Effect of Inhibiting and Activating Wnt Signalling Pathway on NSC67657-inducing Monocytic Differentiation of HL-60 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism.</p><p><b>METHODS</b>The HL-60 cells were treated with 5, 10 and 20 µmol/L XAV-939 (inhibitor of Wnt signalling pathway) for 3 days, and with 10, 20 and 30 mmol/L LiCl (activator of Wnt signalling pathway) for 1 day; the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot, respectively; the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry, moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined. Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 µmol/L for 3 days; the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control (culture mediam) group, simple NSC67657-treated group, NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed.</p><p><b>RESULTS</b>20 µmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively, could significantly down- and up-regulate the expression of cyclinD1, TCF1 and c-Jun genes (P < 0.05) and proteins (P < 0.05); moreover, the number of CD10(+) HL-60 cells in these conditions was below 1%, no early apoptosis of HL-60 cells was found. In the simple NSC67657-treated groups, the expression of cyclinD1, TCF1 and c-Jun proteins was down-regulated (P < 0.05), and the percentage of CD14(+) HL-60 cells accounted for 62.13 ± 9.44; after the HL-60 cells were treated with XAV-939, the NSC67657 could more significantly down-regulate the expression of cyclinD1, TCF1 and c-Jun proteins and the percentage of CD14(+) HL-60 cell accounted for 84.17 ± 5.39%, as compared with simple NSC67657-treated group; as compared with blank controls group, the expression of cyclinD1, TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14(+) HL-60 cells decreased to 33.99 ± 8.37% in NSC67657 combined LiC1 streated group, but which were higher than those in simple NSC67657-treated group (P < 0.05).</p><p><b>CONCLUSION</b>20 µmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins. The influence of XAV-939 and LiC1 on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.</p>

Expressing trend of NME3 protein in acute myeloid leukemia HL-60 cells and patients' bone marrow / 中国实验血液学杂志

To verify the differential expression of non-metastasis cell 3 (NME3) protein in HL-60 cells when they were induced to differentiate into monocyte and granulocyte like cells, and study its value in diagnosis of acute myeloid leukemia, all-trans retinoic acid (ATRA) and a new steroidal drug NSC67657 were employed to induce acute myeloid leukemia HL-60 cells into monocyte and granulocyte like cells. Then the cell differentiating direction was observed by chemical staining, the degree of differentiation was determined by surface antigen CD11b/CD14 detection, and the apoptosis was excluded by phosphatidylserine valgus analysis, by which cellular differentiating model was constructed. Furthermore, RT-PCR and Western blot were employed to verify the differentially expression of NME3 before and after differentiation of HL-60 cells. At last, samples from bone marrow nucleated cells of 26 patients with myeloid leukemia, which were diagnosed definitely by clinical doctors, and 5 normal people were chosen. Then the expressing trend of NME3 protein in these testing groups was analyzed by means of comparison. The results showed that ATRA (2 µmol/L for 5 d) and NSC67657 (10 µmol/L for 5 d) could induce HL-60 cells to differentiate into monocyte and granulocyte like cells above 90% without cell apoptosis. The expression of NME3 gene and protein were down-regulated by the inducers, which was accorded with the screening results that was got using proteomics technology in the former research. The expression of NME3 protein in bone marrow from acute myeloid leukemia patients was elevated significantly as compared to normal persons. It is concluded that the expression level of NME3 protein is down-regulated after cellular differentiation, according with the changing trend in leukemia patients, which imply that NME3 protein may be a potential biomarker for diagnosis of acute myeloid leukemia.

Amino-terminal pro-brain natriuretic peptide and brain natriuretic peptide measurements under various detection conditions in patients with chronic heart failure / 中华心血管病杂志

<p><b>OBJECTIVE</b>To find the potential interference factors for the detection of NT-proBNP and BNP in patients with chronic heart failure.</p><p><b>METHODS</b>EP15-A2 issued by Clinical and Laboratory Standards Institute (CLSI) was employed to compare the precision and accuracy of commercial NT-proBNP and BNP analyzer electrochemiluminescence immunoassay system Cobas E601 and chemiluminescence system ADVIA Centaur. Moreover, NT-proBNP and BNP were detected in different time interval and in different interfered sampling conditions (haematolysis, choloplania, lipemia). NT-proBNP and BNP of 203 patients with heart failure or heart failure complicated with acute cerebral infarction were analyzed to find the deviation caused by patients' endogenous factors.</p><p><b>RESULTS</b>The precision and accuracy were comparable for NT-proBNP and BNP detection using Cobas E601 and ADVIA Centaur (total-CV below 2.9% and 3.5%, the deviation from definite value below 2.38% and 3.91%). The most suitable sample type for NT-proBNP and BNP detection was serum and EDTA-anticoagulant plasma. The detection results of NT-proBNP and BNP were comparable for at least 120 min post sampling and not affected by Hb (2 g/L), DB (428 µmol/L) and chyle (2000 FIU). NT-proBNP was significantly higher in heart failure patients complicated with cerebral infarction (P = 0.003) than in heart failure patients. BNP was significantly higher in heart failure grade III patients complicated with cerebral infarction (P < 0.01).</p><p><b>CONCLUSIONS</b>Cobas E601 and ADVIA Centaur supplied satisfactory detection of NT-proBNP and BNP in patients with chronic heart failure with strong anti-interference capacity. The diagnostic value of NT-proBNP and BNP for chronic heart failure should be analyzed objectively in the presence of complicating diseases.</p>

Effect of C/EBPalpha on the monocytic differentiation of HL60 cells induced by NSC67657 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.</p><p><b>METHODS</b>The differentiation of HL60 cells was induced by NSC67657, and the cell surface antigen CD14 expression was detected by flow cytometry. The gene and protein expressions of CCAAT enhancer binding protein alpha (C/EBPalpha) before and after the induction of cell differentiation were determined by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells, and its expression was verified. The effect of C/EBPalpha overexpression in HL60 cells was assessed by MTT assay, Wright's staining and flow cytometry before and after NSC67657 transfection.</p><p><b>RESULTS</b>HL60 cells could be induced into monocytes by 10 micromol/L ATRA within 5 days, and the coverage of CD14 positive cells reached 93.9% after 5 days of drug treatment. The eukaryotic expressing vector was successfully constructed, and over 90% positive clones were obtained after screening by G418 and electrotransfection. The results of proliferative analysis, chemical staining, ultrastructural observation, and CD11b detection confirmed that HL60 cells could be induced into granulocytic differentiation by overexpression of C/EBPalpha protein. Moreover, in the drug treatment group, transfected cells could not be induced into monocytic differentiation, and their granulocytic differentiation was also inhibited.</p><p><b>CONCLUSION</b>The monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPalpha protein-mediated signal transduction. However, the overexpression of CEBPalpha may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.</p>

Regulation and mechanism of Notch signaling pathway in small cell lung cancer / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the status of Notch signaling pathway in small cell lung cancer (SCLC).</p><p><b>METHODS</b>Expression plasmids of pEFBOS-NIC-MYC and pEFBOS-neo were transfected into NCI-H446 cells. Stably transfected cell lines were selected and their growth rates were examined by MTT method. Expression of downstream genes along the Notch signaling pathway were studied by RT-PCR. Protein expression of euroendocrine markers of CgA and NSE were detected by Western blot analysis and immunocytochemistry.</p><p><b>RESULTS</b>The expression of HES1 was increased in the pEFBOS-NIC-MYC group, but the expression of hASH in the pEFBOS-NIC-MYC group was decreased significantly. The transfected cells with pEFBOS-NIC-MYC plasmid showed a significantly slower growth rate compared with that of two control groups (P < 0.05, Student's t-test). Immunocytochemistry of NSE showed that PUs in the NIC transfected group, sham group and negative control group were 7.21 ± 0.59, 28.25 ± 1.46, 30.57 ± 1.31 respectively, the former one was smaller than the values of the latter two significantly (P < 0.01). Western blot analysis showed the grave scales of CgA in NIC transfected group and sham group to be 0.54 ± 0.03 and 0.99 ± 0.05 respectively (grave scales of the negative control was set as 1.00), the former one significantly smaller than that of the other two groups (P < 0.01). The grave scales of NSE in the NIC transfected group and sham group were 0.43 ± 0.02 and 1.07 ± 0.09 respectively (grave scales of the negative control was set as 1.00) and the former one was significantly smaller than the other two groups (P < 0.01).</p><p><b>CONCLUSION</b>Notch signaling pathway regulates SCLC cells through its inhibitory effect on hASH1 transcription via HES1 along with an expression inhibition of neuroendocrine markers in SCLC.</p>

Effect of Herba ephedrae, honey-fried Herba ephedrae and Maxingshigan decoction on pentobabital sodium sleep experiment in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effects of Herba ephedrae, honey-fried Herba ephedrae and Maxingshigan decoction on pentobabital sodium sleep experiment in mice.</p><p><b>METHODS</b>Male Kunming mice were divided into 11 groups, namely normal saline (NS) group, ephedrine group, 3 Herba ephedrae dose (high, medium, and low) groups, 3 honey-fried Herba ephedrae dose group, and 3 Maxingshigan decoction dose groups. The corresponding drugs were administered intragastrically for 6 consecutive days, and 45 min after the final administration, the mice received intraperitoneal injection of pentobabital sodium, and the latent period and continuous sleeping time were recorded.</p><p><b>RESULTS</b>Compared with high- and low-dose Herba ephedrae groups, Maxinshigan decoction containing equivalent Herba ephedrae significantly increased the sleeping time of the mice (P<0.05). In comparison with NS, the decoction at medium and low doses produced no significant changes in the sleeping time, which, however, was significantly shortened in the other 8 groups (P<0.05). Compared with Herba ephedrae, Maxingshigan decoction and honey-fried Herba ephedrae at equivalent doses showed comparable effects on the sleep latency (P>0.05).</p><p><b>CONCLUSION</b>Under the condition of this experiment and with pentobabital sodium-induced sleeping time as the index, honey-fried Herba ephedrae shows no obvious effect in reducing the excitement of the central nervous system, while Maxingshigan decoction can significantly lower the excitement level, the effect of which is inversely correlated to the dose administered.</p>

Study on the mechanism of THP-1 cell differentiation imduced by a new steroidal drug NSC67657 / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the potential mechanism of the new steroidal drug NSC67657 induced leukemic cells differentiation.</p><p><b>METHODS</b>Cell proliferation was assayed by MTT assay. Surface antigen CD14 on THP-1 cells treated by NSC67657 at different time different concentration, was detected by flow cytometry (FCM). The expression of beta-catenin- interacting protein 1 (ICAT) gene and protein were detected by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cell line. FCM, Wright's staining and electronmicroscope were employed to analyse the differentiation of transfected THP-1 cells after they were treated with NSC67657 for 24 hours.</p><p><b>RESULTS</b>The proliferation of THP-1 cells was significantly inhibited by NSC67657 treatment. The level of CD14 expression was elevated in line with the increasing drug concentration and treatment time. 10 µmol/L NSC67657 treatment for five days was the optimal condition for the induction of THP-1 cells differentiation, when the CD14(+) THP-1 cells were more than 90%. Morphological study indentified the THP-1 cells of monocytic differentiation. The eukaryotic expressing vector pDSRed-ICAT was successfully constructed, and almost 90% positive clone could be obtained after G418 screening. Electro-transfection was employed for transfecting the vector into THP-1 cells. After the transfection the expression of ICAT gene and protein was increased. On the NSC67657 treatment, there was not significant difference in CD14 expression on transfected THP-1 cells compared to that on the control groups. After 24 h treatment, the transfected THP-1 cells remained in early differentiated stage.</p><p><b>CONCLUSION</b>NSC67657 can induce THP-1 cell to monocytic differentiation and activate the expression of ICAT gene, but overexpression of ICAT itself is not sufficient to induce such differentiation.</p>

Study on the expression and significance of Galectin-3 and CDC25B mRNA in human gastric carcinoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the expression of Galectin-3 and CDC25B mRNA in gastric carcinoma and their correlation with clinical-pathological features and the survival time.</p><p><b>METHODS</b>Tissue microarray (TMA) technique and in situ hybridization were used to detect the expression of Galectin-3 and CDC25B mRNA in 220 gastric carcinoma specimens and 31 normal gastric mucosa samples.</p><p><b>RESULTS</b>In situ hybridization results revealed that from the 220 cases, the positive expression rate of Galectin-3 and CDC25B mRNA were 58.6% and 54.1%, respectively. There was significant relationship between the Galectin-3 mRNA expression and tumor diameter, advanced TNM stage, invasion depth, vessel invasion, lymph node and distant metastasis. There was significant relationship between CDC25B mRNA expression and tumor diameter, advanced TNM stage, vessel invasion, lymph node and distant metastasis. In addition, there was apositive relationship of Galectin-3 and CDC25B mRNA expression. Finally, the mean survival time in cases with Galectin-3 and CDC25B mRNA positive expression was significantly shorter than those without Galectin-3 and CDC25B expression.</p><p><b>CONCLUSION</b>The expression of Galectin-3 and CDC25B mRNA appears to act as a promoting factor in the onset and development of gastric cancer. It can be used as a marker of prognosis of gastric carcinoma in clinical practice.</p>

Inhibitory effects of Notch1 overexpression on proliferation and neuroendocrine marker expression in a small cell lung cancer cell line / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of Notch1 signal activation on proliferation and neuroendocrine marker expression in small cell lung cancer cells.</p><p><b>METHODS</b>The active form of Notch1 (NIC) was over-expressed in NCI-H446 cells by constitutive transfection and a stable transfected cell line was established. Proliferation of NCI-H446 cells was analysed by MTT assay on 6 successive days. Expression of neuroendocrine markers (CgA, NSE) was observed by immunohistochemistry and Western blot analysis. Statistical analysis was conducted to compare the results in cells with NIC transfected and those in control groups.</p><p><b>RESULTS</b>MTT assay showed that absorbance (A) of cells overexpressing Notch1 was significantly depressed compared with that of the control cells (P<0.05). Immunohistochemistry of CgA showed that PUs in the NIC transfected group, sham group and negative control group were 8.81 +/- 0.77, 38.10 +/- 1.55, 38.97 +/- 0.80, respectively, the former one was significantly smaller than that of the latter two (P<0.01). Immunohistochemistry of NSE showed that PUs in the NIC transfected group, sham group and negative control group were 7.21 +/- 0.59, 28.25 +/- 1.46, 30.57 +/- 1.31, respectively, the former one was significantly smaller than that in the latter two (P<0.01). Western blot analysis showed that the gray scales of CgA in the NIC transfected group and sham group were 0.54 +/- 0.03 and 0.99 +/- 0.05, respectively, (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P<0.01). The gray scales of NSE in the NIC transfected group and sham group were 0.43 +/- 0.02 and 1.07 +/- 0.09, respectively (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P<0.01).</p><p><b>CONCLUSION</b>Notch1 may behave as a tumor suppressor in small cell lung cancer. Notch1 signal activation can inhibit the proliferation and neuroendocrine marker expression in small cell lung cancer cells, suggesting that Notch1 gene could be a new target for small cell lung cancer treatment and probable relief of paraneoplastic syndrome.</p>

Expression and significance of CD147 and E-cadherin in human gastric carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of CD147 and E-cadherin in gastric carcinoma and their correlation with clinicopathological features.</p><p><b>METHODS</b>The expression of CD147 and E-cadherin in gastric cancer tissue chip (TC) was detected by in situ hybridization (ISH) and immunohistochemistry in 220 cases of gastric carcinoma and 31 cases with normal gastric mucosa.</p><p><b>RESULTS</b>The expression rates of CD147 mRNA, E-cadherin mRNA and E-cadherin protein were 50.5%, 17.7% and 15.5%, respectively. The CD147 expression was negatively correlated with E-cadherin expression. There was a significant relationship between CD147 mRNA expression and tumor diameter, TNM stage, invasion depth, vessel invasion, lymph node and distant metastasis. The E-cadherin mRNA expression was closely related with TNM stage, invasion depth and vascular invasion. There was a significant relationship between E-cadherin protein expression and tumor diameter, TNM stage and vascular invasion. The mean survival time in cases with CD147-positive expression was shorter than that in negative cases, while E-cadherin was in an opposite relationship.</p><p><b>CONCLUSION</b>In gastric carcinoma, up-regulation of CD147 and down-regulation of E-cadherin are in a negative correlation. The examination of both these two factors together is useful for predicting the invasion and metastasis of gastric cancer, therefore, can be used as a significant marker to direct clinic therapy and estimation of prognosis.</p>

Biological appraisal of human bone marrow mesenchymal stem cells during ex-vivo expansion / 中国实验血液学杂志

This study was aimed to investigate the characteristics of human bone marrow mesenchymal stem cells during ex-vivo expansion, MSCs were isolated from human bone marrow. At each passage, the characteristics of proliferation kinetics, osteogenic and adipogenic differentiation potential were analyzed, and cell morphology, surface markers were investigated as well. The karyotype analysis was done in different passage cells. The infection HIV, HCV, HBV and TP were detected by ELISA. Mycoplasma contamination in vitro was detected by PCR method. HLA-SBT was used to reanalyze the results of HLA antigens and alleles. STR genetic loci were detected by PCR in the MSC1, MSC2, MSC3 and MSC4. The results indicated that the proliferative ability and osteogenic potential decreased with the increase of passage number during culture expansion. The multiple differentiation potential of MSCs was maintained during their life span. Karyotype analysis showed that MSCs from 4 groups before passage 8 were normal. The expression of CD29, CD44, CD105, CD166 and CD73 were positive. The expression of CD14, CD34, CD45, CD80, CD86 were all negative. SBT was used to identify HLA-A, B, Cw, DRB1, DRPB1, DQ alleles in the MSC1, MSC2, MSC3, MSC4. The genetype of STR in the MSC1, MSC2, MSC3, MSC4 was different. MSC 3 was examined by TP-ELISA to confirm the infectious disease of TP. MSC2 was contaminated by mycoplasma at passage 5. It is concluded that culture expansion causes MSCs to gradually lose their stem cell properties. During ex-vivo expansion of MSCs, the osteogenic differentiation potential is decreased. MSCs before passage 8 can be a valuable subject for basic research and clinical application.

Establishment and characterization of two new human embryonic stem cell lines, SYSU-1 and SYSU-2 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stem cell lines are highly warranted.</p><p><b>METHODS</b>Surplus embryos (blastocysts) from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation.</p><p><b>RESULTS</b>Two human embryonic stem cell lines (SYSU-1 and SYSU-2) were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA-4, Tra-1-60, Oct-4, Nanog and Rex-1. They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo.</p><p><b>CONCLUSION</b>Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.</p>

Effect of rat mesenchymal stem cells on hematopoietic reconstitution after allogeneic co-transplantation with bone marrow / 中国实验血液学杂志

To investigate effects of rat bone marrow mesenchymal stem cells (rBMMSC) on hematopoiesis after allo-hematopoietic stem cell transplantation (HSCT), allogeneic BMT model from Fischer 344 rats (RT-1Al) to Wistar rats (RT-1Au) was established; effects of MSCs on hematopoietic reconstitution were studied by survival rate, peripheral blood counts, histological analysis and FACS at day 30 after transplantation. The results showed that (1) MSCs from donor Fisher344 could survive in recipient irradiated by lethal dose and could be found in the thymus, spleen and bone marrow of the recipient at 30 days after cotransplantation with BM by measuring EGFP gene. (2) Cotransplanation of MSCs and BM improved hematopoietic reconstitution. Lymphocyte and platelet counts of peripheral blood in cotransplantation group were higher than those in the control group. Active hematopoiesis and increase of bone marrow nucleated cells were observed in cotransplantation group. MSCs significantly enhanced hematopoiesis of B lymphocyte and megakaryocytopoietic lineages by FACS analysis. It is concluded that (1) MSCs of Fisher344 can be found in the thymus, spleen, bone marrow of the recipients at 30 days after cotransplantion by measuring EGFP gene. (2) hematopoietic reconstitution is significantly enhanced by MSCs cotransplanted with BM.

Method performance verification of the clinical chemiluminescence immunoassay / 中华检验医学杂志

Objective To establish a method performance verification project and experimental method for the clinical chemiluminescence immunoassay.Methods Referring to CLSI evaluation protocols and pertinent literature,and by combining our actual works,we designed a verification procedure and experimental method.By Using these above,the precision,accuracy,analytical sensitivity,analytical measurement range,clinical reportable range and biotic interval of AFP on the Bayer Centaur 240 chemiluminescence immunoassay system were verificated.Results would be compared with the declaration of the manufacturer or desirable specifications derived from biologic variation.Results The results showed that the between-day inaccuracy on AFP levels at 77.4 ng/ml and 168.0 ng/ml was 5.70% and 4.84% respectively,these were consistent with manufacturer's inaccuracy claimed.The relative bias between the results measured for calibrator at four levels and target value was less 5.0%,and the relative bias between the results measured for EQA control sample at five levels and target value was-3.4% to 11.9%.Lower limit of detection was 1.04 ng/ml,lower slightly manufacturer's analytical sensitivity claimed.Biologic limit of detection was 2.65 ng/ml-3.53 ng/ml,functional sensitivity was 3.53 ng/ml.Analytical measurement range was 3.53-912.00 ng/ml,within manufacturer's liner range claimed.Clinical reportable range was 3.53-182 400.00 ng/ml.Reference interval was 0.6-7.7 ng/ml,within manufacturer' s claimed.Conclusions The main performances of the detection system are accorded with the declaration of the manufacturer.The performance verification procedure and experimental method of our research ars simple and practical,which has important significations for building medical laboratory and laboratory accreditation, improving quality of the chemiluminescence immunoassay.

The action of S1 nuclease and a cloning strategy for microcircular DNAs / 生物工程学报

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).

Using westgard's method evaluation decision chart for judging method performance of routine biochemistry items on Roche Modular PPI testing system / 中华检验医学杂志

Objective To judge method performance of routine biochemistry parameters on Roche Modular PPI testing system by using westgard's method evaluation decision chart.Methods We assessed imprecision(CV)from internal quality control and inaccuracy(bias)from external quality control evaluation.Combined estimates of imprecision and inaccuracy by plotting imprecision as the x-coordinate and inaccuracy as the y-coordinate to locate an expected operating point of every item on the chart.By comparing this operating point with allowable total errors(TEa),we can decide whether the performance is acceptable or not.Results In the 27 different parameters tested,imprecision and bias of calcium were 0.08 mmol/L and 0.06 mmol/L respectively,its performance was marginal.The imprecision of creatinine,urea,glucose, sodium,chloride and phosphorus were 3.20%,2.13%,1.52%,0.89 mmol/L,1.10% and 1.55%,the bias were 4.79%,0.96%,4.63%,0.80 mmol/L,1.74% and 4.13% respectively,their performance was good.M1 other 20 items were of excellence performance.Conclusions Routine biochemistry parameters on Roche Modular PPI testing system possessed good precision and accuracy,and their performance were acceptable.To judge method performance of biochemistry testing system by using westgard' s method evaluation decision chart was easy to do and suited for clinical laboratory.

Exploring the Antifungal Activity of Bacillus amyloliquefaciens NK10.BAhjaWT / 微生物学通报

Bacillus are well known antibiotic producers. In this study,dozens of Bacillus strains from different sources were screened. Among them,a strain with strong antifungal activity was found. With 16S rDNA test and Biolog assay,this strain was identified to be Bacillus amyloliquefaciens. The fermentation conditions were optimized in small conical flasks. After ammonium sulfate salting out,dialysis,freezing vacuum dehydration,the crude protein extracts were obtained. The thermal stability,pH stability,protease stability,ion stability and antifungal spectrum of this protein were studied further. Scanning electronic microscope was also used to explore the antifungal mechanism.

Osteogenic and neurogenic differentiation of human yolk sacm esenchymal stem cells / 中国病理生理杂志

AIM: To purify human yolk sac mesenchymal ste m cells (hYS-MSC) and investigate its osteogenic and neurogenic differentiation potentials. METHODS: hYS-MSC were separated from yolk sac and purified via p assage culture. The karyotype of hYS-MSCs was analyzed via G-banded characterist ics. Flow cytometric analysis was used to determine the cell cycle and phenotype of hYS-MSC. The AKP expression of hYS-MSC was also tested. Osteogenic different iation of hYS-MSCs was induced by 10 -8mol/L dexamethasone, 10 mmol/L ?-gl ycerophosphate and 50 mg/L vitamin C. Alizarin red S stain was used for identifi cation of mineralization. ?-mecaptoethanol or salviae miltiorrhizae were used t o induce neurogenic differentiation of hYS-MSCs. The expressions of NSE, NF and GFAP were identified by immunohistochemical method. RESULTS: hYS-MSCs could be purified at passages 2 or 3. The cell cycle analysis suggested that hYS-MSCs showed strong proliferational potentials by which the cells kept normal diploid karyotype during the in vitro cultur e. Flow cytometry showed the phenotype of purified hYS-MSCs was uniformly positi ve for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD3 4, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Osteogenic d ifferentiation was appeared after induction of osteogenic differentiation. hYS-M SCs, which were of spindle shape, uniform in size, were induced to pleomorphism osteoblast-like cells which expressed high level of AKP. Aggregates or nodules w ere formed at day 7 and calcium accumulation was detected by alizarin red S stai ning on day 10 or day 14. Neurogenic differentiation of hYS-MSCs was induced by ?-mecaptoethanol or salviae miltiorrhizae. NSE, NF or GFAP positive cells were detected by immunohistochemical staining. CONCLUSIONS: hYS-MSCs have strong proliferation potential and th e normal diploid karyotype is kept during the in vitro culture. The phenoty pe of hYS-MSCs is coincident with adult hMSCs. hYS-MSCs could be induced to dif ferent iate into osteogenic or neurogenic cells.