Management of cytokine release syndrome related to CAR-T cell therapy / 医学前沿

Chimeric antigen receptor T (CAR-T) cell therapy is a novel cellular immunotherapy that is widely used to treat hematological malignancies, including acute leukemia, lymphoma, and multiple myeloma. Despite its remarkable clinical effects, this therapy has side effects that cannot be underestimated. Cytokine release syndrome (CRS) is one of the most clinically important and potentially life-threatening toxicities. This syndrome is a systemic immune storm that involves the mass cytokines releasing by activated immune cells. This phenomenon causes multisystem damages and sometimes even death. In this study, we reported the management of a patient with recurrent and refractory multiple myeloma and three patients with acute lymphocytic leukemia who suffered CRS during CAR-T treatment. The early application of tocilizumab, an anti-IL-6 receptor antibody, according to toxicity grading and clinical manifestation is recommended especially for patients who suffer continuous hyperpyrexia, hypotensive shock, acute respiratory failure, and whose CRS toxicities deteriorated rapidly. Moreover, low doses of dexamethasone (5-10 mg/day) were used for refractory CRS not responding to tocilizumab. The effective management of the toxicities associated with CRS will bring additional survival opportunities and improve the quality of life for patients with cancer.

Screening and identification of RNA interference sequence for human acute monocytic leukemia related antigen 22 / 白血病·淋巴瘤

Objective To screen the optimal RNA interference sequence of acute monocytic leukemia associated antigen 22 (MLAA-22) gene in order to study gene function of it. Methods MLAA-22 coding sequence (CDS) was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the CDS was inserted in to pEGFP-N1-3FLAG vector to construct eukaryotic expression vector of MLAA-22. At the same time, four RNA interference sequences were designed and cloned to the vector. Expression vector and RNA interference vector were co-transfected into 293T cells, and the optimal RNA interference sequence was screened by fluorescence and Western blot analyses. Results MLAA-22 eukaryotic expression vector pEGFP-N1-3FLAG and four RNA interference vectors were successfully constructed. After co-transfected 293T cells, KD2 was selected as the optimal interference sequence of MLAA-22. Conclusion KD2 is an optimal interference sequence for targeting MLAA-22 antigen gene.

Relationship between TRX1 and JAB1 in acute myelogenous leukemia / 西安交通大学学报(医学版)

ABSTRACT:Objective To detect the expressions of thioredoxin (TRX1)and c-jun-activation-domain binding protein-1 (JAB1)in patients with acute myelogenous leukemia (AML)and healthy controls,and measure the TRX1 level in AML patients at different stages for evaluating its clinical significance.Methods The expressions of TRX1 and JAB1 in leukemia samples were analyzed by RT-PCR and Western blot at mRNA and protein levels, respectively.The correlation between TRX1 and JAB1,and the relationship between the gene expression and peripheral blood leukocytes count were also analyzed.Furthermore,serum TRX1 was measured by ELISA.Results TRX1 and JAB1 expressions at both mRNA and protein levels were obviously upregulated in leukemia patients (P<0.05). TRX1 was positively related to JAB1 in both newly diagnosed and recurrent AML patients.And high levels of TRX1 and JAB1 expressions were associated with white blood cell (WBC)counts in AML patients (P<0.05).Moreover, abundance of TRX1 in serum was significantly greater in AML patients,especially in the patients with recurrent AML,than in healthy donors (P<0.05).Conclusion There is a positive correlation between the expressions of TRX1 and JAB1 ,which is closely related to the occurrence and progression of AML.

Relationship of aGVHD with Treg, NK cells as well as their related cytokines in mice with allogeneic bone marrow transplantation / 西安交通大学学报(医学版)

Objective To investigate the relationship of acute graft versus host disease (aGVHD) and the regulatory T (Treg) cells,NK cells as well as their function-related cytokines such as IL-10,TGF-β,and perforin in mice with allogeneic bone marrow transplantation.Methods H-2 completely mismatched C57BL/6→ BALB/c aGVHD mice model was constructed.Flow cytometry analysis was used to detect the proportion of CD4+CD25 + Treg cells and NK-1.1+ NK cells in the spleen of aGVHD mice after transplantation.ELISA method was used to detect the serum levels of IL-10,TGF-β and perforin in the aGVHD mice intervened with CSA prophylactic or not.The normal C57BL/6 mice were used as controls.Results Compared with those of normal mice,the proportion of Treg cells in aGVHD mice after transplantation was decreased (mean 3.6％ vs.1.55％) and the proportion of NK cells increased (mean 3.3％ vs.11.5％).In the aGVHD mice treated with cyclosporine A,serum IL-10 expression level was significantly increased (treated group (125.79 ± 0.27)pg/mL,untreated group [(103.09 ± 3.27)pg/mL,P＜0.01)],TGF-β expression level was increased [(252.05 ±7.84)pg/mL vs.(241.61±15.41)pg/mL,P＞0.05],perforin expression level was significantly increased [(186.97 ± 4.68)pg/mL vs.(144.35 ± 14.42)pg/mL,P＜0.01].Conclusion ① The occurrence of aGVHD is correlated with the decreased number of Tree cells after transplantation in mice.Treg cell function-related cytokines IL-10 and TGF-β are involved in the treatment of aGVHD by cyclosporine A-mediated immunosuppression.②NK cells are involved in the occurrence of aGVHD after allogeneic bone marrow transplantation,and the increased level of perforin is related to the inhibition of aGVHD.

Bioinformatic prediction and functional characterization of human KIAA0100 gene / 药物分析学报

Our previous study demonstrated that human KIAA0100 gene is a novel acute monocytic leukemia-associated antigen (MLAA) gene. But the functional characterization of human KIAA0100 gene has remained unknown to date. Here, firstly, bioinformatic prediction of human KIAA0100 gene was carried out using online software;Secondly, human KIAA0100 gene expression was downregulated by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system in U937 cells. Cell proliferation and apoptosis were next evaluated in KIAA0100-knockdown U937 cells. The bioinformatic prediction showed that human KIAA0100 gene was located on 17q11.2, and human KIAA0100 protein was located in the secretory pathway. Besides, human KIAA0100 protein contained a signal peptide, a transmembrane region, three types of secondary structures (alpha helix, extended strand, and random coil) , and four domains from mitochondrial protein 27 (FMP27). The observation on functional characterization of human KIAA0100 gene revealed that its downregulation inhibited cell proliferation, and promoted cell apoptosis in U937 cells. To summarize, these results suggest human KIAA0100 gene possibly comes within mitochondrial genome; moreover, it is a novel anti-apoptotic factor related to carcinogenesis or progression in acute monocytic leukemia, and may be a potential target for immunotherapy against acute monocytic leukemia.

Preparation of monoclonal antibody against human KIAA0100 protein and Northern blot analysis of human KIAA0100 gene / 药物分析学报

Monoclonal antibodies (MAbs) are important tools for the study of proteins′ function and structure. But there has been no report on the preparation of MAbs against human KIAA0100 protein up to date. Here, first, we generated the mouse MAb against human KIAA0100 protein using purified recombinant 6×Histidinc (6×His)- tagged human KIAA0100 protein segment (1557–2234) as an antigen; then, the mRNA expression of human KIAA0100 gene was detected in U937 cells using Northern blot analysis. The results showed that the mouse MAb against human KIAA0100 protein could sensitively recognize the human KIAA0100 protein using Western blot analysis and immunocytochemistry analysis. Besides, Western blot analysis revealed that human KIAA0100 gene possibly encoded two different protein products (254 kDa and < 250 kDa) in U937 cells. Moreover, Northern blot analysis confirmed that human KIAA0100 gene might produced two different mRNA products (6000–10000 bp and 5000–6000 bp) in U937 cells. The results provide a basis for large-scale production of the MAb against human KIAA0100 protein, which will be useful for the study of human KIAA0100 protein′s function/structure and MAb-targeted drugs in the future.

Application of improved chromosome short-term culture method in the chromosome karyotype analysis of leukemia patients / 西安交通大学学报(医学版)

Objective To make the chromosome karyotype analysis of 130 patients with leukemia by using the improved chromosome short-term culture method.Methods We optimized the main factors with a single factor gradient experiment in short-term culture of bone marrow chromosome, including colchicines concentration, duration of action of colchicines,and hypotonic time.On this basis,we conducted the three-factors and three-level orthogonal experiment to achieve improved bone marrow chromosome preparation system,which was later applied in 130 patients with leukemia in our hospital.Results The orthogonal experiment results showed that the optimum conditions were colchicines concentration of 0.07 μg/mL,colchicines action time of 80 min,and hypotonic time of 35 min during the preparation of the bone marrow chromosome.Using this method,the chromosome preparation success rate reached 97.69% and the detection rate of abnormal karyotype reached 82.3% in the chromosome karyotype analysis.Conclusion Bone marrow chromosome preparation system with colchicines concentration of 0.07 μg/mL and colchicines action time of 80 min,and hypotonic time of 35 min is worthy of clinical promotion.

The role of oxygen free radicals in the pathogenesis of acute mononuclear leukemia / 西安交通大学学报(医学版)

Objective To study the mechanism of oxidative stress involved in the pathogenesis and relapse of acute monocytic leukemia (M5 ).Methods We detected reactive oxide species (ROS)levels,conducted plasma analysis obtained from 76 M5 patients at diagnosis and at relapse,and observed the ultrastructure of mitochondria of mononuclear cells in peripheral blood by transmission electron microscope.Results Compared with that in the control group,the average fluorescence intensity of intracellular ROS was significantly increased in M5 groups, especially in the relapse patients (P < 0.05 ).Low total antioxidative capacity (T-AOC)and antioxidant enzyme activity were characteristic of M5 at both diagnosis and relapse. However, lactate dehydrogenase (LDH ), malondialdehyde (MDA)and 8-hydroxy-2’-deoxyguanine (8-OHdG)increased significantly at both diagnosis and relapse (P < 0.05 ).Prominent ultrastructural abnormalities (mitochondrial swelling,outer membrane blebs,and aberrant cristae disorder)were present in patients with primary M5,and they were obviously abnormal in relapsing M5 patients.Conclusion Oxidative stress is the initiating factor of M5.Mitochondria are the main intracellular location for ROS generation.To maintain the dynamic balance between ROS and antioxidant defence may be the critical factor for preventing relapse.

Application of CellaVision DM96 in nucleated cells classification of serous cavity effusion / 国际检验医学杂志

Objective To explore the capacity of the Sweden CellaVision DM96 automatic digital cell morphology analysis sys‐tem (DM96) in nucleated cell classification of serous cavity effusion .Methods 36 specimens of serous cavity effusion were selected from the inpatients of Second Affiliated Hospital of Xi′an Jiaotong University in March 2015 and performed the Wright staining by the two kinds of method ,the Japanese Sysmex SP‐1000Ⅰ automatic smearing machine and manual smearing ,after staining ,the smear was performed the nucleated cells classification by DM96 .The consistency and relevance of the classification results by DM 96 with those by the Sysmex XT‐4000i were calculated .Results The classification results by DM96 had better consistency with the results by XT‐4000i ,moreover the cell images taking by DM96 were clear with high automatic degree .Conclusion The DM96 auto‐mated digital nucleated cell morphology analysis system is reliable and effective ,and has a significance for improving the cellular morphological analysis of serous cavity effusion specimen .

Expression of WT1 gene in acute leukemia and its clinical significance / 白血病·淋巴瘤

Objective To investigate the expression level and clinical significance of WT1 gene in acute luekemia (AL) patients.Methods WT1 gene level was detected by real time quantitative-polymerase chain reaction in acute myelogenous leukemia (AML) and in acute lymphocytic leukemia (ALL) patients.Then the expression levels of WT1 gene in different subtypes of AML were compared,and the correlation between gene expression and disease courses and prognosis were observed.Moreover,the relationship between disease courses and WT1 expression in patiens after receiving haemopoietic stem cell transplantation were analyzed.Results Among 66 cases,WT1 expression positive rate was 87.5 ％ (14/16) in AML and 76.0 ％ (38/50) in ALL.In AML,the expression level in M3 showed the lowest than that in any other subtypes (compared with M1,M2,M4,M5,P value was 0.040,0.007,0.006 and 0.01,respectively).The expression level of WT1 was closely correlated with leukemia disease courses.The expression level in complete remission (CR) group showed a significant lower expression level than that in non-remission group (P =0.018) and relapse group (P =0.003),and the re-increase of WT1 expression level could predict relapse as early as 1.5 months.Moreover,WT1 expression also showed an close relationship with prognosis of patients receiving haemopoietic stem cell transplantation.Patients whose WT1 was undetectable had a better prognosis than those with persistent expression,and increase again after becoming undetectable.Conclusion WT1 has a high expression level in AL,which can represent minimal residual disease.The expression level in M3 was lowest than that in different AML subtypes,and its expression level has a close correlation with clinical disease course and prognosis of AL.

Allogeneic peripheral blood stem cell transplantation using a low toxicity conditioning regimen with fludarabine and busulfan for 13 patients with hematological malignancies / 白血病·淋巴瘤

[Objective]To discuss the clinical effect of fludarabine and busulfan (Bu+Flu) as a low toxicity myeloablative conditioning regimen for allogeneic peripheral blood stem cell transplantation (allo-HSCT)in leukemia patients.[Methods]Clinical data of 13 patients with hematological malignancies receiving conditioning regimen with Bu+Flu for allo-HSCT were analyzed retrospectively.Conditioning regimen was Bu+Flu,compalriot mismatched and unrelated transplantation combined with rabbit anti-human thymocytes immune globulin (ATG).CsA+short course of methotrexate or CsA + mycophenolate mofetil were used to prevent graft-versus-host disease (GVHD).DNA sequencing of short tandem repeat (STR)polymorphism analysis method was performed for identification of donor stem cells implantation.[Results]13 patients all tolerated with this conditioning regimen well,no serious complications occurred.Neutrophil engraftment was at 9-15 days (median 11 days),platelet engraftment at 8-25 days (median 13 days).10 patients achieved hematopoiesis reconstitution with their full donor chimerisms confirmed by STR-DNA analysis.Acute GVHD occurred in 5 cases,accounting for 38.5％.Chronic GVHD occurred in 4 cases of 10 patients could be assessed,accounting for 40.0％.Severe GVHD more than Ⅱ degree did not happen.1-39 months (median time 11 months)of follow-up revealed the overall survival rate of 76.9％(10/13),disease-free survival of 61.5％ (8/13).The causes for death were relapse in all.[Conclusion]The conditioning regimen with Bu+Flu has low toxicity,well tolerance and better effect.

Clinical observation of HAG regimen in remission inductive treatment for elderly patients with acute myeloid leukemia and myelodysplastic syndrome / 白血病·淋巴瘤

Objective To observe the efficacy of HAG regimen in remission inductive treatment for elderly patients with acute myeloid leukemia (AML) and myelodysplastic syndrome-refractory anemia with excess blasts (MDS-RAEB). Methods The clinical features of 21 cases with AML and 9 cases with MDSRAEB (≥60 year old) treated with HA-G regimen in remission induction were retrospectively analyzed,including the complete remission (CR) rate, efficiency rate as well as their toxicities. Results In 21 elderly patients with AML treated with HAG regimen, the efficiency rate was 66.7 % (14/21), CR rate 47.6 % (10/21).In 9 elderly patients with MDS-RAEB, the CR rate was 55.6 % (5/9). The main toxicity of HA-G regimen was infections secondary to hematopoiesis suppression after chemotherapy. All patients were well tolerated to adjusted regimen. Conclusion The HA-G regimen is much effective in remission induction for elderly patients with AML and MDS-RAEB.

Efficacy of leukemia vaccine combined 1-MT immune treatment of tumor-bearing mice / 白血病·淋巴瘤

Objective To explore the active immunotherapeutic effects of whole-cell leukemia vaccine combined with 1-methyl-tryptophan (1-MT, inhibitor of idoleamine 2,3-dioxygenase, IDO) treatment on leukemia. Methods The tumor-bearing mice model was made by hypodermic injection of FBL-3 cells. Then these mice were divided into 5 groups, normal group, PBS control group, CTX chemotherapy group, vaccine treated group and vaccine combined with 1-MT treated group (1-MT group), respectively. The main outcome measures including general condition, response rate, tumor size, metastasis and survival time were investigated. Results The mice of PBS control group were slow to move and much heavier (including tumor nodules) than the other groups. No obvious difference was observed in activity, eating behavior and weight between normal group, vaccine treated group and 1-MT treated group. The mice of CTX chemotherapy group were observed epilation, arched body and worn, and those weights decreased significantly compared with other group. The treatment-related mortality of vaccine-treated group and 1-MT group was lower than that of CTX chemotherapy group significantly (0, 0 vs 40 %). There were no significant difference in complete remission rates between vaccine treated group and 1-MT group (61.1 % vs 70.0 %, χ2 = 0.221, P >0.05). But the recurrence rate of 1-MT group was lower than vaccine treated group (0 vs 36.36 %). The tumor nodules growth of recurrent mice could be inhibited by 1-MT. The mean survival time of vaccine treated group and 1-MT group were longer than that in CTX chemotherapy group and PBS control group (χ2 = 52.13, P <0.01). Conclusion Whole-cell leukemia vaccine can inhibit tumor growth and prolong tumor-bearing mice survival time with remarkable curative effects and few side effects. Vaccine combined with 1-MT treatment can significantly reduce tumor recurrence rate, and 1-MT was still effective in inhibiting recurrence of tumor nodules growth after vaccine treatment.

Influence of penetration enhancers on in vitro transdermal permeation of L-tethrahydropalmatine / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of different penetration enhancers on the in vitro transdermal permeation of 1-tethrahydropalmatine (L-THP) through rat skin.</p><p><b>METHOD</b>Both natural and chemical synthesis penetration enhancers were applied singly or jointly to investigate the skin permeation rates of l-THP. The skin permeation profiles were evaluated by Valian-Chien permeation cells with isolated rat skin. HPLC-UV method was established to determine the concentration of l-THP in samples.</p><p><b>RESULT</b>As chemical synthesis penetration enhancer was used alone, 8% azone was observed to be the optimal penetration enhancer with the maximal penetration rate of 21.153 microg x cm(-20 x h(-1). Although 2% menthol crystal or 5% eucalyptus oil was effective as a natural penetration enhancer when used alone, the average penetration rate reached only half of that of 8% azone. The penetration potency of either menthol oil or menthol crystal combined with 8% azone was more effective than that of azone alone (P < 0.05).</p><p><b>CONCLUSION</b>Either menthol oil or menthol crystal combined with 8% azone is effective on transdermal penetration of l-THP in vitro. There is significant synergistic effect when natural penetration enhancers combined with chemical synthesis penetration enhancers.</p>

Clinical study on MEAD regimens for relapsed or refractory adult patients with acute lymphocyte leukemia / 白血病·淋巴瘤

Objective To study the clinic effect and safety of MEAD chemotherapy regimen for adult patients with relapsed or refractory acute lymphocyte leukemia. Methods Between July 2006 and July 2009,twenty-two adult patients with relapsed or refractory acute lymphocyte leukemia received MEAD regimen (mitoxantrone 6 mg/d dl-3 iv drip,cytarabine 100 mg/d dl-5 iv drip,etoposide 100 mg/d dl-5 iv drip,dexmethasone 10 mg/d dl-8 iv drip). Results The complete remission (CR) rate of adult patients with relapsed or refractory acute lymphocyte leukemia was 31.8 %,the partial remission(PR) rate was 22.7 % and the overall response (OR) rate 54.5 %. The cumulitive CR rate was 50.0 %,and the PR rate 40.9 % after two times MEAD chemotherapy regimen. The main adverse effect was different level of myelosuppression,and other toxicity of vital organ was mild. Conclusion MEAD regimen is effective and can be tolerated for adult patients with relapsed or refractory acute lymphocyte leukemia,and its side effect is mild.

Effects of Yishen Capsule on sEPCR and CD62P in patients with chronic glomerulonephritis / 西安交通大学学报(医学版)

Objective To explore the effects of Yishen Capsule on the soluble endothelium protein C receptor (sEPCR) and P-selectin (CD62P) as well as the serum levels of immunoglobulin (Ig) and complement 3 (C3) in patients with chronic glomerulonephritis (CGN). Methods The changes of sEPCR and CD62P levels in plasma as well as Ig and C3 levels in serum were assessed in 40 normal subjects and 78 patients before and after treatment by double-antibody enzyme-linked immunosorbent assay, flow cytometry and immunoturbidimetry, respectively. Results Compared with those in normal group before treatment, the levels of sEPCR and CD62P in plasma as well as IgA in serum increased obviously (P<0.01), and the levels of IgG and IgM as well as C3 in serum decreased significantly in CGN patients (P<0.01). After three months' treatment with Yishen Capsule, the levels of sEPCR and CD62P in plasma and IgA in serum decreased significantly (P<0.05 or P<0.01), and the levels of IgG and IgM in plasma as well as C3 in serum increased obviously (P<0.05 or P<0.01). Conclusion The mechanism of Yishen Capsule's effect in treating patients with CGN may be correlated with decreasing sEPCR and CD62P levels in plasma as well as regulating the levels of Ig in serum and C3.

Analysis on curative effects of multiple myeloma using small dose of thalidomide and chemotherapy / 白血病·淋巴瘤

Objective To study the curative effects and adverse effects of the thalidomide combined with COMP chemotherapy in treating multiple myeloma(MM).Methods 42 patients were initially diagnosed as MM and 27 patients were refractory and relapsed MM.The small dose of thalidomide combined with COMP management and COMP management alone were used.The effective rate and adverse effects were analyzed.Changes of M-protein in serum,percentage of plasma cells in bone marrow and the level of hemoglobin were also analyzed in both pre-treatment and post-treatment periods.Results In 42 patients who were initially diagnosed as MM,the effective rate was 40.9% for 22 patients treated by chemotherapy alone and 70.0% for 20 patients treated by the thalidomide combined with chemotherapy.Statistic difference was observed between those two group.As to the 27 patients who were refractory and relapsed MM,the effective rate was 42.9% for 13 patients treated by chemotherapy alone and 84.6% for 14 patients treated by the thalidomide combined with chemotherapy.Statistic significance was present between them.Adverse effects were less and tolerated.Conclusion Treatment of small dose of thalidomide combined with COMP chemotherapy could significantly improve the effective rate with less adverse effects.

Construction and characterization of hGM-CSF-expressing K-562 cell line / 药物分析学报

Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The eukaryocyte expressing plasmid pcDNA3.1/GM-CSF was first constructed and its accuracy was verified through sequencing. The pcDNA3.1/GM-CSF was transfected into COS-7 cells to verify GM-CSF expression and cytokine activity using TF-1 cell line. Then the plasmid was transfected into K-562 cell line using liposome method, and was selected under G-418 and sub-cloned by limiting dilution. GM-CSF product from the monoclone GM-CSF-K-562 cell lines was quantified using ELISA method. Results We successfully constructed the hGM-CSF eukaryocyte expressing plasmid and hGM-CSF expressing K-562 cell line. Conclusion The construction of K-562/GM-CSF line will simplify the preparation of tumor vaccine, thus facilitating the application of tumor vaccination therapy in clinical application.

Cloning and screening of cDNA of Psilgramma menephorn allergen / 药物分析学报

Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

Construction and significance of directional expression cDNA library from human NB4 cells / 华中科技大学学报（医学）（英德文版）

Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.