Quantitative Analysis on Immunophenotype of CD34 / 中国实验血液学杂志

OBJECTIVE@#To investigate the quantitative expression of immunophenotype of CD34@*METHODS@#Multi-parameter flow cytometry (FCM) was used to detect the proportion and mean fluorescence intensity (MFI) of each antigen of bone marrow CD34@*RESULTS@#Bone marrow blast cell proportion (P<0.01), RBC level (P<0.01), and Hb level (P<0.05) of high-risk MDS patients were higher, while EPO level (P<0.05) was lower than those of low-risk patients. The proportion of CD34@*CONCLUSION@#The immunophenotype of CD34

Effect of Signal Transduction Pathway Gene Mutations on the One- course Induced Remission Rate and Analysis of Clinical Characteristics in Patients with CBF-AML / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR) induced by one course of induction chemotherapy in patients with core binding factor-associated acute myeloid leukemia (CBF-AML).@*METHODS@#DNA was extracted from bone marrow or peripheral blood samples of newly diagnosed CBF-AML patients admitted to the Hematology Department of the Second Hospital of Shanxi Medical University from January 2015 to January 2019. Next-generation sequencing was used for detection of 34 kinds of hematologic malignancy-related gene mutations in patients with CBF-AML, the effect of related gene mutations on the first complete remission (CR) rate in one course of induction chemotherapy was analyzed by combineation with clinical characteristics.@*RESULTS@#34 kinds of genes in bone marrow or peripheral blood of 43 patients were detected by high throughput sequencing and the gene mutations were detected in 16 out of 34 genes. The mutation rate of KIT gene was the highest (48.8%), followed by NRAS (16.3%), ASXL1 (16.3%), TET2 (11.6%), CSF3R (9.3%), FLT3 (9.3%), KRAS (7.0%). The detection rates of mutations in different functional genes were as follows: genes related with signal transduction pathway (KIT, FLT3, CSF3R, KRAS, NRAS, JAK2, CALR, SH2B3, CBL) had the highest mutation frequency (72.1% (31/43); epigenetic modification gene mutation frequency was 30.2% (13/43), including ASXL1, TET2, BCOR); transcriptional regulation gene mutation frequency was 7.0% (3/43), including ETV6, RUNX1, GATA2). Splicing factor related gene mutation frequency was 2.3% (1/43), including ZRSR2). The CR rate was 74.4% after one course of induction chemotherapy. At first diagnosis, patients with low expression of WT1 (the median value of WT1 was 788.9) were more likely to get CR (P=0.032) and the RFS of patients who got CR after one course of induction chemotherapy was significantly longer than that of patients without CR [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR in the signal transduction pathway gene mutation group was significantly lower than that in non-mutation group (64.5% vs 100%) (P=0.045), while the level of serum hydroxybutyrate dehydrogenase (HBDH) was significantly higher than that in non-mutation group [(418 (154-2702) vs 246 (110-1068)] (P=0.032). There was no difference in CD56 expression between the two groups (P=0.053), which was limited to the difference between (≥20%) expression and non-expression. (P=0.048).@*CONCLUSION@#CBF-AML patients with signal transduction pathway gene mutation are often accompanied by high HBDH level and CD56 expression, moreover, the remission rate induced by one course of treatment is low.

Clinical Efficacy of Modified BU/CY as Conditioning Regimen Combined with Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation in Young Acute Myeloid Leukemia Patients with Low or Intermediate Risk / 中国实验血液学杂志

OBJECTIVE@#To investigate the safety and efficacy of autologous peripheral blood hematopoietic stem cell transplantation (auto-PBHSCT) using modified BU/CY conditioning regimen for young AML patients of low and middle risk in the first complete remission (CR1).@*METHODS@#Ten young AML patients of low and middle risk who did not want to accept allogeneic hematopoietic stem cell transplantation（allo-HSCT）and underwent auto-PBHSCT in CR1 during May 2013 to December 2016 were retrospectively analyzed. From 3 months after auto-PBHSCT, the maintenance therapy with interleukin-2 (IL-2) or IL-2 combined with histamine dihydrochloride was performed for these patients in the next 18 months. The side effects of the conditioning regimen, hematopoietic recovery time, transplant-related mortality (TRM) within 100 days and 1 year after auto-PBHSCT, relapse rate, leukemia-free survival (LFS) rate at 2 years and 3 years, overall survival (OS) were evaluated at 3 years and 4 years.@*RESULTS@#Gastrointestinal side effects were the major non-hematologic toxicity reaction, among which, 7 cases relatively mild and 3 cases displayed moderate, just one case suffered from severe reaction. In 4 cases, the mild liver damage occurred, but no hemorrhagic cystitis occurred. All the patients experienced different kinds of infection, including 5 cases of bloodstream infection, 2 cases of gastrointestinal infection, 3 cases of crissum infection and 2 cases of oral infection. The myeloablative effect occurred in all ten patients. The median times for absolute neutrophil count (ANC)0.5×10/L at 10 to 19 days, median was 13 days after auto-PBHSCT. The patients achieved platelet count >20×10/L at 10 to 72 days; median was 32 days after auto-PBHSCT. The TRM within 100 days and 1 year after transplantation was 0. The relapse occurred in 2 cases at 6 and 14 months after auto-PBHSCT raspectively. The median follow-up time was 48.1 months, and the median survival time was 54.7 months after transplantation. The 2-year and 3-year LFS were 100% (10 cases) and 80% (8 cases), respectively. The 3-year and 4-year OS were 80% (8 cases) and 70% (7 cases), respectively.@*CONCLUSION@#Modified BU/CY as conditioning regimen for auto-PBHSCT can achieve the myeloablative effect without raising TRM and obtain good LFS and OS. As for young AML patients without high risk, it is a valuable therapeutic option, especially for those lacking the chance of allo-HSCT.

Expression of CD25 in acute B cell lymphoblastic leukemia and its clinical significance / 中国实验血液学杂志

This study was purposed to investigate the relation of CD25 with the acute B cell lymphoblastic leukemia (B-ALL) and its clinical significance. A totol of 88 newly diagnosed B-ALL patients were enrolled in this study. The immunophenotype of leukemic myeloblasts were detected by flow cytometry, including interleukin 2 receptor α chain (CD25), β chain (CD122), γ chain (CD132), CD19, CD20, CD10, CD34, CDIgM, CD79a, CD22 and CDTDT. The expression of BCR/ABL fusion gene was detected by qualitative PCR. The expression of IL2RA (CD25 gene) was detected by real-time qualitative RT-PCR. The results showed that there was no significant statistical difference in WBC count, Hb level, PLT count, marrow blast rate, peripheral blast rate, hepato-lienal infiltration, lymph node infiltration, levels of CD10, CD20, CD22, CD34, CD79a, CDTDT, CDIgM expression between B-ALL patients with CD25(+) and B-ALL patients with CD25(-), while the CD19 expression level in B-ALL patients with CD25(+) was higher than that in B-ALL patients with CD25(-). Out of 88 B-ALL patients, 21 patients showed BCR/ABL(+)(21/88) and their CD25(+) expression level was 66.7% (14/21); 67 patients showed BCR/ABL(-) and their CD25(+) expression level was 4.5% (3/67), there was statistical difference between these two groups (P < 0.05), but the expression level of IL2RA mRNA was not statistical different between CD25(+) and CD25(-) groups (P > 0.05). Among 21 BCR/ABL(+) B-ALL patients the remission rate and relapsed rate were not statistical different between CD25(+) an CD25(-) groups.In BCR/ABL(+) B-ALL patients 8 patients relapsed, the relapsed rate was 38.1% (8/21). In BCR/ABL(-) B-ALL patients 9 patients relapsed, the relapse rate was 13.4% (9/67), there was statistical difference between BCR/ABL(+) and BCR/ABL(-) two groups (P < 0.05). In BCR/ABL(+) group the RFS (relapse free survival) was 21 months, in BCR/ABL(+) CD25(+) patients the RFS was 15 months, while in BCR/ABL(+) CD25(-) patients the RFS was 21 months, in BCR/ABL(-) CD25(-) patients, the RFS was 24 months. It is concluded that the CD25 expresses at high level in B-ALL patients with BCR/ABL(+), which may serve as a predictive marker for the presence of BCR/ABL fusion gene, and relates with relapse, CD25(+) may serve as a adjuvant indicator for poor prognosis.

Detection of intron 22 inversion of factor VIII gene in severe hemophilia A patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the incidence of intron 22 inversion (INV22) of factor VIII (FVIII) gene in severe hemophilia A (HA) patients, clarify its pathological mechanism, and identify INV22 carrier in the female family members.</p><p><b>METHODS</b>One-stage method was used to assay the FVIII activity (FVIII:C)in 126 severe HA patients with a median age of 14 years old (range: 4 months-63 years). INV22 was analyzed by long-distance polymerase chain reaction (LD-PCR) and pulsed field gel electrophoresis (PFGE), and pedigree were conducted in 3 involved HA families.</p><p><b>RESULTS</b>Of all the 126 severe HA, 52 (41.3%) cases had the INV22. Four females including 3 mothers and 1 sister of probands were diagnosed as INV22 carriers among 11 suspected carrier mosaicisms from 3 INV22 positive HA families. In 8 females from one family without HA history, the patient's mother was a INV22 carrier, but her maternal grandmother, 2 maternal aunts, 2 female siblings and 1 elder female cousin were negative.</p><p><b>CONCLUSION</b>LD-PCR and PFGE could be used to diagnose severe HA patients with INV22 and identify the carriers.</p>

The activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator chloride channel / 药学学报

Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.

Construction and identification of recombinant adenoviral vector for antisense klf4 gene / 中国实验血液学杂志

This study was aimed to construct a recombinant adenovirus vector for antisense klf4 gene through AdEasy system. Human klf4 cDNA was reversely inserted into the multiple cloning sites (MCS)of the pShuttle-CMV by using the backbone plasmid AdEasy-l, the antisense klf4 gene was constructed through homologous recombination in E.coli BJ5183, then the adenoviruses were packaged and amplified in the HEK 293 ce1ls. The adenovirus vector for antisense klf4 gene confirmed by PCR, restriction analysis and DNA sequencing. After being transfected with the adenoviruses at 200 MOI for 48 hours, total RNA and protein were extracted from human umbilical vein endothelial cells (HUVEC). Klf4 mRNA and KLF4 protein expression levels were evaluated by Real-time PCR and Western-blot. The results showed that the recombinant adenovirus vector for antisense klf4 gene was successfully constructed, recombinant adenovirus could suppress the expression of klf4 mRNA and KLF4 protein in HUVECs. It is concluded that the adenovirus vector for antisense klf4 gene has been constructed successfully, which provides the material basis for further studying the biologic function and potential application of klf4.

Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells / 中国实验血液学杂志

The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.