Objective To evaluate the effect of driving forces similar to those found in dental plaque fluid on the enamel demineralization and ultrastructure of surface layer.Methods The sections of human enamel were exposed to lactic acid solutions with degree of saturation with respect to enamel and degree of saturation with respect to fluoroapatite for 96 h at 25 ℃,using the deionized water as control in the same conditions.Enamel demineralization was monitored using SEM and confocal laser scanning microscopy(CLSM).Results Only was enamel subsurface demineralization detected in solutions with DS_(EN)(0.1-0.3).Enamel mineral loss and micropores of surface layer decreased significantly with increasing DS_(EN) and DS_(FA) values.However,no mineral loss and micropores of surface layer was observed in sections of enamel exposed to solutions with DS_(EN) values of 0.4 and 0.5,which were covered with homogeneous remineralization layer.Conclusion The demineralization driving forces similar to dental plaque fluid under different DS_(EN) and DS_(FA) have significant effects on the surface layer of enamel and the subsurface demineralization process.

Objective To investigate the MR imaging features of chondroblastoma,and to address the correlation with findings of X-ray radiography and CT.Methods The imaging findings including MRI,X-ray radiography and CT of 16 chondroblastomas proved by surgery and pathology were analyzed and correlated with each other. Results All sixteen chondroblastomas involved the epiphyses of long bones,with varying sizes from 0.8 cm to 5.1 cm and lobulation. They were iso- and hypo-intense on T1WI and had heterogeneous signals on T2WI.They were of soft tissue density on CT,and had areas of calcifications and low density.The rims were hypointense on both T1 WI and T2 WI and showed hyperdensity on CT. The lesions were surrounded by edema of bone marrow which was hypointense on T1 WI and hyperintense on fat suppressed T2WI,while on X-Ray film and CT it was hyperdense sclerotic area.The adjacent soft tissues were swelling.Nine cases had periosteal abnormalities on MRI in which 8 of 9 periosteal abnormalities were distant from the primary lesions,and 6 of them showed hyperdense perosteal new bone on CT.Twelve cases had joint effusion on MRI and CT detected 6 of them.The lesions had heterogeneous enhancement,and there was enhancement in areas of edema within bone marrow,periosteal reaction and adjacent soft tissue.Chondroblastoma was intermediate and hyperintense on DWI,and the intermediate areas on both T1 WI and T2WI,together with areas of bone marrow edema,periosteal reaction and soft tissue swelling,were hyperintense on DWI.Conclusions The MRI,X-ray and CT can reflect the pathological changes of chondroblastoma from different aspects.The characteristics of chondroblastoma can be better appreciated by combining different imaging methods.

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: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.

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.

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 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 observe the effect of 2 Hz electroacupuncture (EA) on long term depression (LTD) of synaptic transmission in the spinal dorsal horn in rats with neuropathic pain, so as to explore the central mechanisms of the antinociceptive effects of 2 Hz electroacupuncture on neuropathic pain. Methods: The neuropathic pain models were produced by tight ligation of the L5/L6 spinal nerves in Sprague Dawley rats. The C fiber evoked field potentials in the spinal dorsal horn were recorded with extracellular recording techniques. The parameters of the electroacupuncture were as follows: frequency of 2 Hz, wavelength of 0.6 ms, intensity of 1,2,3 mA lasting 10 min for each intensity, stimulation time of 30 min. The positive stimulating electrode was placed in acupoint “sanyinjiao” and the negative electrode in “zusanli”. Results: (1) 2 Hz electroacupuncture significantly decreased the amplitudes of C fiber evoked field potentials in the spinal dorsal horn in rats with neuropathic pain to (49.4?0.6)% of the control, compared with that (100.1?1.2) % of the control before EA (unpaired t test, P

Objective:To construct an adenovirus vector harboring human basic fibroblast growth factor (bFGF) cDNA and investigate the expression of bFGF in human umbilical vein endothelial cells (HUVEC) in vitro. Methods: The adenovirus expression vector Ad5-bFGF was constructed by homologous recombination technique. The best value of MOI was tested by transfecting human umbilical vein endothelial cells (HUVEC) with Ad5-GFP. Ad5-bFGF was used to transfect HUVEC at the obtained value of MOI and the expression of bFGF protein was detected by immunocytochemistry method and Western blotting. Results: The best value of MOI for adenovirus 5 to transfect HUVEC was 200 and the transfection rate was 90%. Immunocytochemistry method and Western blotting showed that bFGF was expressed in HUVEC after transfection with Ad5-bFGF and the expression was significantly higher than that in untransfected HUVEC (P

Objective 99Tcm-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime(HL91),a new type of hypoxic agents,accumulates in tumor hypoxic tissue specifically.The aim of this study was to evaluate the value of 99Tcm-HL91 imaging in the diagnosis of bone metastasis.Methods Nineteen cases with bone metastasis(without any treatment)and 8 cases with benign lesions underwent SPECT imaging at 4 h after injection of 740 MBq of 99Tcm-HL91 along with 99Tcm-methylene diphosphonic acid(MDP)imaging.Regions of interest(ROIs)were drawn in tumor tissue and contralateral normal tissue respectively,and the radioactivity ratios of tumor-to-normal(T/N)were calculated.The t-test was used for data analysis with SPSS 11.0.Results There were visible uptake of 99Tcm-HL91 in 79 out of 85 focuses in 19 patients of bone metastasis;however,there was no obvious uptake of 99Tcm-HL91 in 12 focuses of 8 patients of benign lesions.Significant difference existed between the T/N values of malignant(1.877±0.288)and benign lesions[(0.735±0.236);t=13.065,P＜0.05].However,the T/N value of bone metastasis from lung cancer did not differ with that from prostate cancer(1.915±0.344 and 1.825±0.175,respectively;t=1.378,P＞0.05).Conclusion The results indicated that 99Tcm-HL91 was useful in diagnosing the malignant and benign bone lesions.