To investigate the effects of Tongxinluo capsule on sciatic nerve apoptosis in spontaneous type II diabetic KK/Upj-Ay mice, in order to explore its mechanism for improving diabetic peripheral neuropathy (DPN). KK/Upj-Ay mice were selected as the DPN animal model and randomly divided into the model, Tongxinluo low, middle and high group (1, 2, 4 g x kg(-1)). C57BL/6 mice were selected as the control group. Mice were given intragastrically for 12 weeks. Paw withdrawal latency, motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were detected. Apoptotic rate were detected by FCM. Bcl-2, Bax, Caspase-3 mRNA and protein expression in sciatic nerve were examined by Real-time PCR and Western blot. p38MAPK, p-p38MAPK expression were examined by Western blot. In this study,the authors found that Tongxinluo capsule could increase paw withdrawal latency, MNCV and SNCV. Apoptotic rate of sciatic, the expression of Bax and caspase-3 were lower, while Bcl-2 expression was higher in Tongxinluo group than those in model mice. The expression of p-p38MAPK significantly decreased in Tongxinluo group. The results showed that Tongxinluo capsule has protective effects on diabetic peripheral neuropathy of mice via inhibiting cell apoptosis and suppressing the expression of p-p38MAPK.
Animals ; Apoptosis ; drug effects ; Capsules ; administration & dosage ; Diabetic Neuropathies ; drug therapy ; physiopathology ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Sciatic Nerve ; cytology ; drug effects

Objective To observe the serum level of C-reactive protein(CRP) in transient ischemic attack(TIA) patients,and to assess the correlation between CRP and other dangerous factors of cerebral infarction.Methods 102 TIA patients were divided into two groups: TIA deteriorated into cerebral infarction in two weeks(group A,n=40) and TIA could be relieved in two weeks(group B,n=62).Many factors were measured within 24 h,such as CRP,BP,BS,FIB,TC,TG,HDL-L,and LDL-L.67 healthy subjects were usded as control group.Relative analysis was performed between CRP and 8 parameters mentioned above.Results ①The serum CRP level of TIA patients was higher than that of healthy controls(P

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.

BACKGROUND: Stem cells are relatively primitive cells possessing the capabilities of self-renewal, high proliferation and multi-potential differentiation in vivo under certain conditions. Pancreatic stem cells and umbilical cord blood mesenchymal stem cells (MSCs) may serve therapeutic purpose clinically, but they are still difficult to culture in vitro at present.OBJECTIVE: To explore the method for isolation, purification and culture of pancreatic stem cells and umbilical cord blood MSCs in vitro and observe their morphological changes during culture in vitro.DESIGN: Completely randomized experiment with repeated measurement.SETTING: Stem Cell Research Center, Teaching and Research Division of Physiology, Medical School of Zhengzhou University.MATERIALS: This experiment was conducted in the Stem Cell Research Center, Teaching and Research Division of Physiology, Medical College of Zhengzhou University, between April 2004 and January 2005. Ten to fifteen newborn SD rats (1-3 days) were selected for culture in vitro of pancreatic stem cells, and fresh umbilical cord blood was collected from healthy woman (24-35 years old, with informed consent) at full-term delivery for culture in vitro of umbilical blood SMCs.METHODS: The abdomen of the newborn SD rat was opened under aseptic condition to obtain the pancreas, which was cut into small tissue blocks and digested with type-V collagenase for islet isolation. The isolated islets were purified in continuous roller-bottle culture. Umbilical cord blood was freshly collected for isolating the monocytes by means of density gradient centrifugation in lymphocyte separation medium (with density of 1.077 g/cm3). The islet cells and umbilical cord blood monocytes were cultured in the incubator at 37 ℃ with 5% CO2. The morphological changes of the cells were observed at designed time points and flow cytometry was used to determine the expression of cell surface molecules.MAIN OUTCOME MEASURES: The isolation and culture of pancreatic stem cells and umbilical cord blood MSCs, and their morphological changes during culture in vitro.RESULTS: During culture in vitro, the fusiform islet progenitor cells showed adherent polar growth and continuous proliferation, which covered the whole bottom of the flask after 12-14 days and could be subcultured for passages. However round cells appeared after removal of the growth factor and serum in the culture medium. The monocytes isolated from the umbilical cord blood grew initially into numerous hematopoietic cell clones, most of which proved to be granulocyte clones by Switzerland staining. Seven days later, flat flask wall-adhering epithelial cells and long fusiform fibroblasts were observed mixed with a number of osteoclasts. As the cell culture was prolonged, the cell number increased steadily.CONCLUSION: Pancreatic stem cells and umbilical cord blood SMCs can be cultured in vitro for further experiments.

BACKGROUND:Currently,there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells(UCB-MSCs).OBJECTIVE:To investigate the isolation,purification and culture of UCB-MSCs in vitro,and to detect its surface marker variation.METHODS:The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation,followed by incubation in an incubator containing 5%CO2 at 37℃.The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry.RESULTS AND CONCLUSION:The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones,most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid,increasing by(37.1±2.3)and (10.4±1.7),respectively.Switzerland staining showed most of them were granulocyte clones(80,1±85.2)%,next was erythroid clones(14.2±1.8)%.At 7 days after culture,some shuttle fibroblast-like cells and fiat osteogenic-like cell spread the whole plastic well.At 14 days after culture,flow cytometry showed CD38+ cells accounted for 1.64%,and CD34+/CD38+ cells accounted for 1,71%,and CD34+/CD38- were 0.55%.PI+ and Annexin-V+ cells accounted for 0.05% and 0.18% respectively.At 21 days after culture,CD38+,CD34+/CD38+ and CD34+/CD38- cells were 74.32%,1.61%,and 0.24%.The results reveled that UCB-MSCs can be isolated and cultured in vitro.

Objective:To explore the relationship among blood glucose,blood lipid levels,peripheral blood white cells and slow coronary flow (SCF).Methods:Clinical data of 183 patients receiving angiography in our hospital from Apr 2010 to Apr 2013 were retrospectively analyzed.Patients with TIMI grade 2 or lower coronary blood flow were defined as SCF;patients were divided into SCF group (n=93)and normal control group (n=90).Levels of blood lipid,glycosylated haemoglobin (HbA1c),hematocrit,and peripheral blood white cell levels were measured and compared between two groups.Results:Compared with normal control group,there was significant reduction in high density lipoprotein cholesterol (HDL-C) level [(1.21 ± 0.26)mmol/L vs.(1.12 ± 0.28)mmol/L,P =0.043],and significant rise in HbA1c level [(5.41±0.50)% vs.(5.83±0.45)%,P =0.01],hematocrit [(0.41 ±0.04)vs.(0.43±0.07),P =0.01]and white blood cell count [(6.1±1.6)109/L vs.(6.7±1.7)109/L,P <0.05]in SCF group.Logistic analysis indicated that white cell count was the risk factor of SCF (OR 1.920,95% CI 1.234~2.987,P =0.004).Conclusion:Levels of high density lipoprotein cholesterol,glycosylated haemoglobin, hematocrit and white blood cell count are related to slow coronary flow,and elevated white blood cell count may be a risk factor aggravating slow coronary slow.

Objective To establish the biological reference interval of procalcitonin(PCT) applicable for children in Chongqing.Methods Serum PCT level were detected in 120 healthy children with age from 0 to 16 years, including 73 cases of male and 47cases of female children, by using by Maglumi2000 plus PCT analysis system.All data was evaluated according to EP28-A3c document to establish the biological reference interval.Results Data of PCT levels were with non-normal distribution, and without statistical difference between children of different ages and genders(P>0.05).The biological reference interval of PCT was less than or equal to 0.038 μg/L.Conclusion It might be important to establish a usefully biological reference interval in different laboratories with relative detection system, especially for children.

AIM: To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT(hTERT-MSCs) to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity.METHODS: The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-C1.The hTERT segments were subcloned into pLNCX2.The target cells were infected with these retroviral particles.The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT-MSCs was observed.The expression of hTERT in mRNA level was detected by RT-PCR and the telomerase activity was measured by TRAP(PCR)-ELISA assay.The hTERT-MSCs were induced with 5-azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected.RESULTS: The constructed plasmids were digested with restriction endonucleases(BglⅡand NotⅠ).Two characteristic segments including 6.1 kb and 3.6 kb were obtained.The hTERT-MSCs expressed hTERT in mRNA level.The telomerase activity of hTERT-MSCs was positive.The growth kinetics of hTERT-MSCs was higher than those in UCBMSCs.The hTERT-MSCs were induced to myocardiocyte.CONCLUSION: The hTERT recombinant retrovirus vector has been successfully constructed.The hTERT gene activates the telomerase and prolongs the life-span of cells.No effect of hTERT gene on some type of differentiation potential of MSCs is present.

Objective To explore the effect of chemokine CXCL12 and its receptor CXCR4 on proliferation,migration and invasion of epithelial ovarian cancer cells.Methods CXCR4 and CXCL12 mRNA and protein expression of human ovarian cancer cell line CAOV3 was detected by RT-PCR and immunocytochemistry.Integrin ?1 and vascular endothelial growth factor-C(VEGF-C)mRNA expression were detected in CAOV3 cells stimulated by CXCL12.The CAOV3 cells were divided into 6 groups:control group(un-stimulated),experimental group 1(stimulated by 100 ng/ml CXCL12),experimental group 2 (stimulated by 10 ng/ml CXCL12),experimental group 3(100 ng/ml CXCL12 and 10 ?g/ml neutralizing CXCR4 antibody),experimental group 4(100 ng/ml CXCL12 and 1 ?g/ml CXCR4 antagonist AMD3100),experimental group 5(10 ?g/ml neutralizing CXCR4 antibody or ascites).Methyl thiazolyl tetrazolium(MTT)was used to analyze the effects of different concentrations of CXCL12 on CAOV3 cell proliferation.Transwell invasion chamber and reconstructed basement membrane(Matrigel)were used to evaluate effect of various concentrations of CXCL12 and ascites on CAOV3 cell migration and invasion. Results CAOV3 cells expressed CXCR4 mRNA(0.70?0.10)and protein,but did not express CXCL12 mRNA or protein.Immunostaining of CXCR4 was mainly located in cytoplasm.CXCR4 mRNA was up- regulated after 100 ng/ml CXCL12 stimulation(1.24?0.14;t=-7.1088,P=0.0021).Integrin ?1 mRNA was greatly increased at 3 hours by stimulation of 100 ng/ml CXCL12(before and after stimulation 0.53?0.10,1.53?0.16;P0.05).Experimental group 1 stimulated the migration and invasion of CAOV3 cells in chemotaxis assay compared with control group and experimental group 2(number of cell migration respectively 523.3?25.2,108.0?7.2,211.7 ?24.7,number of cell invasion respectively 39.3?4.0,4.0?1.0,15.7?3.1;P

BackgroundSeveral cornea collagen crosslinking methods have been used to treat keratoconus.However,the safety of these methods is dissatisfactory.Glyceraldehyde is a very potent and highly reactive crosslinking agent,with little toxicity,but its effect on corneal biomechanical property is poorly clear.ObjectiveThe aim of this study was to evaluate the biomechanical effects of glyceraldehyde collagen crosslinking on rats cornea.Methods Fifteen clean SD rats were randomly divided into 0.005 mol/L glyceraldhyde group,0.050 mol/L glyceraldhyde group and blank control group.Glyceraldhyde drops was topically administered in the right ryes 2 times per day for consecutive 7 days in the 0.005 mol/L and 0.050 mol/L glyceraldhyde groups,and no any eye drops was used in the blank control group.Seven days later,the rats were sacrificed.Transparency of corneal buttons in these different groups was evaluated.The central corneal strips of 2 mm×6 mm with 2 mm scleral tiasue were obtained for the biomeehanical stress-strain measurement,including ultimate stress ( MPa),ultimate strain (％) and 6％ elastic modulus (MPa).Corneal collagen fibril density was assessed by histological examination under a light microscopy.The use of the animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.ResultsThe words could be clearly displayed transcorneally in all the three groups.When strain was 6％,the stress was (0.463±0.065 ) MPa in 0.005 mol/L glyceraldehyde group,(0.846±0.240) MPa in 0.050 mol/L glyceraldehyde group,both showing a significant increase in comparison with (0.195±0.103 ) MPa of the blank control group (P=0.029,0.000 ).Following the crosslinking treatment,the ultimate stress was significant elevated in 0.050 mol/L glyceraldehydes group compared with the blank control group ( ( 10.759 ± 3.337 ) MPa vs.(5.295± 1.313 ) MPa,P =0.007 ),but no significant change between the 0.005 mol/L glyceraldehydes group and the blank control group ( ( 6.043 ±2.084) M Pa vs.(5.295 ± 1.313 ) MPa,P =0.660 ).Corneal ultimate strain was lower in the 0.005 mol/L glyceraldehyde group and 0.050 mol/L glyceraldehyde group than the blank control group (36.57％ ±3.09％ vs.43.87％ ± 1.89％,P =0.009;28.53％ ±1.89％ vs.43.87％ ± 1.89％,P =0.000).However,significantly increased 6％ elastic modulus were seen in the 0.005 mol/L glyceraldehyde group and 0.050 moL/L glyceraldehyde group compared with the blank control group ( ( 7.718 ± 1.076 ) MPa,( 14.102 ± 4.011 ) MPa vs.( 3.252 ± 1.717 ) M Pa),with statistically significant differences ( P =0.029,0.000).Histological examination showed a increase of collagen fiber density in the 0.050 mol/L glyceraldehyde group.Conclusions Corneal collagen crosslinking induced by glyceraldehyde strengthens biomechanical intensity and increases the density of corneal collagen fiber.But the safety of glyceraldehyde crosslinking for keratoconus needs further study.