Objective To develop ECG Holter system with SD card as storage medium. Methods Data that used MSP430F449 SCM to acquire 3-channel ECG signal to record SD card through serial peripheral interface were reviewed and analyzed on computer. Results Portable ECG Holter System based on SD card is realized. The using of SD card can enhance storage performance. Conclusion Low-cost, and high-performance solution program is observed.

BACKGROUND: There are differences in physical and biological activity between the antibody from mammals and egg yolk antibody (IgY) from chicken. IgY is acid- and heat-resistant, and can prevent and cure the infectious diseases in animals and human being, which is also benefit to develop routine diagnostic immunoassays. Conventional ELISA assay for IgY takes much more time than dot-immunobinding assay.OBJECTIVE: To detect the IgY stability byusing dot-immunobinding assay.DESIGN: Open trail.SETTING: Department of Transfusion, Kunming General Hospital of Chinese PLA.MATERTALS: The experiment was completed in the Kunming General Hospital of Chengdu Military Area Command of Chinese PLA from January to June 2006. Two White Leghorn hens (30 weeks old) were selected. HLA-A*0201 α chain served as the antigen. The total protein concentration of the purified antigen was 0.04 g/L with the molecular mass of 32 000(self-prepared); nitrocellulose filter (NC, import and divided); nonfat dry milk (Anyi Corp. No. 20051220); DAB (Boshide Corp.);caprylic acid (made by Shanghai Xinghuo Chemical Factory); ammonium sulfate (Shantou Guanghua Chemical product).METHODS: ①HLA-A*0201 α chain with the total protein concentration of 0.04 g/L was purified with egg yolk antibody,and identified by SDS-PAGE. ②1 μL antigen was spotted into the center of NC membrane and dried in the incubator at 37 ℃. Then the NC membrane was blocked in 1 mL PBST and put in the incubator at 37℃ with shaking in 90 r per minute for 15 minutes. Then the liquid was exchanged with 1 mL PBST and added the primary antibody at a final concentration of 10 mg/L. After 30 minutes shaking in the incubator at 37 ℃, the NC membrane was washed in PBST for three times. The second antibody, mouse anti-chicken IgY conjugated to horseradish peroxidase (HRP) was added and after 30 minutes incubation, the NC membrane was washed three times in PBST. Binding was revealed by incubation with a DAB reagent. A positive reaction was represented by adeep brown spot,Irdlcating that IgY had better activity; if the spot became lighter IgY lost part activity, and when the spot disappeared, the IgY lost a the activty.According to intensity (gray degree)of the dot compared tothe standard, the remained percent of activity of the IgY was calculated. ③IgY was adjusted to three different protein concentrations with PBS: 1, 0.1, 0.01 g/L and stayed at room temperature for four months. 10 μg lgY was taken out from each concentration sample every month to detect the activity by dot-immunobinding assay. ④IgY was put into seven EP tubes with 100 μL per tube and numbered 1-7. Number 1 to 3 was adjusted pH to 5, 3 and 2, respectively with 1 mol/L HCI; Number 4 to 6 was to 9, 11 and 12, respectively with 1 mol/L NaOH. The pH of number 7 was neutral without adding acid or base. The samples were stayed in incubator at 37 ℃ for 3 hours. 10 μg IgY from each tube was taken every hour to detect the stability at different pH by dot-immunobinding assay. ⑤IgY was added to six EP tubes (10 μL per tube) and numbered 1-6. Number 1-6 was put into waterbath at 30, 40, 50, 60, 70 and 80 ℃ for 15 minutes. After cooled in refrigerator at 4 ℃, 10 mg samples from each tube and standard sample (untreated sample) taken to check the thermal stability by dot-immunobinding assay.MAIN OUTCOME MEASURES: ①SDS-PAGE of IgY. ②IgY stability at room temperature. ③IgY stability at different pH. ④ Detection of IgY thermal stability.RESULTS: ①Purified IgY after SDS-PAGE had two major binds, the molecular mass of the heavy chain was 66.000,and the light chain was 25 000. ②1, 0.1, 0.01 g/L IgY still had partial activity after staying at room temperature for four months. ③When pH ranged from 5 to 9, IgY still had partial activity after staying in 37 ℃ for 3 hours. If pH was lower than 5 or higher than 9, it lost the whole activity in above condition. ④Purified IgY was added to six EP tubes, the number 1-4 still had partial activity, but number 5 and 6 showed some white precipitate, which was caused by protein denaturation at higher temperature.CONCLUSION: IgY stability is higher than others. The dot-immunobinding assay described a rapid and simple method for the demonstration and characterization of functional activity of egg yolk antibody. With only small volume antigen and antibody, and specific dot, the dot-immunobinding assay method could process many samples at the same time.

BACKGROUND: Antiviral drugs can reduce the incidence of early-onset cytomegalovirus(CMV)disease,but are associated with strong toxicity and the development of late-onset CMV disease.In order to prevent CMV disease better,cytotoxic T lymphocytes(CTL)may play a critical role in controlling CMV reactivation.Fluorescent HLA-peptide tetramers are used to monitor the recovery of CMV CTL in recipients of allogeneic transplants.OBJECTIVE: To explore the effect of HLA-peptide tetramers and adoptive immunotherapy in treating CMV disease.METHODS: A computer-based online search of Pubmed and Wanfang databases was performed for articles related to CTL detection,application of antiviral drugs and HLA-peptide tetramers,and adoptive immunotherapy with key words"HLA-peptide tetramers,cytomegalovirus,specific CTL,adoptive immunotherapy"in English and Chinese.Repetitive articles were excluded and 29 articles were included.RESULTS AND CONCLUSION: Adoptive immunotherapy with CMVs cytotoxic T cells as preemptive therapy is a very elegant strategy; however,generation of these cells is costly and time-consuming,and therefore the therapy is not available at every transplantation center.Magnetic selection of CMVs CD8+T cells from peripheral blood of CMV-seropositive donors by using HLA-peptide tetramers may be very hopeful,which simplifies adoptive immunotherapy.

The culture of umbilical cord-derived mesenchymal stem cells is extremely important for studies on umbilical cord mesenchymal stem cells. Optimization of cellculture technology is crucial for clinical application of mesenchymal stem cells and even celltherapy. Meanwhile, the labeling and tracer technique of umbilical cord-derived mesenchymal stem cells is a hotspot in stem celltransplantation. OBJECTIVE:To review the research and development of the cellmarkers and tracer methods of umbilical cord-derived mesenchymal stem cells. METHODS:A computer-based search of VIP, CNKI, Medline, Highwire and Foreign Journals Integration System databases was performed for articles concerning culture and labeling of umbilical cord-derived mesenchymal stem cells published from January 2001 to October 2013. The keywords were“stem cells, mesenchymal stem cells, umbilical cord-derived mesenchymalstem cells, cellculture, labeling methods”in Chinese and English, respectively. Final y, 35 articles were included in result analysis. RESULTS AND CONCLUSION:Umbilical cord-derived mesenchymal stem cells have not yet been widely used, mainly because of the immature isolation, culture and staining techniques of umbilical cord-derived mesenchymal stem cells. These techniques are worthy of further optimization studies. Although in recent years, cellmarkers and tracer technology of umbilical cord-derived mesenchymal stem cell s have made great progress, there are stil many problems need to be solved.

BACKGROUND:There are less studies addressing whether bone marrow mesenchymal stem cells from systemic lupus erythematosus patients are different from healthy people.
OBJECTIVE:To compare the multi-differentiation capacity of bone marrow mesenchymal stem cells isolated from systemic lupus erythematosus model mice and normal control mice.
METHODS:Bone marrow mesenchymal stem cells of systemic lupus erythematosus model mice and C57BL mice were isolated and cultured fol owed by osteogenic and adipogenic differentiations, respectively. Differentiation abilities of two kinds of mouse bone marrow mesenchymal stem cells were observed.
RESULTS AND CONCLUSION:Passaged bone marrow mesenchymal stem cells from C57BL mice were long spindle-shaped and evenly distributed, while bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice showed slow growth and were relatively smal er than those from C57BL mice. After osteogenic induction, the amount of calcium salt and calcium nodules were significantly less in the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice than from normal control mice. PCR detection showed that expressions of Runx2, alkaline phosphatase and osteocalcin were also significantly decreased in the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice. After adipogenic induction, the number of lipid droplets in the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice was significantly less than the control group, and PCR detection also showed significantly decreased expression of adipogenic genes, including PPARγ2 and lipoprotein lipase. These findings suggest that the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice exhibit lower osteogenic and adipogenic capacities than those from normal C57BL mice, and also have the impaired multi-differentiation ability.

BACKGROUND: The inoculation of hybridoma cell strain onto mouse abdominal cavity may obtain ascites containing mass antibody. Previous method to purify monoclonal antibody in ascites is complex and difficult to operate.OBJECTIVE: To prepare, purify and label anti-human leukocyte antigen (HLA)-I class molecule light chain monoclonal antibody, and to detect the expression of tumor cell surface HLA-I class molecules. METHODS: Hybridoma cells were inoculated onto the mouse abdominal cavity. Ascites containing anti-human light chain beta2-microglobulin antibody were obtained and purified with the modified caprylic acid-ammonium sulfate method. The purified monoclonal antibody was labeled with fluorescein isothiocyanate to detect peripheral blood mononuclear cells, T2 cells expressing blank HLA-A2 molecule and K562 cells surface HLA-I class molecules. The expression of HLA-I class molecules was determined by using flow cytometry and fluorescent microscopy. RESULTS AND CONCLUSION: The purified anti-human light chain beta2-microglobulin-fluorescein isothiocyanate monoclonal antibodies accounted for 96% purity. Flow cytometry results showed that, the HLA-I class molecules were highly expressed in peripheral blood mononuclear cells surface, lowly expressed in T2 cells, and not expressed in K562 cells surface. It is a simple and convenient method to purify ascites with the modified caprylic acid-ammonium sulfate method, and according prepare anti-human light chain beta2-microglobulin-fluorescein isothiocyanate. This method is effective to distinguish the levels of HLA-I class molecules expressed in various cells.

BACKGROUND: Studies have shown that umbilical cord mesenchymal stem cells are ideal seed cells for tissueengineering research.OBJECTIVE: To isolate, culture and identify tree shrew umbilical cord mesenchymal stem cells, in order toestablish a standardized tree shrew umbilical cord mesenchymal stem cell lines.METHODS: Caesarean-isolated tree shrew umbilical cord samples were used to isolate and culture umbilical cordmesenchymal stem cells using tissue explant adherent method. Flow cytometry assay was used to detect cellsurface markers. Osteogenic and adipogenic induction media were used to induce umbilical cord mesenchymalstem cells to differentiate into osteoblasts and adipocytes.RESULTS AND CONCLUSION: The cultured umbilical cord mesenchymal stem cells expressed CD90 and CD105 with the positive rate of 99.9% and 99.8% respectively. Hematopoietic stem cell marker CD34 expression ratewas 0.0% and the endothelial cell marker CD31 expression rate was 0.7%, in line with the characteristics of umbilicalcord mesenchymal stem cell surface markers. Calcium nodules by alizarin red staining and lipid droplets by oil red Ostaining were observed in the induced cells. These experimental findings indicated that umbilical cord mesenchymalstem cells from tree shrews capable of osteogenic and adipogenic differentiation were successfully isolated and cultured.

AIM:To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells ( SMCs) .METH-ODS:Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 ( containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1 ×105 units/L of penicillin and streptomycin, respectively).EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding.Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats.Vascular SMCs were cultured by tissue explant method and identified byα-SM-actin immunofluorescence.In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower cham-ber.The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE).The protein expression ofα-SM-actin and osteopontin was detected by Western blotting.[3H]-TdR incor-poration assay was used to determine the proliferation.SMC migration was analyzed by scratch wound healing assay.RE-SULTS:Compared with P3 group,α-SM-actin expression in P4 group significantly decreased and osteopontin protein ex-pression obviously increased, whereas no significant change was found in P4YE group.Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs.Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phe-notype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION:Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs pro-liferation and migration.Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.

Objective To establish a method for isolation of cynomolgus monkey umbilical cord mesenchymal stem cells.Methods Fresh cynomolgus monkey umbilical cord was directly minced into pasty fine pieces, and the pieces were cultured in tissue flask with DMEM/F12 medium supplemented with 10% fetal bovine serum.The morphological characteristics of the resulting cells were examined, and their expression of mesenchymal cell surface markers were analyzed by flow cytometry.The multidifferentiation potential was examined in vitro, too.Results The fibroblast-like cells were successfully isolated from the fresh umbilical cord by an adherent culture procedure.These adherent cells expressed mesenchymal markers including CD29, CD44, and CD90, and also could be induced to differentiate into adipocytes, osteoblasts and chondrocytes.Conclusion Mesenchymal stem cells can be isolated from fresh cynomolgus monkey umbilical cord by using an adherent culture procedure.

BACKGROUND:If an extract can prolong the S phase and reduce the percentage of apoptosis after co-culture with cels, it is proved that the extract is able to promote cel proliferation. OBJECTIVE: To prove the effects of chicken egg-white extracts with different components on the proliferation, cel cycle and apoptosis of 293T cels. METHODS: An ultrafiltration unit was used to separate chicken egg-white extracts into different components that were > 10 ku, < 10 ku, > 3 ku and < 3 ku to co-culture with cels for 3 days. Then, cel proliferation, cel cycle and cel apoptosis were detected. RESULTS AND CONCLUSION:Chicken egg-white extract components of < 10 ku and < 3 ku could promote cel proliferation, increase the percentage of S-phase cels and reduce the percentage of apoptosis. In conclusion, chicken egg-white extract components that are < 10 ku and < 3 ku can promote cel proliferation, as wel as increase the percentage of S-phase cels and reduce apoptosis percentage.