Significance of Targeted Sequencing Assay for Patients with Suspected Myeloid Malignancies / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of the targeted next-generation sequencing assay for patients with suspected myeloid malignancies.@*METHODS@#A total of 39 hematopenia patients with suspected myeloid malignamies in Department of Hematology of The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University from January 2018 to April 2019 were treated, 20 hot spot genes of myelodysplastic syndrome (MDS) were detected.@*RESULTS@#Regarding the diagnostic type, there were 7 cases of idiopathic cytopenia of undetermined significance (ICUS), 8 cases of clonal cytopenias of undetermined significance (CCUS) and 24 cases of myeloid myeloid malignancies which included 18 cases of MDS, 4 cases of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 2 cases of acute myeloid leukemia. Positive mutation was detected in 70.8% (17/24) of myeloid malignancy patients , and 72.7% (16/22) in MDS and MDS/MPN patients. The main mutation types were ASXL1, TET2 and RUNX1. Compared with gene negative group, there were no significant differences in sex, age (＜60 years old or ≥60 years old), proportion of bone marrow blast cells (＜5% or≥5%) and cytogenetics (good, medium and poor) (P＞0.05). Furthermore, all 8 CCUS patients showed positive mutation, and the incidence of double or multiple mutation in CCUS group was significantly lower than that of the MDS and MDS/MPN group (37.5% vs 54.5%) (P=0.002). The mutation types between the two groups were similar, and there was no significant difference in variant allele frequency (P＞0.05).@*CONCLUSION@#Our results suggest that there are high rates of double or multiple mutations in myeloid malignancies, especially in patients with MDS and MDS/MPN. Targeted sequencing assay can improve the diagnosis of myeloid malignancies, and guide clinical treatment.

Clinical Analysis of 66 Patients with Essential Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics of essential thrombocytopenia (ET) patients with positive mutations including JAK2, CALR, MPL, or negative mutations.@*METHODS@#A total of 66 newly diagnosed ET cases from January 2016 to December 2018 in Department of Hematology, Huaian No.1 People's Hospital affiliated to Nanjing Medical University were analyzed. Statistical analysis data included the patient's sex, age, symptoms, thrombosis and embolism events, spleen omegaly, platelet count (Plt), leukocyte (WBC) count, hemoglobin (Hb), fibrinogen (FIB), thrombus elastic diagram (TEG), serum potassium, blood glucose (GLU), lactate dehydrogenase (LDH), JAK2, CALR and MPL mutations, treatment options, and efficacy.@*RESULTS@#All the patients were not MPL-positive, and divided in three groups: JAK2 mutation (46 cases, 69.7%), CALR mutation (9 cases, 13.6%) and gene negative mutation (11 cases, 16.7%) group. The average age of patients in the JAK2 mutation group was 63.2 years old, and significantly higher than that in the CALR mutation group (51.8 year) and gene negative group (50.2 year) (P＜0.05). Compared with the JAK2 mutation group and gene negative group, the CALR mutation group had lower WBC count (6.3×10/L vs 13.79×10/L) (P=0.003) (6.3×10/L vs 9.70×10/L) (P=0.009). Also the Hb level of patients in CALR mutation group was lower than the JAK2 mutation group (121.22 g/L vs 136.2 g/L) (P=0.036). However, there was higher tumor burden in the CALR mutation group, compared with the gene negative mutation group (300.11 U/L vs 227.4 U/L) (P=0. 033). There was no significant difference among the three groups, such as the Plt counts, serum potassium level, GLU level and FIB level (P＞0.05). In addition, thrombus and embolism appeared in 30.3% (20/66) cases. 18.2% (12/66) cases were complicated with hyperkalemia, which significantly correlated with Plt counts (r=0.518). TEG was performed in 34 patients, of which 41.2% (14/34) had abnormal TEG and 55.9% (19/34) were accompanied by Plt count ＞ 1 000 ×10/L, but there was no significant correlation between them (r=0.134). After routine clinical treatment, all the 66 cases achieved partial or complete hematological remission, but the disease usually repeated. Until now 4.5% (3/66) cases had been converted to myelofibrosis (MF) all with JAK2 mutation, but without advancing to acute myeloid leukemia.@*CONCLUSION@#ET patients with JAK2 mutation have higher incidence, moreover were in older age. However, the patients with CALR mutations display lower WBC count and Hb level, but higher tumor burden. In short, the multiple gene mutations of ET showed different clinical features closely relates with the prognosis, thus providing guidance for the clinical diagnosis and treatment.

Post-translational Modifications of PML in Regulating the Functions of Nuclear Bodies --Review / 中国实验血液学杂志

Abstract　　The promyelocytic leukemia (PML) gene encoded PML protein as a tumor suppressor protein, plays important roles in the occurrence and development of various cancers including acute promyelocytic leukemia. Recent studies have indicated that there are a variety of post-translational modifications of the PML protein, such as SUMOylation, ubiquitination, phosphorylation, and acetylation in cells. These modifications of the PML protein can directly affect the formation of PML nuclear bodies (PML-NBs), repair DNA damage, and modulate cell apoptosis. Furthermore, the abnormal modifications of PML not only result in the occurrence of hematopoietic tumors, but also are closely related to the drug-resistance of cancer. Therefore, investigating the post-translational modifications of PML is significant to uncover the mechanism of formation and functions of PML-NBs, thus contributing to the prevention and treatment of related hematopoietic tumors. In this review, the characteristics of the post-translational modifications of PML protein and the relationship between these modifications and functions of PML-NBs are summarized so as to provide the potential targets for the treatment of related cancers.

Therapeutic Response and Prognosis of Adult Acute Myeloid Leukemia with Chromosome Karyotype Abnormalities / 中国实验血液学杂志

OBJECTIVE@#To investigate the efficacy and prognosis of acute myeloid leukemia (AML) patients with chromosome karyotype abnormalities.@*METHODS@#The clinical features and treatment responses of 91 patients with AML were collected and analyzed retrospectively. The efficacy and survival rate of the AML patients with normal and abnormal chromosome karyotype were compared.@*RESULTS@#Chromosome translocations and monosomal karyotypes were the main heterogeneity of AML. There was no significant difference in complete remission rate and overall response rate between the normal and abnormal karyotype groups, but the recurrence rate was higher in abnormal karyotype group. There was no significant difference in response of AML patients received the standard "3+7 regimen" and pre-excitation chemotherapy in the treatment of normal and abnormal karyotype groups. The relapse free survival time (RFS) was longer in the normal karyotype group, but there was no significant difference in overall survival time (OS).@*CONCLUSION@#The abnormal karyotype of AML is an independent prognostic factor, monosomal karyotype shows a poor prognosis, and the recurrence rate in AML patients with monosomal karyotype is higher.

Mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns / 中华烧伤杂志

Objective@#To investigate the mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns.@*Methods@#Seventy-two Sprague-Dawley rats were collected and divided into sham injury group, simple burn group, burn+ phosphate buffer solution (PBS) group, and burn+ 3-methyladenine (3-MA) group according to the random number table, with 18 rats in each group. Rats in simple burn group, burn+ PBS group, and burn+ 3-MA group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns). Rats in sham injury group were sham injured. Immediately after burns and fluid resuscitation, rats in burn+ PBS group were intraperitoneally injected with 1 mL PBS, and rats in burn+ 3-MA group were intraperitoneally injected with 1 mL 3-MA (125 g/L). On post injury day 3 and 7, the weights of anterior tibial muscle of right hind limbs and body of rats were measured to calculate percentage of anterior tibial muscle of right hind limbs weight. Protein expressions of microtubule related protein 1 light chain 3A (LC3A) and Beclin-1 of anterior tibial muscle were observed by immunofluorescence method and detected by Western blotting, and ratio of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ was calculated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, t-test and Bonferroni correction.@*Results@#On post injury day 3 and 7, percentages of anterior tibial muscle of right hind limbs weight of rats in simple burn group were (0.148±0.009)% and (0.134±0.018)%, respectively, which were significantly lower than those in sham injury group [(0.203±0.009)%, (0.181±0.015)%, t=10.585, 4.913, P<0.01]. Percentages of anterior tibial muscle of right hind limbs weight of rats in burn+ 3-MA group were (0.187±0.004)% and (0.192±0.009)%, respectively, which were obviously higher than those in burn+ PBS group [(0.162±0.005)%, (0.167±0.005)%, t=9.564, 5.948, P<0.01]. On post injury day 3 and 7, protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group, while protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group. Ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group (t=3.461, 3.353, P<0.05), while ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group (t=3.129, 3.977, P<0.05).@*Conclusions@#Cell autophagy induced by severe burns is involved in the process of skeletal muscle wasting of rats, and inhibition of cell autophagy may contribute to the remission of skeletal muscle wasting of rats induced by burns.

Morphology of intervertebral disc in Chinese adults evaluated using computer tomography three-dimensional imaging / 中国组织工程研究

BACKGROUND: The artificial disc requires a height, a width, and a shape as much as possible to be similar to the original intervertebral disc in order to perfectly distribute the load. At present, most of the data are from foreign countries or measured using X-ray and magnetic resonance imaging, but there are some shortcomings. OBJECTIVE: To diagnose the spinal disease and provide data for the design of a native lumbar disc device by measuring the normal lumbar intervertebral disc using computer tomography (CT). METHODS: A total of 2 235 patients who underwent lumbar CT examination in Liuzhou Worker's Hospital from January 2012 to May 2017 were collected and analyzed. There were 62 cases, including 45 males and 17 females, after being strictly met the inclusion and exclusion criteria. From the age of 20, they were divided into four groups: 20-30, 31-40, 41-50 and more than 50 years old. The range was from L1to S1. The measurement index include intervertebral disc anteroposterior diameter, transerverse diameter, disc volume, sagittal anterior, middle and posterior height, coronal left and right height, and interverterbral angle. RESULTS AND CONCLUSION: (1) In terms of age, there was a statistically significant difference in measurement indexes between L1/2, L2/3 and L3/4(P < 0.05). Therefore, the factor of age should be taken into account. However, there was no statistically significant difference between L4/5and L5/S1segments (P > 0.05). (2) There was a statistical difference between the anterior and middle height of the sagittal position in L4/5and L5/S1(P < 0.05). (3) There were statistical differences between the angles of L2/3, L3/4, L4/5, L5/S1intervertebral space (P <0.05), but the difference of angle between L4/5and L5/S1was the most. (4) There was no statistical difference in the height between the left and right sides of the coronal position (P > 0.05). (5) There was statistical difference between the anteroposterior and transverse diameters of the disc on the adjacent cross section (P < 0.05). (6) The results showed that the lower the segment, the smaller the statistical difference between each group. It is indicated that the age difference should be considered on the L1/2, L2/3, and L3/4segments in the design of lumbar disc or interbody fusion. The lumbar artificial intervertebral disc can be placed in the middle or side. The artificial disc should be designed into the wedge shape instead of a rectangle. These will provide a good anatomical basis for the design of domestic lumbar artificial intervertebral discs.

Application Research on BP Algorithm in Pulse Recognition Based on Hadoop Environment / 中国中医药信息杂志

Objective To analyze the factors of errors in the pulse recognition; To improve the speed of processing massive data; To explore the method of reducing the subjective errors in pulse recognition. Methods BP algorithm based on distributed MapReduce in Hadoop environment was optimized. Optimized BP algorithm was used to self-learn pulse-sequence data to reduce fitting errors. The pulse-counting data collected by TCM electronic pulse diagnosis instrument were used as input layer of neural network. Momentum-learning rate adaptive fast BP algorithm was adopted to train neural network. Results In the training set (75%) of 768 M, a total of 35 890 data were collected, and 29 150 items were correctly predicted in stand-alone mode, with the correct rate of 81.22%. MapRedece parallel improved BP algorithm model correctly predicted 35 841 items, with the correct rate of 99.86%. Conclusion Compared with traditional BP algorithm, BP algorithm based on distributed MapReduce in Hadoop environment has smaller fitting errors, with higher accuracy.

Influence of Co-inhibiting mTORC2 and HSP90 on Proliferation Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

<p><b>UNLABELLED</b>Objective：To explore the influence of co-inhibiting mTORC2 and HSP90 on the proliferation and apoptosis of multiple myeloma(MM) cell line U266.</p><p><b>METHODS</b>During culture, the human MM cell line U266 were treated with 20 nmol/L of rapamycin, 600 nmol/L 17-AAG, 20 nmol/L of rapamycin + 600 nmol/L 17-AGG and phosphate-buffered saline (PBS), then the growth inhibition rate, morphologic changes, apoptosis rate and the expression of caspase 3 and ATK protein in U266 cells were compared and analyzed.</p><p><b>RESULTS</b>The rapamycin and 17-AAG both could inhibit the growth of U266 cells, while the inhibitory effect of rapamycin in combination with 17-AAG on growth of U266 cells was significantly higher them that of rapamycin and 17-AAG alone and control (PBS); the apoptosis rate of U266 cells treated with rapamycin, 17-AAG and their combination was higher than that of control PBS groups, and the efficacy of 2 drug conbination was higher than that of control PBS group, and the efficacy of 2 drug combination was superior to single drug. The expression levels of caspase 3 and ATK in U266 cells treated with rapamycin, 17-AAG and their combination were higher and lower than those in control group respectively, and the efficacy of 2 drug combination was superior to signle drug. There were significant difference between them (P<0.05).</p><p><b>CONCLUSION</b>The co-inhibition of mTORC2 and HSP90 can suppress the proliferation and induce the apoptosis of MM cells.</p>

HSP90 Inhibitor 17-AAG Inhibits Multiple Myeloma Cell Proliferation by Down-regulating Wnt/β-Catenin Signaling Pathway / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism.</p><p><b>METHODS</b>The multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively.</p><p><b>RESULTS</b>The 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P < 0.05 and r = -0.473, P < 0.05), while the culture medium without 17-AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P < 0.05); At same time of culture, the more high of 17-AAG concentration, the more high of cell ratio in G1 phase (P < 0.05), at same concentration of 17-AAG, the more long time of culture, the more high of cell ratio in G1 phase (P < 0.05).</p><p><b>CONCLUSION</b>The HSP90 inhibitory 17-AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.</p>

Treatment of 568 patients with frostbite in northeastern China with an analysis of rate of amputation / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the key points of treatment and amputation in patients with frostbite, so as to increase the successful rate of the treatment.</p><p><b>METHODS</b>Five hundred and sixty-eight patients with frostbite admitted to our department from January 2005 to December 2014. (1) For the patients admitted to our department within one week after injury, the frostbite wounds were soaked in 42 °C herbal fluid (twice per day, 30 min for each time) and irradiated with infrared or red light (three times per day, 40 min for each time) from the day of admission to the 7th day after injury. Meanwhile, treatment for improvement of microcirculation, vasodilation, and anti-infection were also given. Then they received infrared or red light irradiation to the wound sites. For the patients admitted to our department longer than one week after frostbite, the frostbite wounds were irradiated with infrared or red light, and treated with antibiotics if inflammation was found around the wound. Among all the patients, 5 cases suffered from frozen stiff, and they were given fluid resuscitation as well as above-mentioned treatments after admission. (2) All patients were given wound treatment immediately after admission. The superficial partial-thickness wounds and deep partial-thickness wounds of 264 patients were given routine dressing change. The full-thickness wounds in 79 patients were treated with exposure therapy after routine dressing change first, and then granulation tissue of these wounds were grafted with autologous thigh split-thickness skin grafts. After debridement and exposure therapy, amputation was done in 225 patients 3 to 4 weeks after injury when the underlying bone was exposed. In 4 patients with exposure of calcaneus, the wounds were covered with reverse sural nerve nutrient vessels island flap. Mean healing time of superficial partial-thickness wound and deep partial-thickness wound, survival rate of skin graft in full-thickness wound, and survival rate of flap covering wound deep to bone at the heel were all recorded. The amputation rate of patients injured in December, January, February, and other months, that of patients admitted shorter than 1 day after frostbite, 1 to 3 days after frostbite, longer than 3 days and shorter than or equal to 5 days after frostbite, and longer than 5 days after frostbite, that of patients caused by drunkenness, mental disorders, improper protection, going astray, and trauma including traffic accident etc., and that of patients treated with rewarming under room temperature, rubbing with snow, wrapping with quilt, and soaking in warm water before admission were all recorded and analyzed. Parts of the data were processed with χ(2) test.</p><p><b>RESULTS</b>All patients were survived after treatment. Average wound healing time of superficial partial -thickness wound and deep partial-thickness wound was respectively 10 and 23 days. The survival rate of skin graft on full-thickness wound was about 95%. Survival rate of flap on wound deep to bone at the heel was 100%. Amputation rates of patients injured in December and January were respectively 47.46% (84/177), 42.56% (103/242), and both were significantly higher than those of patients injured in February and the other months [respectively 29.55% (26/88), 13.11% (8/61), with χ(2) values from 42.595 to 220.900, P values below 0.01]. Amputation rate of patients with admission time shorter than 1 day after frostbite was 32.06% (84/262), which was obviously lower than that of patients with admission time from 1 to 3 days after frostbite, longer than 3 days and less than or equal to 5 days after frostbite, or longer than 5 days after frostbite [respectively 40.48% (68/168), 49.02% (50/102), 52.78% (19/36), with χ(2) values from 107.284 to 165.350, P values below 0.01]. Amputation rates of patients with frostbite occurring after getting drunkenness, mental disorders, and trauma including traffic accident etc. were respectively 42.06% (106/252), 43.48% (60/138), and 53.12% (17/32), and they were all significantly higher than those of patients with frostbite caused by improper protection and going astray [respectively 27.45% (28/102), 22.73% (10/44), with χ(2) values from 187.260 to 209.738, P values below 0.01]. Amputation rates of patients undergoing treatment of rewarming under room temperature, rubbing with snow, wrapping with quilt before admission were respectively 44.29% (62/140), 48.28% (84/174), and 35.38% (46/130), and they were significantly higher than the amputation rate of patients who received the treatment of soaking in warm water [23.39% (29/124), with χ(2) values from 97.364 to 136.189, P values below 0.01].</p><p><b>CONCLUSIONS</b>Early diagnosis and treatment, properly rewarming at early stage, and correct wound treatment are the key points for reducing amputation rate of patients after frostbite. Attention should be paid to the occurrence of frostbite in December and January, and also to protection of high-risk groups (patients with mental disorders and drunker).</p>

Unraveling the chromosome higher-order structures in the transcriptional regulation of hematopoiesis by chromosome conformation capture / 中国实验血液学杂志

Emerging evidence has demonstrated that genomes are organized into higher-order structures in vivo and long range interactions between genomic regions largely contribute to the regulation of gene expression. Hematopoiesis, orchestrated by the precise spatial regulation and organization of hematopoietic transcription factors, serves as a good model for exploring these issues. The chromosome conformation capture (3C) methodology and its high throughput based technology provide an innovative solution for analyzing the regulation of functional elements through inter-chromosomal and intra-chromosomal interactions, and contacts of functional components in nuclei, thus leading to a more comprehensive understanding of human genome and gene expression. This review focuses on the recent progress of 3C and its derivatives, and their applications in unraveling the mechanisms of transcriptional regulation in hematopoiesis.

Transcriptional regulation of BHLHB2 induced by PML-RARα / 中国实验血液学杂志

Objective of this study was to investigate the transcriptional regulation of BHLHB2 gene by the PML-RARα fusion protein in APL cells and reveal the pathogenesis of APL. RT-PCR was performed to detect the expression change of BHLHB2 before and after the induction of PML-RARα in PR9 cells, and its expression level after the treatment of ATRA in PR9 and APL patient derived NB4 cells. Chromatin immunoprecipitation (ChIP)-based PCR was used to analyze whether the BHLHB2 promoter could be bound by PML-RARα in vivo. A large-scale gene expression profile dataset was used to observe the expression pattern of BHLHB2 in AML. The results showed that the expression level of BHLHB2 was significantly reduced with the induction of PML-RARα and ATRA could reverse this inhibition in both PR9 and NB4 cells and increase the expression of BHLHB2. However, the expression of BHLHB2 could not be induced by ATRA in U937 cells which do not express PML-RARα. Mechanism study revealed that PML-RARα could bound to the promoter of BHLHB2 in vivo to regulate the the expression of BHLHB2. It was found that the expression of BHLHB2 was relatively lower in APL as compared with other subtypes of AML and normal bone marrow cells. It is concluded that BHLHB2 is the target of PML-RARα, and the expression of BHLHB2 is inhibited by PML-RARα through binding to its promoter in APL.

Genomic distribution of AML1-ETO fusion protein binding sites / 中国实验血液学杂志

This study was purposed to characterize the genomic distribution of the binding sites for AML1-ETO fusion protein on chromosome 2, 9 and 19, and to further gain insights into the characteristics of transcriptional regulation by AML1-ETO in acute myeloid leukemia so as to provide theoretical basis for the development of targeted therapy and optimization for treatment. Chromatin immunoprecipitation (ChIP) coupled with high density tiling arrays (chip), also known as ChIP-chip, was utilized in this study. ChIP-DNA enriched by an anti-ETO antibody and total genomic DNA of Kasumi cells were hybridized to tiling arrays, tiled through chromosome 2, 9 and 19. The ChIP enriched regions were identified using a model based analytical tool (MAT). Genomic distribution of the ChIP regions was analyzed using publicly available CEAS web server. The Gene Ontology (GO) enrichment analysis was performed to excavated the biological significance. The results indicated that a total of 588 enriched regions were identified on chromosome 2, 9 and 19 by the anti-ETO antibody. A number of the identified regions were located within enhancers (48.86%) or introns (37.35%), much smaller fractions were within proximal promoters (5.96%) or exons (5.49%). Functional enrichment analysis showed that cell proliferation and signal transduction biological pathways were enriched in potential genes of AML-ETO. It is concluded that half of the AML1-ETO binding sites are located within known transcriptional regulatory regions (promoter, 5' UTR and enhancer), while almost another half were within the sequences which were not previously reported as regulatory regions. The potential target molecular network of AML1-ETO is involved in several essential biological processes.

Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line / 中国实验血液学杂志

Jagged-1 protein is one of the ligands belonging to Notch signaling pathway. Notch signaling pathway is one of the major signaling pathways mediated by contact between cells and plays an important role to regulate the process of proliferation and differentiation of hematopoietic cells in the hematopoietic microenvironment. To study the biological effect after the combination of receptor and ligand in Notch signaling pathway and the mechanism of Notch signaling pathway in bone marrow stromal cells mediated-drug resistance, a NIH-3T3 cell line over-expressing Jagged-1 protein was constructed for further research purposes. A full coding region of Jagged-1 gene was cloned and inserted into eukaryotic expression plasmid to construct pEGFP-IRES2-Jagged-1 eukaryotic expression vector, then transfected into NIH-3T3 cell line, a mammalian cells. As a result Western blot analysis confirmed that the transfectant NIH-3T3 cells highly expressed Jagged-1 protein and flow cytometry analysis confirmed that the NIH-3T3-pEGFP-IRES2-Jagged-1 cell line over-expressed Jagged-1 protein was monoclonal after screened by selective medium and limiting dilution analysis. It is concluded that the pEGFP-IRES2-Jagged-1 eukaryotic expression vector and a stable transfectant monoclonal NIH-3T3 cell line are successfully established. The construction of the stable transfectant monoclonal NIH-3T3 cell line which overexpressed Jagged-1 protein, provides the conditions to further study the mechanism of the bone marrow stromal cell-mediated drug resistance and to discover the new drug targets.

Influence of AML1 and AML1-ETO on transcriptional regulation of nucb2 gene / 中国实验血液学杂志

This study was purposed to investigate the effect of AML1-ETO fusion protein resulted from hematopoietic transcription factor (AML1) and acute myeloid leukemia M(2b)(AML-M(2b)) on transcription activity of nucleobindin 2 (nucb2) promoter, and to explore the role of AML1-ETO in molecular pathogenesis of AML-M(2b). The real-time RT-PCR was used to study the regulation of AML1-ETO on nucb2 at transcription level in AML1-ETO inducible leukemia cell line, the chromatin immunoprecipitation (ChIP)-based qPCR was used to investigate the direct in vivo interaction between the AML1, AML1-ETO and nucb2 promoter in AML1-ETO positive leukemia cell line, the luciferase report gene assay was used to detect the regulation of AML1, AML1-ETO on the transcription activity of nucb2 promoter. The results showed that the expression level of nucb2 was reduced with the increase of AML1-ETO. The promoter of nucb2 could be bound by both AML1 and AML1-ETO. The promoter of nucb2 was trans-repressed by AML1 and AML1-ETO respectively. It is concluded that the nucb2 is the direct target gene of AML1 and AML1-ETO, the transcription regulation of AML1, AML1-ETO on nucb2 is carried out via repressing its promoter activity.

Analysis of gene expression profiles in gastric cancer cell cycle / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the gene expression profile in gastric cancer cell cycle and explain the mechanism of gastric cancer cell proliferation by a genomic study.</p><p><b>METHODS</b>Gastric cancer cells MKN45 were synchronized at G2/M and G1/S point by nocodazole-thymidine and double thymidine methods. The synchronizing degree of cells was monitored by flow cytometry. The gene expression profiles at G2/M point, M/G1 transition, G1 early phase, G1 late phase, G1/S point, S early phase, S late phase, G2 early phase and G2 late phase in MKN45 cell cycling were examined using cDNA microarray chips. Hierarchy analysis was conducted with a professional software package and the up-regulated genes at G1 late and G2 phase were analyzed according to gene database. Furthermore, the mRNA level of cyclin E, cyclin B, plk1 and STK15 in above mentioned nine points were measured by quatitative PCR.</p><p><b>RESULTS</b>2001 genes were detected to be available at all 9 points via software processing, out of which 959 appeared up-regulated or down-regulated. 379 genes showed to be up-regulated at late G1 (147) or G2 phases (232), 40 at S and M phases (also up-regulated at G1 late and G2 phases). The 147 up-regulated genes at G1 late phase are involved in DNA metabolism, transcription and translation, protein transportation, ubiquitination and signal transduction, etc. The 232 up-regulated genes in G2 phase are involved in RNA synthesis and processing, intracellular protein transportation, cytoskeleton synthesis, signal transduction, apoptosis and anti-apoptosis, transcription regulation, ubiquitination, mitosis regulation and oncogene expression, etc. The mRNA level of 4 genes detected by quantitative PCR during cell cycle was in agreement with that detected by microarray.</p><p><b>CONCLUSION</b>During MKN45 cell cycling, the preparation for DNA synthesis and chromosome separation are conducted in G1 and G2, which are implicated in multiple genes, may be the main impetus of driving MKN45 cell cycle. Some of these genes may be related to tumor over-proliferation. The cDNA microarray technique has characteristic features such as reliability and can provide a great deal for future research on cell cycle related genes in gastric cancer.</p>

Mechanisms of fenretinide-triggered apoptosis in leukemic cells / 中国实验血液学杂志

The retinoid N-4-hydroxyphenyl retinamide (4-HPR also known as fenretinide), a synthetic derivative of all trans retinoic acid (ATRA), has shown as an efficient chemopreventive, chemotherapeutic agent and a potent inducer of apoptosis in various cancer cell types in vitro, including leukemic cells. However the mechanisms by which 4-HPR has the apoptotic effects is not completely elucidated. This study was aimed to investigate the effect of 4-HPR on several leukemic cells and explore its mechanisms of effect on U937 cells. The cell growth and proliferation experiments were performed [corrected] cell apoptosis was detected by annexin V; reactive oxygen species (ROS) and mitochondrial transmembrane potential (DeltaPsim) were determined; protein [corrected] expression was detected by Western blot. The results showed that 4-HPR inhibited the proliferation of U937 cells in a dose- and time-dependent manner. 4-HPR markedly [corrected] induced apoptosis in U937 cells, triggered the generation of ROS, induced the loss of mitochondrial transmembrane potential, decreased the expression of procaspase-8 and procaspase-3. Pretreatment of L-ascorbic acid suppressed the generation of ROS, disruption of mitochondrial potential, activation of caspases and apoptosis. It is concluded that the generation of ROS followed by the disruption of mitochondrial transmembrane potential plays an important role on 4-HPR-induced apoptosis in leukemic cells, suggesting that 4-HPR may be one of mitochondrial-targeted agents with clinical potential in treating cancer.

Analysis of gene expression profile of G1/S transition in gastric cancer cell cycle / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To detect the variation of gene expression profile of G1/S transition and elucidate the role of related genes regulating cell cycle from G1 to S phase in gastric cancer.</p><p><b>METHODS</b>Nocodazole-thymidine and double thymidine methods were used to synchronize gastric cells at G2/M and G1/S point, cDNA microarray chips was applied to examine the gene expression profile at G1 early and late phase, S early and late phase during the cell cycle, hierarchy analysis was conducted by a professional software package.</p><p><b>RESULTS</b>A total of 2001 genes were detected available, 959 genes appeared to be upregulated or downregulated, including 147 genes upregulated at G1 late phase. These 147 genes are involved in DNA metabolism, transcription and translation,posttranslational modification, ubiquitination, signal transduction etc, which all affected cell cycle from different aspects.</p><p><b>CONCLUSION</b>Complex multiple gene processes, such as DNA metabolism, transcription and translation, posttranslational modification, ubiquitination, signal transduction etc,are implicated in and also essential for G1/S transition during gastric cancer cell cycle, part of these genes are significantly associate with overproliferation in gastric cancer.</p>

Establishment of CPP-SOM integrated cDNA microarray technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To get an insight into the molecular mechanisms of diseases development and targeted therapy at the transcriptome level and search for potential therapeutic targets.</p><p><b>METHODS</b>The present researchers established a cDNA microarray platform and applied component plane presentation integrated self-organizing map (CPP-SOM) to the microarray data obtained from a differentiation model, all trans retinoic acid-induced differentiation in NB4 cells.</p><p><b>RESULTS</b>The platform included 12630 unique clones, including 9436 known genes. By CPP-SOM, the researchers were able to not only well classify the regulated genes into functionally distinct categories but also depict transcriptional changes throughout the process of the development of diseases or drug treatment.</p><p><b>CONCLUSION</b>The platform has proven to be steady and reliable, and the CPP-SOM could serve as an important and good tool for analysis of microarray data.</p>