BACKGROUND:Recently, there is more and more attention on uric acid (UA), especially the correlation with cardiovascular disease in the filed of epidemiology; however, researches of the effect of UA on endothelial cells are lack based on cytology.OBJECTIVE:To explore the effects of UA in different concentrations on human umbilical vein endothelial cells line (ECV304), malondialdehyde (MDA), nitric oxide (NO) and 3-(4,5-dimethyl thiazole-2)-2, 5-diphenyl thiazolyl blue (MTT)in vitro.DESIGN: Randomized controlled study based on ECV304.5ETTING: Department of Endocrine, Xijing Hospital, the Fourth Military Medical University of Chinese PLA.MATERIALS:ECV304 was provided by Department of Immunology, the Fourth Military Medical University of Chinese PLA; DMEM Iow-glucose medium by Gibco Company, USA; bovine serum by Beijing Yuanheng Shengma Biotechnology Researching Institute; trypsin by Gibco Company, USA; MTT by Huamei Company; dimethyl sulfoxide (DMSO) analytical pure by Tianjin Bodi Chemical Engineering Co. Ltd.; UA by Sigma Company; NO kit and MDA kit by Nanjing Jiancheng Company; ordinary invert microscope and enzyme-linked immune detector IX70 invert microscope by Olympus, Japan;enzyme-linked immune detector by Eastern China Electron Tube Factory.METHODS: The experiment was carried out in Department of Endocrine and Burning, the Fourth Military Medical University of Chinese PLA from December 2003 to April 2004. Resuscitation, culture, regeneration and inoculation of endothelia cells were undertaken Cell Culture published by Situ et al. Endothelia cells were cultured with non-serum DMEM for 24hours so as to maintain synchronization at the phase of G0/G1 and divided into 4 groups, including control group, low-concentration UA group, moderate-concentration UA group and high-concentration UA group. Each group was divided into 3time points, including 24 hours, 48 hours and 72 hours with 8 samples in each time point. Samples were added with 5％serum medium containing 0 mmol/L, 0.1 mmol/L, 0.2 mmol/L and 0.4 mmol/L UA in control group, low-concentration UA group, moderate-concentration UA group and high-concentration UA group, respectively, and incubated in box with the volume fraction of 0.05 CO2 at 37 ℃. Twenty-four hours later, MDA and MTT were detected; additionally, MTT was detected once more after 48 and 72 hours.MAIN OUTCOME MEASURES: Proliferation of ECV304 and content of NO and MDA.RESULTS:With the increasing concentration of UA, content of MDA was decreased to (2.97±0.05),(2.89±0.09),(2.78±0.10) and (2.44±0.03) μmol/L, respectively; content of NO was (6.86±1.41), (12.5±2.7), (18.9±1.8) and (21.1±1.4) μ mol/L. Absorbencies of NO and MTT were increased and proliferation was increased remarkably at 48 hour.CONCLUSION: A which is characterized by anti-oxidation may promote proliferation of vascular endothelia cells and release of NO.

To investigate the optimum transfection condition in transfecting human keratinocytes with plasmid-liposome complexes in vitro,the cultured human keratinocytes at 60% ～ 100% confluences were exposed to the eukaryotic expression plasmid,pCMV*SPORT-β-gal,coated with LipofectAMINE in different DNA/liposome mixing concentration ratios.After cultured for another 48 hours following the ends of 6 ～ 24 hours exposures to the DNA-liposome complexes,the transfected human keratinocytes were visualized by β-galactosidase staining.Then,the transfection efficiency was determined by calculating the rate of β-galactosidase staining positive cells.β-galactosidase expression was showed clearly in human keratinocytes transfected with the DNA-liposome complexes.The highest efficiency was achieved with cultured cells at 80% and 90% confluences,demonstrating by the transfection rates of (31.35±1.35)% and (32.32±2.47)% respectively.Meanwhile,the essential transfection conditions for these efficiencies were in coating pCMV*SPORT-β-gal DNA of 1.5μg/100μL with LipofectAMINE of 12.5μL/100μL,and exposing the cells to the DNA-liposome complexes for 8 hours.These results indicate that LipofectAMINE could effectively transfer eukaryotic expression plasmid into human keratinocytes in vitro,for which the optimization of transfection conditions involve in cells growth state,DNA/liposome mixing concentration ratio,and exposure time of cells to DNA-liposome complexes.

ObjectiveTo investigate the effects of bFGF on human hypertrophic scar in a nude mouse model. MethodsHuman hypertrophic scars excised in operation for burned patiens were grafted onto the back of nude mice subcutaneously and the mice were randomly divided into: control group, collagenase group, bFGF group, and bFGF plus collagenase group. Two weeks after grafting, preparations were injected into the scars for three times. The size, hardness, and morphological changes of the scars were examined. Biopsies were done 3 weeks after first injection and the histological changes were examined. ResultsIt was found that in bFGF group the size of the grafted scars reduced to 1/2～1/3 of their original size, the majority of the coarse and dense scar collagen bundles resolved and became soft loosed lump. In bFGF plus collagenase group, two thirds of the grafted scar disappeared. ConclusionTopical injection of bFGF into hypertrophic scar degrades scar collagen.

BACKGROUND: Schwann cells play an important role in regeneration of peripheral nerves and the extracellular matrix(ECM) is also important to growth of Schwann cells.OBJECTIVE: To explore the effects of laminin and type Ⅳ collagen on the adhesion and proliferation of Schwann cells.DESIGN: A controlled trial in groups with Schwann cell as subject.SETTING: Plastic Surgery Laboratory of the Fourth Military Medical University of Chinese PLA.MATERIALS: The trial was conducted in the Plastic Surgery Laboratory of Xijing Hospital Affiliated to the Fourth Military Medical University of Chinese PLA from January through April in 1995. The Schwann cells were extracted from newborn rabbits.METHODS: The culture plates were coated with laminin and type Ⅳ collagen. While in the control group, the antibody of laminin and collagen Ⅳ,collagen Ⅰ and polylysine were added respectively. The Schwann cells of the same concentration were cultured in plates of each group. The cells attachment rate in each group ,was determined after 2, 6, 24 hours respectively. After 72 hours 3H-TdR were incorporated into culture matrix fluid and double channel liquid scintillation counter was used for measurement of radiactivity count per minute.MAIN OUTCOME MEASURES: ① Attachment rate of Schwann cell. ②3H-TdR counting.RESULTS: The attachment rates in laminin and collagen Ⅳ groups (66%and 59% ) were higher than those of the type Ⅰ collagen and polylysine groups(45% and 43% ) after 2-hour culturing. Those in laminin antibody and collagen Ⅳ antibody groups were lower. The incorporation value were (10.0±2.7)×103, (1.3±0.3)×103, (10.4±2.4)×103, (1.4±0.5) ×103, (5.8±2.7)×103, (3.3±1.0)×103 per minute respectively in laminin, laminin antibody, collagen Ⅳ, collagen Ⅳ antibody, collagen Ⅰ and polylysine groups. The incorporation values in laminin and collagen Ⅳgroups were higher than controls.CONCLUSION: Laminin and type Ⅳ collagen promote the attachment and proliferation of Schwann cells in vitro. This study provides experimental basis for applying the action of Schwann cell to the nerve tissue engineering.

Objective To analyze the clinical manifestations and treatment for toxic megacolon induced by drastic cathartics inpatients with an unknown history of ulcerative colitis. Methods The clinical data of 5 patients with toxic megacolon induced by ulcerative colitis with initial onset type from June 2003 to October 2008 were analyzed retrospectively. Results In 5 cases, the first symptom was abdominal pain and distention. After taking cathartics, these 5 cases were complicated with toxic megaeolon and 2 cases suffering from intestinal perforation. Four female patients suffered from transient unconsciousness, in which 3 patients were found with cerebral lacunal infarction identified by magnetic field diffusion-weighted imaging. All 5 cases underwent exploration, colectomy and ostomy, one patient died perioperatively, anastomotic fistula and anastomotic constriction developed in one each cases. Conclusions The most common clinical manifestations of toxic megacolon induced by ulcerative colitis are abdominaigia, abdominal distention. Emergency therapeutic strategy consists of partial culectomy and ostomy.

Objective To explore the relationship between hypothyroidism and myocardial injury in patients with idiopathic dilated cardiomyopathy (IDC) by 99Tcm-MIBI SPECT/18 F-FDG PET and late-gadolinium enhancement cardiac magnetic resonance imaging (cMRI-LGE).Methods Sixty-three consecutive patients (42 males and 21 females,(52±11) years) with IDC were enrolled from October 2010 to December 2012.Serum TT3,TT4,FT3,FT4 and TSH were determined using a fully automated chemiluminescence immunoassay.All patients underwent 99Tcm-MIBI myocardial perfusion SPECT/18F-FDG myocardial metabolism PET imaging and cMRI-LGE.Seventeen segments model was used for segmental analysis.Patterns of perfusion/metabolism were classified as normal,mismatch,mild-to-moderate match and complete match.cMRI-LGE was classified into 3 categories (non-LGE,mid-wall LGE and transmural LGE).x2 test was used for data analysis.Results All patients were divided into euthyroid group (n =53) and hypothyroidism group (n =10) according to the levels of serum thyroid hormones.The percentage of normal perfusion/metabolism segments in the euthyroid group was apparently higher than that in the hypothyroidism group:71.8％ (647/901) vs 57.6％ (98/170),x2 =13.50,P＜0.001 ; whereas the percentage of perfusion/metabolism mismatch segments in the euthyroid group was significantly lower than that in the hypothyroidism group:17.8％ (160/901) vs 31.2％ (53/170),x2=16.20,P＜0.001.The euthyroid group had a higher percentage of non-LGE segments (88.0％ (793/901) vs 69.4％ (118/170),x2 =35.70,P＜0.001) and a lower percentage of mid-wall LGE segments (4.8 ％ (43/901) vs 24.1％ (41 / 170),x2 =74.70,P＜ 0.001) compared to hypothyroidism group.Conclusions Hypothyroidism has a detrimental effect on myocardium.99Tcm-MIBI SPECT/18F-FDG PET imaging is sensitive in detecting viable/ischemia myocardium,and cMRI-LGE is good at detecting moderate fibrosis.Combining SPECT/PET imaging and cMRI-LGE for assessing myocardial injury would provide more comprehensive information.

Objective To construct tissue-engineered skin via in vitro inoculation of epidermal stem cells(ESCS)onto de-epidermized dermis.Methods Skin tissue was obtained from the foreskin of a healthy 6-year-old child.and keratinocytes were isolated by two-step trypsinization method followed by the collection of ESCS via rapid adhesion by collagen Ⅳ.The ESCS were identified by morphological observation and immunohistochemical staining with K19 and integrin β1.To construct tissue-engineered skin,selected ESCS were seeded onto the surface of de-epidermized dermis followed by a one-week culture immersed in the medium and a subsequent 4-week culture at the air-medium interface.The tissue-engqneered skin was evaluated with haematoxylin & eosin(HE)staining as well as keratin immunohistochemistry.Results Micro scopically,cultured ESCs showed a paving stone-like appearance and grew into colonies.Immunohistochemistry revealed that the ESCs were positive for integrin-β1 and keratin 19.After 5 weeks of culture,3-6 layers of epidermal cell were observed on the dermis with the formation of stratum corneum.Keratin protein was observed in the artificial epidermal skin.Conclusion Tissue-engineered skin is successfully constructed with epidermal stem cells and de-epidermized dermis in vitro.

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on cultured anoxic-reoxygenation human umbilical vein endothelial cells. METHODS:In the present experiment, an anoxic-reoxygenation model was established by using cultured human umbilical vein endothelial cells. The uptake rate of trypan-blue and calcium and magnesium contents of endothelial cells and lactic dehydrogenase (LDH) activity in medium and malondialdhyde (MDA) content of endothelial cells were measured 60,120 and 180 min after anoxia and 30 and 60 min after reoxygenation, and the effects of 1?10 -8 mol/L CGRP on anoxic-reoxygenation endothelial cells were studied. RESULTS: The findings showed that, as anoxia prolonged, the uptake rate of trypan-blue and LDH activity and MDA content gradually elevated and, during reoxygenation, these parameters sharply increased. Calcium and magnesium levels gradually declined as anoxia prolonged, and during reoxygenation calcium content significantly increased. Meanwhile, 1?10 -8 mol/L CGRP might significantly reduce the uptake rate of trypan-blue and LDH activity and MDA content during anoxia and reoxygenation and lessen the increase in calcium content and the loss of magnesium during reoxygenation. CONCLUSION: These results showed that CGRP might have a direct protective function to endothelial cells afflicted with anoxic-reoxygenation injury by inhibiting lipid peroxidation, attenuating calcium overload and loss of magnesium and enzyme.

OBJECTIVETo observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

METHODSFive SD rats were collected to prepare ADSCs with over-expression of GDNF. One hundred and fifty SD rats were divided into normal control group (N), GDNF-ADSCs group (GA), ADSCs group (A), GDNF group (G), and physiological saline group (P) according to the random number table, with 30 rats in each group. Rats in group N were routinely fed without treatment, and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg. Rats in groups GA, A, G, and P were respectively injected with 100 µL suspension of ADSCs with over-expression of GDNF (1 x 10(7) cells per mL), 100 [µL ADSCs suspension (1 x 10(7) cells per mL), 100 µL GDNF solution (100 mg/L) , and 100 µL physiological saline to the surface of the injured nerves immediately after injury. Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8, and morphology of the sciatic nerves was observed in PIW 8. In PIW 4, the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting. Data were processed with one-way analysis of variance, analysis of variance of repeated measurement, and SNK test.

RESULTSCompared with that of group N, the hind limb stride values in groups GA, A, G, and P were significantly lower at each time point (with P values below 0.05). Compared with those of group P, the hind limb stride values in group GA from PIW 3 to 8, in group A in PIW 3, 5, and 7, and in group G in PIW 3, 5, 7, and 8 were significantly longer (with P values below 0.05). The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97), (13.25 ± 1.40), (12.86 ± 1.42), (14.06 ± 1.50), and (15.09 ± 1.17) cm, which were significantly longer than those in group A [(8.90 ± 0.82), (9.03 ± 0.57), (9.27 ± 0.36), (9.86 ± 0.36), and (9.52 ± 0.58) cm] and group G [(8.87 ± 0.69), (8.51 ± 1.18), (9.34 ± 0.87), (9.76 ± 0.67), and (9.50 ± 1.22) cm], with P values below 0.05. Compared with that of group N, the number of myelinated nerve fibers of sciatic nerves was obviously decreased in group P but obviously increased in groups GA, A, and G; the diameter of axons was obviously shorter, and the myelin thickness was obviously increased in groups GA, A, G, and P in PIW 8 (with P values below 0.05). The number of myelinated nerve fibers in group GA was 31.2 ± 0.8, which was significantly higher than that in group A (23.7 ± 2.7), group G (22.3 ± 2.7), or group P (9.3 ± 2.8), with P values below 0.05. The diameter values of axons among groups P, A, G, and GA were similar (with P values above 0.05). The myelin thickness of rats in group GA was (3.41 ± 0.34) µm, which was significantly thicker than that in group A [(2.64 ± 0.37) µm] or group G [(2.41 ± 0.34) µm], with P values below 0.05. In PIW 4, the protein expression of GDNF of sciatic nerves was significantly higher in groups P, A, G, and GA than in group N (with P values below 0.05), and the protein expression of GDNF in group GA was significantly higher than that in group P, A, or G (with P values below 0.05).

CONCLUSIONSADSCs over-expressing GDNF protein can obviously promote the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

Adipose Tissue ; Animals ; Electrophysiology ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Nerve Crush ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; physiology

Objective To investigate the ability of magnetic resonance imaging (MRI) in tracking magnetically labeled mesenchymal stem cells (MR-MSCs) in a swine myocardial infarction (MI) model.Methods Adult Chinese mini-pigs (n = 6) were subjected to open-chest experimental MI operation.Their antegeneic bone marrow-derived mesenchymal stem cells ( MSCs ) was cultured and doubly labeled with ferumoxides and DAPL On the 14 th day after MSCs transplantation, the size and location of the myocardial infarction were assessed by using delayed-enhancement MRI (DE-MRI). Then the labeled MSCs were injected intramyocardially into peri-infarct zone and normal myocardium. At 24 hrs and 3 weeks after injection, the contrast and the volume of the MR-MSCs hypointense lesion from the MR images were acquired, and the contrast was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.After humane euthanasia, the heart was excised and histology corresponding to MRI slices that demonstrated MR-MSCs lesions was performed.Repeated-measures ANOVA and a paired t test were used for comparison of the contrast and the volume of the MR-MSCs hypointense lesion at different time points. Comparisons between independent groups were performed with the standard Student t test.Results The labeling efficiency of ferumoxides and DAPI was 100% . On the 14 th day after the MI operation, the average percentage of infracted myocardial area was (33.6±8.9)% .Twenty- four hours after MSCs transplantation, MSCs injection sites appeared as ovoid hypointensive lesions with sharp border on T2 * images. At 24 h after injection, the signal contrast [(67.00±5.48)% vs (61.92 ±7.76)%,t = 1.65,P =0.1158] and the size [(0.56 ±0.24) cm2 vs (0.52 ± 0.25 ) cm2, t = 0.39, P = 0.7044 ] of the lesions showed no statistical difference between the peri infarct zone and the normal myocardium.At 3 weeks after injection, the signal contrast decreased and the size diminished both in the peri-infarct zone and in the normal myocardium. Moreover, the contrast of the lesions in peri-infarct zone decreased more significantly than that in normal myocardium [(26.88 +7.27)%vs (15.00 :t:4.51)%, F =20.08, P =0.0003].Post mortem analysis found the fluorescontly labeled MSCs demonstrated on histological sections.There were much more dense fluorescently labled MSCs per high power fields at injection sites of normal myocardium than at injection sites of peri-infarct zone [ (106 ±25 )/HPF vs ( 143 ± 31 )/HPF, t = - 2.47, P = 0.0293 ].In MSCs injection sites of the peri-infarct zone,the capillary density was significantly higher than that in control sites [ (13.4 ± 4.0 )/HPF vs (9.4 ±3.1 )/HPF, t = 2.49, P = 0.0229].At 3 weeks after injection, ferumoxide was contained within partial original MSCs.Conclusion Magnetic resonance imaging of MSCs is a feasible method for the in vivo tracking of transplanted stem cells and could reflect the tendency of the local stem cell quantity, but there still has limitation for the semi-quantitation of the transplanted stem cells.