BACKGROUND:Adult central nervous system lacks the ability to regenerate,so it is of great significance to find a new source of neural stem cells.OBJECTIVE:To investigate the differences between basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF)in inducing bone marrow mesenchymal stem cells(MSCs)to differentiate into neurons in vitro.METHODS:MSCs were isolated from normal human bone marrow using density gradient centrifugation and cell attachment method.MSCs were plated in 96-well culture plates at a density of 0.25×108/L and cultured with 200 μL DMEM/F12 for 0,1,2,3,4,5,6,and 7 days,with 20 μL MTT(5 mg/mL)in 5 wells at each time point.The supernatant was removed and 100 μL dimethyl sulphoxide was added to each well for 4 additional hours of incubation.In addition,some cells were exposed to bFGF and EGF.Growth curve was determined with MTT method.Telomerase activity were examined by TRAP(PCR)-ELISA.Additionally,the functional differences of the two cytokines were checked by RT-PCR.RESULTS AND CONCLUSION:RT-PCR revealed that nestin,glial fibrillary acidic protein(GFAP)and neurofilament subunit M (NF-M)mRNA were expressed in un-induced MSCs of passage 4.Nestin expression reduced at 7 days.The expression of micro-tubule-associated protein-2(MAP2)mRNA was not detected until the induction,and increased thereafter.The expression of MAP2 mRNA was greater in bFGF+EGF and bFGF alone groups compared with EGF alone group,and the expression of GFAP in EGF alone group was greater than other groups.Results showed that MSCs can be cultivated,proliferated and differentiated into neural stem cells in vitro.The differentiated neural stem cells have the activity of proliferation,but not have the ability of infinite proliferation as tumor cells.

Disorders of gastrointestinal motility are common complications in patients with diabetes mellitus and may involve the whole alimentary tract. Dyspepsia, gastroparesis and constipation are very common. Gastric dysfunction in diabetes may influence the pharmocokinetics of hypoglycemic and other drugs. This article reviews the disorders of gastrointestinal motility in diabetes and possible mechanisms.

The advantages of intense pulsed light therapy in the treatment of Meibomian gland dysfunction include non-invasive, painless and good results, which has been greatly developed in the ophthalmology field.This article reviews the current situation, mechanism of action, operating procedures, treatment outcomes, safety, and other aspects of intense pulsed light therapy in the treatment of Meibomian gland dysfunction.

The patients of chronic lymphocytic leukemia(CLL)have great individual differences.It is important for clinicians to determine the prognosis at the beginning of diagnosis and choose proper treatment for the patients.The recent advances in biological prognostic indicators of CLL were reviewed,including IgVH gene mutation status,ZAP70,CD_(38),chromosome abnormality[t(11q;v),del(11q),del(17p),+12 and del(13q)].telomere and telomerase.

BACKGROUND:Human mesenchymal stem cells (hMSCs) become aging and even die after several passages. Some investigations have shown that telomere has a close correlation with life span of the cells. Whether the ectopic expression of human telomerase reverse transcriptase (hTERT) could induce the activity of the telomerase, maintain the length of telomere, and finally prolong the life cycle of MSCs without losing their multipotent differentiation capacity is still uncertain.OBJECTIVE: To observe the influence of the ectopic expression of hTERT on the telomerase activity and cell life cycles of hMSCs.DESIGN: Repetitive measurement trails.SETTING: Research Center of Stem Cell, Zhengzhou University Medical College.MATERIALS: The experiment was conducted in the Research Center of Stem Cell, Zhengzhou University Medical College from October 2003 to December 2005. hMSCs were obtained from 20 healthy donators from the Department of Pediatric Surgery and Outpatient, the Third and First Affiliated Hospitals of Zhengzhou University. Enhanced green fluorescent protein plasmid (pEGFP-C1) and pEGFP-hTERT were provided by Dr. Chantal Autexier of Canada. DH5α strain provided by Dr. Hou Wei-hong, the Key Molecular Medical Laboratory of Zhengzhou University Medical College.METHODS.: Under sterile condition, 2 mL bone marrow of sternum of healthy donors were harvested, and prepared after centrifugalization,dilution and passage.① Transfection of pEGFP-hTERT into hMSCs and the screening and amplification of resistance cloning:The 5th passage cells were seeded in a 24-well plate,and transfected by pEGFP-hTERT with lipofectamine method.The cells were divided into four groups including untransfected group,lipofectamine group,pEGFP-C1 group and pEGFP-hTERT group. Resistance cloning screen and amplification was performed by G418. ②hTERT mRNA expression and detection of telomerase activity:RT-PCR and PCR-ELISA were used to detect the hTERT mRNA expressions of the fifth passage hMSCs transfected with pEGFP-hTERT, and pEGFP-C1, the untransfected tenth passage hMSCs and K562 cells (positive control), and the telomerase activity of the fifth and thirtieth passage hMSCs transfected with pEGFP-hTERT,the fifth pEGFP-C1-transfected cells and the tenth passage untransfected cells. ③Karyotype analysis of hTERT-transfected MSCs: Chromosome analysis was performed by conventional Giemsa staining.④Inducement of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification:The transfected MSCs were cultured in a medium containing epidermal growth factor and basic fibroblast growth factor, which could induce the cells differentiate into neuron-like cells. The culture solution was changed every 3 days, and the changes in cell growth condition and morphologic characteristics were observed under an inverted microscope. The microtubule associate protein (MAP2) and neurofliament subunit M (NF-M) were identified by RT-PCR.MAIN OUTCOME MEASURES:①hMSCs transfection with different kathion liposomes and the screening and amplification of resistance cloning; ②hTERT mRNA expressions of each group and detection of telomerase activity; ③Karyotype analysis of pEGFP-hTERT-transfected MSCs; ④ Induction of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification.RESULTS: ①With the decrease of G418 concentration, the cells in the untransfected and lipofectamine groups died, and stably EGFP expressed MSCs were obtained; after G418 screening, there was a cell clone undergone 35 passages and continued to proliferate, whose appearance and growth characteristics were similar to the untransfected MSCs observed under inverted microscope. ②The fifth passage pEGFP-C1-transfected hMSCs and tenth passage untransfected hMSCs remained telomerase-negative, but the K562 and fifth passage hTERT-transfected cells showed positive telomerase activity. ③The telomerase activity of the fifth and thirtieth passage hTERT-transfected cells was positive. ④The hTERT-MSCs at passage 10, 20 and 30 had 23 pairs of chromosomes, and two X chromosomes. So they were still normal diploid with normal chromosome appearance and number. ⑤Many hTERT-transfected MSCs had the typical appearance of neuron-like cells. RT-PCR analysis showed that th expressions of MAP2 and NF-M were increased.CONCLUSION:Ectopic expression of the hTERT gene is found in hMSCs,and can induce the telomerase activity of hMSCs.The ectopic expression of the hTERT gene in hMSCs could extend the life spans of cells and maintain their multipotent differentiation capacity.

Objective To investigate the genetic stability, immunogenicity and protective efficacy of AdC68-rab. gp, a novel rabies vaccine based on the replication-defective chimpanzee adenoviral vector AdC68-ept. Methods The recombinant adenovirus AdC68-rab. gp expressing the glycoprotein of rabies vi-rus ERA strain was constructed. Genomes of the AdC68-rab. gp of different generations were extracted and analyzed. HEK293 and Huh7 cells were infected with the AdC68-rab. gp of different generations. ICR mice were immunized with the AdC68-rab. gp and blood samples were collected 4 weeks or 6 months after immuni-zation. Rapid fluorescent focus inhibition test ( RFFIT) was performed to detect the neutralizing antibody against rabies virus in mice serum samples. ICR mice were challenged with lethal dose of rabies virus 4 weeks after the immunization with AdC68-rab. gp to evaluate the protective efficacy of AdC68-rab. gp. Re-sults The genome of AdC68-rab. gp was stable after 15 passages, which was identical to that of the 5th and 1st generations. High levels of neutralizing antibody against rabies virus in serum samples were detected in mice immunized with AdC68-rab. gp and maintained for a long period of time. Immunization mice with one dose of AdC68-rab. gp could protect all mice from the lethal dose challenge of rabies virus. Conclusion The novel AdC68-rab. gp was characterized by good genetic stability and ideal protective effi-cacy. The adenoviral vector based vaccine could be further developed as a potential candidate for the substi-tute of current rabies vaccine.

Objective To explore the clinical features, diagnosis, and treatment of neonatal necrotizing pneumonia. Methods The clinical data of two cases of neonatal necrotizing pneumonia were retrospectively analyzed. The clinical features, diagnosis, and treatment of neonatal necrotizing pneumonia in literatures were summarized. Results Two cases were diagnosed of community-acquired Staphylococcus aureus necrotizing pneumonia and had the onset with fever. The chest X-ray showed exudative change with cystic shadow. The chest CT showed multiple cavity changes. The sputum and blood cultures were positive for Staphylococcus aureus. Both of them were effectively treated by vancomycin. The imaging was improved during the follow-up. Searching the database, 4 related literatures were being found, and there were totally 7 cases of neonatal necrotizing pneumonia including current 2 cases. The main features were as follows: The pathogenic bacteria in all cases include Staphylococcus aureus. One case was combined with pseudomonas aeruginosa. Six cases were community-acquired infections. All of them were non-immune deficiency newborn. Six cases were primary necrotizing pneumonia. Six cases were unilateral lung involvement. Five cases got fever, 5 cases had septicemia, 3 cases had pleural effusion, 2 cases had aerothorax, one case had bronchial chest and 2 cases had extrapulmonary infection. The C-reactive protein was increased in all cases. Three cases need mechanical ventilation. Six cases had a good prognosis. Conclusions The main pathogenic bacterium in neonatal necrotizing pneumonia was Staphylococcus aureus. The diagnosis was mainly depends on the typical imaging and pathogenic examination. The treatment is mainly the use of antibiotic for gram positive cocci.

Objective To discuss the influence of psychological nursing on the therapeutic effect of patients with newly diagnosed type 2 diabetes. Methods Patients (60 cases) who received psychological nursing were set as the nursing group. Patients (65 cases) who did not received psychological nursing were set as the control group. The number of patients who received early insulin treatment and whose fasting plasma glucose return to normal value on the 7th and 30th days after treatment was observed. Results The number of patients who received early insulin treatment and whose fasting plasma glucose return to normal value on the 7th and 30th days after treatment in the nursing group was higher than that of the control group (P<0.01). Conclusions Psychological nursing of patients with newly diagnosed type 2 diabetes could make patients obey doctors' advice and accept early insulin treatment to control plasma glucose and delete the toxicity of high concentration of plasma glucose as soon as possible.

AIM: To selecte the best process for Kangfuxin Drop Pills. METHODS: With appearance,weight coefficient of variation and dissolution time for index sign,orthogonal design was applied. RESULTS: The results indicated that the excellent factors and levels were as follows: the scale between PEG6000 and drug extraction was four to one,the temperature of drug fluid was at 85 ?C,drip rate was 40 drops/min. CONCLUSION: The method adopted is steady and feasible.