ABSTRACT
Objective:To select human chronic myeloid leukemia (CML) cell line K562 as the experimental object, and use lentivirus mediated CRISPR/Cas9 gene editing technology to construct a stable CML cell line K562/TCRP1-KO that knocks out the tongue cancer resistance related protein 1 (TCRP1) gene; and through functional tests such as cell proliferation, apoptosis, and drug sensitivity, compare the phenotypic differences between K562/TCRP1-KO and control cells (K562/cas9-CTL), and preliminarily explore the possible mechanism of TCRP1 gene involvement in the pathogenesis of CML.Methods:The small guide RNA (sgRNA) targeting TCRP1 was designed at a specific location. After annealing, the oligonucleotide fragments were recombined with the linearized Cas9 expression vector, and the lentivirus packaging system was transfected into 293T cells. The purified virus was collected and infected with K562 cells. Positive polyclons were screened for puromycin pressure, and monoclonal K562/TCRP1-KO was further screened by limited dilution method. Stable cell lines were successfully knocked out by sanger sequencing and Western blot detection; Simultaneously, K562 cells transfected with lentiCRISPR vector were constructed as control cell lines (K562/cas9-CTL); Using cell counting method, cell counting kit 8 (CCK8) method, imatinib (IM) gradient dilution method, and flow cytometry cell proliferation, drug sensitivity, and apoptosis analysis were performed on K562/TCRP1-KO and K562/cas9-CTL, respectively.Results:The sgRNA-Cas9 recombinant plasmid vector for TCRP1 knockout was successfully constructed, and after transfection into 293T cells, TCRP1 knockout monoclonal cell lines were successfully screened using limited dilution method. Compared with K562/cas9-CTL cells, the proliferation ability of K562/TCRP1-KO cells was significantly reduced, IM drug sensitivity was significantly enhanced, and the process of cell apoptosis was significantly accelerated (all P<0.05). Conclusions:A CML cell line with TCRP1 knockout was successfully constructed using CRISPR/Cas9. TCRP1 may act as a cancer related gene to affect the proliferation, IM resistance, and apoptosis process of CML cells.
ABSTRACT
OBJECTIVE@#To explore the coincidence rate of G-banding karyotype analysis and fluorescence in situ hybridization (FISH) for the diagnosis of children with sex chromosome mosaicisms.@*METHODS@#A retrospective analysis was carried out for 157 children with suspected sex chromosome abnormalities who had presented at Shenzhen Children's Hospital from April 2021 to May 2022. Interphase sex chromosome FISH and G-banding karyotyping results were collected. The coincidence rate of the two methods in children with sex chromosome mosaicisms was compared.@*RESULTS@#The detection rates of G-banding karyotype analysis and FISH were 26.1% (41/157) and 22.9% (36/157) , respectively (P > 0.05). The results of G-banding karyotype analysis showed that 141 cases (89.8%) were in the sex chromosome homogeneity group, of which only 5 cases (3.5%) were inconsistent with the results of FISH. There were 16 cases (10.2%) in the sex chromosome mosaicism group, of which 11 cases (68.8%) were inconsistent with the results of FISH. There was a statistical difference between the two groups in the coincidence rate of the results of the two methods (P < 0.05).@*CONCLUSION@#No significant difference was found between G-banding karyotype analysis and FISH in the detection rate of chromosome abnormalities. The coincidence rate in the mosaicism group was lower than that in the homogeneity group, and the difference was statistically significant. The two methods should be combined for clinical diagnosis.
Subject(s)
Humans , Mosaicism , In Situ Hybridization, Fluorescence/methods , Retrospective Studies , Karyotyping , Chromosome Aberrations , Sex Chromosome Aberrations , Karyotype , Chromosome Banding , Sex ChromosomesABSTRACT
Objective:To explore the epidemiological characteristics of Streptococcus pyogenes, namely β-hemolytic Group A Streptococcus (GAS) in children in Shenzhen. Methods:Multilocus sequence typing (MLST) data on the epidemic clonal population of GAS infection in children in Shenzhen Children′s Hospital from January 2016 to December 2018 were retrospectively analyzed.In the present study, 32 GAS strains belonging to 7 different emm types were from 32 children′s with impetigo, cellulitis, scarlet fever, sepsis, pneumonia, obstructive sleep apnea hypopnea syndrome, bronchitis, allergy with rhinitis, buttock abscess, allergic purpura or pharyngeal tonsillitis, which were isolated from 23 throat swabs, 5 sputum samples, 3 pus and 1 blood.Using polymerase chain reaction technology, 7 pairs of allelic housekeeping genes ( gki, gtr, murI, mutS, recP, xpt and yqiL) of 32 GAS isolates were analyzed, and the target gene products were subjected to sequencing.Then the obtained gene sequences of each allele were submitted to the MLST database to obtain the allele profile.Finally, the allele profiles were introduced in the MLST database again to confirm the sequence typing (ST). Results:The GAS clone groups of emm 1.00 and its subtypes, emm 4.00, emm 12.00 and its subtypes, emm 22.00, emm 28.00, emm 75.00, and emm 89.00 belonged to the sequence typing ST28, ST39, ST36, ST46, ST52, ST49, and ST921, respectively. Conclusions:From 2016 to 2018, the MLST clone populations of GAS isolates causing infections in children in Shenzhen are classified as ST28, ST39, ST36, ST46, ST52, ST49 and ST921.
ABSTRACT
Objective:To establish the biology reference interval (RI) of peripheral blood procalcitonin (PCT) for children between 3 days and 6 years old in China.Methods:Totally 3 353 reference individuals with apparent health or no specific diseases were recruited in 18 hospitals throughout the country during October 2020 to May 2021. Reference individuals were divided into four groups: 3-28 days, 29 days - 1 year, 1-3 years and 4-6 years. Vein blood or capillary blood were collected by percutaneous puncture from every reference individual. The PCT level in serum and the capillary whole blood were assayed by Roche Cobas e601 and Norman NRM411-S7 immunoanalyzer. Outliers were deleted and 95th percentiles of every group were provided as RIs. Man-Whitney U test or Kruskal-Wallis test were used performed to assess the difference among different gender, age or method groups. Results:The difference of PCT distribution between male and female is not statistically significant, but the difference between serum and capillary whole blood is statistically significant. The differences between age groups are significant too. For Roche e601, serum PCT RI of 3-28 days group is <0.23 μg/L, 29 days - 6 years are <0.11 μg/L. For NRM411, Serum PCT RI of 3-28 days group is <0.21 μg/L, 29 days - 1 year: <0.09 μg/L, 1 - 6 years: <0.10 μg/L. For whole blood PCT, RI of 3-28 days group is <0.26 μg/L, 29 days - 6 years is <0.15 μg/L.Conclusions:Serum and capillary whole blood PCT have different RIs, however, capillary whole blood PCT testing is valuable in pediatric application. Children in 3-28 days show higher PCT levels than other age group. To establish the RIs and understand the differences among different groups are essential for the interpretation and clinical application of peripheral blood PCT testing results.
ABSTRACT
Objective:To investigate the clinical characteristics, drug resistance and serotypes of children′s invasive pneumococcal disease(IPD) in Shenzhen.Methods:Clinical data and drug sensitivity results of IPD children enrolled in Shenzhen Children′s Hospital, from January 2012 to December 2018, were analyzed retrospectively, and serotypes of the retained strains were identified by capsule swelling method or polymerase chain reaction(PCR) method.Results:One hundred and forty-one cases were enrolled, majority of them were less than 2 years old (86 cases, 61.0%). A total of 99 cases(70.2%) had onset in autumn and winter.The clinical manifestation included single bloodstream infection(62 cases, 45.4%), purulent meningitis(30 cases, 21.3%), pneumonia with bacteremia(28 cases, 19.9%), bone and joint infection(12 cases, 8.5%), purulent pleurisy (4 cases, 2.8%), peritonitis (3 cases, 2.1%), and infective endocarditis (2 cases, 1.4%). Underlying diseases were found in 33 cases(23.4%), co-infection in 14 cases (9.9%), complications in 39 cases (27.7%). After active treatment, 5 cases (3.5%) who were all under 2 years old died, and all of the isolates had multi-drug resistance.Four cases (2.8%) were discharged without recovery, and the rest cases were improved.The incidence of Penicillin insensitive Streptococcus pneumoniae (PNSP) with underlying diseases (30.7% vs.15.4%, χ2=3.956), meningitis(32.0% vs.9.2%, χ2=10.722) and multiple drug resistance (86.7% vs.63.1%, χ2=10.538)were higher than those of Penicillin sensitive Streptococcus pneumo- niae(PSSP)(all P<0.05). The serotypes of 97 invasive Streptococcus pneumoniae strains were identified.Types of 14 and 19F (21 strains for each type, 21.6%) were the most common, followed by type 19A (15 strains, 15.5%), type 6B and 23F (13 strains for each type, 13.4%), and type 3 (3 strains, 3.1%). The serotype coverage of 13-valent pneumococcal conjugate vaccine (PCV13) was 92.8% (90/97 strains). Conclusions:Children under 2 years old are prone to IPD and death.The IPD distribution varies in different seasons, and single bloodstream infection is the most common clinical manifestation; PNSP is more likely to occur in children with underlying diseases and meningitis, and the multi-drug resistance of pathogenic strains may be related to poor prognosis.PCV13 can cover most IPD serotypes.
ABSTRACT
Objective@#To analyze the features of Norovirus outbreaks in schools and kindergartens and the associated factors in Chengdu, 2017, and to provide the scientific basis for Norovious outbreaks prevention and control.@*Methods@#A total of 75 schools and kindergartens in Chengdu of 2017 participated in the study about Norovirus outbreaks. ANOVA and binary Logistic regression models were used to analyze the influencing factors and duration of Norovirus outbreaks.@*Results@#Overall 710 cases were included. There was an average of 9.47 cases and 27.52 hours for each outbreak,decreasing by 1.06 cases and 10.56 hours compared with those of 2016. Most outbreaks happened in kindergartens and in the firstlevel economy regions from January to March, with GII type as the main outbreak. Compared with the first case of vomiting at home, vomiting in public area (OR=11.76, 95%CI=1.63-84.69) was much more serious, and compared with the active report of school/ community, being informed of the outbreak passively (OR=4.09，95%CI=1.04-16.03) was positively associated with outbreak severity.@*Conclusion@#To prevent Norovirus infection, specific development and training of dealing with vomiting and feces should be introduced, and measures to increase the ability to surveil and report Norovirus outbreaks should be enhanced.
ABSTRACT
Objective@#To study the characteristics of clinical diagnosis and treatment for 3 children with phytosterolemia. @*Methods@#The different clinical manifestations of 3 children with phytosterolemia were retrospectively reviewed. The case 1 and case 2, who were 7 years and 2 months old twin sisters, hospitalized for frequent epistaxis and abdominal pain. The case 3, who was 5 years and 7 months old male, came to the hospital for cutaneous xanthoma. The phytosterol levels in serum of the children were analyzed by gas chromatography-mass spectrometry, and the second generation sequencing method was used to analyze the disease-causing gene. Sanger sequencing method was used to verify the ABCG5 gene mutation and parental source. @*Results@#(1) The case 1 and case 2 showed moderate anemia, raised reticulocytes, total bilirubin and indirect bilirubin as well as splenomegaly. The blood smear showed that there were more irregular red blood cells, such as oral red blood cells, increased large/giant platelets, and ristomycin-induced platelet aggregation test was decreased. The urine routine examination indicated that there was bleeding in the urinary system. The results of blood lipid test were almost normal. The case 3 showed mild anemia with normal shape of erythrocyte and normal size of spleen. The large/giant platelets increased. The results of platelet aggregation test, bilirubin and urine routine examination were in normal range, but the levels of total cholesterol and low-density lipoprotein cholesterol increased significantly. (2) The levels of serum phytosterol were significantly increased in all the 3 children. (3) Two heterozygous mutations were detectable in ABCG5 gene of case 1 and 2 which were complex heterozygous mutation, i.e., c.9041G＞A and c.751C＞T. The variations were from their father and mother respectively. In case 3, only one homozygous mutation was detectable in ABCG5 gene which originated from their parents. @*Conclusion@#When the child showed increased large/giant platelets, hemolytic anemia, erythrocytosis or xanthoma of skin and rised total cholesterol and low-density lipoprotein cholesterol at first visit, the possibility of phytosterolemia should be considered. The blood phytosterol content and gene detection should be carried out as early as possible in order to treat early and improve prognosis.
ABSTRACT
Objective To analyze the difference of serum levels of anti-Mullenan hormone (AMH) in chil-dren with different ages and different types of cryptorchidism,and to explore its role in the evaluation of tes-ticular development.Methods 60 children with simple cryptorchidism were selected as case group and 52 healthy children were selected as control group.The levels of serum AMH in two groups of children were measured and the differences were compared.Results (1)The level of AMH in the case group was lower than that in control group (P < 0.05),and there was no statistical significance between two subgroups of >6 to 11 years old children with cryptorchidism and healthy children (P>0.05).(2)The level of AMH in bi-lateral cryptorchidism group was lower than that in unilateral cryptorchidism group (P<0.05),and there was no significant difference between two subgroups of >6 to 11 years old children with bilateral cryptorchidism and unilateral cryptorchidism (P>0.05).(3)The level of AMH in the high level cryptorchidism group was lower than that of the low level cryptorchidism group (P<0.05),and there was no statistical difference be-tween between two subgroups of 3~11 year old children with cryptorchidism and low level cryptorchidism (P>0.05).(4)AMH level was negatively correlated with age,and positively correlated with testicular devel-opment.Conclusion AMH can be used as an important indicator of testicular development in children with cryptorchidism.
ABSTRACT
Objective To investigate the histone acetylation status of forkhead box P3(Foxp3)gene and its roles in immunological pathogenesis of Kawasaki disease(KD). Methods Forty - two children with KD and 32 age -matched healthy children consented to participate in this study as a control group. Co - immunoprecipitation and real -time PCR were performed to determine acetylation levels of histone H4 associated with Foxp3 gene and binding abilities of Kruppel like factor 10(KLF10)and p300 / CBP - associated factor (PCAF)with Foxp3 gene in CD4 + T cells. The proportion of CD4 + CD25 high Foxp3 + cells (Treg)and protein levels of Foxp3,KLF10,PCAF,phosphated SMAD family member 2 / 3(pSmad2 / 3)and itchy E3 ubiquitin protein ligase(Itch)were analyzed by flow cytometry. Quantitative real - time PCR was used to evaluate the levels of Foxp3,transforming growth factor - β1 receptor Ⅱ (TGF - βRⅡ), transforming growth factor - β1 receptor Ⅰ(TGF - βR Ⅰ),tyrosine kinase 2 (Tyk2),SH2 domain - containing protein - tyrosine phosphatase - 2(SHP2)and cytotoxic T - lymphocyte - associated protein 4 (CTLA4)mRNA in Treg. Plasma concentrations of TGF - β and interleukin - 6(IL - 6)were measured by enzyme - linked immunosorbent assay. Results (1)Compared with the control group,the proportion of Treg,expression levels of Foxp3 and TGF - β, and histone acetylation levels of Foxp3 promoter decreased remarkably during acute KD,and the differences were all statistically significant(all P < 0. 05),and restored after IVIG therapy(all P < 0. 05). Meanwhile,the 5 indexes afore-mentioned in KD patients with coronary artery lesions (KD - CAL +)were lower than those without coronary artery le-sions(KD - CAL -),and the differences were all statistically significant (all P < 0. 05). (2)Compared with the control group,protein levels of KLF10 and PCAF in Treg cells,and their binding abilities with Foxp3 gene were significantly down - regulated during acute KD,and the differences were all statistically significant(all P < 0. 05),and restored to some extent after IVIG treatment(all P < 0. 05). Positive correlations between protein levels of KLF10 and PCAF,and mRNA level of Foxp3 were detected in acute KD patients (r = 0. 47,0. 59,all P < 0. 05). Furthermore,protein levels of KLF10 and PCAF and their binding abilities with Foxp3 gene in KD - CAL + group were lower than those in KD -CAL - group,and the differences were all statistically significant(all P < 0. 05). (3)Compared with the control group, the plasma concentration of TGF - β and expressions of TGF - βRⅡ/ Ⅰ,pSmad2 / 3,Itch,CTLA4 and SHP2 in Treg were down - regulated during the acute phase of KD,and the differences were all statistically significant(all P < 0. 05), as well as plasma concentration of IL - 6 and expression its downstream molecule Tyk2 increased remarkably in patients with acute KD(all P < 0. 05). All the indexes mentioned before restored significantly after IVIG treatment (all P <0. 05). Simultaneously,the 7 former indexes in KD - CAL + group were found to be lower than those in KD - CAL -group,while 2 indexes of the latter in KD - CAL + group were higher than those in KD - CAL - group,and the differ-ences were all statistically significant(all P < 0. 05). Conclusion Histone hypoacetylation of Foxp3 promoter might be one of the important factors contributing to insufficiency and dysfunction of regulatory T cells during acute Kawasaki dis-ease.
ABSTRACT
Objective To evaluate the creditability of warning message of white differential count (WDF) and white precursor cell (WPC) channels in Sysmex XN-3000 hematology analyzer,and verify its optimal threshold and adjust the alarm threshold.Methods A total of 61 EDTA-K2 anticoagulated blood samples without abnormal warning and 521 EDTA-K2 anticoagulated blood samples with abnormal warning were simultaneously detected in WDF and WPC channels.After the smear specimens of blood sample were automatically prepared by the instrument,microscopic examinations were performed manually.The results of microscopic examination were considered as the gold standard to determine the reliability of the warning message from the instrument and verify the reasonability of initial warning threshold value provided by the manufacture.Consequently,the threshold values were adjusted based on the requirements in practical work.Results The warning messages of atypical lymphocytes and blasts/abnormal lymphocytes in WDF channel were higher sensitive (95.8％ and 100％ respectively),but lower specific (34.7％ and 23.5％ respectively) compared with microscopic examination.The warning messages of atypical lymphocyte,blasts and abnormal lymphocytes in WPC channel were lower sensitive (81.3％,66.7％,and 76.5％ respectively) but higher specific (61.9％,55.5％ and 88.3 ％ respectively) compared with microscopic examination.According to the ROC curve analysis,the prognostic values of warning message of microscopic examination were of medium level,except the warning message for abnormal lymphocytes was poor compared with WPC channel.Combining the practical retest rules,the optimal critical threshold values of atypical lymphocytes and blasts/Abn lymph in WDF channel were adjusted as 120,and they were adjusted as 140 in WPC channel.Conclusion The high sensitive WDF channel should first be used for screening,and the detectable warning message could be retested by using high specific WPC channel to shorten the turnaround time of the test results and improve the working efficiency.The initial critical warning threshold provided by the manufacture should be verified and adjusted to the optimum critical threshold in order to ensure the accuracy of test results.
ABSTRACT
To study the current situation of Chinese medicine services from both perspective of supply and demand, looking to promote the healthy development of Chinese medicine community health service strategy. Questionnaire survey was conducted to practitioners of 14 community health service centers in Longhua district (80) and service objects (800). Ratios and SWOT methods were used for analysis. 75 qualified practitioners and 736 qualified service objects questionnaire recoveries showed that there are different degrees of commands from both sides, different options on therapeutic methods and lack of practitioners. It is suggested that community health service center should supply TCM service according to different time, different place and different patient on different type and levels.
ABSTRACT
Objective To improve the detection of G6PD heterozygotes in female patients by the optimum experimental factors of G6PD/6PGD specific value assay.Methods (1)Identifying the mutations of G6PD gene with the use of amplified refractory mutation system (ARMS).(2)Measuring the G6PD/6PGD specific value.Results According to the data analysed by statistics and ROC curve, the optimum experimental factors included that the incubation temperature was 37℃,the substrate concentrations were 0.78 mmol/L G6PNa2 and 0.195mmol/L NADP+, the reaction time was 10min.Conclusion The optimum experimental factors of G6PD/6PGD specific value assay may be used to improve the detection rate of G6PD heterozygotes in female patients.
ABSTRACT
Objective:To compare microcolumn gel Coombs test and test tube Coombs test for IgG anti-A and anti-B titre in serum of pregnant women with blood type O.The critical titre for IgG anti-A and anti-B should be established in domestic microcolumn gel Coombs' test.Methods:524 blood samples of pregnant women with blood type O,whose husbands were of blood type A or B,were detected simultaneously by domestic microcolumn gel Coombs' test and test tube Coombs test.The results were analyzed using paired "t " test,"?2 " test and regression analysis.Results:IgG anti-A mean titres determined by the two methods separately were 249.98 and 120.85,and IgG anti-B mean titre were 156.98 and 76.38.IgG anti-A and anti-B titration in domestic microcolumn gel Coombs'test showed significantly higher titres(mean 2.07 fold and mean 2.06 fold) than in test tube Coombs test in all samples studied(t=19.64,P
ABSTRACT
OBJECTIVE To investigate the distribution of bacteria detected from blood culture of pediatric patients and to observe the antibiotic resistance of these bacteria. METHODS The BacT/Alert blood culture system was applied for culture.Species identification and antibiotic resistance tests were performed by the VITEK automicroscan system. RESULTS Coagulase-negative staphylococcus(CNS) and Staphylococcus aureus accounted for 67.2% and 7.8%, respectively.The proportion of Gram-negative bacteria was 14.4%.The resistant rate of CNS and S.aureus to oxacillin was 85% and 36%,respectively.The strains producing the extended spectrum beta-lactamases(ESBLs) of Gram-negative bacteria were often detected. CONCLUSIONS Gram-postive bacteria are the main pathogens detected from the blood culture of pediatric patients.Detection rate of CNS is the highest.Multiresistant strains are common.
ABSTRACT
Objective To understand the spectroscopic property of AO-H2SO4-KBrO3-naphthol system and the enhanced mechanism of resonance light scattering(RLS),and to develop a method for the determination of trace naphthols in urine.Methods In the dilute H2SO4 medium,naphthols could react with KBrO3 and AO to form ion-association complexes,which produced a new RLS spectrum and resulted in the great enhancement of RLS.The characteristics of RLS spectrum,three-dimensions fluorescence spectrum,absorption spectrum and fluorescence spectrum and the optimum conditions of reaction were studied.Results The enhanced intensity of RLS was 468 nm.The linear range was at 1.41?10-7-2.80?10-5 mol/L for ?-naphthol,1.28?10-7-3.00?10-5 mol/L for ?-naphthol.The relative coefficient and the limits were 0.999 6 and 0.422?10-7 mol/L,0.999 3 and 0.385?10-7 mol/L for ?-naphthol and ?-naphthol,respectively.The urine samples analysis of the relative standard deviation was 4.8%-7.3% and the average recovery rate was 90.7%(n=6).Conclusion This method is sensitive,simple,rapid and applicable to the determination of trace naphthols in human urine.
