Objective To establish a method of culturing mesenchymal stem cells from umbilical cord blood. Methods The mononuclear cell fraction of umbilical cord blood was isolated with the aid of a Ficoll/Hypaque gradient, then MSC were cultivated in serum containing medium or serum-free medium. The serum containing medium (SC) consisted of LG-DMEM supplemented with 10% fetal calf serum and 10-6M hydrocortisone, with a final cell concentration of 1?106 cells/ml. The serum-free medium (SF) was supplemented with 1?10 -6M hydrocortisone and 40ng/ml fibronectin with a final cell concentration of 1?106 cells/ml. After a week, 50% of the media were renewed. Before reaching the monolayer phase cells were trypsinised and re-cultured under similar conditions for the another 3 weeks. Cells were counted at the beginning and the end of the culturing period. Phenotypes were identifiad by PAS, NAE, ALP staining. Identification of surface markers and cell cycle analysis of MSCs were performed with flow cytometry. Results Adherent cells appeared 24h after plating of mononuclear cells. The cells formed adherent heterogeneous cell populations after 4-7 days in culture, and they consisted of round and spindle-like cells. In the primary passage of culture, the cells proliferated slowly and became confluent in 14-20 days. When subcultured, the heterogeneous cell populations became homogeneous assuming flat and fibroblast-like shape. Though colonies in the medium containing serum formed earlier than those in the serum-free medium, passage times and cell morphology were the same. The total cell numbers in SF group were lower than those of SC group. The percentage of cells at G0-G1 cell cycle was higher in SF group than that of SC group with significant difference. By flow cytometry analysis, cells of two groups were negative for CD34, CD13 and CD45, but strongly positive for specific surface markers such as CD166, CD29, CD105 and CD54. Conclusion Serum-free medium was superior to medium containing serum for culturing MSCs.

Objective To make molecular diagnosis for puerile spinal muscular atrophy(SMA).Methods Genomic DNA was extracted directly from the blood of both the case group(10 children with SMA) and the control group(including 19 parents of SMA patients and 20 healthy individuals).Two methods,polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and allele-specific PCR,were used to analyze exon 7 of SMN gene from genomic DNA,and consequent electrophoresis of PCR products on agarose gel was performed.Results Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed.In conventional PCR-RFLP,part of the PCR products(189bp) from genomic DNA of all 39 members in the control group remained intact after digestion with Dra I,while the PCR products from genomic DNA of all 10 SMA children in the case group was completely digested by Dra I.In allele-specific PCR,exon 7 of both SMN1 and SMN2 could be seen when genomic DNA of all 39 members in the control group was used,while only SMN2's exon 7 could be seen when genomic DNA of all 10 SMA children in the case group was used.Conclusion Homozygous deletion of SMN1 was present in all 10 SMA children in the case group,while homozygous deletion of SMN1 was not detected in all 39 members in the control group.The combination of PCR-RFLP and allele-specific PCR,both their results can be references for each other,offers efficient and accurate methodology for molecular diagnosis of SMA.

Objective To investigate the effects of quercetin on the growth of lung cancer cell line A549 and the expression of hTERT gene. Methods The number of viable cells was ascertained by trypan blue dye exclusion test. Morphological changes of apoptotic cells were observed by electronic microscopy and DNA ladder assay. The telomerase activity was analyzed by PCR-TRAP assay and hTERT mRNA expression was detected by quantitative RT-PCR. Results Quercetin had a significant inhibition on the proliferation of A549 cells in a dose-dependent manner. The IC50 was 22.5 ?mol/L after exposure to quercetin for 48 h. The results from electron microscopy and DNA ladder showed that apoptosis occurred in the A549 cells of treatment groups. The results of quantitative RT-PCR and PCR-TRAP revealed that the expression of hTERT mRNA was significantly inhibited by quercetin and telomerase activity was decreased. Conclusion Quercetin inhibits the growth of lung cancer cell line A549 in a dose-dependent manner,and induce their apoptosis. The down-regulated expression of hTERT,suppression of telomerase activity and destruction of telomere stability may all contribute to the mechanism of apoptosis induction.

ObjectiveTo investigate the effects of combination of dexmedetomidine and sufentanil preconditioning on ischemia-reperfusion (I/R) injury in isolated rat hearts.MethodsForty male SD rats weighing 180-220 g, aged 8-10 weeks, were anesthetized with intraperitoneal 3％pentobarbital 60 mg/kg and heparin 3000 U/kg.The hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％O2-5％CO2 at 36.5-37.5 ℃.Forty isolated rat hearts were randomly divided into 5 groups ( n =8 each):control group (group C),I/R group,dexmedetomidine preconditioning group (group DP),sufentanil preconditioning group (group SP),and dexmedetomidine + sufentanil preconditioning group (group DS).I/R injury was induced by 30 min of ischemia followed by 120 min of reperfusion.Groups DP,SP and DS received 30 min of perfusion with K-H solution containing dexmedetomidine 2.3 ng/ml,sufentanil 3.77 ng/ml and dexmedetomidine 2.3ng/ml + sufentanil 3.77 ng/ml respectively at 30 min before ischemia.Coronary flow (CF),left ventricular developed pressure (LVDP),± dp/dtmax and HR were measured at 15 min of equilibration,immediately before ischemia,at 30 min of ischemia and at 120 min of reperfusion.The superoxide didmutase (SOD) and myeloperoxidase (MPO) activities and the malondialdehyde (MDA) content in myocardial tissues and infarct size were determined at 120 min of reperfusion.Results Compared with group C,CF,LVDP, ± dp/dtmax,HR and SOD activity were significantly decreased,and MPO activity,MDA concent and infarct size were significantly increased in the other 4 groups ( P ＜ 0.05).Compared with group I/R,CF was significantly decreased in groups DP and DS,HR was significantly decreased in groups SP and DS,and LVDP, ± dp/dtmax and SOD activity were significantly increased,and MPO activity,MDA content and infarct size were significantly decreased in groups DP,SP and DS ( P ＜0.05).Compared with group DS,CF was significantly decreased and HR increased in group DP,and SOD activity was significantly decreased,CF,MPO activity,MDA content and infarct size were significantly increased in group SP (P ＜ 0.05 ),and no significant change was found in SOD and MPO activities,MDA content and infarct size in group DP ( P ＞ 0.05).Conclusion Dexmedetomidine combined with sufentanil preconditioning can reduce I/R injury in isolated rat hearts,but the myocardial protection is not further enhanced.

AIM To establish the method for the enzyme assay of dimethylarginine dimethylaminohydrolase (DDAH) in rabbit kidney and to determine the optimal condition for the assaying. METHODS Five healthy Japanese rabbits weighing 3 0 to 3 5 Kg were killed by air embolism,kidneys were harvested and then were homogenized. Asymmetric dimethylarginine (ADMA) was used as the substrate for DDAH. UV 265 spectrophotometer was applied to determine the amount of the enzymatic product L Citrulline( L Cit).The amount of L Cit produced under different conditions was compared and the optimal condition was screened. The kinetic parameters of ADMA degraded by DDAH were calculated. RESULTS The kinetic parameters of ADMA degraded by DDAH were as follows: K m=(0 28?0 10) mmol?L -1 , V m=(1 36?0 42) mmol?L -1 ?g -1 ?min -1 . The optimal conditions for the enzyme assay of DDAH in rabbit kidney determined in this study follow: The concentration of the enzyme protein was 12 g?L -1 ,the optimal pH of the buffer was 7 4,the final concentration of ADMA was 2 mmol?L -1 ,and the reaction time was 30 min. CONCLUSION The method offered here is easily done. The concentration of the substrate determined in this study was based on the value of Km,thus it was beneficial to the accurate assay of DDAH.

Transforming growth factor-?_1 (TGF-?_) mRNA or protein expression in renal cortex of diabetic rats was assessed by real-time quantitative RT-PCR with SYBR Green,immunohistochemistry or Western blot.After melatonin treatment,the expressions of TGF-?_1 mRNA and protein were decreased,suggesting that beneficial effect of melatonin may result from its antioxidative property and inhibiting TGF-?_1 expressions.

Objective To establish a continuous monitoring assay of serum argininosuccinate lyase (ASL) activity with automatic biochemistry analyzer, and perform methodology validation and preliminary clinical application.Methods According to the chemical reaction catalyzed by ASL and the working characteristics of automatic biochemistry analyzer, an enzyme coupled reaction system with high specificity was set up, and the methodology validation was performed.Three hundred and nine patients with various liver diseases, 269 non-liver disease patients and 40 healthy controls were enrolled in this study.Serum ASL, ALT, and AST level were determined in all subjects.Results A new kinetics assay of ASL activity was set up with automatic biochemistry analyzer.The methodological validation demonstratod that inter-assay and intra-assay coefficient of variation were 4.0% and 5.9% respectively and the mean recovery was 100.5%.The linear range was 0-167.7 U/L.The lowest detection limit was approximately 0 U/L.The interference test showed that there is no significant interferences while the concentration of bilirubin is less than 342 μmoL/L or commonly used anticoagulants is employed at their routine concentrations.However,interference was significant when Hb level is more than 0.06 g/L.Preliminary study of clinical application showed that there was no significant difference of serum ASL level between non-liver disease group and healthy group ( q = 0.027, P = 0.979 ), but there was significant differences for both serum ALT and AST levels (ALT:q =6.461,P =0.000;AST:q =6.481,P =0.000).Conclusions A continuous monitoring assay for the determination of serum ASL activity is successfully established. Serum ASL may be a good biomarker for liver injury.

Humans ; Macrophage Activation Syndrome ; genetics ; therapy

Objective To investigate the mechanism of extremely low frequency electromagnetic field (ELF-EMF) and X-ray irradiation on liver carcinoma cell lines BEL-7402. Methods Liver carcinoma cell lines BEL-7402 had been incubated with ELF-EMF (100 Hz, 0.7 mT, 30 min, 3 days) after X-ray irradiation at different doses (0, 2, 4, 6, 8,10 Gy). The cells were observed on morphologic changes with scanning electron microscope. Flow cytometry and gene microarray were used to investigate the mechanism of cell apeptosis. Results ELF-EMF plus X-ray induced apoptosis on BEL-7402 was observed under scanning electron microscope.When X-irradiation was 2, 4, 6, 8 and 10 Gy, the apoptosis ratios of combined group and only X-irradiation group were 10.0%, 14.5%, 4.3%, 5.1%, 7.1% and 0.1%, 8.1%, 0.1%, 0.4%, 2.2% (P < 0.05) on flow cytometry. The result of microarray indicated that 1465 genes were up-regulated and 1108 down*regulated in the ELF-EMF plus X-ray group in comparison with the control group. The change rates of 110 apoptosis related genes were above 2 times, which including 71 up-regulated and 39 down-regulated. Gene microarray showed that ELF-EMF and X-ray irradiation had a mainly effect on different gene of apoptosis paths (CDC25 and CHKI, ATM, p38, PTEN, p53, G1/S, Fas, G2/M, Cell Cycle, Apoptosis and Caspase). The same genes of ELF-EMF plus X-ray group were showed 13 in ELF-EMF group and 42 in X-ray group. Conclusions Apoptosis paths were significantly different between ELF-EMF and X-irradiation. ELF-EMF cooperates with X-irradiation on inducing BEL-7402 cell apoptosis.

Objective To evaluate the effects of endogenous NO on the chemosensitivity of human breast cancer cell. Methods MCF-7 cells were cultured as monolayer, incubated with cytokine IL-1β. The pro-duction of NO was detected by NO assay. The expression of iNOS protein was measured by Western blotting. Establishing control group and experimental group, the chemosensitivity of MCF-7 cells incubated by L-NMMA and L-Arg to ADM and 5-Fu was studied by MTT assay. Results There was a positive correlation of dose-dependence between NO production and IL-1β concentration. MCF-7 cells expressed plenty of iNOS by induetion of IL-1β. There was no significant difference on iNOS whether L-NMMA and L-Arg existed or not. Incubating MCF-7 cells with 0. 5 μmol/L or 1 μmol/L ADM, the survival rate of experiment group was remarkablely decreased(P < 0.05) ; L-NMMA significantly increased survival rate of experiment group(P < O. 05) ; L-Arg decreased survival rate of experiment group(P < 0.05). Conclusion The induction of IL-113 in MCF-7 cells can increase the production of endogenous NO, which increases MCF-7 cells' sensitivity to chemotherapy.