ABSTRACT
OBJECTIVE@#To establish a method for rapid detection and typing of NPM1 mutations in acute myeloid leukemia (AML) by fluorescence melting curve analysis technology.@*METHODS@#A pair of primers and a fluorescent single-stranded probe (molecule beacon) were designed for the mutant genes mutA, mutB, mutD in exon 12 of nucleopsin (NPM1) and wild type. With a real-time qPCR, the A, B, and D gene mutations of NPM1 were detected and typed by different-melting curve peak value of the probe through RT-PCR.@*RESULTS@#This method could detected the mutations of A, B, and D in NPM1 effectively with a sensitivity of 1%. Furthermore, 62 AML clinical samples were evaluated by the method. In the results, the detection rate and typing of NPM1 mutations were consistent with the sequencing results of clinical samples.@*CONCLUSION@#There are three features in the method of fluorescence melting curve analysis: stable PCR system, easy to operate, and the easily distinguishable results. The method might meet the demand for rapid typing of NPM1 gene mutation in early diagnosis or concomitant diagnosis of AML.
Subject(s)
Humans , Exons , Genotype , Leukemia, Myeloid, Acute/genetics , Mutation , Nuclear Proteins/geneticsABSTRACT
Objective: To investigate the effects of hepatic stem cells survival in the pathological microenvironment of cholestatic cirrhosis, and the effects of sodium glycosaminodeoxycholate (GCDC) on apoptosis of hepatic stem cells in vitro. Methods: Balb/c mice were subjected to bile duct ligation (BDL) to simulate the pathological environment of cholestatic cirrhosis; Liver stem cells HP 14-19 were transplanted back into liver by the splenic vein and survival of cell colonization was detected; Effects of sodium glycosaminodeoxycholate on the viability of HP 14-19 cells at different concentrations by cell counting kit-8(CCK-8) and crystal violet staining, flow cytometry was used to detect the apoptosis of HP14-19 cells treated with 600 μmol/L GCDC 24 hours later, and the expression of Bax, Bcl-2, Capase-3, phosphorylated adenosine 5′-monophosphate-activated protein kinase α (p-AMPKα) and adenosine 5′-monophosphate-activated protein kinase α (AMPKα) were detected by Western blotting. Results: The results of CCK-8 and crystal violet staining showed that the proliferation of HP 14-19 cells was inhibited with the increase of the concentration of sodium glycosaminodeoxycholate. Flow cytometry showed that the apoptosis rate of GCDC treated group was higher than that of control group. Compared with the control group, the expression of Bax, Capase-3 was up-regulated and the expression of Bcl-2 protein was down-regulated in the experimental group. The results showed that GCDC could induce apoptosis of HP14-19 (P<0.05) AMPK was activated. Conclusion: Microenvironment of cholestatic cirrhosis induced apoptosis of liver stem cells.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the changes of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in mice with cholestatic cirrhosis and their role in regulating the balance of liver stem cell differentiation.</p><p><b>METHODS</b>Balb/c mice were subjected to bile duct ligation (BDL), and serum biochemical parameters were measured and hepatic histopathology was observed using HE staining to evaluate the modeling of cholestatic cirrhosis. Immunohistochemistry and Western blotting were used to detect the changes of TNF-α and TGF-β in the mice after modeling. Mouse embryonic hepatic stem cells (HP14-19) were treated with different concentrations of TNF-α and TGF-β, and the cell differentiation was assessed using Western blotting, real-time PCR, and PAS staining.</p><p><b>RESULTS</b>The mice receiving BDL showed significantly increased blood biochemical parameters (P<0.05), and HE staining revealed obviously increased collagen fibers in the liver with significantly increased expressions of TNF-α and TGF-β (P<0.05). In HP14-19 cells, induction with TNF-α and TGF-β for 3 days did not cause significant changes in cell differentiation, but induction for 5 days resulted in significantly increases intensity of PAS staining in the cells. The cells induced with 20, 40, and 80 ng/mL TNF-α for 5 days exhibited a significantly stronger expression of cytokeratin 18 than cytokeratin 19 (P<0.05), while induction with 20, 40, and 80 ng/mL TGF-β produced opposite changes in cytokeratin 18 and cytokeratin 19 expressions. Further induction of the cells with TNF-α and TGF-β for 10 days, did not alter the expression patterns of cytokeratin 18 and cytokeratin 19 observed on day 5, but their protein expression levels and PAS staining intensity of the cells were enhanced and their mRNA expressions became lowered.</p><p><b>CONCLUSION</b>Common bile duct ligation can induce conditions simulating cholestatic cirrhosis in mice. TNF-α and TGF-β are elevated in cholestatic cirrhosis and play opposite roles in regulating the differentiation balance of liver stem cells: the former promotes the differentiation of liver stem cells into hepatocytes, while the latter promotes the cell differentiation into colangiocytes.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the curative effect of fascia reconstruction of annular ligament combined with internal fixation for the treatment of Monteggia fracture.</p><p><b>METHODS</b>From December 2014 to October 2016, 30 cases with Monteggia fracture were treated by fascia reconstruction of annular ligament combined with internal fixation including 18 males and 12 females with an average age of 34.6 years old ranging from 6 to 50 years old. Elbow joint function were evaluated according to Mackay efficacy evaluation criteria.</p><p><b>RESULTS</b>All 30 patients were followed up for an average of 12.5 months. No radial head re-dislocation occurred. Internal fixation of reconstructive plate of ulna were all bone healing. According to the evaluation standard of Mackay curative effect, 23 cases were excellent, 5 cases were good, and 2 cases were poor. There were 4 cases of mild elbow pain and no pain in the wrist joint. Functional recovery was the fastest and most satisfactory in the forearm. Eight cases had dysfunction of elbow extension with an average limitation of 9.1 degrees. Six cases delayed injury of radial nerve were recovered for half a year.</p><p><b>CONCLUSIONS</b>For the Monteggia fracture, fascia reconstruction of annular ligament combined with internal fixation can effectively restore the forearm rotation function, the recent effect is satisfactory, further observation is needed for the long-term effect.</p>
ABSTRACT
Objective To evaluate the methods for treatment of paediatric stone disease in middle and lower ureter with fascia dilator and ureteroscopic holmium laser. Methods For 35 child patients with middle and lower ureteral calculi, the dilation of orifice was taken by 6.0 ～ 8.0 F fascia dilator, then ureteroscopic holmium laser lithotripsy followed. Results Among the 35 cases, 33 cases were the successful gravel, with a total rate of 94.3％ gravel. In other two cases, the broken stones were rushed into the renal pelvis and ESWL was successfully carried. Conclusion Fascia dilator and ureteroscopic holmium laser in paediatric middle and lower ureteral calculi can improve the successful rate of operation and reduce the operation risks. It is safe and effective.
ABSTRACT
The relationship of interleukin-4 (IL-4) C-33T and C-590T (C-589T) gene polymorphisms with allergic rhinitis was analyzed. Data about the case control studies of IL-4 gene promoter polymorphisms [C-33T and C-590T (C-589T)] and their association with allergic diseases and correlation between serum IL-4 levels and allergic rhinitis were retrieved. The Stata 12.0 statistical software was applied to analyze the correlation between IL-4 gene polymorphisms and allergic rhinitis. The meta-analysis result of TT/CC genotype of -590 (-589) polymorphism showed a significant association with allergic diseases [OR=1.93, 95% CI (1.61-2.31), P=0.00]. Meta-analysis of the TT+TC versus CC genotype of IL-4 C-33/T polymorphism revealed significant associations with allergic diseases [OR=3.23, 95% CI (1.13-9.25), P=0.03]. Meanwhile, there was a significant correlation between serum IL-4 levels and allergic rhinitis [OR=2.52, 95% CI=(1.80-3.23), P=0.00]. IL-4 gene -590 TT genotype may increase the risk of allergic rhinitis and the T allele mutation of -33 might be correlated with allergic rhinitis.
Subject(s)
Humans , Gene Frequency , Genotype , Interleukin-4 , Blood , Genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Genetics , Rhinitis, Allergic , Rhinitis, Allergic, Perennial , Blood , Genetics , Risk FactorsABSTRACT
The relationship of interleukin-4 (IL-4) C-33T and C-590T (C-589T) gene polymorphisms with allergic rhinitis was analyzed. Data about the case control studies of IL-4 gene promoter polymorphisms [C-33T and C-590T (C-589T)] and their association with allergic diseases and correlation between serum IL-4 levels and allergic rhinitis were retrieved. The Stata 12.0 statistical software was applied to analyze the correlation between IL-4 gene polymorphisms and allergic rhinitis. The meta-analysis result of TT/CC genotype of -590 (-589) polymorphism showed a significant association with allergic diseases [OR=1.93, 95% CI (1.61-2.31), P=0.00]. Meta-analysis of the TT+TC versus CC genotype of IL-4 C-33/T polymorphism revealed significant associations with allergic diseases [OR=3.23, 95% CI (1.13-9.25), P=0.03]. Meanwhile, there was a significant correlation between serum IL-4 levels and allergic rhinitis [OR=2.52, 95% CI=(1.80-3.23), P=0.00]. IL-4 gene -590 TT genotype may increase the risk of allergic rhinitis and the T allele mutation of -33 might be correlated with allergic rhinitis.
ABSTRACT
This study was aimed to establish an efficient method to detect 10 common MLL fusion genes in patients with acute leukemia. Firstly, the relevant references and databases were searched to thoroughly investigate all fusion breakpoints; the primers and probes were designed according to nearly all the involved fusion types of gene. Then the multiplex real-time PCR system was established and optimized by using the established 16 positive plasmids and negative cell lines. Finally, the detection system was clinically evaluated by means of collected 54 samples of leukemia. The results indicated that the established detection system could efficiently detect all positive plasmids with sensibility to 10 copies. Four kinds of fusion gene types such as MLL-AF4, MLL-AF9, MLL-AF10, MLL-ELL could be detected in 54 samples, the sequencing of positive samples showed consistency of sequencing results with detection results. It is concluded that a novel multiplex real-time PCR detection method is established which can detect 10 common MLL fusion genes covering about 90% of the cases harboring MLL fusions. This method is fast, sensitive, specific and reliable, and should be an useful clinical tool for identification and management of leukemia patients with MLL fusions.
Subject(s)
Humans , Acute Disease , Cell Line , Gene Rearrangement , HL-60 Cells , Leukemia , Genetics , Myeloid-Lymphoid Leukemia Protein , Genetics , Oncogene Proteins, Fusion , Genetics , Real-Time Polymerase Chain ReactionABSTRACT
This study was purposed to establish new method for detecting CBFB-MYH11 fusion gene in acute myeloid leukemia (AML) and to evaluate its value in clinical use. All fusion types of reported CBFB-MYH11 fusion gene were defined by search of references and databank, then the primers and probes were designed on this basis, and 3 positive plasmids and negative cell line as control were established. GUSB gene was also amplified as an internal reference. The primer/probe sets were tested with 3 positive plasmids and HL-60 cDNA using quantitative real-time PCR (qPCR) assays, which were then combined as a multiplex qPCR for simultaneous detection of CBFB-MYH11 and GUSB. After optimization, the multiplex qPCR assay demonstrated both high sensitivity (10 copies for all the 3 plasmids) and high specificity. Finally, the multiplex qPCR assay was clinically evaluated with 58 AML patients, and 4 CBFB-MYH11-positive cases (6.9%) were detected, involving A type (3 cases) and J type (1 case). By comparison, the multiplex qPCR assay showed results concordant with sequencing results, and detected one case that was missed by cytogenetic analysis. It is concluded that a novel qPCR method for screening of CBFB-MYH11 fusion gene in AML is established. This method is fast, comprehensive, sensitive, specific, reliable, and should consider to be a robust tool for identification and management of AML patients with CBFB-MYH11 fusion gene.
Subject(s)
Humans , Case-Control Studies , Core Binding Factor beta Subunit , Genetics , HL-60 Cells , Leukemia, Myeloid, Acute , Diagnosis , Genetics , Myosin Heavy Chains , Genetics , Oncogene Proteins, Fusion , Genetics , Real-Time Polymerase Chain ReactionABSTRACT
Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML. A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3, with the aim of detecting almost all reported mutations. After optimization, a touchdown PCR was chosen for the multiplex PCR procedure, with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively. The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products. The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio. All the positive mutated samples were confirmed by sequencing. The results showed that 17 patients with NPM1 mutation, 15 patients with FLT3-ITD mutation, 6 patients with both NPM1 and FLT3-ITD mutations were found among 93 patents. 7 patients with M₂, 4 patients with M₄, 5 patients with M₅ and 1 patients with M₆ were found out of 17 patients with NPM1 mutation, in which 10 patients were male and 7 patients were female, 15 patients were with type A, 1 patients was with type B and 1 patients was with type Nm, strikingly 1 CML patient in blast crisis was found to carry a type A mutation. Among 15 patients with FLT3-ITD mutation 1 patient with M₁, 8 patients with M₂, 2 patients with M₂, 2 patients with M₃, 1 patient with M₄, 3 patients with M₅ were found, in which 5 patients were male and 10 patients were female. Sequencing results further confirmed the accuracy and reliability of this method. It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated. This method is fast, easy, accurate and capable to calculate the mutant/wild-type ratio.
Subject(s)
Female , Humans , Male , Exons , Genotype , Karyotyping , Leukemia, Myeloid, Acute , Diagnosis , Genetics , Mutation , Nuclear Proteins , Genetics , Polymerase Chain Reaction , Methods , fms-Like Tyrosine Kinase 3 , GeneticsABSTRACT
The purpose of this study was to establish real-time based methods for detection of NPM1 gene mutation in acute myeloid leukemia (AML). Primers/probes were designed according to the clustered region of NPM1 mutations on exon 12. Two real-time PCR assays, including high resolution melting curve (HRM) and allele-specific PCR (AS-PCR), were developed and clinically evaluated with 89 AML samples, which were parallelly detected by capillary electrophoresis (CE) and sequencing. The results showed that a total of 17 mutation-positive samples were detected, including type A (15 cases), type B (1 case) and type Nm (1 case). HRM assay could detect all mutant types, and the analytical sensitivity was around 5%. In contrast, AS-PCR assay detected only 95% mutant types, but its sensitivity was as high as 0.01%. It is concluded that considering the characteristics of each method as well as the clinical evaluation results, HRM may be used for screening of NPM1 mutations at diagnosis, while the AS-PCR can be used for the MRD quantification during follow-up.
Subject(s)
Humans , Alleles , DNA Mutational Analysis , Electrophoresis, Capillary , Methods , Genome , Leukemia, Myeloid, Acute , Diagnosis , Genetics , Mutation , Neoplasm, Residual , Diagnosis , Nuclear Proteins , Genetics , Plasmids , Polymerase Chain Reaction , Methods , Sensitivity and SpecificityABSTRACT
<p><b>OBJECTIVE</b>To understand the mechanism of effect of conditioned immune response in curing bronchial asthma.</p><p><b>METHODS</b>An experimental asthma modal was produced on healthy BALB/C mice (female, 4 - 6 weeks old) by sensitization and stimulation with ovalbumin (OV A). Totally 105 mice were divided into 7 groups randomly with 15 in each and treated differently: in group CIR(1), noise was used as conditioned stimulus (CS) and budesonide and salbutamol as unconditioned stimulus (UCS) respectively, a conditioned immune response model of mice with asthma was established by the combination of CS and UCS 7 times (7 days), then the mice were given CS only, and the combination were given once a week for 20 weeks. In group CIR(2) saccharin (SAC) was taken as CS, and the other treatments were the same as the group CIR(1). In the group of conventional therapy, the mice were given inhalation of nebulized budesonide and salbutamol only for 20 weeks. In the group of lower dose conventional therapy, the mice were given nebulized inhalation of budesonide and salbutamol for the first 7 days, then once a week for 20 weeks. In the noise group the mice were given noise only everyday for 20 weeks. In SAC group the mice were treated with SAC only everyday for 20 weeks. In the blank control group the mice were treated with placebo for 20 weeks. The mice in all the groups were stimulated with OVA once a day. The mice in the healthy control group were given PBS inhalation for 20 weeks. After 20 weeks therapy, the bronchoalveolar lavage fluid (BALF) was taken for eosinophils (EOS) counting. The spleens were taken to obtain CD4(+)T lymphocytes and the expression of neuronal acetylcholine receptor alpha 7 (nAChRalpha7), IL-4, IFN-gamma and IL-17 were detected by flow cytometry.</p><p><b>RESULTS</b>(1) The percent of EOS of groups CIR(1), CIR(2), conventional therapy and healthy control was much lower than that of blank control (P < 0.01), and there was no significant difference among groups CIR(1), CIR(2) and conventional therapy (P > 0.05). (2) The expression of nAChRalpha7, IL-4 and IL-17 of groups CIR(1), CIR(2), conventional therapy and healthy control was much lower than that in blank control group, IFN-gamma was much higher (P < 0.01), and no significant difference was found among groups CIR(1), CIR(2) and conventional therapy (P > 0.05). There was a positive correlation between nAChRalpha7 and IL-4 (r = 0.76, P < 0.01), nAChRalpha7 and IL-17 (r = 0.46, P < 0.01). There was a negative correlation between nAChRalpha7 and IFN-gamma (r = 0.69, P < 0.01). (3) In the groups treated with lower dose of conventional therapy, noise, SAC and blank control, the epithelial tissue of airway were much thicker, the lumens were much narrower, and inflammatory cells and collagen fibers were much more than in the healthy control group, and after therapy, the inflammation in groups CIR(1), CIR(2) and conventional therapy was significantly improved.</p><p><b>CONCLUSION</b>The conditioned immune response models established by both noise and SAC as CS and budesonide and salbutamol as UCS can downregulate nAChRalpha7 on CD4(+)T lymphocytes, regulate the function of CD4(+)T lymphocytes, and achieve the same therapeutic efficacy in treatment of asthma.</p>