[Cerebrospinal Fluid ß2-Microglobulin for Prognostic Evaluation of Patients with Central Nervous System Infiltration in Acute Lymphoblastic Leukemia].

OBJECTIVE: To investigate the prognostic value of serum and cerebrospinal fluid ß2-microglobulin (ß2-MG) in acute lymphoblastic leukemia (ALL) with central nervous system invasion after chemotherapy. METHODS: 40 patients with leukemia who had been confirmed to have central nervous system infiltration were selected for treatment at the Second Affiliated Hospital of Chongqing Medical University from January 2015 to May 2017, and the serum levels of ß2-MG and CSF-ß2MG were dynamically monitored and performed statistical analysis. RESULTS: After chemotherapy, the changes in serum ß2-MG were not statistically significant (P>0.05); the absolute level of CSF-ß2MG and the percentage of relative baseline changes were statistically different in different clinical outcome groups(P<0.05), and the decreasing CSF-ß2MG levels suggest a better prognosis, with cut-off values of 1.505 and -25%, respectively. CONCLUSION: The best cut-off point may be a predictor of complete remission; the reduction of the absolute and relative levels of CSF-ß2MG can suggest a good prognosis for patients.

[Metabolic Characteristics of Leucocyte-deplated and Suspended RBC Produced by Using Lipid Whole Blood During Routine Storage].

OBJECTIVE: To investigate the differences of metabolic pathways of leucocyte-deplated RBCs prepared by using lipid whole blood and nomal blood during routine storage so as to provide some reference for clinical blood use. METHODS: Twenty U whole blood from 20 donors, including 10 U lipid blood and 10 U normal whole blood, were selected for preparing leukodepleted red blood cells, red blood cells were taken from storage bags on day 0, 14 and 35, respectively. Metabolites in the red blood cells were analyzed, red blood cell metabolic extracts were detected by UPLC-MS/MS. The metabolite data of RBC from 2 groups were analyzed by SIMCA-P 13.0 software using OPLS-DA and by SPSS 19.0 using Mann-Whitney U test. Difference of metabolic pathways was described according to different metabolites. RESULTS: The glucose, adenine, pyruvic acid, GSH, GSSG and niacinamide levels on day 0 in lipid RBCs were higher than those in the control group(P<0.05). The glucose, pyruvic acid and GSH levels on day 14 in lipid RBCs were lower than those in the control group (P<0.05), and the levels of adenine, GSSG and niacinamide were higher than that in the control group (P<0.05). The glucose level on day 0 was lower than that in the control group (P<0.05), and the levels of adenine and niacinamide were higher than those in the control group (P<0.05). but the pyruvic acid, GSH and GSSG levels were not significantly different between 2 groups (P>0.05). CONCLUSION: Compared with the normal red blood cells, the energy metabolism pathway decreases in lipid red blood cells within the storage period and pentose phosphate pathway increases.

[Expression of BAG3 Gene in Acute Myeloid Leukemia and Its Prognostic Value].

OBJECTIVE: To investigate the expression of BAG3 gene in acue myeloid leukemia (AML) and its prognostic value. METHODS: Real-time quantitative RT-PCR was used to detect the expression of BAG3 mRNA in 88 previously untreated AML patients. The corelation of BAG3 expression level with clinical characteristics and known prognostic markers of AML was analyzed. RESULTS: In 88 patients with AML, the expression of BAG3 mRNA in NPMI mutated AML patients was obviously lower than that in NPMI unmutated patients (P = 0.018). The expression level of BAG3 mRNA did not related to clinical parameters, such as age, sex, FAB subtype, WBC count, extra-modullary presentation, and to prognostic factors including cytogenetics, FLT3-ITD, c-kit and CEBPα mutation status (P > 0.05). The expression level of BAG3 had no obvious effect on complete remission (CR) of patients in first treatment. The expression level of BAG3 in non-M3 patients was higher than that in relapsed patients (P = 0.036). The expression level of BAG3 had no effect on overall survival (OS) of patients. CONCLUSION: The expression level of BAG3 does not correlated with known-prognostic markers of AML, only the expression level of BAG3 in NPM1 mutated patients is lower than that in NPM1 unmutated patients. The expression level of BAG3 has no effect on OS of AML patients, the BAG3 can not be difined as a prognostic marker in AML.

Effect of osteoprotegerin in combination with interleukin-6 on inhibition of osteoclast differentiation.

OBJECTIVE: To observe the effect of recombinant interleukin-6 (IL-6) and osteoprotegerin (OPG) on inhibiting bone absorption induced by receptor activator for nuclear factor-kB ligand (RANKL) in murine osteoclast precursor cells (OCPs) model. METHODS: RAW 264.7 cells were solely treated with 50 ng/ml RANKL for 1 day, and then they were divided into three groups: RANKL (control group), RANKL+IL-6 (IL-6 group) and RANKL+IL-6+OPG (combination group). These cells were harvested and investigated by means of HE staining under light microscope after consecutive 9 days. Furthermore, staining tartrate-resistant acid phosphatase(TRAP)-positive multinucleated cells were detected by inverted phase contrast microscope. The absorption pits of bone slices were observed under scanning electron microscope. RESULTS: The number of mature osteoclast cells in control group was more than that in IL-6 alone or IL-6 combined with OPG group (P less than 0.05). Interestingly, this experiment has also demonstrated that there was a large number of TRAP-positive multinucleated osteoclasts (more than 3 nuclei) and several bone absorption formation in the control group, whereas the outcome was completely different in both IL-6 group and IL-6+OPG group (P less than 0.05). CONCLUSION: IL-6 can suppress the differentiation of mature osteoclasts as directly adding it into the RAW 264.7 cells induced by 50 ng/ml RANKL, and further the effect of osteolysis is remarkably reduced. When treatment with IL-6 combined with OPG, a more effective strategy for the treatment of osteoporosis is reached.

[Construction of lentivirus-mediated short hairpin RNA targeting human STAT3 gene].

AIM: To construct and identify a lentivirus-mediated short hairpin RNA (shRNA) targeting human signal transducer and activator of transcription 3 (STAT3) gene. METHODS: The shRNA chains targeting to human STAT3 gene were designed and synthesized, and then inserted into lentivirus expression vector pSicoR containing U6 promoter and green fluorescent protein (GFP) gene by gene recombination technique. The constructed recombinant plasmid pSicoR-STAT3-shRNA was identified by double restriction enzyme digestion and DNA sequencing, and then mixed with the 3rd generation of lentiviral packaging system and co-transfected to HEK293 cells using new generation of Roche X-tremeGENE HP DNA Transfection Reagent mediated transfection method. RT-PCR and Western blotting were employed to detect the expressions of STAT3 at mRNA and protein levels, respectively. Negative plasmid transfected into the same cell line was used as a control group. RESULTS: Restriction analysis and sequencing proved that the recombinant plasmid pSicoR-STAT3-shRNA was constructed correctly. Lentivirus particles were successfully packaged in HEK293 cells with high titer. The expressions of STAT3 at mRNA and protein levels in the transfected HEK293 cells were weaker than those of the control group (P<0.05). CONCLUSION: The lentivirus-mediated shRNA targeting human STAT3 gene is successfully constructed.

A novel immobilization multienzyme glucose fluorescence capillary biosensor.

Based on the immobilization enzyme technology and the fluorescence capillary analysis method, the authors have developed a highly sensitive fluorescence reaction system and a novel immobilization multienzyme glucose fluorescence capillary biosensor for determining glucose contents. Reaction principle of the system is that under the catalysis of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized on inner surface of a medical capillary, beta-D-glucose reacts with dissolved oxygen to form gluconic acid-delta-lactone and hydrogen peroxide, and then the latter reacts with l-tyrosine to produce a tyrosine dimer, which has maximal excitation and emission wavelengths at 320 nm and 410 nm, respectively. Fluorescence of the dimer is proportional to the concentration of the beta-D-glucose. Optimization conditions suitable for the reaction system and the biosensor were as follows. Concentration of the L-tyrosine used as fluorescence reagent was 0.15 mol L(-1), the active concentrations of the GOD and the HRP for the immobilization were 15 kU L(-1) and 8 kU L(-1), respectively. Consumptions of the sample and reagents in one determination were 5.0 microL and 15 microL, respectively. Quantitative range of the biosensor for the glucose was in the range 1-10 micromol L(-1), its relative standard deviation was less than 4.9%, and its detection limit was 0.62 micromol L(-1). The biosensor's recovery was in the range 96-105%. Results of some serum determined with the biosensor and with a commercial glucose-kit were well coincident to each other. Accordingly, the biosensor can be applied to the determination of serum glucose contents in the diagnosis of diabetes.

Study of serum argininosuccinate lyase determination for diagnosis of liver diseases.

The objectives of this research were to establish an automatic analysis method for the determination of serum argininosuccinate lyase (ASL) and to investigate the value of serum ASL test in the diagnosis of various liver disorders. According to the chemical reaction catalyzed by ASL, an enzyme-coupled reaction system was designed, and a methodology evaluation of this method was performed. A total of 291 patients with various liver diseases, 247 patients with nonliver disease and 32 healthy controls, were recruited, their serum levels of ASL and traditional hepatopathy markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and total bilirubin (TBil), were all determined, and their diagnostic values in liver diseases were analyzed and compared. Liver biopsy and the score of histopathological inflammation grading were performed in 31 patients with hepatopathy to explore the correlation between serum ASL level and hepatic histopathological change. A continuous monitoring assay method of serum ASL activity was established, which could be performed with automatic biochemistry analyzer. Methodological evaluation exhibited that the precision of this method was good indicated by the 4.0% intraassay coefficient of variation (CV), and 5.9% interassay CV. The mean recovery was 100.5%, linear range was from 0 to 167.7 U/L, and the lowest detection limit was approximately 0 U/L. All of the tested hepatopathy markers listed above were significantly increased in the liver disease group. However, levels of traditional markers of hepatopathy were all significantly increased at different degrees (all P<0.001) in patients with nonliver diseases; in contrast, there were no significantly increased ASL levels in all non-hepatopathy groups (P=0.335). The receiver operating characteristic (ROC) curve showed that the sensitivity and specificity of ASL were 100% and 91.1% (cutoff value=8 U/L), respectively, in the assessment of liver diseases. In contrast, ALT levels were 97.6% and 24.7%, and AST levels were 83.8% and 28.3% (both cutoff values=40.0 U/L), respectively. A positive correlation (r=0.417, P=0.019) was observed between serum ASL levels (86.9+/-26.5) and scores of histopathological inflammation grading (SHIG) (9.83+/-3.36). The sensitivity and specificity of ALS is much higher than that of ALT and AST for the diagnosis of liver diseases. ASL may be a more valuable marker for estimating hepatopathy.

[Determination of serum argininosuccinate lyase in diagnosing liver diseases].

OBJECTIVE: To investigate the value of the determination of the levels of serum argininosuccinate lyase (ASL) in diagnosing various liver diseases. METHODS: Two hundred and ninety-one patients with various liver diseases, 257 patients with non-liver disease, and 32 healthy controls were recruited for this study and their serum ASL, ALT, AST, GGT, LDH, ALP, and total bilirubin (TBil) levels were determined. Liver biopsies were performed on 31 patients with hepatopathy. RESULTS: Receiver operating characteristic (ROC) curve analysis showed that the sensitivity and specificity of ASL in assessing liver diseases were 100% and 91.1% (at cut-off values of 8 U/L), those of ALT were 97.6% and 24.7% and those of AST were 83.8% and 28.3% (both at cut-off values = 40.0 U/L), respectively. The levels of ASL in various liver disease patients were: in liver cancer - acute hepatitis - liver cirrhosis - chronic hepatitis. A positive correlation (r = 0.417) was observed between serum ASL levels (86.9+/-26.5) and scores of histopathological inflammation grading (9.83+/-3.36). CONCLUSION: ASL is of higher sensitivity and specificity than those of ALT and AST for diagnosing liver diseases. ASL may be used as a useful marker in estimating hepatopathy.