective, less toxic and cheaper agents as the inductor of gamma-globin gene.
Many gamma-globin gene inductors were found from antitumor agents, includingchemical compounds (e.g. 5-azacytidine, arabino-furanosyl-cytosine, hydraxyurea,tallimustine, cisplatin analogs and doxorubicin derivatives), herbs (harringtonine,meisoindigo and vinblastine) for chemotherapy of leukemia. In our study, 11 effectiveparts or simple substances of Chinese medicines that are anti-leukemic confirmed by invitro and in vivo studies are used to induce hemoglobin production of human chronicmyelogenous leukemia (CML) cell line K562, by benzidine staining. Matrine wasfirstly screened out as an inductor for erythroid differentiation of K562 cells.Meanwhile, effects of matrine on K562 cells proliferative inhibition were determinedby trypan-blue dye exclusion test and the colorimetric (XTT) assay, followed bywestern blotting and real time fluorescence quantitative RT-PCR to investigate theexpression of gamma-globin gene at protein and mRNA level, respectively. We attemptto screen out a new gamma-globin inductor for the treatment of beta-thalassemia.
Methods
1. Methods for proliferation inhibition and erythroid differentiation of K562 cellsby matrine
1.1 Methods for proliferation inhibition of K562 cells by matrine: K562 cells werecultured in RPMI-1640 containing 10% fetal bovine serum, 100U/mL penicillin,100U/mL streptomycin and 2 mmol/L glutamine in a 5% CO_2 incubator at 37℃.
1.1.1 Trypan-blue dye exclusion test: K562 cells at the exponential growth phase wereharvested to be inoculated into the culture flask of 25 cm~2 at the density of 5×10~4 cells/mL, 10mL per culture flask. According to the final mass concentration of matrine,three experimental groups were established at the matrine final mass concentrations of0.05g/L, 0.10g/L and 0.20g/L. Instead of matrine, partes aequales of RPMI-1640culture fluid and 0.5mmol/L sodium butyrate were taken as the negative and thepositive controls, respectively. Trypan-blue dye exclusion test was performed atintervals of 24 h from the time of inoculation and aliquots were removed daily for up to6 days.
1.1.2 Colorimetric (XTT) assay: K562 cells at exponential growth phase wereharvested and re-suspended at a density of 1×10~4 cells/mL. Six group including threecontrols (RPMI-1640 culture medium only for a blank, a negative and a positive withaddition of 0.5mmol/L sodium butyrate) and three experimental group (with finalmatrine mass concentrations of 0.05g/L, 0.10g/L and 0.20g/L, respectively) wereinvolved in this study. At day 3, the dosage effect of matrine on K562 cellsproliferative inhibition was recorded.
1.2 Methods for erythroid differentiation of K562 cells by matrine:
1.2.1 Benzidine staining: K562 cells culture procedures and experimental design werethe same as those in Trypan blue exclusion assay. Trypan blue exclusion assay wasperformed at the intervals of 24 h from the time of inoculation. Benzidine staining wascarried out only in case of the cell viability greater than 95%. The percentage ofbenzidine-positive cells (visualized as cells with blue crystals) was calculated for up to6 days.
1.2.2 Wright-Gimesa staining: K562 cells of negative control group and the matrinegroup at the final mass concentration of 0.10g/L at day 4 was centrifuged respectively for the preparation of cell smears and then stained with Wright-Gimesa for subsequentmorphological observations.
2. Methods for matrine-induced gamma-globin gene expression in K562
K562 cells culture procedures and experimental design were performed asdescribed in the section of Trypan blue exclusion assay. Western blotting techniquesand real time fluorescence quantitative RT-PCR was used to detect levels of proteinsynthesis and mRNA expression, respectively.
2.1 Methods for matrine-induced fetal hemoglobin (α_2γ_2) synthesis in K562 cells
For dose-dependent effects of matrine, K562 cells were collected fromexperimental group at the final matrine concentrations of 0.05g/L, 0.10g/L and 0.20g/L,from the positive control group at day 4 and from the negative control group,respectively. For time-dependent effects ofmatrine, K562 cells were collected from the0.10g/L matrine group at day 3, 4 and 5, from the positive and the negative controls.The total protein was extracted from the collected K562 cells and firstly was performedprotein quantitation, taking beta-actin as the house-keeping gene. These proteinblotting results of Fetal hemoglobin (α_2γ_2) and the corresponding of beta-actin fromeach group were separted by Western blotting techniques and analyzed by Gel analysissoftware package, Gel-pro analyzer 3.1, was used to determine the gray scale of theprotein bands. The ratio of each gray scale of fetal hemoglobin band to itscorresponding gray scale of beta-actin band was used for correction of protein samplequantities. The corrected expression of the negative control was taken as "1" forcalculating relative fold of each other groups, which were used for subsequentstatistical comparisons.
2.2 Methods for matrine-induced ~Gγglobin gene and ~Aγglobin gene mRNA expressionin K562 cells
Quantitative of mRNA expression was performed with relative quantitationanalysis. The standard curve was used for inter-sample amplifying efficiencycorrection. All these samples were performed normalization (quantitative correction ofRNA), taking beta-actin, as the house-keeping gene. When making the standard curve,cDNA sample, reverse transcription for total RNA at high concentration, was diluted10 times by specific agent, EASY Dilution. Five Ct values of amplified gradientconcentration samples were obtained through real time fluorescence quantitativeRT-PCR. Based on the 5 points, the linear relative quantitation standard curve wasfinally constructed with Ct values of each gradient samples as the y axis and with Logvalues of corresponding original template concentrations as the x axis (y=ax+b). Fordose-dependent effects, K562 cells from all groups at day 3 were collected. Fortime-dependent effects of matrine, K562 cells from the experimental group (0.10g/L ofmatrine) at day 2, 3 and 4, positive and negative controls were harvested, respectively.The extracted total RNA was firstly identified by agarose gel electrophoresis (AGE)and ultraviolet spectrophotometry, and then by real time fluorescent quantitativeRT-PCR to regulate the sample concentration for placement of Ct values on thestandard curve. The samples to be assayed and the standard sample were spontaneouslyamplified in different tubes for respective destination gene. The Ct values of eachdestination gene mRNA and house-keeping gene mRNA were then substituted into thecorresponding standard curve. The quantified house-keeping gene mRNA was taken as"1" to calculate the relative times of each destination gene mRNA as the errorcorrected total RNA. Subsequently, the corrected expression of negative control wastaken as "1" for calculating the relative expression of other group, which were used forstatistical comparisons.
Results
1 Effects of matrine on proliferation inhibition and erythroid differentiation inK562 cells
1.1 Results of matrine on proliferation inhibition in K562 cells by trypan-blue dyeexclusion test and XTT assay: After pretreatment with matrine, decreased K562 cellscounts have statistical significance not only at different time points (P=0.000), but atdifferent matrine concentrations (P=0.000). K562 cells were inhibited after treatedwith each matrine concentration, the more proliferation inhibit strong and the moreincreasing matrine concentrateion, matrine can inhibit proliferation of K562 cells bydose-dependent way.
1.2. Result of matrine on erythroid differentiation in K562 cells:
After pretreatment with different concentrations of matrine, the percentage ofbenzidine-positive cells (BZ%) of K562 cells increased statistically not only atdifferent time points (P=0.000) in a time dependent way, but at different matrineconcentrations (P=0.000) in a dose dependent way. The reault indicates that matrineinduce hematoglobin production of K562 cells by dose-dependent and time-dependentway. The peak value reached 15.7% on day 4 at 0.10g/L ofmatrine.
Morphological assessment of the K562 cells stained with Wright-Gimesa: theuntreated K562 cells showed features of undifferentiated progenitor in shape, whilematrine-induced K562 cells on day 4 were characterized of erythroid differentiation.
2 Matrine-inducedγ-globin gene expressions in K562 cells
2.1. Matrine-induced fetal hemoglobin (HbF) synthesis in K562 cells by Westernblotting: HbF production incresed were 1.08-, 1.53-, 1.36- and 1.52- fold of K562 cellsin the presence of 0.05g/L, 0.10g/L and 0.20g/L of matrine and sodium butyrate,respectively. K562 cells which induced by 0.10g/L and 0.20g/L matrine have statistical significance relative to negative control (P＜0.01). HbF production incresed were 1.38-,1.81-, 1.54- and 1.92- fold on day 3, 4 and 5 after matrine treatment and on day 3 afterincubation with sodium butyrate, respectively, as compared to negative control (P＜0.01). Matrine-induced fetal hematoglobin synthesis of K562 cells by dose-dependentand time-dependent way. The action reached its peak of K562 cells on day 4 at 0.10g/Lof matrine.
2.2 Matrine-induced accumulation ofγ-globin gene mRNA in K562 cells by real timeFQ-RT-PCR.
No statistical significance of ~Aγ-globin gene mRNA expression with differentconcentration matrine and on different time points among all experimental group ascompared to negative control (P＞0.05).
Accumulation of ~Gγ-globin gene mRNA expression in K562 cells were 1.40-,2.72-, 2.20- and 3.39- fold on day 3 in the presence of 0.05g/L, 0.10g/L and 0.20g/L ofmatrine and sodium butyrate, respectively. ~Gγ-globin gene mRNA expression inducedby 0.10g/L and 0.20g/L of matrine have statistical significance relative to negativecontrol (P＜0.05); Accumulation of ~Gγ-globin mRNA were 1.57-, 3.08-, 2.54- and3.45-fold on day 2, 3 and 4 in the presence of matrine and on day 3 after incubationwith sodium butyrate, respectively. ~Gγ-globin gene mRNA expression induced ondifferent time have statistical significance relative to negative control (P＜0.05);Matrine-induce K562 cells accumulate ~Gγ-globin gene mRNA in a dose-dependent andtime-dependent way. The effect reached its peak on day 3 at 0.10g/L of matrine. Theseresults suggested that matrine-induced accumulativeγ-globin gene mRNA expressionwas associated with accumulation ~Gγ-globin gene mRNA expression.
Conclusions:
1. Matrine (at 0.05g/L, 0.10g/L and 0.20g/L) can inhibit K562 cells proliferation and induce erythroid differentiation by increasing hematoglobin synthesis in adose-dependent and time-dependent way. The effect reached its peak on day 4 at0.10g/L of matrine.
2. Matrine can induce K562 cells to synthesis fetal hematoglobin in a dose-dependentand time-dependent way. The effect reached its peak on day 4 at matrine concentrationof 0.10g/L.
3. Matrine-induced accumulativeγ-globin gene mRNA expression was asscciated withaccumulation ~Gγ-globin gene mRNA expression. The effect reached its peak on day 3 atmatrine concentration of 0.10g/L.
4. The induction ofγ-globin genes expression by matrine is similar to that of sodiumbutyrate, which was used as positive control.
5. The study paves the experimentation base for pharmacologically mediatedupregulation expression of humanγ-globin genes and production of fetal hemoglobinas a potential therapeutic strategy in theβ-thalassaemia.
Main new ideas:
1. 11 kinds of effective parts and simple substances of Chinese medicines that areanti-leukemic confirmed by in vitro or in vivo studies are used to induce hemoglobinproduction of K562 cells by benzidine staining, and matrine was firstly screened out asan inducer for erythroid differentiation of K562 cells.
2. 0.05g/L, 0.10g/L and 0.20g/L of matrine can inhibit proliferation and erythroiddifferentiation in K562 cells and firstly verity that matrine can induce fetal hemoglobinsynthesis.
3. Firstly verity that accumulation ofγ-globin gene mRNA after treatment of humanK562 cells with matrine and was associated with the increase ~Gγ, globin gene mRNAexpression.
4. Matrine is a hypo-toxious and effective inducer ofγ-globin gene and similar tosodium butyrate.
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[1] Arthur Bank. Regulation of human fetal hemoglobin: new players, new complexities [J].Blood. 2006, 107(2):435-441.
[2] Rund D, Rachmilewitz E. β-Thalassemia [J]. N. Engl. J. Meal, 2005, 353(11): 1135-1146.
[3] Chakalova L, Osborne CS, Dai YF, et al. The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression [J]. Blood. 2005, 105(5):2154-2160.
[4] Weinberg RS, Ji X, Sutton M, et al. Butyrate increases the efficiency of translation of γ-globin mRNA [J]. Blood 2005, 105(4): 1807-1809.
[5] Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation [J]. Exp Hematol. 2005, 33(3):259-271.
[6] O'Neill D, Bornschlegel K, Flamm M, et al. A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine-rich domain upstream from the human delta-globin gene. Proc Natl Acad Sci U S A. 1991;88(20):8953-8957.
[7] Gribnau J, Diderich K, Pruzina S, et al. Intergenic transcription and development remodeling of chromatin subdomains in the human β-globin locus. Molecular Cell, 2000, 5(2):377-386.
[8] Enver T, Raich N, Ebens AJ, et al. Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice. Nature. 1990, 344 (6264):309-313.
[9] O'Neill D, Yang J, Erdjument-Bromage H, et al. Tissue-specific and developmental stage-specific binding of a SWI/SNF complex associated with human fetal to adult globin switching. Proc Nat Acad Sci U S A. 1999, 96 (2):349-354.
[10] Szulawska A, Arkusinska J, Czyz M. Accumulation of gamma-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives [J]. Biochem Pharmacol. 2007, 73(2):175-184.
[11] Watanapokasin R,Sanmund D, Winichagoon P, et al. Hydroxyurea responses and fetal hemoglobin induction in P-thalassemia/HbE patients' peripheral blood erythroid cell culture [J]. Ann Hematol. 2006, 85(3): 164-169.
[12] Fibach E, Bianchi N, Borgatti M, et al. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells [J]. Blood. 2003, 102(4):1276-1281.
[13] Bianchi N, Osti F, Rutigliano C, et al. The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells [J]. Br J Haematol, 1999,104(2): 258-265
[14] Bianchi N, Chiarabelli C, Borgatti M, et al. Accumulation of γ-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine [J]. British Journal of Haematology, 2001, 113(4):951-961.
[15] Lampronti I, Bianchi N, Borgatti M, et al. Accumulation of γ-globin mRNA in human erythroid cells treated with angelicin [J]. Eur J Haematol. 2003, 71(3): 189-195
[16] Lampronti I, Bianchi N, Zuccato C, et al. Effects on erythroid differentiation of platinum(Ⅱ) complexes of synthetic bile acid derivatives [J]. Bioorg & Med Chem. 2006, 14(15):5204-5210.
[17] Saunthararajah Y, Lavelle D, DeSimone J. DNA hypo-methylating agents and sickle cell disease [J]. Br J Haematol. 2004, 126(5):629-636
[18] Lal A, Vichinsky E. The role of fetal hemoglobin-enhancing agents in thalassemia [J]. Semin Hematol. 2004, 41(Suppl 6): 17-22.
[19] Witt O, Monkemeyer S, Ronndahl G, et al. Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin[J]. Blood. 2003, 101(5):2001-2007.
[20] 吴志奎，张新华，李敏，等。益髓生血颗粒治疗β-地中海贫血156例临床观察[J]．中国中西医结合杂志，2006，26(4)：352-354．
[21] 吴志奎，张新华，王蕾，等。益髓生血灵治疗β-地中海贫血症92例临床研究[J]。中国中医药信息杂志，2004，11(4)：292-293．
[22] 杨克恭，何平，陈松森，等。两种新的激活γ珠蛋白基因的中药—盐酸山莨菪碱(654—2)和三尖杉酯碱[J]。生物化学杂志，1994，10(3)：285-289．
[23] 黄有文，王荣新，蔡利群，等。山莨菪碱(654-2)治疗地中海贫血的临床研究[J]。中国优生与遗志，1997，5(6)：93-94．
[24] Xiao-Dong CHENG, Chun-Hui HOU, Ruo-Lan QIAN, et al. Effects of Huangqi (Hex) on Inducing Cell Differentiation and Cell Death in K562 and HEL Cells [J]. Acta Biochimica et Biophysica Sinica, 2004, 36(3):211-217.
[25] Normile D. Asian Medicine. The new face of traditional Chinese medicine [J]. Science, 2003, 299(5604): 188-190.