Objective To investigate the expression and clinical significance of early B-cell factor 3 (EBF3) mRNA and protein in hepatocellular carcinoma(HCC) and the effect of EBF3 overexpression on HepG2 cell proliferation. Methods The expression levels of EBF3 mRNA in 20 pairs surgical specimens of HCC and their distant noncancerous tissues were detected by fluorescence quantitative real-time polymerase chain reaction(FQ-RT-PCR). Western blot was used to detect the expression levels of EBF3 protein in 5 pairs surgical specimens of HCC and distant noncancerous tissues. The fusion protein EBF3-EGFP was ex-pressed in HepG2 cells by transfection of pEGFP/EBF3 into the synchronized HepG2 cells using lipo-fectAMINE 2000 regent. Expression of EBF3-EGFP fusion protein was observed under the inverted fluores-cence microscope. S-phase fraction(SPF) and proliferating index(PI) were analyzed with flow cytometry (FCM). Results The ratio of EBF3 mRNA to β_2 mRNA in HCC tissues was significantly higher than that in distant liver non-cancerous tissues(0.55 ±0.12 versus 0. 22 ± 0.23, t = 5.69, P < 0.001 ). EBF3 protein in nuclear extracts of HCC tissues was about 4 fold that in distant non-cancerous tissues (26.35 ±14.06 versus 7.86 ± 8.47, t = 2.52, P = 0.036). Fluorescence microscopy revealed that 24 h after trans-fection of pEGFP/EBF3 into hepatoma HepG2, the fluorescence of EBF3-EGFP fusion protein was mainly observed in the nucleus. After transfection for 24 h and 48 h, SPF and PI were markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfeeted cells. Conclusion The expression of EBF3 at both mRNA and protein levels was up-regulated in HCC tissues. EBF3 promotes HepG2 cells proliferation through DNA replication, effect of EBF3 in ttCC needs to be further investigated.

Objective To discover and certify the minimum required months of IQC ( Internal Quality Control ) data which were used to quantify the imprecision To identify the impact of test items , different concentrantions and years on the defective rate .Methods IQC data involving 20 analysis items ( including Albumin, Creatine kinase, Total bilirubin, Alanine aminotransferase, Aspartate aminotransferase, Lactate dehydrogenase,γ-Glutamyl transferase, Alkaline phosphatase, Calcium, Chlorine, Creatinine, Glucose, Potassium, Sodium, Phosphorus, Triglyceride, Total cholesterol, Total protein, Uric acid, Urea) and six measurement systems (including Roche, Architect, Hitachi, Dade/Behring, Beckman, Olympus) were collected from hospitals in Shanghaibetween 2009 and 2011.A total of 3 534 groups, referred to one year laboratory′s IQC data of one concentration range , were analysed to find the minimum required months in each group when the cumulative months were compared with the population by using T test.The correlation coefficient of hospital′grades, measurement levels and test items were evaluated by u test, and the percentage of groups of P>0.05 were collected.The cumulative IQC data′coefficient variation ( CV) of six months and eleven months were compared with the total CV, respectively .Imprecision higher than the professional specification was regarded as unqualified .Difference of unqualified rate among test items, concentrations and years were expolored .Results Rates of hospital′grades, measurement levels , items are 94.2%,100%, 100%respectively , 88%of groups′imprecision became stable in ten months .25%groups reach the criteria that the relative bias is ≤5% when calculated the cumulative IQC data′CV of six months, while 88%groups do when calculated the cumulative IQC data′CV of ten months.The unqualified rate is 20%and the most unqualified item is Ca , the unqualified groups of low level are larger .With increase of year , the unqualified rate showed a downtrend .Conclusions Ten months′IQC data is more reliable to quantify imprecision .Unqualified rates are different according to test items , measure levels and years , the establishment of professional specification should consider them.

Objective:To clone the encoding sequence of human EBF3 gene,construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3,and study the effect of EBF3 on HepG2 cell cycling.Methods:Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR,cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced.The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot.Cell cycles were analyzed with flow cytometry analysis.Results:Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank,and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly.24 h after transfected by pEGFP/EBF3,the fusion protein EBF3-EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF3 for 24 h or 48 h.Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells.These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase.Conclusion:The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established.The percentage of cells in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells.It is likely that EBF3 promotes HepG2 cells proliferation through DNA replication.

Objective To establish a sensitive and specific liquid chromatography method for the determination of loganin and morroniside in rat serum containing Liuwei Dihuang Formula(LDF).Methods Loganin and morroniside were extracted from the rat serum using SPE column,then separated on a C_(18)column.A HPLC method for determining serum concentration of mor- roniside and loganin in rats before and after administration LDF was established.Results A good linear calibration curve of lo- ganin and morroniside was obtained.The recovery of the method was in the range of 95 %～105 %.The extracted recovery was more than 75 % ,and within-day and intra-day precisions were less than 12 %.Conclusion This method is sensitive, simple and rapid for monitoring of drug level in the study on pharmacokinetics and different combination of LDF.

Objective To study the influence of short hairpin RNA-mediated inhibition of APRIL gene on proliferation,invasion and metastasis of human colon carcinoma SW480 ceils in vitro. Methods After treatment with shRNA, the expression level of APRIL protein in human colon carcinoma SW480 cells was detected by western blotting. And cell adhesion, cell migration and cell invasion of SW480 cells transfected with sh637 were analyzed respectively as compared with non-transfected SW480 cells and mocktransfectant. Results The expression of APRIL protein in SW480 transfected with sh637 were significantly lower than mock-plasmid groups and untransfected ones (F=42.15, P=0.00). Cell proliferation was markedly inhibited, compared with untransfected groups and negative control ones (F=24.76, P<0.05).And the average absorption of cell adhesion in transfected groups, mock-plasmid and non-transfected ones were 0.343, 0.409, 0.412, respectively. Cell adhesion decreased 39% compared with untransfected groups. Similarly, there was significant difference for cell migration (F=65.53, P<0.01) between transfected groups (The average ceil number in exposed area to migrate was 47.89±13.16) and nontransfected (98.78±23.26) as well as mock-plasmid ones (108.01±39.11). Then, in view of the average absorption of cell invasion between transfected groups(0.58±0.10) and non-transfected (0.94±0.23) as well as mock-plasmid ones(1.11±0.21), a prominently difference for cell invasion was also found between them (F=5.771, P<0.05). Conclusion The results suggest APRIL has a proliferation-inducing effect and probably be involved in cell invasion and cell metastasis of colon carcinoma and it could enhance capacity of invasion and metastasis of SW480 cells.

Objective To perform the methodological evaluation on Beckman Immage 800 automatic special protein analyzer for determining serum freeκ,λlight chains (sFLC) .Methods The Beckman Immage800 automatic special protein analyzer was used to quantitatively detect sFLC values for investigating its precision and accuracy ,analyzing its detecting range ,interference and residual contamination rate ,meanwhile verifying its reference intervals .Results The low and high values of within‐run precision coefficients of variation(CV) forκsFLC were 7 .84% and 2 .95% respectively ;which of between‐run precision CV were 7 .38% and 5 .57% re‐spectively .The within‐run precision CV for λFLC were 4 .59% and 3 .94% respectively ,which of between‐run precision CV were 3 .97% and 2 .01% respectively ;bias for detecting theκFLC andλFLC fixed quality serum and the target values was less than 5% , when the analytical detection ranges of κFLC andλFLC were 10 .8-128 .0 mg/L and 10 .1-121 .0 mg/L ,a value was 0 .95-1 .05 , r2 ≥0 .98;the carry‐over rates of κFLC andλFLC were 0 .411% and 0 .216% respectively .The interference test results showed that when free hemoglobin≤342 .0 μmol/L ,conjugated bilirubin ≤342 .0 μmol/L ,hemoglobin≤5 g/L and chyle ≤2 400 turbidness , which had no influence on sFLC results ;among 40 healthy subjects undergoing the physical examination ,1 case of κFLC detection results exceeded the reference interval recommended by the manufacturer ,while theλFLC detection values in these 40 cases were all within the reference interval recommended by the manufacturer .Conclusion The main analytical performance of the Beckman Im‐mage800 automatic special protein analyzer for detecting sFLC meets the requirements of quality objectives and can provides the sci‐entific and precise evidence of diagnosis and treatment for clinic .

Objective To investigate the action and mechanism of NF-κB pathway in up-regulating B cell-activating factor receptor (BAFF-R) expression in multiple myeloma cells induced by IFN-γ.Methods Activated NF-κB were detected with Western blot, while the expression of BAFF-R were measured with RT-PCR and ELISA, and investigated the effect of BAY11-7082 on transcription of BAFF-R mRNA and translation of protein in multiple myeloma cells stimulated by IFN-γ. Results IFN-γ can induce the degradation of IκB-α in time-dependent and dosage-dependent manner, and up-regulated BAFF-R expression in multiple myeloma cells. BAY11-7082, an NF-κB inhibitor, inhibited not only the transcription of BAFF-R mRNA but also the protein of regulated by IFN-γin dosage-dependent manner. Conclusion NFκB may play an important role in high expression of BAFF-R in multiple myeloma cells induced by IFN-γ.

Objective To construct and screen siRNA targeting a proliferation-inducing ligand (APRIL) gene in a mouse colorectal cancer celline, CT-26. To investigate the effects to the cell growth and migrant capacity of CT-26 after knockdown APRIL gene, lay the foundation for molecular targeted therapy to colorectal cancer. Methods Four pairs of APRIL siRNA were designed and chemically synthesized. And disorder sequences were synthesized as a negative control. These sequences were transfected with LipofectAMINE 2000 into CT-26 cells, which high-expressed APRIL gene. The transfection efficency rate of 6-FAM labelled control siRNA was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and Western blot, respectively. Cell proliferation activity was analyzed by cell counting kit-8, cell migration capacity was detected by the repair of cell damage, and MMP-2 together with TIMP-1, two important regulatory genes in cell metastasis, were measured by RT-PCR.Results The different kinds of APRIL siRNA effectively suppressed the level of APRIL mRNA and the protein expression in CT-26 (P ＜ 0.05 ). Cell proliferation and metastasis ability were repressed after APRIL siRNA transfection( P ＜ 0.05 ), compared with random siRNA control and nontransfected control. The mRNA levels of MMP-2 and TIMP-1 genes wre significantly altered among APRIL siRNA groups and two control groups ( P ＜ 0.05). Conclusion We have constructed and screened a kind of siRNA (APsi737) targeting APRIL gene in a mouse colorectal cancer cell line, CT-26. APRIL siRNA can effectively inhibit the cell growth and migration capacity, maybe be regulated by MMP-2 and TIMP-1.

Objective To explore the status of infection, biotype and resistant background of epi-demic strains of Haemophilus influenza ( Hi ) in neonates, and the clinical features of neonatal pneumonia caused by Hi. Methods The multicenter prospective epidemiological cross-sectional design was used; four hospitals in west Sichuan China were chosen as research field,sputum bacterial culture was done and biologi-cal typing,PCR identification and drug sensitivity test of Hi epidemic strains were carried out among 0 to 28 days hospitalized neonates with infectious pneumonia in four hospitals located in west Sichuan China. The ca-ses with discharge diagnosis of neonatal infectious pneumonia with Hi positive separation were assumed as case group,and the same number of cases with Hi negative separation were assumed as control group accord-ing to 1∶1 extraction at the same time. Results Totally 757 cases with admitting diagnosis of neonatal infec-tious pneumonia in four hospitals were investigated in west Sichuan from November 2014 to October 2015, and the rate of sputum culture was 95. 51%(723/757). The total pathogenic bacteria positive rate of sputum culture was 15. 63%(113/723),and Hi positive rate was 1. 94%(14/723),Hi accounting for 12. 39%(14/113) of the pathogenic bacteria in respiratory system. All the Hi strains(100%) were non-typeable Hae-mophilus influenzae( NTHi) indentified by PCR. The main biotypes of 14 Hi strains were typeⅠwith 57. 1%(8/14),type Ⅲ with 14. 3%(2/14) and type Ⅳ with 28. 6%(4/14). The total of 35. 7%(5/14) bacterial strains of β-lactamase distributed in four hospitals,7. 1%(1/14) bacterial strains of β-lactamase-nonproduc-ing-ampicillin-resistant,and 35. 7%(5/14) bacterial strains of β-lactamase-positive-ampicillin-resistant were found in four hospitals. The rate of resistance and mediation to cefuroxime were 14. 2%(2/14) respectively, the resistance rate to cefaclor was 35. 7%( 5/14 ) , and 21. 4%( 3/14 ) to ofloxacin. None of the 14 strains was resistant to amoxicillin clavulanic acid and cefotaxime. The 1∶1 matching analysis had been done for 10 cases with discharge diagnosis of neonatal pneumonia caused by Hi. There were no statistical differences in general conditions,main symptoms, lung signs, X-ray appearance, classification of leukocyte and C-reactive protein levels between case group and control group(P>0. 05). Conclusion All the Hi isolated from spu-tum were NTHi among 0 to 28 days inpatients with neonatal pneumonia and the main biotype were typeⅠ, type Ⅲand typeⅣin west Sichuan China. There were no significant differences in the clinical manifestations of neonatal pneumonia with NTHi infection and other infectious pneumonia.

Objective To investigate the effects of a proliferation-inducing ligand(APRIL)gene silencing by small interfering RNA(siRNA)on cell cycle and proliferation of colon carcinoma SW480 cells.Methods The siRNA plasmid vector targeting APRIL gene,named as siRNA-APRIL,was transfected into SW480 cells,transfected with scrambled vector as a nontargeting control and nontransfected group as another control.APRIL mRNA and protein expression were examined by real-time PCR and Western blot,respectively.Cell proliferation activity was analyzed by cell counting kit-8(CK-8),cell cycle was detected by flow cytometry,and p21 together with p27,two important regulatory genes in cell cycle,were measured by RTPCR.Results Compared with nontargeting control and nontransfected control,APRIL expression was inhibited significantly at both mRNA and protein level by siRNA-APRIL being transfected in SW480 cells(P ＜0.05).Cell proliferation ability was drastically repressed after siRNA-APRIL being transfected at 48 h,72 h and 96 h(P ＜ 0.05).After transfected 48 h,the percent of Go/G1 phase cell was significantly increased,S and G2/M phase cell were significantly decreased,the number of cell in apoptosis was increased and the expression of p21 and p27 mRNA were up-regulated(P ＜ 0.05).There was no significant difference when compared the two control groups each other(P ＞ 0.05).Conclusion siRNA-APRIL can effectively knockdown the expression of APRIL gene in SW480 cells,moreover,it can inhibit the cell proliferation and induce G0/G1 phase cell cycle arrest,which occurrence may involve in upregulation the mRNA expression of p21 and p27.