OBJECTIVETo study the effect of liposomal transfection of antisense oligonucleotide (ASON) on the erythroid cell alpha-globin gene in the patients with severe beta-thalassemia, and provide a new idea for beta-thalassemia gene therapy.

METHODSA highly effective ASON targeting alpha-globin gene was transfected into severe beta-thalassemic erythroid cells cultured in vitro by liposomal at an optimal concentration. The expression level of alpha, beta, gamma-globin gene, the level of hemoglobin, and the excess alpha-globin chains precipitates in ASON group and control group were carefully analyzed by quantitative real-time PCR(Q-RT-PCR), high performance liquid chromatography (HPLC), and electron microscope, respectively.

RESULTSThe mRNA expression of alpha-globin gene was significantly lower in ASON group (9.04 +/- 0.29) than in control group (24.23 +/- 0.29) (P<0.01). Simultaneously, the disequilibrium between alpha- and beta-, gamma-globin gene expression was partly modified by ASON, the ratios of ASON group and control group being 0.79 +/- 0.02 and 2.26 +/- 0.06 respectively (P<0.01). HPLC demonstrated that the levels of HbA2 and HbF increased with downregulation of alpha-globin gene in beta-thalassemic erythroid cells, particularly HbF. The precipitates of alpha-globin chains in ASON group were lessened under electron microscope, particularly in early erythroblast while no change in the control group.

CONCLUSIONThe high effective ASON contributes to inhibit the alpha-globin gene expression of severe beta-thalassemic erythroid cells, partly modify the disequilibrium between alpha-, beta- and gamma-globin gene expression and obviously reduce the precipitates of alpha-globin chains in erythroid cells. It might provide a new idea for gene therapy of beta-thalassemia.

Cells, Cultured ; Child ; Genetic Therapy ; Humans ; Liposomes ; Oligonucleotides, Antisense ; genetics ; Transfection ; alpha-Globins ; genetics ; metabolism ; beta-Globins ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; therapy ; gamma-Globins ; metabolism

β-thalassemia is a chronic hemolytic anemia characterized by the reduction or absence of synthesis of β-globin chains because of the β-globin gene mutations. β-thalassemia belongs to the inherited hemoglobin disease, and occurs in some provinces of China, such as in Guangdong, Guangxi, Fujian, its prevalence is about 2%. The treatment of this disease include transfusion, iron chelating agent, hematopoietic stem cell transplantation, splenectomy, induced expression of Fetal Hemoglobin (HbF) and gene therapies. However, the mortality rate of this disease is still higher, thus some new treatments are urgently needed. In recent years, the study was mainly concentrated in 2 aspects: the normal β-globin gene transfer and endogenous γ-globin re-activation. Some studies showed that the expression of miRNAs was dysregulated in β-thalassemia. Some miRNAs could regulate γ-globin at posttranscriptional level, thus, the clarification of relationship between miRNAs and β-thalassemia is expected to provide experimental bases to β-thalassemia therapy. In this review, the induced therapy of γ-globin for β-thalassemia and its relationship with the miRNA are summarized.
China ; Fetal Hemoglobin ; metabolism ; Genetic Therapy ; Humans ; MicroRNAs ; metabolism ; beta-Globins ; genetics ; beta-Thalassemia ; therapy ; gamma-Globins ; therapeutic use

OBJECTIVETo investigate the effects of Astragalus polysaccharides (APS) in inducing the mRNA expression of Agamma- and Ggamma-globin in K562 cells.

METHODSK562 cells were treated with APS at the concentration of 150, 300, and 450 mg/L, with Na-butyrate (NaB)-treated cells serving as the positive control and untreated cells as the blank control. Benzidine staining was used to examine the changes in hemoglobin synthesis in K562 cells after the treatments, and RT-PCR was employed to investigate the mRAN expression of Agamma- and Ggamma-globin.

RESULTSCompared with the untreated cells, APS treatment (300 mg/L) for 48 h resulted in a significant increase of the percentages of benzidine-positive cells from (4.37-/+0.58)% to (15.67-/+1.80)%, and also in significantly increased expression of Agamma-globin and Ggamma-globin mRNAs by 3.59-/+0.16 and 5.02-/+0.81 folds, respectively (P=0.000).

CONCLUSIONAPS potently enhances the mRNA expression of Agamma- and Ggamma-globin in K562 cells and warrants further evaluation as a potential therapeutic agent for beta-thalassemia.

Astragalus membranaceus ; chemistry ; Humans ; K562 Cells ; Polysaccharides ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; gamma-Globins ; genetics ; metabolism

We studied the function and mechanism of miR-24 in regulating beta-like globin gene expression. We first detected the expression of miR-24 during erythroid differentiation and also detected the globin gene expression in miR-24 overexpressing K562 cells through q-PCR. Dual-luciferase reporter assay and Western blotting were used to identify target genes of miR-24. "Rescue experiment" was further used to investigate the regulation of miR-24 on globin gene expression whether depending on targeting Sp1 or not. We found that miR-24 increased during hemin-induced K562 cells and EPO-induced HPCs (hematopoietic progenitor cells) erythroid differentiation. Overexpression of miR-24 in K562 cells promoted the epsilon- and gamma-globin gene expression during hemin-induced erythroid differentiation through targeting the negative globin regulator Sp1. These results suggested that miR-24 can improve the expression of beta-like globin gene through targeting Sp1.
Cell Differentiation ; Gene Expression Regulation ; Hematopoietic Stem Cells ; metabolism ; Humans ; K562 Cells ; MicroRNAs ; genetics ; Sp1 Transcription Factor ; genetics ; epsilon-Globins ; genetics ; gamma-Globins ; genetics

This study was aimed to analyze the hematologic and molecular biologic characteristics of 14 Wenzhou patients with minor beta-thalassemia, to find out the mutation sites responsible for the disease by detecting sequences of PCR products and to analyze the single nucleotide polymorphism. The peripheral blood of patients was collected intravenously and was anticoagulated with EDTA-K(2); then the templates from blood samples were extracted, the related primers were designed for sequencing the products amplified by PCR; finally mutation sites resulting in beta-thalassemia were found through comparison and analysis of sequences. The results indicated that the C-->T heterozygous mutation occurred at the IVS-2 -654 site in 4 cases; the TTCT deficiency appeared at CD41/42 site in 1 case; in 2 sites existed single nucleotide polymorphisms occurring at the 59th site of exon 1 (T/C, CAT/CAC, His) and IVS-2 nt 665 (T/C). It is concluded that single nucleotide polymorphism of minor beta-thalassemia patients born in Wenzhou had specificity, this study found too kinds of gene mutations which are IVS-2 -654 C-->T heterozygous mutation and CD41/CD42 site-TTCT deficiency.
Base Sequence ; China ; Humans ; Mutation ; Polymorphism, Single Nucleotide ; beta-Globins ; genetics ; beta-Thalassemia ; genetics ; metabolism

The discovery of neuroglobin (Ngb), a brain- or neuron-specific member of the hemoglobin family, has revolutionized our understanding of brain oxygen metabolism. Currently, how Ngb plays such a role remains far from clear. Here, we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia. We found that Ngb was present in, co-localized to, and co-migrated with mitochondria in the cell body and neurites of neurons. Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane (CM) or cell surface in living neurons, and this was accompanied by the mitochondria. In vivo, hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio. Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase (SDH) and ATPase activity in neuronal N2a cells. Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia. Mutation of Ngb at its oxygen-binding site (His⁶⁴) significantly increased SDH activity and reduced ATPase activity in N2a cells. Taken together, Ngb was physically and functionally linked to mitochondria. In response to an insufficient oxygen supply, Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation. This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer's disease and diseases that cause hypoxia in the brain such as anemia.
Rats ; Animals ; Neuroglobin/metabolism* ; Globins/metabolism* ; Nerve Tissue Proteins/metabolism* ; Neurons/metabolism* ; Hypoxia/metabolism* ; Brain/metabolism* ; Oxygen ; Anemia/metabolism* ; Adenosine Triphosphatases/metabolism*

The objective of study was to investigate the effect of liposomal transfection of antisense oligodeoxynucleotide (ASON) on alpha-globin gene expression and proliferation of K562 cells, to explore the new way of gene therapy in beta-thalassemia. Targeted ASON of alpha-globin was designed and synthesized, and compared with positive control [sense oligodeoxynucleotide (SON) group] and blank control. By liposomal transfection, ASON, SON was co-cultured with K562. The efficiency of transfection was assayed by fluorescence microscopy and flow cytometry (FCM), the alpha-globin gene expression of K562 was measured by real-time PCR, and the proliferation of K562 was determined by Cell Count Kit-8 assay. The results indicated that the highest efficiency was at 24 hours after liposomal transfection, the gene expression level of alpha-globin in ASON group was significantly lower than that in SON group and blank control (p < 0.01). The proliferation of K562 cells was obviously inhibited, meanwhile the above effect showed the dose-dependent manner. It is concluded that the liposomal transfection of ASON inhibits the alpha-globin gene expression of K562 cells, which may be the new target for gene therapy in beta-thalassemia.
Cell Proliferation ; drug effects ; Gene Expression ; Humans ; K562 Cells ; Liposomes ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; Transfection ; alpha-Globins ; metabolism

OBJECTIVETo investigate the incidence and the gene mutation frequencies and patterns of α-thalassemia in preschool children in Chongqing city.

METHODSCluster random sampling was used. A total of 1057 preschool children in three areas of Chongqing were screened by using routine blood test and hemoglobin electrophoresis analysis. Molecular analysis carried out for all the samples.

RESULTSOf the 1057 samples, 55 cases were diagnosed as being carriers of α-thalassemia, which included 80 allele genes. Therefore, the frequency of α-thalassemia carriers in Chongqing was 5.20%. Of the 55 α-thalassemia carriers, five different deletions of α-thalassemia were identified, the three most common deletion types and proportions were 54.55% for the -α(3.7) deletion, 18.18% for --(SEA) deletion, and 9.08% for the -α(4.2) deletion, respectively; eight types of nondeletion defects were determined, containing one case of Hb Quong Sze and seven novel mutations of a-globin gene. Furthermore, 24 cases of α-Triplication were detected with the α-Triplication carrier rate of 2.55%. In addition, in this study we also found two cases of abnormal hemoglobin disorders occurred on α-globin gene, Hb J-Wenchang-Wuming and Hb Arya. Hb Arya was characterized in the Chinese population for the first time confirmed by literature retrieval.

CONCLUSIONIn this study, we have clarified the carrier frequency and molecular spectrum of α-thalassemia in Chongqing, and we first reported the carrier incidence of α-Triplication in Chongqing. The materials obtained from this study would be of valuable reference for genetic counseling and the examination instruction of children in this area.

Asian Continental Ancestry Group ; Child, Preschool ; China ; epidemiology ; Humans ; Incidence ; Mutation ; Prevalence ; alpha-Globins ; metabolism ; alpha-Thalassemia ; epidemiology ; metabolism

OBJECTIVETo study the expression changes of neuroglobin in rats with the model of diffuse traumatic brain injury and explore the relationship between the neuroglobin and neuron apoptosis in traumatic brain injury.

METHODSThe diffuse traumatic brain injury of rats was induced by the Marmarou's 'weight-drop' device. And the immunohistochemical technique was used to detect the expression changes of neuroglobin and neuron apoptosis in rat brain at different time points post-injury.

RESULTSThe expression of neuroglobin increased twice and reached peaks at 2 hours and 72 hours post-injury respectively. And the increased expression of neuroglobin from 30 minutes to 1 hour post-injury and from 48 hours to 72 hours post-injury accompanied with the decreased expression ratio of Bax to Bcl-2.

CONCLUSIONThe increased expression of neuroglobin in traumatic brain injury informed us that neuroglobin had anti-apoptosis action in post-injury neuron. It could protect the neuron from traumatic stress and secondary ischemia and hypoxia insults during ultra-early and acute stages.

Animals ; Apoptosis ; physiology ; Brain ; metabolism ; pathology ; Brain Injuries ; metabolism ; pathology ; Globins ; metabolism ; Male ; Nerve Tissue Proteins ; metabolism ; Neurons ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the expression of neuroglobin (Ngb) in frontal cortex, cerebrospinal fluid (CSF) and serum in rats with brain injury induced by LPS and to elucidate its significance.

METHODSSeventy adult Sprague-Dawley rats were randomly divided into LPS (n = 60, with injection of 0.1 mg/kg LPS into cistern) and control (C, n = 10, with injection of equal volume of isotonic saline into cistern) groups, with 10 rats in each group. The plasma, CSF as well as frontal cortex from sacrificed rats were collected in LPS group at 3, 6, 12, 24, 48, 72 post injection hour (PIH), with 10 rats at each time point. The protein expression of Ngb was measured by enzyme-linked immunosorbent assay (ELISA) and Western blot. Expression of Ngb in frontal cortex was determined by SP. Water content of frontal cortex was assessed by drying method. The correlation among Ngb contents in serum, CSF, frontal cortex and water content of frontal cortex was analyzed with multiple comparison.

RESULTSNgb expression (by ELISA): Ngb expression of frontal cortex, CSF, and serum in LPS group was higher than that in C group at each time point, and it peaked at48 PBH (35.4 +/- 3.9, 22.7 +/- 3.1, 14.4 +/- 2.8 ng/mL, respectively, P < 0.01). Ngb expression (by Western blot): Ngb protein with relative molecular mass of 17 x 10(3) was observed in each group. Ngb expression detected by Western blot was similar to that detected by ELISA. Brain water content was significantly greater in LPS group than that of C group at 6-72 PIH (P < 0.01), and it peaked at 48 PBH (83.3 +/- 1.9)%. Ngb contents in serum, CSF, frontal cortex, and water content of frontal cortex were positively correlated with multiple comparison ( y = 0.631-0.719, P < 0.01).

CONCLUSIONSUp-regulation of Ngb expression in brain injury induced by LPS is correlated with duration after challenge of LPS.

Animals ; Brain Injuries ; chemically induced ; metabolism ; Disease Models, Animal ; Female ; Globins ; metabolism ; Lipopolysaccharides ; adverse effects ; Male ; Nerve Tissue Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Up-Regulation