Expression and Clinical Significance of Heat Shock Protein 90 in Multiple Myeloma Patients / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of HSP90 in bone marrow samples of multiple myeloma (MM) patients and explore its clinical significance.@*METHODS@#Maxvision immunohistochemistry technique was used to detect the protein expression level of HSP90 76 MM patients and 29 normal healthy donors. The clinical characteristics of the patients were collected, and the correlation between the HSP90 expression and the clinical characteristics was analyzed.@*RESULTS@#The count of MM patients with positive HSP90 protein was significantly higher than that of normal healthy donor, and there were no significant correlation between HSP90 expression and age, sex, hemoglobin (Hb), creatinine (CREA), blood calcium, lactate dehydrogenase (LDH), bone marrow plasma cell proportion and MM subtypes (P>0.05), but HSP90 expression was correlated with β@*CONCLUSION@#HSP90 protein was over-expressed in MM patients, and was correlated with β

Clinical Characteristics and Prognostic Factors of Primary FL Patients with Grade 3 or Large B Cell Transformation / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics and prognostic factors of primary follicular lymphoma (FL) patients with grade 3 or large B cell transformation, so as to provide more reference for the subsequent clinical diagnosis and treatment.@*METHODS@#Forty-seven primary FL patients with grade 3 or large B cell transformation from March 2010 to March 2018 were selected, the clinical characteristics and survival of patients were analyzed. Cox regression model were used to evaluate the related prognostic factors.@*RESULTS@#The cumulative progression-free survival rate and cumulative overall survival rate of 47 patients in 3-year follow-up reached to 55.32% (26/47) and 80.85% (38/47) respectively. There were significant differences in cumulative progression-free survival rate and cumulative overall survival rate among different subgroups of IPI, FLIPI-1 and FLIPI-2 in 3-year follow-up (P3 cm lymph node-involved site number≥3, extranodal lesion site number≥2, IPI score=2-3, FLIPI-1 score and FLIPI-2 score≥3 were the risk factors for progression-free survival (P<0.05); LDH≥240 U/ml, IPI score=2-3 and FLIPI-2 score≥3 were risk factors for overall survival (P<0.05). Cox regression model multivariate analysis showed that IPI score=2-3 was the independent risk factor for progression-free survival and overall survival (P<0.05). FLIPI-2 score≥3 was the independent risk factor for overall survival (P<0.05).@*CONCLUSION@#Primary FL patients with grade 3 or large B cell transformation by using the existing treatment regimen might be possibly curable, and the current treatment strategies and IPI score can be used to predict the clinical prognosis of patients.

Effect of MiRNA-145 on Apoptosis of Leukemia HuT 78 Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and mechanism of miRNA-145 on leukemic cell apoptosis.@*METHODS@#After transfection of miRNA-145 mimic and negative control mimic in leukemia cells by Lipofectamine 2000 liposome, the MTT assay was used to detect the effect of miRNA-145 on cell proliferation. Flow cytometry was used to detect the effect of miRNA-145 on cell cycle and apoptosis. Western blotting assay was used to detect the expression levels of BCL-2, CDK6, Cyclin D1, BAX, PI3K p-PI3K, p-AKT and AKT.@*RESULTS@#The relative level of microRNA in HuT 78 cells transfected with miRNA-145 was 2.3±02, which was significantly higher than that in blank control group and miRNA-NC group (P＜0.05). MTT assay showed that the proliferation level of HuT 78 cells transfected with miRNA-145 mimic was significantly lower than that of blank control and miRNA-NC group (P＜0.05). Flow cytometry showed that the cells at G/G, S and G2/M phase of HuT 78 cells were significantly decreased after transfection with miRNA-145 mimic (P＜0.05). Annexin V/PI double staining assay showed that the apoptosis rate of HuT 78 cells was 17.6%±3.4%，which was significantly higher than that in blank control group and miRNA-NC group (P＜0.05). Western blot showed that the expression levels of BCL-2, CDK6 and Cyclin D1 in HuT 78 cells were significantly lower than those in blank control and miRNA-NC group (P＜0.05), and BAX expression in HuT 78 cells was significantly higher than that in blank control and miRNA-NC group (P＜0.05). Western blot showed that expression of PI3K, p-PI3K, AKT and p-AKT in HuT 78 cells transfected with miRNA-145 mimic were significantly lower than that in blank control and miRNA-NC group (P＜0.05).@*CONCLUSION@#Upregulation of miRNA-145 may inhibit the proliferation of leukemia cells and promote the apoptosis, which may be related with the inhibition of PI3K/AKT signaling pathway.

Regulatory effects of miR-195 on biological behaviors of lung cancer A549 cells / 中国病理生理杂志

AIM: To investigate the regulatory effects of microRNA(miR)-195 on the biological behaviors, such as viability,apoptosis and migration, of lung cancer A549 cells, and to explore the related mechanisms.METH-ODS:After miR-195 mimics were transfected into the A549 cells,the cell viability, cell cycle distribution and apoptosis were measured by CCK-8 assay and flow cytometry.Transwell assay was used to detect cell migration ability.Furthermore, the protein levels of cyclin D1,CDK2,Bcl-2 and p-Rb/Rb were determined by Western blot.Dual-luciferase reporter as-say was used to screen and identify the possible target genes of miR-195.RESULTS: Over-expression of miR-195 in the A549 cells inhibited the cell viability and induced cell cycle arrest,accompanied with the decrease in the cell migration a-bility and the increase in the apoptotic rate(P<0.05).Furthermore,the protein levels of cyclin D1,CDK2,Bcl-2 and p-Rb were significantly decreased(P<0.05).Dual-luciferase reporter assay demonstrated that MYB was a potential target gene of miR-195.Over-expression of MYB in the A549 cells partially reversed the effects of miR-195 on the cell viability, apoptosis and migration.CONCLUSION: miR-195 inhibits lung cancer A549 cell growth and migration, and promotes cell apoptosis by targeting MYB gene.

Clinical Significance of Corrected Serum Calcium in 320 Patients with Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical significance of corrected serum calcium(CSCa) in patients with multiple myeloma.</p><p><b>METHODS</b>The serum calcium levels of 320 patients with initial multiple myeloma were measured and corrected by serum albumin and its levels measured simultaneously. The differences of serum calcium levels were analyzed before and after the correction by serum albumin.</p><p><b>RESULTS</b>There was a significant difference between serum calcium and CSCa in MM patients (2.34±0.15 vs 2.6±0.17 mmol/L). The constituent ratio of patients with hypercalcemia was from 11.3% to 23.1% after correction, the MM patients with hypocalcemia was decreased from 42.8% to 7.8% after correction, and the patients with normal calcium level were increased. There was a significant difference between serum calcium level and CSCa in I, II, III stages of MM patients respectively(P<0.05). In the 320 patients, the incidence of anemia was 80%, renal failure was 20.9%, and myeloma bone disease was 68.8%. Calcium concentration in both anemia and renal insufficiency was higher than the normal group, and the difference was more significant after correction. In 220 cases of MM receiving chemotherapy, the median progression-free survival (PFS) was 15 months, and overall survival(OS) time was 20 months. The PFS and OS time of the patients with hypercalcemia were shortened, and the difference was very significant after correction(P<0.01).</p><p><b>CONCLUSION</b>Corrected serum calcium can more sensitively to reflect the diseases serious extent, thus indicating prognosis has better effect.</p>

Infection Status and Prognosis Analysis of Epstein-Barr Virus in Patients with Newly Diagnosed Hodgkin's Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the Epstein Barr virus(EBV) positive rate in newly diagnosed Hodgkin's lymphoma(HL) and the prognostic significance of EBV status.</p><p><b>METHODS</b>A total of 120 previously untreated patients with histologically confirmed Hodgkin's lymphoma were enrolled in this study. The EBV infection status was confirmed through examining EBV-RNA(EBER) or EBV latent membrane protein-1, and these patients were divided into EBV positive group and EBV negative group. The correlation of clinical features and EBV infection status was analyzed. For analysis of prognostic significance of EBV infection, the patients were divided into dead and survival groups, and the factors affecting the living conditions were analyzed.</p><p><b>RESULTS</b>Among 120 patients with HL, 36 patients(30.0%) were found with EBV infection. In EBVHL group patients were male, aged 6-15 and 61-74, the proportion of patients with mixed cellular sybtype was significantly higher than that in EBVHL group(P<0.05). The 1 year and 2 year total survival rate of patients in EBVgroup were 88.9% and 83.3%, and significantly lower than 97.6% and 95.2% in EBVgroup. Out of the 120 patients with HL, 10 patients died(8.3%). In death group, patients aged 61-74 and did not received radiotherapy, their proportion of EBVinfection was significantly hyher than that in survival group (P<0.05). Multiriabl analysis showed that the age 61-74 and EBVinfection were the risk factors for survival coditions of patients (P<0.05).</p><p><b>CONCLUSION</b>The EBV infection relates with HL, the clinical featuses of HL patients with EBVor EBVare different, the total survival time of patients in EBVgroup is shorter than that of patients in EBVgroup, the EBVinfection is a risk factor for total survival time of patients with HL.</p>

Analysis of Indicators Related with Cell Proliferation and Apoptosis in the Patients with B Cell Non-Hodgkin's Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the indicators related with cell proliferation and apoptosis in patients with B cell non-Hodgkin's lymphoma(B-NHL).</p><p><b>METHODS</b>Seventy-two cases of B-NHL and 50 cases of reactive hyperplasia of lymph nodes were entrolled in the experimental group and control group respectively. The expression levels of proliferating cell nuclear antigen(PCNA), X-linked inhibitor of apoptosis(XIAP) and B cell lymphoma leukemia-2(BCL-2) in paraffin-embedded tissue samples of 2 groups were detected by immuno-histochemistry staining, and their ralationship with pathologic factors was analyzed.</p><p><b>RESULTS</b>The positive cell rates of PCNA, XIAP and BCL-2 in experiment group were significantly higher than those in control group (P<0.05); the positive cell rate of PCNA in B-NHL patients at III-IV stage was higher than that in B-NHL patients at I-II stage(P<0.05), however, the positive cell rates of XIAP and BCL-2 in B-NHL patients with different pathologic factors were not significantly different(P>0.05). The progression-free survival(PES) time in PCNA low positive expression group was longer than that in PCNA high positive expression group(P<0.05), but the PFS time between B-NHL patients with XIAP positive and negative expression was not significantly different(P>0.05); the PFS time also was not significantly different between B-NHL patients with BCL-2 positive and negative expression(P>0.05).</p><p><b>CONCLUSION</b>All the PCNA, XIAP and BCL-2 participate in the pathngenesis of B-NHL, among them the positive level of PCNA obviously influences the clinical staging of B-NHL.</p>

Effect of G Protein-coupled Receptor Kinase 6 on Proliferation and Apoptosis of Multiple Myeloma Cells and Its Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effect of G protein-coupled receptor kinase 6(GRK6) on proliferation and apoptosis of multiple myeloma(MM) cells and its mechanisms.</p><p><b>METHODS</b>The samples were collected from MM patients and healthy people for study in vivo. The plasma cells isolated from multiple myeloma patients, as well as U266 and NCI H929 myeloma cell lines were used for study in vitro. Western blot and quantitative real-time PCR were used to evaluate the protein and mRNA of expression of GRK6 in multiple myeloma, cell proliferation and apoptosis were tested by BrdU and Annexin V-FITC/PI assays.</p><p><b>RESULTS</b>The protein and mRNA expression of GRK6 in multiple myeloma was higher than those in control group, and the expression level of GRK6 in stage I of MM was higher than that in control group, while the expression level of GRK6 in stage II was higher than that in stage I, but lower than that in stage III (P<0.05). U266 and MM cells showed high-sensitivity to CX-4945, except NCI H929. GRK6 expression level in U266, NCI H929 and MM cells treated with siRNA and CX-4945, significantly decreased as compared with those cells treated by CX-4945 alone. Cell proliferations of U266, NCI H92 and MM groups treated with CX-4945 were (58.25±18.24)%, (64.32±20.03)% and (45.42±25.01)% respectively, moreover, their apoptotic rates were (62.82±53.21)%, (43.25±47.05)% and (85.67±40.32)% respectively.</p><p><b>CONCLUSION</b>The expression level of GRK6 in multiple myeloma increases, and GRK6 inhibitor CX-4945 inhibits proliferation and promotes apoptosis of myeloma cells; GRK6 regulates Rac1 and involves in the proliferation and apoptosis pathway of multiple myeloma cells.</p>

Clinical Efficiency and Safety of the First-line CHOP Regimen Containing PLD Applied to Treat Aged Patients with Advanced DLBCL / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical efficiency and safety of CHOP regimen containing pegylated liposomal doxorubicin (PLD) for the aged patients with advanced diffuse large B-cell lymphoma (DLBCL).</p><p><b>METHODS</b>Fifty aged patients with advanced DLBCL treated in our hospital from February 2010 to February 2014 were selected and divided into two groups. Out of 50 cases, 25 cases received standard CHOP regimen (sCHOP group), other 25 cases received CHOP regimen containing PLD at dose of 30 mg/m2 (PLD+CHOP). These patients were followed up for 18 months, and the total effective rate, the survival rate and the adverse reaction rate were compared between these two groups.</p><p><b>RESULTS</b>After receiving different treatments, the survival rate of patients on 6, 12 and 18 months in PLD+CHOP group was 88.0%, 80.0% and 76.0%, respectively, and the survival rate of 18 month was significantly higher than that in the sCHOP group (P<0.05); The total effective rate in the PLD+CHOP group was statistically higher than that in the sCHOP group (P<0.05); and all the incidences of non-hematological toxicity, peripheral sensory neuropathy, lung infection, gastrointestinal reaction and hepatotoxicity were not statistically different between two groups (P>0.05), while the incidence of cardiac toxicity including acute myocardial infarction, congestive heart failure, atrioventricular block (AV block) and paroxysmal atrial tachycardia significantly decreased in the PLD+CHOP group (P<0.05).</p><p><b>CONCLUSION</b>The efficiency of CHOP regimen containing PLD for the aged patients with advanced DLBCL has been confirmed to be significant, and its cardiac toxicity is low, thus being worth to be popularized and applied for the treatment of advanced diffuse large B-cell lymphoma.</p>

Clinical Comparative Study of Two Kind Doses of Bortezomib Combinated with Bisphosphonates for Treating Patients with Multiple Myeloma Ostespathy / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical efficacy and safety of conventional dose and reduction dose of bortezomib in combination with bisphosphonates for treating patients with multiple myeloma ostespathy.</p><p><b>METHODS</b>A total of 150 patients with multiple myeloma ostespathy were chosen in the period from March 2011 to July 2015 and randomly were divided into 2 groups: A group (75 cases) and B group (75 cases). The patients in A and B groups were treated with conventional dose of bortezomib and reduction dose of bortezomib on the basis of bisphosphonates respectively and the clinical efficacy, the improvement rate of life quality, NRS score, levels of IL-6 and CRP before and after treatment, and the adverse effects of 2 groups were compared.</p><p><b>RESULTS</b>There was no significant difference in the clinical efficacy between 2 groups (P<0.05). The improvement rate of patients life quality in B group was significantly better than that in A group (P>0.05). There was no significant difference in the NRS score, levels of IL-6 and CRP after treatment between 2 groups (P>0.05). There was no significant difference in the incidence of neutrophil reduction and thrombocytopenia between 2 groups (P<0.05). The incidence of BiPN, nausea and vomiting, herpes zoster and fatigue of B group was significantly lower than that in A group (P<0.05).</p><p><b>CONCLUSION</b>Conventional dose and reduction dose of bortezomib in combination with bisphosphonates for treating patients with multiple myeloma ostespathy possess the same effects, including pain relief and disease progression control; but the reduction dose of bortezomib application can efficiently improve the life quality of patients and reduce the risk of adverse reactions.</p>

Growth Inhibition of Multiple Myeloma Cells Caused by MicroRNA-15a and Its Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect and possible mechanisms of miR-15a on growth of multiple myeloma(MM) cells.</p><p><b>METHODS</b>MM cell lines (U266 and RPMI8226) were transfected by lentiviral particles. MM stable cell lines were selected and collected by flow cytometry (FCM). Proliferation of MM cells before and after miR-15a high expression was detected by CCK-8 method. Apoptosis of MM cells before and after miR-15a high expression was detected by AO/EB dying, Hoechst 33258 dying and FCM, respectively. Cell cycle of MM cells before and after miR-15a high expression was detected by FCM. The expressions of miR-15a, BMI-1 and BCL-2 mRNA of MM cells before and after miR-15a high expression were detected by real-time PCR. The expressions of BMI-1 protein of MM cells before and after miR-15a high expression were detected by Western blot.</p><p><b>RESULTS</b>MM stable cell lines with miR-15a high expression was acquired. CCK-8 result showed that high expression of miR-15a could inhibit growth of MM cells (U266 and RPMI8226). AO/EB dying, Hoechst 33258 dying and FCM testing results showed that high expression of miR-15a could significantly induce apoptosis of MM cells (U266 and RPMI8226). The apoptosis rates of U266 and RPMI8226 cells in high expression group and control group were 90.52% vs 37.08% and 59.40% vs 44.17%, respectively. Meanwhile, FCM testing results showed that high expression of miR-15a could induce G1 arrest of MM cells (U266 and RPMI8226), which proportion of G1 phase were 41.50%±0.64%, 45.31%±0.77%, respectively. Real-time PCR results showed that during the growth inhibition process of MM cells caused by miR-15a high expression, the expression of BCL-2 mRNA decreased, but there was no significant changes in the expression of BMI-1 mRNA, while the expression of BMI-1 protein decreased significantly.</p><p><b>CONCLUSION</b>High expression of miR-15a can induce cell cycle arrest and apoptosis of MM cells, then inhibit their growth. The mechanisms may be related with the negative regulation of BMI-1 and BCL-2 genes in post-transcription level caused by miR-15a.</p>

Expression and Clinical Significance of Bmi-1 and P14 in Extranodal NK/T-cell Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the expression and clinical significance of Bmi-1 and P14 in extranodal NK/T-cell lymphoma (ENKTCL) tissue.</p><p><b>METHODS</b>Maxvision immunohistochemistry technique was used to detect the expression level of Bmi-1 and P14 in the tissues of 21 patients with ENKTCL and 11 normal lymph nodes. The correlation of Bmi-1 or P14 expression with the clinical features and the correlation between Bmi-1 and P14 expression were analyzed.</p><p><b>RESULTS</b>The expression of Bmi-1 protein was higher in tissues of ENKTCL than that in tissues of lymph nodes, and the Bmi-1 expression levels did not correlate with patients' sex, age, lactate dehydrogenase (LDH), International Prognostic Index (IPI) scores and B symptoms (P > 0.05), except for clinical stage (P < 0.05). The P14 protein expression level was lower in ENKTCL tissues than in normal lymph node tissues, which did not correlate with age, sex, LDH, IPI scores, clinical stage and B symptoms. Correlation test showed a negative correlation between Bmi-1 and P14 (r = -0.472, P = 0.031).</p><p><b>CONCLUSION</b>Bmi-1 protein over-expresses in ENKTCL tissues that may display a negative-regulation effect on P14 in the genesis and progress of ENKTCL.</p>

Multicenter survey of surgical site infection following clean incision breast surgery under non-local anesthesia / 中国感染控制杂志

Objective To investigate the incidence of surgical site infection(SSI)following clean incision breast surgery under non-local anesthesia,and evaluate risk factors for SSI.Methods Clinical data of 3 327 patients who underwent clean incision breast surgery under non-local anesthesia in 22 hospitals in Fujian Province were surveyed retrospectively,SSI and risk factors were analyzed.Results Among 3 327 patients,1 502(45.19%)were with malignant tumors,the average dura-tion of surgery were (101.18 ±8.04)minutes;a total of 24 cases of SSI occurred,incidence of SSI was 0.72%;253 (7.60%)patients received pre-operative antimicrobial prophylaxis,62.66% used antimicrobial agents within 0.5-2 hours before surgery.The main pathogenic bacteria was Staphylococcus aureus .Univariate and logistic regression analysis re-vealed that malignant tumor,diabetes mellitus,and use of immunosuppressants were all risk factors for SSI (all P <0.05). Conclusion SSI following clean incision breast surgery under non-local anesthesia is well controlled,risk factors for SSI should be evaluated before operation,comprehensive preventive measures should be taken to reduce the incidence of SSI.

Anti-leukemia mechanism of miR-17 and miR-20a silencing mediated by miRNA sponge / 中国实验血液学杂志

This study was aimed to quantitatively detect the expression levels of pre-miR-17 and pre-miR-20a in acute leukemia patients and eight kinds of leukemia cell lines, and to investigate the anti-leukemia mechanism of miR-17 and miR-20a silence mediated by miRNA Sponge. Quantitative real-time PCR was used to detect the mRNA expression levels of pre-miR-17 and pre-miR-20a in patients with various types of leukemia and leukemia cell lines. The Jurkat cells over-expressing miR-17 and miR-20a were transfected with recombinant lentivirus-transfecting units targeted at miR-17 and miR-20a plus 6 µg/ml of polybrene. Then the proliferation ability and cell cycle of Jurkat cells was evaluated by CCK-8 and flow cytometry respectively. The results showed that the expression level of pre-miR-17 and pre-miR-20a in all leukemia patients was significantly higher than that in normal group(P < 0.05), the expression of pre-miR-17 and pre-miR-20a in acute lymphoid leukemia was significantly higher than that in acute myeloid leukemia(P < 0.05), and the pre-miR-17 and pre-miR-20a expression level did not correlate significantly with high white blood cell count>20.0×10(9)/L(P > 0.05). The miR-17 and miR-20a silencing mediated by miRNA Sponge led to a significant decrease of cell growth, restored G1 accumulation and increase of cell apoptosis. It is concluded that the expression of miR-17 and miR-20a is upregulated in leukemia patients, which may contribute to leukemogenesis. Over-expressed miR-17 and miR-20a promote cell growth and cell cycle progression, and inhibit apoptosis through negatively-regulating P21 and E2F1 after-transcriptionally.

Expression of MCL-1 and microRNA-29a in extranodal NK/T-cell lymphoma tissue / 中国实验血液学杂志

This study was aimed to explore the expression level and correlation of MCL-1 and miR-29a in extranodal NK/T-cell lymphoma (ENKTCL) tissue. Maxvision immunohistochemistry technique and real time fluorescent quantitative PCR were used to detect the expression level of MCL-1 and miR-29a in tissue of 20 patients with ENKTCL and 10 patients with proliferative lymphadenitis, respectively. The results showed that the expression of MCL-1 protein were higher in patients with ENKTCL than that in patients with proliferative lymphadenitis, but there were no significant correlation between MCL-1 overexpression and age, sex, Ann Arbor stage and International Prognostic Index (IPI), respectively. Correlation analysis indicated that there was significant negative correlation between miR-29a expression and MCL-1 expression (r = -0.59, P = 0.016). It is concluded that miR-29a may target MCL-1 gene, regulate its expression, then participate in tumorigenesis and development of ENKTCL.

Establishment and application of lentivirus luciferase reporter-mediated miRNA target gene screening system / 中国实验血液学杂志

This study was aimed to establish a high-throughput luciferase reporter system, through which to screen and identify miRNAs directly targeting p21, and to explore the biological function and significance of these miRNAs. Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors-pWPXL-Luc and pWPXL-Luc-P21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-P21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pDM2G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and P21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following experiments. Finally luciferase activity of the latter stable cells was measured by using the luciferase reporter assay system. The results showed that high-titre recombinant lentivirus was produced and two stable cell lines were constructed, also to some certain, the luciferase activity was in direct proportion to the number of cells. In conclusion, the high-throughput luciferase reporter system is established successfully; using this system, the 28 miRNA that directly target P21 Cip1/Waf1 are screened experimentally.

A case of Richter syndrome transformed from chronic lymphocytic leukemia with karyotype aberration of trisomy 12 / 中国实验血液学杂志

This study was aimed to investigate the relationship between Richter's syndrome (RS) transformation and clinical characteristics as well as karyotype of patient with chronic lymphocytic leukemia (CLL). By the follow-up of a patient with CLL, the clinical characteristics, karyotype, treatment pattern and its effect, as well as disease progression were monitored regularly with serological test, flow cytometry and FISH technique. The results indicated that the patient typically presented with history of CLL at initial diagnosis, with expression of CD5(+), CD19(+) and CD23(+), Binet stage C, as well as karyotype aberration of trisomy 12, and poorly responded to 4 cycles of standard chemotherapy of FCR regimen. The disease progression was confirmed at 5 months with the symptoms of fever in the absence of infection, elevated lactate dehydrogenase level and rapidly enlarging lymphnodes which showed typically diffuse large B cell lymphoma by the biopsy. It is concluded that karyotype aberration of trisomy 12 is one of the risk factors for RS transformation, and treatment pattern of the patient with CLL may be associated with the transformation of RS.

Establishment of a U266 cell line with stable Bmi-1 silencing by lentivirus-mediated RNA interference / 中国实验血液学杂志

This study was aimed to construct lentivirus-mediated shRNA expression vector targeting Bmi-1 and establish a stable cell line U266-li, so as to pave the way for further research on function of Bmi-1 and application of shRNA to gene therapy. One pair of oligonucleotide sequences targeted at human Bmi-1 mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pLVTHM vector. Virus particles were collected after the control or shRNA vectors were co-transfected with the psPAX2 packaging plasmid and the plasmid pMD2.G was enveloped into HEK-293T cells by using Lipofectamine2000. The U266 cells were transduced with 5 × 10(6) recombinant lentivirus-transducing units plus 6 µg/ml of polybrene. Real-time PCR and Western blot were used respectively to detect the expression of Bmi-1 and P14 after lentivirus transduction. DNA sequencing demonstrated that the lentivirus RNAi vector of Bmi-1 was constructed successfully and the virus was packaged in 293T cells. The titer of virus was 5 × 10(7) TU/ml. Stable transfected U266 cell line was established. As was expected, the mRNA and protein levels of Bmi-1 was reduced significantly in U266 cells after lentivirus transduction, whereas the mRNA and protein levels of P14 was upregulated. It is concluded that the lentiviral RNAi vector of Bmi-1 is constructed, and U266 stable cell line is established.

Clinical analysis on 40 patients with chronic lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum β2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum β2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.

Construction of miRNA sponge targeting miR-20a and stable expression in Jurkat leukemia cell line / 中国实验血液学杂志

This study was aimed to construct miRNA sponge targeting miR-20a and to establish a stable cell line Jurkat-S, paving the way for further research on function of miR-20a and application of RNAi in gene therapy. One pair of two-repeated oligonucleotide sequences containing bulged sites that are mispaired opposite miR-20a positions 9-12 was designed and synthesized with enzyme cutting sites. The annealed oligonucleotide fragments were subcloned into pCDNA3.0-L expressing vector. After double-enzyme cutting, the vector was ligated to the annealed oligonucleotide fragments again. Enzyme cutting and luciferase activity assay were performed for identification after four repeats. Then the ligated fragment was subcloned to lentivirus expressing vector. Virus particles were collected after the control or sponge vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into HEK-293T cells using Lipofectamine 2000. The Jurkat cells were transfused with recombinant lentivirus-transfusing units plus 6 µg/ml of Polybrene. Real-time PCR and Western blot were used to detect the mRNA and protein expression of P21 and E2F1 after lentivirus transfusion respectively. As a result, luciferase activity assay demonstrated that the sponge targeting miR-20a was constructed successfully and the virus was packaged in 293T. The titer of virus was 5×10(7) TU/ml. Stable transfected Jurkat-S cell line was established. As was expected, the mRNA and protein level of P21 and E2F1 was upregulated significantly in Jurkat-S cells. It is concluded that the miR-20a sponge is constructed successfully, and Jurkat-S stable cell line is established, in which the expression of miR-20a is inhibited stably.