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Objective:To verify the performance of the next-generation sequencing (NGS) platform and evaluate the application of NGS, droplet digital PCR (ddPCR) and super amplification refractory mutation system (super-ARMS) in the detection of circulating free DNA (cfDNA) mutations in patients with non-small-cell lung cancer (NSCLC).Methods:A total of 75 patients with NSCLC in the respiratory department of Zhongshan Hospital Affiliated to Fudan University were enrolled. The standards, cfDNA from 25 patients with newly diagnosed and untreated NSCLC, and self-made mixed samples mixed with hemoglobin (1 000 mg /dl), bilirubin (500 mg/l), fat emulsion (2%), enterococcus gDNA and Escherichia coli gDNA were used to verify the blank limit, analytical sensitivity, precision, accuracy and specificity of NGS platform. The cfDNA mutations of 75 NSCLC patients were detected by ddPCR and NGS, and the mutation positive rates of the two platforms were compared. The linear relationship between the two platforms was compared by Pearson correlation test. 12 patients were selected by simple random sampling for the detection of plasma super-ARMS platform. The performance of three platforms in the detection of plasma cfDNA mutation in patients with NSCLC was compared.Results:The blank limit of NGS platform was set to 0.00%, the analytical sensitivity was 0.2%, the intra-assay precision and inter-assay precision were 100%. The test results were not affected by endogenous hemoglobin, bilirubin or fat emulsion in plasma or exogenous DNA interference, and the analysis specificity was good. The mutation positive rates of plasma cfDNA in 75 NSCLC patients detected by ddPCR and NGS were 61.33% and 60.00%, respectively. The complete coincidence rate was 89.33%, which suggests there was a positive correlation between the mutation abundance of NGS and ddPCR ( r=0.984, P=0.001). Among the plasma of 12 NSCLC patients, the results of NGS, ddPCR and super-ARMS were completely consistent in 7 cases, including 2 wild-types and 5 mutants. Conclusion:The NGS platform was verified to be useful for cfDNA mutation detection in patients with NSCLC. The ddPCR, NGS and super-ARMS have their own advantages in detecting cfDNA mutations in patients with NSCLC.
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Exosomes are cell-derived nanovesicles with diameters from 30 to 150 nm, released upon fusion of multivesicular bodies with the cell surface. They can transport nucleic acids, proteins, and lipids for intercellular communication and activate signaling pathways in target cells. In cancers, exosomes may participate in growth and metastasis of tumors by regulating the immune response, blocking the epithelial-mesenchymal transition, and promoting angiogenesis. They are also involved in the development of resistance to chemotherapeutic drugs. Exosomes in liquid biopsies can be used as non-invasive biomarkers for early detection and diagnosis of cancers. Because of their amphipathic structure, exosomes are natural drug delivery vehicles for cancer therapy.
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Glioblastoma multiforme (GBM) in the central nervous system is the most lethal advanced glioma and currently there is no effective treatment for it. Studies of sinomenine, an alkaloid from the Chinese medicinal plant,
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Objective To investigate the therapeutic effect of goal-directed fluid therapy on perioperative volume therapy in patients with placenta previa during perioperative period. Methods Eighty patients undergoing cesarean section were randomly divided into 2 groups(n=40 in each group):the conventional therapy group(N group)and the target management of resuscitation group(G group). Patients in G group were connected the Flo-Trac/Vigileo monitoring system to obtain the information of△SV and CI which were used to guide goal-directed flu-id therapy. Hemodynamics,blood coagulation indexes and lactic acid content in different periods of operation were recorded. Results Compared with N group,colloid infusion volume,HR,△SV,the amount of phenylephrine, PT,APTT and TT were decreased,but urine volume,CVP,CI and MBP of each time point and FIB were mark-edly increased(P < 0.05,respectively). Conclusion The application of FloTrac/Vigileo monitoring system for goal-directed fluid therapy,real-time monitor and effectively maintain hemodynamics in patients with dangerous placenta previa during perioperative period,which can improve the postoperative coagulation function and is better than the conventional rehydration capacity.
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Objective·To investigate the effects of interleukin-1β (IL-1β) on neonatal rat alveolar arrest induced by intra-amniotic injection of lipopolysaccharide (LPS). Methods·A neonatal SD rat model of bronchopulmonary dysplasia (BPD) was constructed by intra-amniotic injection of LPS in pregnant rats. The pregnant rats (E19) were randomly assigned to Saline group, LPS group and LPS+anti-IL-1β group. The lungs of the neonatal rats were randomly collected 1, 3 and 7 days after birth. Pathological changes in the lungs were observed by hematoxylin-eosin (H-E) staining, and expression of IL-1β mRNA and protein was detected by real-time PCR and ELISA, respectively. Rat bone marrow derived primary macrophage was cultured in vitro, and given LPS intervention, then genes related with IL-1β were detected through whole transcriptome sequencing. Results·Compared with the Saline group, the alveolar counts and secondary septa counts significantly decreased, and mean liner intercept significantly increased in LPS group. Moreover, the expression of IL-1β mRNA and protein in lungs significantly increased in LPS group. The LPS-induced pathological changes of lung tissues in neonatal rats were improved by anti-IL-1β. LPS could up-regulate the expression of genes including Gbp5, Ccl3, Nod2, Ccr5, Mefv, Casp4 and Ifnar1, but down-regulate Lgals9 and Gstp1. Among these genes Gbp5, Ccl3, Nod2, Ccr5, Casp4, Ifnar1 and Lgals9 could positively regulate IL-1βproduction. Conclusion·LPS can induce alveolar arrest through up-regulating the expression of IL-1β in macrophages in neonatal rat BPD model. Whole transcriptome sequencing reveals that LPS can regulate the expression of IL-1β in macrophages through several paths.
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Objective To explore the changes of platelet count in children with bronchial pneumonia and its clinical significance in the disease.Methods 320 cases of bronchial pneumonia were retrospectively analyzed after hospitalization.According to the number of platelet count,the cases were divided into increased platelet count group (>400 ×109 /L)and normal platelet group(100 -400 ×109 /L),and the clinical data of two groups were statistically analyzed,such as age,gender,dyspnea,treatment,white blood cell count,hemoglobin,C -reactive protein.Results In 320 cases,62 cases'platelet count increased,258 cases were normal.Compared with the normal platelet group,the cases in increased platelet count group had the following characteristics:younger(<1 year old mainly,accounted for 46.77%;the former was between 1 and 3 years old mainly,accounted for 40.70%;Z =2.08),treatment time pro-longed (7.37 ±1.90)d,the former was (6.79 ±1.51)d,white blood cell count increased [(10.71 ±3.91)×109 /L,the former was (9.37 ±3.56)×109 /L,t =2.61,P <0.05];the incidence rate of dyspnea was higher (74.19%, the former was 52.33%,χ2 =9.73),hemoglobin decreased [(104 ±14)g/L,the former was (111 ±13)g/L,t =3.34],C -reactive protein increased [(24.42 ±12.73)mg/L,the former was (18.31 ±8.86)mg/L,t =3.58,P <0.01].There was no significant difference in gender between the two groups.Conclusion Children with bronchial pneumonia who platelet count increased are younger and their diseases are more serious.Observation the platelet count change can help to rightly judge and effective treat.
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Objective To evaluate the role of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in cardio-protection by ischemic or pinacidil postconditioning ( IP,PP) against ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Fifty-six male SD rats of both sexes weighing 200-250 g were anesthetized with intraperitoneal amobarbital sodium.The isolated rat hearts were perfused in a Langendorff apparatus with Krebs-Hensleit buffer (K-H).Fifty-six isolated rat hearts with I/R injury were randomly divided into 7 groups ( n =8 each):normal control group (group C) ; group I/R; group IP and group PP1-4 postconditioning with 4 different concentrations of pinacidil.After 20 min of equilibration,the perfusion was suspended for 40 min (global ischemia) followed by 60 min of reperfusion in group I/R.In group IP after 40 min of global ischemia,the isolated hearts underwent 6 cycles of 10 s reperfusion and 10 s ischemia followed by 58 min of reperfusion.In group PP1-4 at the end of 40 min of global ischemia,the isolated hearts were perfused with K-H containing pinacidil 5,10,30 and 50μmol/L for 5 min respectively followed by 55 min reperfusion with regular K-H.Left ventricular developed pressure (LVDP) and LVEDP were measured immediately before global ischemia and at the end of 60 min reperfusion.Myocardial specimens were obtained at the end of reperfusion for detection of Nrf2,quinopeoxidoreductase (NQO1),HO-1 and SOD1 mRNA (by RT-PCR) and protein (by Western blot) expression.Results I/R significantly up-regulated Nrf2,NQO1,HO-1 and SODI mRNA and protein expression,decreased LVDP and increased LVEDP in group I/R as compared with group C.IP and 30,50 μmol/L pinacidil postconditioning further significantly increased Nrf2,NQO1,HO-1 and SOD1 mRNA and protein expression and IP,5,10,30,50 μmol/L pinacidil postconditioning significantly increased LVDP and decreased LVEDP as compared with group I/R.Conclusion Ischemic or pinacidil postconditioning can attenuate I/R injury by activating Nrf2-ARE pathway in isolated rat hearts.
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Objective To research the expression of the estrogen receptor alpha ( Erα) and estrogen receptor beta ( Erβ) in uvea tissues of the female rats, and to provide molecular biology data for further studies of the relation estrogen to uvea diseases. Methods Twenty-two adolescent SD female rats were selected. All rats were killed by dislocation of cervical vertebra, the eyeballs were in paraffin imbedding and made to a series of sections, using immunohistochemical method;Erα and Erβ distribution were investigated in uvea tissue of rats;and quantitied by Tanaka scores analytical method. The uteri of rats was used as positive control and PBS as negative control. The level of estradiol in serum of the rats were examined by radioimmunoassay. Results The expression level of Erβ was moderate or highter in stroma cell, anterior pigment epithelium as well as pasterior pigment epithelium of the iris, unpigmented epithelium, pigmented ciliary epithelium and vascular endocemet of the choroid layers. But Erα was not obviously expressed in uvea tissues. The expression rate of Erβ was higher than Erα in these tissues(P＜0.05). Immnoreactivity positive substance was granule, which was distributed in the cytoplasm or in the nucleus. The level of estradiol in serum of the rats was (22.13±3.54)ng/L.Conclusion The expression of either Erα or Erβ in uvea tissues of rats is mainly in Erβ. The results indicate that uvea tissue is regulated directly by estrogen throught Erβ.
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Objective To explore the consistency of the clinical serum creatinine results in Shanghai district and investigate the population distribution of apparently healthy people, modified MDRD formula adapted to Chinese population was used to estimate glomerular filtration rate and its distribution for further assessment of the clinical applicability of the eGFR.Methods A fresh pooled human serum sample with given IFCC's creatinine level from c.f.a.s.(calibration for automatic system) ,was used to calibrate the creatinine detecting systems of each participating hospital laboratory before every examination. Fourteen hospital laboratories successively conducted 6 experiments, and the test results were almost identical.They tested and studied the creatinine values of 6 837 ( male 3 289, female 2 132, children and teenagers 1 416)apparently healthy individuals, age from 0 to 99 years old, and estimated eGFR value of these apparently healthy individuals according to the documented eGFR formula [ eGFR = 175 × (Scr) -1.154 × (age) -0.203 ×0.742 (female) × 0.827 ] which was applied especially to Shanghai people.Results Before calibration,the inter-laboratory CVs of creatinine results varied from 3.1％ -9.1％, and after calibration, CVs decreased to less than 5％.A good consistency of the creatinine results was established among all these hospitals.The result of population distribution study of creatinine for men was 63.0-102.8 μmol/L,for women was 45.0-76.0 μmol/L, and for children and teenagers was 0-1 year old 11.0-77.0 μmol/L, 2 years old 15.5-33.3 μmol/L,3-5 years old 19.0-42.0 μmol/L, 6-19 years old 41.4-62.0 μmol/L.The Cr value were different between the male and femal [ male: ( 82.1 ± 10.9 ) μmol/L, femal: ( 59.4 ± 8.4 ) μmol/L, t =94.3 ,P <0.01 ＝ ;The eGFR value could decrease the sexual difference[ male: (79.1 ± 13.5) ml · ( min ·1.73 m2 ) -1, femal: (79.2 ± 13.6) ml · ( min · 1.73 m2 ) - 1, t = 0.266, P > 0.05 ].The difference of Cr and eGFR could not be eliminated between the groups divided by every 10 years(x2Cr =2 601 ,P <0.01 ;x2eGFR2= 1 105 ,P <0.01 ＝.Conclusions The pooled patients' sera could be used as calibrator for harmonizing of creatinine results among the laboratories. The reference rang of Cr should be differentiated by age and sex.Although eGFR can decrease the difference of sex, it cannot eliminate the difference of age.