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1.
Chinese Traditional and Herbal Drugs ; (24): 3659-3666, 2018.
Article in Chinese | WPRIM | ID: wpr-851809

ABSTRACT

Objective: To clone novel member of alkaline/neutral invertase (NI) gene in a rare and endangered medicinal plant of Dendrobium officinale, conduct bioinformatic analysis and detect the quantitative expression in different organs. Methods: Primers were designed according to NI gene segment which was selected from leaf transcriptome sequencing results of D. officinale. The full-length cDNA of NI gene was cloned via homology-based cloning and rapid amplification of cDNA ends (RACE) approach. The physical and chemical properties, secondary structure and tertiary structure of NI protein were forecasted and analyzed using related software. The expression levels of NI gene in roots, stems, and leaves of D. officinale were detected using real-time PCR. Results: A novel gene encoding a NI protein was cloned from D. officinale. This gene (named as DoNI2, GenBank accession number: KY794404) had a total length of 2 397 bp with an open reading frame of 1 836 bp, and encoded a predicted polypeptide of 611 amino acids with a molecular weight of 69 050. Bioinformatics predicted that the isoelectric point of DoNI2 gene encoding protein was 6.38, the instability coefficient was 44.95, and the hydrophobic coefficient was −0.232. RT-PCR showed that DoNI2 gene expressed in all organs with highest expression level in stems and the lowest in roots. DoNI2 gene expression was significantly positively correlation with NI enzymatic activities at different growth years of D. officinale. Conclusion: The full length cDNA sequence in a mitochondrial DoNI2 gene was identified, facilitating future functional analysis of the gene involving in the regulation of sugar metabolism in D. officinale.

2.
Academic Journal of Second Military Medical University ; (12): 1246-1248, 2018.
Article in Chinese | WPRIM | ID: wpr-838117

ABSTRACT

In Afghanistan and Iraqi war, the war mortality has been greatly reduced due to the upgrading of United States medical technology and equipment and the optimization of the rapid evacuation of the wounded. With the improvement of survival rate, pain management is more important in the combat casualty care in United States army. The United States army implements a grading pain management strategy in the 5-level combat casualty care system, which may be an important reference for Chinese military. This paper reviews the current pain management for combat casualty care of United States army, analyzes the advantages and disadvantages of therapeutic drugs and protocols, and highlights the enlightenment on improving pain management in Chinese military in future.

3.
Medical Journal of Chinese People's Liberation Army ; (12): 12-16, 2018.
Article in Chinese | WPRIM | ID: wpr-694069

ABSTRACT

Objective To investigate the relationship between the expression of linc-VLDLR in extracellular vesicles (EVs) and the development and drug resistance of esophageal carcinoma.Methods Fifty percent of inhibitory concentration (ICs0) ofadriamycin (ADM) for Eca109 cells was detected by MTT assay,after the treatment of esophageal squamous cell carcinoma Eca109 cell line with different concentrations of ADM for 24h.The culture medium was treated with 3 concentrations of ADM based on the IC50 for 24h for extracting EVs in Eca109 cells.linc-VLDLR mRNA expression in EVs was detected by qRT-PCR assay.ICs0 of ADM for Eca109 cells intervened by EVs for 48h was detected by MTT assay.Cell cycle was detected by FCM and linc-VLDLR and ABCG2mRNA expressions in Eca109 cells were detected by qRT-PCR after the treatment of the EVs for 48h.Results ICs0 of ADM acting on Eca109 cells for 24h was 0.44 ± 0.02μg/ml,so ADM concentrations of 0.2,0.4,0.8μg/ml were choosed in the following studies.EVs were extracted from the supernatant after the treatment of 0,0.2,0.4,0.8μg/ml ADM for 24h and were labeled as EVs1,2,3 and 4 respectively.LincVLDLR mRNA expression in EVs4 was significantly higher than that in EVs1-3 (P<0.01).ADM ICs0 for Eca109 cells in EVs4 group was significantly higher than that in other groups after the treatment of EVs1-4 on Eca109 cells for 48h (P<0.05).Flow cytometry results showed that the proliferation index of Eca109 cells in EVs4 group was significantly higher than that in EVs 1-3 and control groups (P<0.01).Linc-VLDLR and ABCG2 mRNA expression levels in Eca109 cells of EVs4 group were significantly higher than these of EVs1-3 and control groups (P<0.05).Conclusions High expression of linc-VLDLR and ABCG2 gene in esophageal cancer cells is involved in the formation of esophageal cancer resistance.EVs released by drug-resistant cells can upregulate the expression of ABCG2 in esophageal cancer cells and regulate the drug resistance of esophageal cancer cells,which is related to the linc-VLDLR gene carried by EVs.

4.
Modern Clinical Nursing ; (6): 60-62, 2017.
Article in Chinese | WPRIM | ID: wpr-614215

ABSTRACT

Objective To investigate the effect of Mepilex on pressure sores in patients at lateral position after craniocerebral surgery.Methods Toally 60 patients lying at lateral position after craniocerebral surgery were randomized into two groups in equal number with random digit table:the control group and experiment group.In the control group,Gel pad was used to prevent and treat the pressure sores and in the experiment group Mepilex was used between the compressed skin and operation table before setting the position.The skin conditions of the two groups were observed after operation.Result The prophylactic effect of pressure sore in the experiment group was significantly better than that in the control group (P<0.05).Conclusion Mepilex can prevent the skin pressure sores in the patients at lateral position after cerebral surgery.

5.
Tumor ; (12): 1102-1108, 2014.
Article in Chinese | WPRIM | ID: wpr-848836

ABSTRACT

Objective: To investigate the effect of phospholipase C epsilon 1 (PLCε1) gene silencing on apoptosis and invasion of esophageal carcinoma Eca109 cells and its possible mechanism. Methods: The recombinant vectors targeting short hairpin RNA (shRNA) of PLCε1 (pGenesil-1-PLCε1-shRNA, pGenesil-1-PLCε1-shRNA2 and pGenesil-1-PLCε1-shRNA3) were transfected into Eca109cells by cationic liposome method and to screen out the expression vector with best interference effect. The apoptosis rate and invasive ability of Eca109 cells 48 h after transfection were determined by flow cytometry (FCM) and Transwell chamber method, respectively. After PLCε1 gene silencing, the expression levels of factorassociated suicide (Fas), Fas ligand (FasL), CD44, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) mRNAs were detected by semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR). Results: The pGenesil-1-PLCε1-shRNA2 had the best interference effect on PLCε1 gene. The apoptosis rate of Eca109 cells 48 h after transfection with PLCε1-shRNA2 was (30.27±5.13)%, which was significantly higher than that of the cells transfected with pGenesil-1-NC-shRNA (NC group) [(22.06±4.47)%, P < 0.05]. The number of Eca109 cells across the polycarbonate membrane of Transwell chamber in the NC group and PLCε1-shRNA2 group 48 h after transfection were 82.00±2.00 and 62.67±3.06, respectively. The number of Eca109 cells across the polycarbonate membrane in PLCε1-shRNA2 group was lower than that of the NC group (P < 0.05). The expression level of Fas mRNA in Eca109 cells of PLCε1-shRNA2 group was significantly up-regulated as compared with that of the NC group (P < 0.05), while the expression levels of FasL, MMP-9 and VEGF mRNAs in PLCε1 - shRNA2 group were significantly down-regulated (all P < 0.05). There was no significant difference in CD44 mRNA expression level between the NC group and PLCε1-shRNA2 group. Conclusion: Silencing PLCε1 gene might promote the apoptosis of Eca109 cells by up-regulating the expression of Fas and down-regulating the expression of FasL, and it can also suppress the invasive ability of Eca109 cells by down-regulating the expressions of MMP-9 and VEGF.

6.
Chinese Journal of Medical Library and Information Science ; (12): 44-48, 2014.
Article in Chinese | WPRIM | ID: wpr-454843

ABSTRACT

After a description of subject service by revealing Nature/Science contents and pushing Information Bulletin of Nature/Science-special issue of medical and life sciences in Library of Nantong University, its utilization was inves-tigated with questionnaire, and some novel ideas were put forward for deepening subject service in libraries.

7.
China Journal of Chinese Materia Medica ; (24): 3757-3763, 2014.
Article in Chinese | WPRIM | ID: wpr-310994

ABSTRACT

This study left flavonoids and alkaloids Chinese herbal monomer with common parent nucleus as cream base carriages drug respectively, cream base were prepared with stable span 60-tween 80 emulsification system. The near-infrared stability analysis technology was performed to quantitatively characterize the physical stability of cream. Base on the theory of gel network structure, theory of emulsification, theory of solubility parameter and theory of double layer, the influence mechanism of Chinese herbal monomer on physical stability of cream was discussed. The results showed that tetrahydropalmatine, matrine and naringenin had similar solubility parameter value with cream base material, creams prepared with those Chinese herbal monomer have higher Zeta potential value and stronger physical stability, and that those creams had similar microstructure information with cream base. However, a larger solubility parameter difference exists between baicalin, baicalein, berberine, palmatine and cream base material. Creams prepared with those Chinese herbal monomers had lower Zeta potential value and poorer physical stability, and that those creams had great different microstructure information with cream base.


Subject(s)
Drug Stability , Drugs, Chinese Herbal , Chemistry , Emulsions , Chemistry , Kinetics , Skin Cream , Chemistry , Solubility
8.
Modern Clinical Nursing ; (6): 37-38, 2013.
Article in Chinese | WPRIM | ID: wpr-441073

ABSTRACT

Objective To investigate the surgical collaboration in laparoscopic live donor nephrectomy.Method A retrospective analysis was made on the clinical data of 18 cases of laparoscopic live donor nephrectomy.Result All laparoscopic live donor nephrectomy were completed successfully,without severe complications caused by surgical collaboration.Conclusion The key points of successful operation are delicate mental care,sufficient preoperative preparation,good cooperation during the operation and related health education.

9.
Chinese Journal of Gastrointestinal Surgery ; (12): 1191-1194, 2013.
Article in Chinese | WPRIM | ID: wpr-256834

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression of vascular endothelial growth factor D (VEGF-D) in human esophageal squamous cell carcinoma (ESCC) and its significance.</p><p><b>METHODS</b>The expression of VEGF-C mRNA in tumor tissues and non-cancer tissues from 39 ESCC patients in our hospital from March 2009 to February 2010 was detected by in situ hybridization (ISH) method. The expression of D2-40 was detected by immunohistochemistry,and microlymphatic vessel density (MLVD) was determined by lymphatic endothelial specific marker D2-40. The associations of VEGF-C mRNA expression with clinical data and MLVD were analyzed.</p><p><b>RESULTS</b>Positive ISH VEGF-D mRNA was observed in tumor tissue samples of 22 cases (56.4%, 22/39) and non-cancer tissue sample of 1 case (2.6%, 1/39), whose difference was statistically significant (P<0.05). The expression of VEGF-D mRNA in ESCC was significantly associated with lymph node metastasis [92.9% (13/14) vs. 36.0% (9/25), P<0.05] and MLVD [(8.20±1.22) vs. (5.31±0.97), P<0.01], but not significantly associated with age, sex, pathological grade and depth of infiltration.</p><p><b>CONCLUSION</b>VEGF-D can promote lymphatic metastasis of ESSC by induction of lymphangiogenesis.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Carcinoma, Squamous Cell , Metabolism , Esophageal Neoplasms , Metabolism , Immunohistochemistry , Lymphatic Metastasis , Vascular Endothelial Growth Factor D , Metabolism
10.
Chinese Medical Journal ; (24): 3915-3920, 2013.
Article in English | WPRIM | ID: wpr-236139

ABSTRACT

<p><b>BACKGROUND</b>Carotid artery stenting (CAS) as a competing treatment modality has had to adhere to limits to gain widespread acceptance in some studies. This study analyzed the clinical data of 1700 consecutive patients after CAS to retrospectively evaluate the 30-day outcome of CAS for internal carotid artery stenosis in a Chinese population.</p><p><b>METHODS</b>Medical records of 1700 patients who underwent CAS at Xuanwu Hospital affiliated to Capital Medical University between January 2001 and August 2012 were reviewed. Postoperative 30-day complication rates were analyzed and compared with those of other studies. Univariate and multivariate Logistic regression analyses were used to identify factors associated with perioperation myocardial infarction (MI), stroke, and death.</p><p><b>RESULTS</b>The overall 30-day rate of MI, stroke, and death after CAS was 2.53%. In univariate analysis, patients who were symptomatic, had a neurological deficit (modified Rankin score (mRS) ≥3; P = 0.001), and who were not taking statins experienced a significantly increased rate of MI, stroke, and death (P = 0.017). In multivariate Logistic regression analysis, the presence of symptoms (odds ratio (OR) = 2.485; 95% confidence interval (CI) = 1.267-4.876; P = 0.008) and a neurological deficit (mRS ≥3) (OR = 3.025; 95% CI = 1.353-6.763; P = 0.007) were independent risk factors for perioperative MI, stroke, and death.</p><p><b>CONCLUSIONS</b>According to this single-center experience, CAS may effectively prevent and treat carotid artery stenosis that would otherwise lead to stroke. Being symptomatic and having a neurological deficit (mRS ≥3) increased the risk of perioperative MI, stroke, and death.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Carotid Stenosis , General Surgery , Multivariate Analysis , Myocardial Infarction , Stents , Stroke , General Surgery , Treatment Outcome
11.
Journal of Experimental Hematology ; (6): 53-56, 2012.
Article in Chinese | WPRIM | ID: wpr-331023

ABSTRACT

This study was aimed to quantify plasma circulating DNA level in patients with acute myeloid leukemia (AML) and to evaluate its clinical significance. 66 AML patients and 100 controls (60 healthy subjects for health examination, 20 cases of benign hematopathy, and 20 cases of solid tumors) were enrolled in this study. Blood samples were collected from AML patients at different status of disease and control groups. Circulating DNA were drew by using the BILATEST DNA Kit. The level of plasma DNA was determined by using duplex real-time quantitative PCR. The results showed that the median value of plasma DNA level in AML patients at diagnosis was 168.5 (73.4 - 245.1) ng/ml, significantly higher than those in three control groups, and the median level in male patients was significantly higher than that in female patients (P = 0.019). No significant difference was found in plasma DNA level of the patients at different ages and with different FAB subtypes. Compared with level before chemotherapy, the plasma DNA levels in complete remission patients and partial remission patients decreased significantly, and with no statistical difference from level of healthy controls, but was significantly different from level of non-remission patients (P < 0.05). Following up of 31 remission patients showed that the plasma DNA level increased in 5 out of 6 (83.3%) relapsed patients, but no increase was found in 22 out of 25 (88.0%) non-relapsed patients. It is concluded that the quantification of plasma DNA may be useful for evaluating therapeutic effects and monitoring relapse in AML patients.


Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Case-Control Studies , DNA , Blood , Leukemia, Myeloid, Acute , Blood , Pathology , Prognosis
12.
Journal of Experimental Hematology ; (6): 303-307, 2011.
Article in Chinese | WPRIM | ID: wpr-244934

ABSTRACT

This study was purposed to investigate the mutational status of DNA methyltransferase (DNMT3a) gene and the clinical features of AML patients with DNMT3a mutations. Using PCR combined with directly sequencing, the somatic mutations of DNMT3a involving residue of amino acid 882 were detected in 77 AML patients. Furthermore, the clinical features of these patients were also studied. The results showed that the DNMT3a mutation were detected in 7 out of 59 patients with de novo AML (11.9%), which included 4 patients with DNMT3a R882C, 2 patients with DNMT3a R882H and 1 patient with DNMT3a Y874C. Morphology examination indicated that 2 patients were M(2), 1 patient was M(4) and 4 patients were M(5). Cytogenetic analysis revealed that karyotype in 5 out of 7 patients with DNMT3a mutation were normal. In total of 27 patients with normal karyotype 5 patients (22.7%) were found harboring DNMT3a mutation, while no DNMT3a mutation was found in 21 patients with abnormal karyotype. The mutation rate in patients with positive CEBPA was obviously higher than that in patients with negative CEBPA (p = 0.002). Immunophenotype analysis showed that 4 patients (4/7, 57.1%) with DNMT3a mutation expressed lymphoid antigens including CD4 or/and CD7. There were no statistical significance in age, gender, blast cells of bone marrow, white blood cell and platelet counts, hemoglobin level, ratio of CR, mutations of FLT3-ITD, NPM1 and c-kit between patients with DNMT3a mutation and patients with wild DNMT3a (p > 0.05). It is concluded that the DNMT3a mutations are more prevalent in AML patients with normal karyotype accompanying with positive NPM1 and/or CEBPA mutation, the role of DNMT3a mutation in AML prognosis needs to be further studied.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Humans , Male , Middle Aged , Young Adult , CCAAT-Enhancer-Binding Proteins , Genetics , DNA (Cytosine-5-)-Methyltransferases , Genetics , Leukemia, Myeloid, Acute , Genetics , Mutation , Nuclear Proteins , Genetics
13.
Journal of Experimental Hematology ; (6): 983-986, 2011.
Article in Chinese | WPRIM | ID: wpr-261945

ABSTRACT

The aim of this study was to explore cytogenetic characteristics of acute promyelocytic leukemia (APL) and compare the interphase fluorescence in situ hybridization (I-FISH) with conventional cytogenetic (CC) analysis. A total number of 157 APL patients were recruited in this study, and the I-FISH and CC were applied to analyze cytogenetic features. Chromosome samples of bone marrow cells were prepared by short-term culture. Out of all 157 cases, 136 were observed with CC assay, 66 with I-FISH, of which 45 samples were analyzed with both methods. The results showed that among all 136 CC samples, t(15;17)(q22;q21) was found in 120 cases, of which 107 cases was isolated t(15;17)(q22;q21) abnormality, 13 cases was complex abnormalities and 12 case without mitotic figure. Among all 66 cases of I-FISH group, PMI/RARα fusion gene was found in 64 cases (97.0%), suggesting that I-FISH group was more sensitive than CC group (p = 0.041). It is concluded that combination of I-FISH and CC techniques plays a pivotal role for diagnosis and detection of minimal residual disease in APL.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult , Cytogenetic Analysis , Methods , In Situ Hybridization, Fluorescence , Methods , Karyotyping , Leukemia, Promyelocytic, Acute , Diagnosis , Genetics
14.
Journal of Experimental Hematology ; (6): 1145-1149, 2011.
Article in Chinese | WPRIM | ID: wpr-261912

ABSTRACT

This study was aimed to investigate the expression level of murine double minute 4 (MDM4) mRNA in chronic lymphocytic leukemia (CLL) and its prognostic value in CLL. By means of β-actin as internal reference, the real-time quantitative RT-PCR was set up. The expression of MDM4 mRNA in 66 CLL patients was measured by fluorescence dye SYBR Green I. The dispersion of MDM4 expression ratio of groups with different prognostic factors was described by using Mann-Whitney U test. The results showed that the median MDM4 mRNA expression level was 0.037098 (0.088245-0.014875) in CLL patients. The expression level of MDM4 mRNA was significantly higher in patients with P53 gene deletion than that in patients without P53 gene deletion (0.13167 vs 0.030927) (p < 0.001), and also significantly higher in patients with P53 mutation than that in patients without P53 mutation (0.13167 vs 0.03077) (p < 0.001). MDM4 expression was also associated with Binet stages (p = 0.044) and ATM gene deletion (p = 0.046), but was not associated with LDH (p = 0.216), β(2)-MG (p = 0.314), TK1 (p = 0.300), ZAP-70 (p = 0.559), CD38 (p = 0.513) and IgVH mutation status (p = 0.333). It is concluded that the expression level of MDM4 is significantly higher in patients with P53 deletion or mutation. MDM4 expression is significantly associated with Binet stages and ATM gene deletion. MDM4 may be an important prognostic factor in CLL.


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Leukemia, Lymphocytic, Chronic, B-Cell , Diagnosis , Genetics , Metabolism , Nuclear Proteins , Genetics , Metabolism , Prognosis , Proto-Oncogene Proteins , Genetics , Metabolism , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53 , Genetics
15.
Chinese Journal of Hematology ; (12): 3-7, 2011.
Article in Chinese | WPRIM | ID: wpr-252025

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the efficacy of combination chemoimmunotherapy of fludarabine, cyclophosphamide and rituximab (FCR) in chronic lymphocytic leukemia (CLL).</p><p><b>METHODS</b>Twenty-one patients with CLL were treated with FCR regimen which consisted of fludarabine (25 mg/m(2), days 2 to 4), cyclophosphamide (250 mg/m(2), days 2 to 4) and rituximab (375 mg/m(2), day 1) in a course of 28 days. The minimal residual disease (MRD) was determined by multiparameter flow cytometry. The correlation between the pretreatment characteristics and complete remission (CR) rate was analyzed.</p><p><b>RESULTS</b>Eleven patients (52.4%) achieved CR, 7 (33.3%) achieved partial remission (PR) with a overall response (OR) rate of 85.7%. With a median follow-up time of 19 (7 - 73) months, the overall survival (OS) was 86.0%, and the progression-free survival (PFS) was 72.0%. Pretreatment parameters independently associated with higher CR rates were Binet stage A + B, IgVH mutated and ZAP-70 less than 20%. MRD was less than 1% in 6 patients. The most common toxicities were myelosuppression and gastrointestinal reaction.</p><p><b>CONCLUSION</b>FCR is an effective regimen for CLL patients.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Antibodies, Monoclonal, Murine-Derived , Antineoplastic Combined Chemotherapy Protocols , Therapeutic Uses , Cyclophosphamide , Leukemia, Lymphocytic, Chronic, B-Cell , Drug Therapy , Rituximab , Treatment Outcome , Vidarabine
16.
Journal of Experimental Hematology ; (6): 570-574, 2010.
Article in Chinese | WPRIM | ID: wpr-243311

ABSTRACT

This study was aimed to explore the prognostic significance of telomere length in patients with chronic lymphocytic leukemia (CLL) and to analyze relation of telomere length with Binet stage, IgVH mutation status, CD38, ZAP-70 expression as well as other clinical features. 35 CLL patients who contained 80% or more tumor cells in the peripheral blood or bone marrow samples were selected as objects studied, while 13 healthy donors were served as normal controls. The telomere relative length was detected by using a real-time fluorescent quantitative polymerase chain reaction method (qPCR); the expression of CD38 and ZAP-70 protein were detected by flow cytometry, the IgVH mutation was detected by multiplex PCR. The results showed that the mean telomere relative length in CLL patients and normal controls were 0.384 and 0.443 respectively, but the difference between them was not significant (p > 0.05). The telomere length was significantly correlated with Binet stages and IgVH mutation status. Patients in Binet stage B and C showed significantly shorter telomeres than those in Binet stage A (p = 0.001). Mean telomere relative lengths in patients without IgVH mutation were shorter than those in patients with IgVH mutation (p = 0.015). No relation of telomere length with sex, age, ZAP-70 protein and CD38 were found (p > 0.05). It is concluded that telomere length may have a prognostic significance for CLL patients. Combining telomere length and IgVH mutation status may achieve a better prognostic subclassification for CLL patients.


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , ADP-ribosyl Cyclase 1 , Metabolism , Case-Control Studies , Leukemia, Lymphocytic, Chronic, B-Cell , Genetics , Metabolism , Mutation , Prognosis , Telomere , Chemistry , Genetics , Metabolism , ZAP-70 Protein-Tyrosine Kinase , Metabolism
17.
Journal of Experimental Hematology ; (6): 757-761, 2010.
Article in Chinese | WPRIM | ID: wpr-237657

ABSTRACT

This study was purposed to establish a method for detecting miRNA expression by using fluorescent quantitative polymerase chain reaction (RQ-PCR), and to investigate the application value of this method in quantitative detection of miRNA. Total RNA was extracted from the peripheral blood or bone marrow of 82 patients with chronic lymphocytic leukemia (CLL) and 70 patients with acute leukemias (AL). Standard curves of U6 and miRNA were constructed from 10-fold serial dilutions of the cDNA, the quantitative detection was performed by using real-time quantitative PCR with SYBR Green by Roche Light Cycler. U6 RNA was used as the reference, the relative expression levels of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in patients with CLL, while miR-128-1, miR-223, let-7b, miR-155 and miR-181a in patients with AL were analyzed by 2((-DeltaDeltaCT)) method. The relative parameters including specificity, linearity range, sensitivity and repeatability were evaluated. The results showed that the RQ-PCR assay could precisely detect the mature miRNA in as few as 10-50 ng of total RNA with the sensitivity of 0.05 ng RNA. Ct values of miRNA and U6 were all within 15 to 30. A good linear relationship (R(2) > 0.980) was obtained from the standard curves, also the efficiency of amplification was above 90%. Only a specific peak was shown on the melting curve diagram. The coefficients of variation (CV) of interrun and intrarun assay was < 4.8% and 6.3% respectively. It is concluded that the RQ-PCR is a sensitive and fast method for the detection of miRNA with its high-flux and low cost, this method will facilitate the research with detection of miRNA.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Humans , Middle Aged , Young Adult , Leukemia , Genetics , MicroRNAs , Genetics , Polymerase Chain Reaction , Methods , Sensitivity and Specificity
18.
Journal of Experimental Hematology ; (6): 843-848, 2010.
Article in Chinese | WPRIM | ID: wpr-237639

ABSTRACT

This study was aimed to investigate the expression level of puma (p53 up-regulated modulator of apoptosis) mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. The puma mRNA expressions in 100 CLL patients and 11 normal controls were measured by relative quantification RT-PCR with fluorescent dye SYBR Green I, the beta-actin was used as internal reference. The difference of puma expression rate between groups with different prognostic factors was described using the Mann-Whitney U test. The relative quantitative value of puma expression was calculated by means of 2 (-ΔCt). The results indicated that the correlation coefficients of the standard curves in qRT-PCR were ≥ 0.99. The coefficients of variations (CV) within group or between groups were < 5%, and the sensitivity reached 10² copies/microg RNA. The median puma mRNA expression level was 1.038 x 10⁻³ (4.106 x 10⁻⁴ - 2.806 x 10⁻³) in CLL patients, which was 1.220 x 10⁻³ (7.233 x 10⁻⁴ - 1.405 x 10⁻³) in normal controls. There was no difference of puma mRNA expression between CLL patients and normal controls (U = 544.5, p = 0.957). Puma expression was significantly correlated with Binet stages (p < 0.001), expression of CD38 (p = 0.002), ZAP-70 protein (p = 0.012), LDH levels (p = 0.009) and beta₂-MG (p = 0.046). The puma expression level in patients with earlier Binet stage (Binet stage A) was obviously higher than that in patients with later Binet stage (Binet stage B, C). The puma expression levels in patients with positive expression of CD38 and ZAP-70 protein, elevating levels of LDH and beta₂-MG were sharply lower than those in patients without above-mentioned unfavorable factors. The puma expression was also correlated with molecular cytogenetic abnormalities, the puma expression levels in patients with trisomy 12 (p = 0.003) and 14q32 translocation (p = 0.045) detected by FISH were significantly lower than those in patients without above-mentioned molecular cytogenetic abnormalities. It is concluded that the qRT-PCR assay is reliable and sensitive. Puma mRNA expression is significantly correlated with a great deal of prognostic factors, and may be a prognostic marker of CLL.


Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , ADP-ribosyl Cyclase 1 , Metabolism , Apoptosis Regulatory Proteins , Genetics , Leukemia, Lymphocytic, Chronic, B-Cell , Diagnosis , Genetics , Metabolism , Prognosis , Proto-Oncogene Proteins , Genetics , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Methods , ZAP-70 Protein-Tyrosine Kinase , Metabolism
19.
Journal of Experimental Hematology ; (6): 1499-1504, 2010.
Article in Chinese | WPRIM | ID: wpr-332330

ABSTRACT

In order to evaluate the clinical, biological features and prognostic factors of Richter's syndrome (RS), 8 RS patients were analyzed retrospectively. The serological test, multiplex parameter flow cytometry, conventional cytogenetic analysis, FISH technique and PCR combined with sequence detection were used to detect the LDH, β(2)-MG, TK1, SF, CA125, ZAP-70, chromosome karyotype, ATM and p53 gene deletion, as well as +12 abnormality and IgVH mutation. The results indicated that 7 out of 8 patients transformed to diffuse large B cell lymphoma (DLBCL) and 1 patient transformed to Hodgkin lymphoma (HL). Among 8 patients, LDH level in 7 patients, β(2)-MG level in 4 patients, SF level in 7 patients, CA-125 level in 4 patients and TK1 level in 1 patient exceeded the normal range. Meanwhile, ZAP-70 and CD38 were expressed positively in 4 and 7 out of 8 patients respectively. Unmutated IgVH was found in 5 patients, and 4 patients had the complex chromosome abnormalities. +12 and p53 deletion was found in 1 patient. 8 patients were divided into two groups (Binet A + B and Binet C), the mean time from diagnosis to progression was 98.5 months in Binet A + B group, compared with 38.3 months in Binet C group, there was significant difference between two groups (p = 0.021). Mean overall survival was 123.8 months and 49.8 months in two groups, respectively (p = 0.049). The mean survival after transformation was 34.5 months in Binet A + B group and 10.3 months in Binet C group. In conclusion, the level of LDH, β(2)-MG and SF are higher in RS patients in Binet C group, and so are the incidence of high expressed ZAP-70 and CD 38, unmutated IgVH. The clinical stage may be the risk and prognostic factors for RS transformation.


Subject(s)
Aged , Female , Humans , Male , Middle Aged , ADP-ribosyl Cyclase 1 , Metabolism , L-Lactate Dehydrogenase , Blood , Leukemia, Lymphocytic, Chronic, B-Cell , Blood , Diagnosis , Lymphoma, Large B-Cell, Diffuse , Blood , Diagnosis , Prognosis , Retrospective Studies , Syndrome , ZAP-70 Protein-Tyrosine Kinase , Metabolism , beta 2-Microglobulin , Blood
20.
Journal of Experimental Hematology ; (6): 36-39, 2010.
Article in Chinese | WPRIM | ID: wpr-328577

ABSTRACT

The study was purposed to explore the relationship between the expression of deoxycytidine kinase (dCK) gene and fludarabine (Flud) resistance in patients with chronic lymphocytic leukemia (CLL). The real time quantitative polymerase chain reaction (RQ-PCR) technique was used to detect the mRNA expression of dCK gene in the bone marrow or peripheral blood mononuclear cells from 30 CLL patients, the difference of dCK expression levels between CLL patients sensitive to Flud and patients resistant to Flud was analyzed. The results showed that dCK expression level in the Flud-sensitive patients was much higher than that in the Flud-resistant ones (p = 0.001); dCK expression level had no relationship with sex, age, Binet stage, IgVH mutations, CD38, ZAP-70 or p53 gene mutation (p > 0.05). It is concluded that low expression or deficiency of dCK in patients may contribute to resistance against Flud- and the prognostic significance of dCK expression remains to be further studied.


Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Case-Control Studies , Deoxycytidine Kinase , Genetics , Drug Resistance, Neoplasm , Genetics , Gene Expression , Leukemia, Lymphocytic, Chronic, B-Cell , Drug Therapy , Genetics , Vidarabine , Pharmacology , Therapeutic Uses
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