Pediatric reference intervals for plasma and whole blood procalcitonin of in China: a multicenter research / 中华检验医学杂志

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.

Pathogen and clinical characteristics of bacterial enteritis in a third class children′s hospital in Shanghai city from 2016 to 2020 / 中国小儿急救医学

Objective:To investigate the pathogens and drug resistance of bacterial enteritis in children, analyze the clinical characteristics of bacterial enteritis in children, and provide basis for clinical diagnosis and treatment.Methods:The fecal culture strain and drug sensitivity of patients with bacterial enteritis admitted to our hospital from January 2016 to December 2020 were analyzed and summarized, and the clinical characteristics of patients who were infected by Salmonella and Escherichia coli were compared.Results:There were a total of 173 patients, aged from 21 days to 15 years, with a median age of 2.00(1.10, 3.54)years.Bacterial enteritis was most likely to occur in summer and autumn, and the incidence rate was 40.5% and 29.5%, respectively.One hundreds and seventy-three strains of bacteria were cultured in feces, including 148 strains of Salmonella(85.5%), 18 strains of Escherichia coli(10.4%), five strains of Staphylococcus aureus and two strains of Shigella.One hundreds and one of 141 patients who were infected with Salmonella were detected for leukocytes of in feces(71.6%), and four of 16 patients with Escherichia coli were detected for leukocytes(25.0%). The difference was significant( χ2=14.1, P<0.001). Eighty-eight of 113 patients(77.9%) who were infected by Salmonella with increased CRP(CRP>8 mg/L)and the proportion in Escherichia coli infection cases was 6/13(46.2%). There was significant difference( χ2=4.63, P=0.03). The drug sensitivity of Salmonella and Escherichia coli was summarized.There was no carbapenem resistant strain cultured; The sensitivity to piperacillin/tazobactam and cefoperazone/sulbactam was higher than 85%; The sensitivity to cefepime, ceftazidimeand ceftriaxone was higher than 75%; The sensitivity to ampicillin was lower than 30%, and the sensitivity to quinolones was between 20%-40%. Conclusion:Children aged 1-3 years are prone to bacterial enteritis in summer and autumn.The most common pathogens causing bacterial enteritis are Salmonella and Escherichia coli.White blood cells are more easily detected in feces of patients with Salmonella infection, and the increase rate of C-reactive protein in peripheral blood is higher.Patients with bacterial enteritis are recommended to use the third-generation cephalosporins and aforementioned antibiotics and piperacillin/tazobactam for empirical treatment.The sensitivity to quinolones is reduced, and may not be suitable for clinical application.

Evaluation of the performance of systems for whole blood C-reactive protein detection: a multi-center study / 中华检验医学杂志

Objective:To explore the performance of the commonly used whole blood C-reactive protein (CRP) detection systems and give related recommendation on the performance requirements of detection systems.Methods:A total of 7 540 venous blood samples from 26 maternal, child and children′s hospitals were collected to conduct this multi-center study on the analytical performance of 5 commonly used whole blood CRP detection systems from March to April in 2019. The blank check, carryover, repeatability, intermediate precision, linearity, sample stability, influence of hematocrit/triglyceride/bilirubin, comparison with SIEMENS specific protein analyzer and trueness were evaluated. The 5 systems included BC-5390CRP autohematology analyzer, AstepPLUS specific protein analyzer, Ottoman-1000 Automated Specific Protein POCT Workstation, i-CHROMA Immunofluorometer equipment Reader and Orion QuikRead go detecting instrument. The 5 systems were labeled as a, b, c, d and e randomly.Results:Within the 5 systems, all values of blank check were less than 1.00 mg/L, the carryovers were lower than 1.00%. The repeatability of different ranges of CRP concentrations including 3.00-10.00, 10.00-30.00 and>30.00 mg/L were less than 10.00%, 6.00% and 5.00%, respectively, and the intermediate precision was less than 10.00%. The linearity correlation coefficients of the 5 systems were all above 0.975, while the slope was within 0.950-1.050. Whole blood samples were stable within 72 hours both at room temperature (18-25 ℃) and refrigerated temperature (2-8 ℃). The CRP results were rarely influenced by high triglyceride or bilirubin, except for the immmunoturbidimetric test based on microparticles coated with anti-human CRP F(ab) 2 fragments. When triglyceride was less than 15.46 mmol/L, the deviation of CRP was less than 10.00%. When bilirubin was less than 345.47 μmol/L, the deviation of CRP was less than 10.00%. CRP was more susceptible to Hct on the systems without Hct correction. The deviation of CRP between different Hct dilution concentration and 40% dilution concentration can reach as high as 67.48%. The correlation coefficients ( r) of 5 systems were all more than 0.975 in the range of 0-300.00 mg/L compared with Siemens specific protein analyzer. All systems passed the trueness verification using the samples with specified values of 12.89 and 30.60 mg/L. Conclusion:The performance of 5 systems can basically meet the clinical needs, but it is suggested that the whole blood CRP detection system without automatic Hct correction should be modified manually.

Effectiveness of platelet transfusion in children receiving extracorporeal membrane oxygenation / 中国输血杂志

【Objective】 To explore the clinical effectiveness of platelet transfusion during pediatric extracorporeal membrane oxygenation (ECMO). 【Methods】 Neonates and children treated with ECMO and received platelets transfusion at least once in the Department of Cardiothoracic Surgery, Shanghai Children′s Medical Center in 2020 were enrolled in our research. Platelet count was measured 24 hours before and after each platelet transfusion, and the corrected count increment (CCI) was calculated for effectiveness estimation of platelet transfusion. The research objects were divided into three groups based on the platelet count before transfusion, and the effective rates of each group were calculated. 【Results】 Thirty-seven patients received 79 platelet transfusions (2.14±1.21 on average), among which 39 were effective (49.37%) and 40 were refractory (50.63%). The effective rates of group 1, group 2 and group 3 were 1, 67.74%, 40.91%, and 34.62%, respectively. 【Conclusion】 The restricted platelet transfusion strategy, on the premise of ensuring life safety, is preferred for children treated with ECMO.

Laboratory diagnosis of inherited metabolic liver disease in children / 中华检验医学杂志

Children with inherited metabolic liver disease appear liver damage caused by metabolic disorders and accumulation of substrate or abnormal metabolite in liver due to genetic defects . Because of complex clinical manifestations and non-specificity of routine examination, its definite diagnosis depends on laboratory special examinations. Early treatment is closely related to the prognosis of children so that the importance of early diagnosis of inherited metabolic liver disease is emphasized. This article described the laboratory diagnosis of three common inherited metabolic liver diseases and screening for childhood with inherited metabolic liver disease.

Role of long non-coding RNA in osteoblast differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2 / 中国组织工程研究

BACKGROUND:Long non-coding RNAs (lncRNAs) have became the hot topic in current studies and play an important role in the tumorigenesis. However, lncRNAs involved in the osteoblast differentiation remain poorly reported. OBJECTIVE:To investigate the role of human LncRNAs in osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2 and explore action mechanism. METHODS:The induction of bone morphogenetic protein-2 was validated by alkaline phosphatase staining and the expression of corresponding genes was detected. The lncRNA expression profile was analyzed using the Arraystar lncRNA array in C3H10T1/2 MSCs undergoing early osteoblast differentiation. The expression with or without bone morphogenetic protein-2 induction was compared with high-flux sequencing, and the down-regulated genes were screened. The effect of lncRNA overexpression on osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2 was observed. RESULTS AND CONCLUSION:The bone morphogenetic protein-2 induced C3H10T1/2 cells led to increased alkaline phosphatase activity. After 72 hours of bone morphogenetic protein-2 induction, alkaline phosphatase, Id1,osteocalcin, Runx2, sp7 expression were increased (P<0.05). There were 24 down-regulated lncRNAs identified between bone morphogenetic protein-2 treated and untreated groups, the decrease of expression was 1.5 folds, and among them, only AK035085 contained intron. Compared with control group with no AK03508 expression, over-expression lncRNA AK035085 decreased the expression of alkaline phosphatase, Id1, osteocalcin, Runx2, sp7 (P<0.05). Experimental findings indicate that bone morphogenetic protein-2 induces osteogenic differentiation of C3H10T1/2 cells and AK035085 inhibits the osteogenic differentiation.

Bone morphogenetic protein 2-induced C2C12 and MC3T3-E1 osteoblast differentiation and autophagy / 中国组织工程研究

BACKGROUND:A series of studies indicate that autophagy is closely linked with differentiation. Bone morphogenetic protein 2 (BMP-2) is the classical pathway for C2C12 and MC3T3-E1 osteoblast differentiation, and the ideal model to study osteogenic differentiation process. OBJECTIVE:To observe the relationship between autophagy and BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. METHODS:Real-Time PCR was applied to detect osteogenic differentiation and autophagy related index after C2C12 and MC3T3-E1 were induced with BMP-2 (100μg/L) for 72 hours. The osteogenic index alkaline phosphatase in BMP-2-induced MC3T3-E1 and C2C12 cultured with different concentrations of 3-methyladenine (0, 1, 5, 10 mmol/L) was determined with alkaline phosphatase staining. Western blot analysis was applied to detect LC3-I/II expression levels in C2C12 and MC3T3-E1 induced with BMP-2 for different time points (0, 12, 24, 48, 72, 96 hours). RESULTS AND CONCLUSION:The autophagy-related mRNA and protein expression showed an increasing tendency and autophagy-related protein LC3 levels was increased, which was associated with the time, during the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. Meanwhile, alkaline phosphatase expression levels were inhibited by autophagy in the process of osteogenic differentiation. Therefore, there is a close relationship between autophagy and the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation.

Osteogenic differentiation induced by bone morphogenetic protein 2 and long non-coding RNA AK007000 / 中国组织工程研究

BACKGROUND:Long non-coding RNA (lncRNA) regulates a series of physiological processes and it is considered to play important roles in the gene regulation of development, differentiation and metabolism. MC3T3-E1, C2C12 and C3H10T1/2 cells are able to differentiate into different celllineages, such as bone cells and muscle cells, and they can be used in the study of musculoskeletal diseases. OBJECTIVE:To study the role of lncRNA in osteogenic differentiation induced by bone morphogenetic protein 2. METHODS:Osteogenic differentiation of MC3T3-E1, C2C12 and C3H10T1/2 cells was induced by bone morphogenetic protein 2, and microarray expression profiling of lncRNA was undertaken in osteogenic differentiation. LncRNA simultaneous changes in three cells were found out. The siRNA interference of the lncRNA was used to study its effects on the osteogenic differentiation induced by bone morphogenetic protein 2. Real-time PCR and alkaline phosphatase staining were applied to detect osteogenesis related indicators. RESULTS AND CONCLUSION:In the process of osteogenic differentiation induced by bone morphogenetic protein 2, osteogenic differentiation indicators were increased, while myogenic differentiation indicator myogenin was reduced. LncRNA AK007000 was screened out to play a role in osteogenic differentiation induced by bone morphogenetic protein 2. Knockdown of lncRNA AK007000 decreased the expression of osteogenic differentiation indicators, while increased the expression of myogenin. Therefore, AK007000 may play a role in promoting osteogenic differentiation and inhibiting myogenic differentiation.

Effect of TNF-α on production and activation of caspase-3 in primary rat renal proximal tubule cells / 中国病理生理杂志

AIM:To investigate the production and activation of caspase-3 in primary rat renal proximal tubule cells in response to tumor necrosis factor-α(TNF-α) and the implication of nuclear factor-κB (NF-κB) in the process. METHODS:Isolated rat renal proximal tubule cells (PTCs) from male adult Sprague Dawley rats were treated with TNF-α according to the indicated time courses. A specific NF-κB inhibitor,Bay11-7082,was used alone or as a pretreatment for 1 h followed by exposure to TNF-α for 24 h.The protein levels of cleaved caspase-3,caspase-3,I-κBα,phosphorylated I-κBα,and GAPDH were detected by Western blotting using specific antibodies. RESULTS:The protein level of cleaved caspase-3 relative to caspase-3 was significantly increased in the presence of TNF-α for 6 h,12 h,and 24 h. Protein levels of caspase-3 were significantly decreased by 12 h and returned to baseline by 24 h in the presence of TNF-α. Treatment with Bay11-7082 for 25 h alone or pretreatment with Bay11-7082 for 1 h followed by addition of TNF-α for 24 h caused a remarkable reduction in both cleaved caspase-3 and caspase-3 as compared to control and TNF-α treated groups. An increase in phosphorylated I-κBα was observed from 15 min to 60 min after treatment with TNF-α at a dose of 10 μg/L in PTCs. CONCLUSION:NF-κB is not only associated with the activation of caspase-3 but also the production of caspase-3 in primary rat renal proximal tubule cells in response to TNF-α.

Nude Mouse Models for Green fluorescence Human Endometriosis / 中山大学学报(医学科学版)

[Objective]To establish a novel noninvasive fluorescent animal model for endometriosis in vitro and in vivo.[Methods]Adenovirus encoding enhancing green fluorescent protein(Ad-eGFP)was used to transfect endometrial glandular cells and stromal cells(cells transfection and injection,Method No.1),and fragments(tissues transfection and injection,Method No.2).Transfection efficiencies were compared between the two methods in vitro.Then GFP transfected glandular cells and stromal cells suspension were injected into nude mice subcutaneously(Method No.1),taking Method No.2 as a comparison.In vivo observation last for 25 days,and positive rates and duration times of fluorescent lesions were calculated.Histological examination was used to confirmed lesion formation.[Results]On the fifth day after injection,lesion positive rate of Method No.1 was 88.9%,which was statistically significantly higher than that of Method No.2(22.2%),P=0.015＜0.05.The fluorescent positive duration of Method No.1 and No.2 were 12 ± 8 days and 7±4 days.The structures of lesions were all identified as human original endometrium by histological examination,including HE staining and immunofluoresceney.[Conclusion]Noninvasive animal model of endometriosis can be built up by subcutaneously injection of Ad-EGFP transfected endometrial glandular cells and stromal cells suspension with higher positive rate and longer observation time

Expression of microRNA let-7 in bladder cancer / 中华泌尿外科杂志

Objective To detect the differentially expressed microRNAs (miRNAs) in bladder cancer tissue and normal bladder tissue. Methods Total RNA was extracted from bladder cancer tissue and normal bladder tissue by Trizol, and then miRNAs were isolated and enriched from the total RNA. Mammalian miRNA microarrays were used to analyze the differentially expressed miRNAs between the bladder cancer tissue and normal tissue. Data analysis was performed by software of LuxS-can3. 0 and SAM version 2. 1. Choose let-7 gene which was interesting to us, and validation of mi-croarray results was carried out by northern blotting. Results Compared with normal bladder tissues, there were 71 differentially expressed miRNAs in bladder cancer tissue, of which there were 38 down-regulated ones and 33 up-regulated ones. Among these miRNAs, 26 miRNAs were the most significant with 12 up-regulated and 14 down-regulated. The expression of let-7 gene in bladder cancer was down-regulated to normal bladder tissue by northern blotting, which was in agreement with the results of the miRNA microarrays. Conclusion There are differentially expressed miRNAs between bladder cancer tissue and normal bladder tissue, and let-7 gene is probably as a tumor suppressor in bladder cancer.

Expression of BMP-2/7 heterodimers with potent ability to stimulate osteogenic differentiation in CHO cells / 生物工程学报

Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230.75+/-13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coli. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased (P<0.05), and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers (P<0.05). The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.

Correlation between intracellular magnesium and expression of beta 2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice / 中国病理生理杂志

0.05,respectively).Plasma Mg2+,intracellular Mg2+,the beta 2-AR mRNA and protein in lung tissue in group C at 21st d and 34th d were significantly higher than those in group A at 21st d and 34th d 21st d:(0.84?0.09)mmol/L vs 0.57?0.10)mmol/L,(2.39?0.14)mmol/L vs(2.11?0.08)mmol/L,(0.75?0.09)pmol/g vs(0.59?0.06)pmol/g,(88.50?8.50)pmol/g vs(60.10?7.70)pmol/g,P

Effects of blocking the expression of PKARⅠ? gene with antisense nucleotide on Shuanglong-Jiegu pill (SLJGP) containing serum-induced osteoblast proliferation / 中国病理生理杂志

AIM: To further investigate the role of PKARⅠ? in the growth-promoting effects of Shuanglong-Jiegu pill (SLJGP), a Chinese medicine, on cultured osteoblasts. METHODS: pcDNA-antiPKARⅠ?, a recombinant expressing the antisense sequence of PKARⅠ?, was constructed and transformed HFOB1.19 by lipofectin. MTT was undertaken to assess the cell growth with the treatment of high dosage of SLJGP containing serum. RESULTS: Antisense gene blocked the growth-promoting effects of SLJGP containing serum on HFOB1.19. CONCLUSION: The function of SLJGP is closely related to cAMP-dependent protein kinase A. [

Construction and identification of eukaryotic expression vector of bcl-2 siRNA / 中国病理生理杂志

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.

Effect of insulin glargine and human insulin on proliferation of a human breast cancer cell line MDA-MB-231 / 中国药理学通报

Aim To investigate the effect of insulin glargine and human insulin on proliferation of a human breast cancer cell line MDA-MB-231 and the role of ERK in the process.Methods MDA-MB-231 cells were incubated with insulin glargine and human insulin at different concentrations and for different time courses.A specific ERK1/2 inhibitor,PD98059,was used either alone or in combination with insulin glargine or human insulin to test the involvement of ERK pathway in cell growth.Cell proliferation was evaluated using cell counting kit-8 reagents.Cell cycle distribution was analyzed by flow cytometry.Results Both insulin glargine and human insulin dose-dependently enhanced MDA-MB-231 cell proliferation at the concentrations from 1 to 100 IU?L-1 after treatment for 96 h.At the concentration of 10 IU?L-1,both drugs promoted cell growth at 48,72,and 96 h.The percentage of S+G2/M cells was significantly increased in both insulin glargine and human insulin treated groups as compared to untreated controls.No significant difference was observed between insulin glargine and human insulin in their effects on cell proliferation and cell cycle distribution.Cell proliferation was significantly inhibited by PD98059.However,in the presence of PD98059,both drugs still promoted cell proliferation significantly as compared to untreated controls.Conclusions Insulin galrgine and human insulin similarly promote proliferation of MDA-MB-231 cells independent of ERK activation.