Screening and expression of CD34(+) cell-specific microRNA in acute myelogenous leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen and analyze CD34(+) cell specific microRNAs (miRNAs) from the patients with acute myelogenous leukemia (AML) and their expression.</p><p><b>METHODS</b>CD34(+) cells were sorted from AML patients or the mobilized peripheral blood of the donors of hematopoietic stem cell transplantation (normal control subjects) and followed by the extraction of the cell total RNAs. The differentially expressed microRNAs (miRNAs, miR) were selected after hybridizing with miRNA microarray, real time polymerase chain reaction (real-time PCR) was subsequently applied to confirm the expression of the selected miRs, and PCR products were further cloned and sequenced to check their specificity.</p><p><b>RESULTS</b>Of the differentially expressed miRNAs, 191 were found to be at least one-fold change in the CD34(+) cells between the AML patients and the normal control subjects. Of the 191 miRNAs, the expression difference of 94 was significant (P < 0.05). Among these 94 miRNAs, the expression of 44 miRNAs was increased and the other 50 miRNAs was decreased in the CD34(+) cells from the bone marrow of AML patients compared with the CD34(+) cells from the mobilized peripheral blood of the normal control subjects. Real time PCR verified that the expression level of miR-10a and miR-220c in the CD34(+) cells from the bone marrow of AML patients was 19.6% and 19.0% of that of CD34(+) cells from mobilized peripheral blood of the normal control subjects. DNA sequencing and BLAST DNA database searching results indicated that the PCR products were really miR-10a and miR-220c.</p><p><b>CONCLUSION</b>A variety of differentially expressed-miRNAs are existed between AML and normal control subjects CD34(+) cells, the expression of miR-10a and miR-220c was significantly down-regulated in the CD34(+) cells from the bone marrow of AML patients.</p>

Chimeric Ad5F35 adenoviral vector-mediated expression of mutant IκBα induces apoptosis of leukemia cells / 中国实验血液学杂志

Constitutive activation of nuclear transcription factor-κB (NF-κB) exists in a variety of leukemia, and induction of apoptosis through blocking NF-κB activation may be an alternative strategy for leukemia treatment. The aim of this study was to investigate the inducing effect of modified adenovirus 5-based adenovirus vector (i.e. chimeric Ad5F35 Vec)-mediated expression of mutant IκBα (IκBαDN) on apoptosis of HL-60 cells. The recombinant Ad5F35-IκBαDN Vec carrying IκBαDN cDNA which deleted the first 1-70 amino acids coding sequences at 5' terminal of human IκBα was transfected into HL-60 cells. The apoptosis, NF-κB DNA binding activity, the expressions of IκBα, cIAP-2 and xIAP in HL-60 cells were detected by DNA binding assay, flow cytometry, real-time quantitative polymerase chain reaction and Western blot respectively. The results showed that apoptosis rates were 22.53 ± 2.999%, 6.08 ± 2.464% and 4.86 ± 1.366% for Ad5F35-IκBαDN Vec-infected or blank vector of Ad5F35-EGFP Vec-transfected and untransfected HL-60 cells respectively, which showed a significant difference between Ad5F35-IκBαDN Vec-transfected and untransfected cells (p < 0.001) and between Ad5F35-IκBαDN Vec-transfected and Ad5F35-EGFP Vec-transfected cells (p < 0.001, p < 0.002), while NF-κB DNA binding activity was decreased, the truncated IκBα was expressed, and IκBα mRNA expression was up-regulated, but the expression of cIAP-2 and xIAP mRNA was down-regulated after transduction for 48 hours. It is concluded that the chimeric Ad5F35 Vec can effectively mediate the expression of IκBαDN cDNA in HL-60 cells, leading to the inhibition of NF-κB DNA binding activity and inducing apoptosis of HL-60 cells.

Screening and structure analysis of nucleic acid aptamers binding to surface of CD33(+)/CD34(+) cells from patients with acute myeloid leukemia subtype M₂ / 中国实验血液学杂志

A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M₂) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M₂ CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M₂ were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M₂ CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M₂ leukemia.

Gene expression of livin and survivin in adult patients with acute lymphoblastic leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expressions of livin and survivin in adult patients with acute lymphoblastic leukemia (ALL) and clinical significance. The expressions of livin and survivin mRNA in bone marrow mononuclear cells of 95 adult ALL patients including 52 de novo patients, 23 relapsed patients and 20 patients with complete remission (CR), and 20 healthy adults as normal controls were detected by using RT-PCR. The results indicated that the expression of livin and survivin mRNA in de novo and relapsed patients was higher than that in normal controls and patients with CR. There were no significant relation of expression level with clinical features such as age, sex, type and de novo leukocyte level. The CR rate in de novo adult ALL patients with positive expression of livin and survivin was lower than that in adult ALL patients with negative gene expression. No relation of mRNA expression between livin and survivin was found in de novo adult ALL patients. It is concluded that genes livin and survivin may be involved in the pathogenesis and progression of adult ALL. Overexpression of livin or survivin may show poor progression. There is no relation of expression between genes livin and survivin in de novo adult ALL patients.

Expression of angiotensin-II type 1 receptor at1 mRNA in myeloid leukemia / 中国实验血液学杂志

This study was aimed to explore the expression level of angiotensin-II type 1 receptor (AT1) mRNA in bone marrow of myeloid leukemic patients, and its correlation with the proportion of leukemia cells in samples and Hb, WBC, Plt counting in peripheral blood. 51 samples, including 36 AML, 7 CML, and 8 samples of non-malignant hematological diseases as control group were collected. The expression of at1 mRNA was detected by real time-PCR; the expression levels of at1 gene in AML and CML groups were relatively quantitatively analyzed by using 2(-ΔΔCT) and were compared with control group. The results showed that the expression levels of at1 mRNA in AML, CML and control groups were 0.038 ± 0.076, 0.033 ± 0.039, 0.281 ± 0.366, respectively. at1 gene expression in the myeloid leukemic group was significantly lower than that in the control group. The expression level of at1 mRNA in AML was negatively correlated with the proportion of leukemia cells and positively with Hb level in peripheral blood. It is concluded that at1 gene may play a minor role in leukaemogenesis, however, may promote erythropoiesis.

Effect of acetyl-11-keto-β-boswellic acid on proliferation, apoptosis and cell cycle of human acute myeloid leukemia cell line HL-60 / 中国实验血液学杂志

The aim of this study was to investigate the effect of 3-O-acetyl-11-keto-β-boswellic acid (AKBA) on the proliferation, apoptosis and cell cycle of human acute myeloid leukemia (AML) cell line HL-60. HL-60 cells were treated by AKBA at various concentrations. The inhibitory effects of AKBA on the proliferation of HL-60 were analyzed by MTT assay. Morphologic changes of HL-60 cells were observed by fluorescence microscopy with Hochest33342 staining. Cell apoptosis rate was determined by flow cytometry with Annexin-V-FITC/PI double staining. The cell cycle was measured by flow cytometry with PI staining. The results showed that AKBA inhibited the proliferation of HL-60 and the apoptosis rate of HL-60 cells was gradually enhanced when AKBA dose increased. AKBA changed the cell cycle of HL-60, resulting in cell increase at G(1) phase and decrease at S phase. It is concluded that the AKBA has anti-proliferation and apoptosis-inducing effects on HL-60 cells, that seems a promising therapeutical approach for AML.

Novel frame-shift mutation of 540 A deletion in GP IIb gene from a patient with Glanzmann thrombasthenia / 中南大学学报(医学版)

OBJECTIVE@#To explore the molecular mechanism of Glanzmann thrombasthenia (GT).@*METHODS@#All 45 exons of alphaIIb and beta3 subunit genes as well as their splicing sites were amplified by polymerase chain reaction(PCR) with 40 primer pairs, and then the PCR products were used to screen the gene mutation by single strand conformation polymorphism-polyacrylamide gel electrophoresis (SSCP-PAGE). The mutation was further confirmed by direct DNA sequencing.@*RESULTS@#A DNA band alterated migration was detected after SSCP-PAGE. DNA sequencing showed that a base deletion within the band at the site of 540 in GPIIb gene(540A) was found.@*CONCLUSION@#The frame-shift mutation caused by the deletion of 540A in GPIIb gene is a novel mutation which is a genetic defect in patients with GT.

Effect of ribosomal protein L6 on drug resistance and apoptosis in K562/A02 cells / 中国实验血液学杂志

The objective of study was to investigate the effect of ribosomal protein L6 (RPL6) gene expression on the drug resistance of leukemia cells and its possible mechanism. RPL6 cDNA was obtained by RT-PCR, both sense and antisense cDNA recombinants of RPL6-encoding gene were constructed with pcDNA3. 1 (+) expression vector. Subsequently, the sense RPL6 cDNA recombinant was transfected into K562 cells while the antisense one into K562/A02 cells by liposomal reagent. The chemosensitivity, apoptosis and caspase-3 activity of K562 and K562/A02 cells were evaluated by MTT assay, flow cytometer and fluorometer respectively. The results indicated that expression of RPL6 in K562/A02 was higher than that in K562; resistance of sense-transfected K562 cells to doxorubicin was 325% of control cells, apoptosis and caspase-3 activity decreased (P<0.005); whereas resistance of antisense-transfected K562/A02 cells to adriamycin was 38% of control cells, apoptosis and caspase-3 activity significantly increased (P<0.005). It is concluded that RPL6 gene plays an important role in the development of drug resistance in K562/A02 cells by changing drug-induced apoptosis.

IkappaB alpha mRNA expression and its DNA sequence in nasopharyngeal carcinoma cell lines / 中南大学学报(医学版)

OBJECTIVE@#To examine the expression of the inhibitor alpha of nuclear transcription factor kappaB (IkappaBalpha) mRNA expression and its sequence characteristics in human nasopharyngeal carcinoma cell (NPC) lines CNE1, CNE2, HNE1 and HNE2.@*METHODS@#Reverse transcription was performed with the total RNAs isolated from the NPC cell lines CNE1, CNE2, HNE1 and HNE2, as well as the transplanted tumor tissues with HNE1 cells. Then IkappaBalpha cDNA was amplified by PCR, and the products were used to examine IkappaB alpha mRNA expression and DNA sequencing, or the DNA sequencing after the products were cloned into plasmid vector.@*RESULTS@#IkappaB alpha mRNA was expressed in all the 4 nasopharyngeal carcinoma cell lines. DNA sequencing showed that polymorphisms and 5 mutations (A825G, A975G, G576A, A655G and C653A) existed in IkappaB cDNA from the transplanted tumor tissues with HNE1 cells, CNE1 and CNE2 cells.@*CONCLUSION@#The expression of IkappaBalpha mRNA not only exists, but DNA polymorphisms and some additional mutations in IkappaBalpha cDNA are also detected in the nasopharyngeal carcinoma cells.

CD34(+)/CD123(+) cell sorting from the patients with leukemia by Midi MACS method / 中国实验血液学杂志

The aim of this study was to sort the CD34(+)/CD123(+) cells from the bone marrow cells of patients with acute myeloid leukemia (AML) by Midi MACS method. Firstly, the bone marrow mononuclear cells (BMMNC) were isolated from the patients with AML with Ficoll Paque, CD34(+) cells were then isolated by Midi MACS method followed by the isolation of CD34(+)/CD123(+) cells from the fraction of CD34(+) cells. The enrichment and recovery of CD34(+) and CD34(+)/CD123(+) cells were assayed by FACS technique. The results showed that the enrichment of CD34(+) cells was up to 98.73%, its average enrichment was 95.6%, and the recovery of CD34(+) was 84.6%, its average recovery was 51% after the first round sorting, by the second round sorting, the enrichment of CD34(+)/CD123(+) cells was up to 99.23%, its average enrichment was 83%. With regard to BMMNCs before sorting, the recovery of CD34(+)/CD123(+) was 34%. But, on the CD34(+) cells obtained by the first round sorting, its recovery was 56%. In conclusion, these results confirmed that the method of Midi MACS sorting can be applied to sort CD34(+)/CD123(+) cells from the bone marrow cells of AML patients, which give rise to the similar enrichment and recovery of the sorted cells with that of literature reported by the method of FACS.

Revision of submucous resection of nasal septum / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the clinical significance and surgical technique for revisionary submucous resection of nasal septum.</p><p><b>METHODS</b>Thirty-two patients who had undergone nasal septal resection were evaluated rhinologically and the causes of unsuccessful septoplasty were analysed . Based on the location and extent of deviation, the status of residual cartilage and bone, and the age of the patients, one of three incisions was chosen during septal surgeries: (1) For 5 cases with anterior, 1 with inferior and 14 with superior deviation, a "U" shaped incision at the left side of anterior edge of septum cartilage was used. (2) For 8 cases with posterior and 2 with superior deviation, the site of the incision was located just anterior to the deviation, with the aid of endoscope. (3) For 2 cases with anteroinferior deviation, because of their young age, a sublabial incision was made to surge the mucosa of the septum and base of nasal cavity, the otological electronic drill was then used.</p><p><b>RESULTS</b>Revision nasal septoplasty was successful in all cases. The symptoms resulting from septal deviation disappeared or significantly relieved. Following successful revision surgery, the treatment outcomes of concomitant nasal and/or sinusal diseases also significantly improved.</p><p><b>CONCLUSION</b>Revision nasal septoplasty requires different approaches depending on different clinical characteristics. A successful correction of septal deviation can not only relieve the symptoms derived from deviation, but also be of significance for the treatment of concomitant nasal and/or sinusal diseases.</p>

Advances of study on activation of nuclear factor kappaB in hematological malignancies and its mechanism--review / 中国实验血液学杂志

The nuclear factor kappa B(NF-kappaB) plays a crucial role in inflammatory, immune response, embryo development, cell proliferation and apoptosis, cell cycle control as well as tumorgenesis. In recent years, a variety of investigations have demonstrated that NF-kappaB was closely associated with the pathogenesis of hematological malignancies such as leukemia, lymphoma and multiple myeloma. Nowadays, increasingly attention has been paid to the studies on the activation and its mechanism of NF-kappaB in the hematogenic malignancies. So that, in this article, progress on these aspects is reviewed.

Expression of coagulation factor IX in human CD34 + hematopoietic stem cells by adeno-associated virus 2 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of human coagulation factor IX (hFIX) gene in human umbilical cord blood (CB) CD34+ cells which was transfected with recombinant adeno-associated virus 2 (rAAV-2).</p><p><b>METHOD</b>The CD34+ cells were transfected with rAAV-2/hFIX and cultured for 21 days for inducing differentiation into myeloid, erythroid and megakaryocytes, respectively. The expression of hFIX was studied at mRNA, protein and biological activity levels. The cytotoxicity of rAAV-2 to CD34+ cells was evaluated by cell proliferation, cell vitality, CD antigen expressions and CFU yields.</p><p><b>RESULTS</b>The hFIX mRNA was expressed in the cultured cells which was verified by RT-PCR and DNA sequencing. An elevated level of hFIX expression and biological activities were detected with a maximum amount of 14.10 ng/10(6) cell x 24 h. During the period of 21 day culture, the cell vitality, cell proliferation, CD antigen expression and CFU yields between the transfected and un-transfected groups had no difference(P > 0.05).</p><p><b>CONCLUSION</b>The human CB CD34+ cells are able to produce functional hFIX after transduction by rAAV-2/hFIX. The cell proliferation and differentiation capacities of the host CD34+ cells were not affected by rAAV-2.</p>

Correction of posttraumatic deviated nose in 136 cases / 中华创伤杂志

Objective To explore a good of treating posttraumatic deviated nose. Methods Clinical data of 136 patients with posttraumatic deviated nose were analyzed.Closed nasal bone replacement was employed in 34 patients with the disease history of 20-30 days,while open rhino- plasty approach was employed in 102 patients with the disease history over 6 months to correct their postt- raumatic deviated nose,and straightening the septum and ectomizing the inferior turbinate were done if necessary.Results The follow-up was over one year.In the 34 patients with the disease history of 20- 30 days,the outcome was excellent in 28 cases and good in 6.In the 102 patients with the disease history over 6 months,the outcome was excellent in 81 cases and good in 21.The deformity of nose was corrected satisfactorily.Normal nasal shape and good ventilation were obtained.Conclusion Posttraumatic devi- ated nose deformities are often caused by delayed and inaccurate treatment.Closed nasal bone replace- ment can be employed for the patients with trauma history less than one month,and open rhinoplasty ap- proach and straightening the septum and restoration of the nasal shape are employed for other patients.In this way good results can be obtained.