Objective To study the feasibil ity of preparation of the poly-D, L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide l inkage formation. Methods PDLLA scaffolds (8 mm diameter, 1 mm thickness) were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma. The consequent scaffolds were labeled as aminated PDLLA (A/ PDLLA). The pore size, porosity, and surface water contact angle of groups 0 (un-treated control), 5, 10, and 20 minutes A/ PDLLA were measured. A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solutionwhich contain 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide(NHS), the molar ratio of peptides/EDC.HCL /NHS was 1.5 ∶ 1.5 ∶ 1.0, then brachytely sloshed for 24 hours in roomtemperature. The consequent scaffolds were labelled as peptides conjugated A/PDLLA (PA/PDLLA). The scaffolds in groups 0, 5, 10, and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy (XPS). The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance l iquid chromatography (HPLC), un-treated and un-conjugated scaffolds employed as control. Bone marrow mesenchymal stem cells (BMSCs) from SD rats were isolated and cultured by whole bone marrow adherent culture method. BMSCs at the 3rd–6th passages were seeded to the scaffolds as follows: 20 minutes ammonia plasma treatment (group A/PDLLA), 20 minutes ammonia plasma treatment and conjugation of GRGDS (group PA/PDLLA), and untreated PDLLA control (group PDLLA). After 16 hours of culture, the adhesive cells on scaffolds and the adhesive rate were calculated. After 4 and 8 days of culture, the BMSCs/scaffold composites was observed by scanning electron micorscope (SEM). Results No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments (P gt; 0.05). With increased time of ammonia plasma treatment, the water contact angle of A/PDLLA scaffolds surface was decreased, and the hydrophil icity in the treated scaffolds was improved gradually, showing significant differences when these groups were compared with each other (P lt; 0.001). XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma. With time passing, the peak N1s became more visible, and the ratio of N/C increased more obviously. AfterPDLLA scaffolds treated for 0, 5, 10, and 20 minutes with ammonia plasma and subsequent conjugation of peptides, the ratio of N/C increased and the peak of S2p appeared on the surface. The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time. The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully, showing significant differences between 10 minutes and 20 minutes groups (P lt; 0.001). However, the amount of peptides conjugated in un-treated control and 0, 5 minutes PA/PDLLA scaffolds was too small to detect. After 16 hours co-culture of BMSCs/scaffolds, the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds, showing significant difference between every 2 groups (P lt; 0.01). Also, SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group, especially in PA/PDLLA group. Conclusion Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide l inkage formation. This new type of biomimetic bone has stablized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.
Objective To observe the curative effect on non-obese type 2 diabetes and the effect on change of glucagon-like peptide-1 (GLP-1) of gastric bypass operation. Methods Thirty-two cases of gastric ulcer with non-obese type 2 diabetes were suffered gastric bypass operation. Plasma glucose concentrations, insulin and GLP-1 were measured respectively in fasting and postprandial conditions before operation and in week 1, 2, 3 and month 1, 3, 6 after gastric bypass operation, and the body mass index (BMI), homeostasis model assessment β cell function index (HBCI) and glycosylated hemoglobin (HbA1c， the index was detected only before operation and in month 3, 6 after operation) were also measured. The turnover of the diabetes condition in the 6th month after surgery was observed. Results Compared with the levels before operation, the fasting and postprandial plasma glucose levels were descending (P＜0.05), fasting and postprandial plasma insulin and GLP-1 levels were ascending (P＜0.05), HBCI was ascending and HbA1c was descending significantly after operation respectively (P＜0.05), while BMI changed un-significantly after operation (Pgt;0.05). The diabetes control rate was 78.1%(25/32) overall six months after operation. Level of GLP-1 was negatively correlated with level of plasma glucose (P＜0.05) and positively correlated with level of insulin (P＜0.05). Conclusions Gastric bypass operation can markedly reduce plasma glucose level on the type 2 diabetes patients with non-obese, and the hypoglycemic effect may be contributed by more GLP-1 secretion that caused more insulin secretion, which doesn’t depend on the loss of weight.
Objective We investigated the effect of supplementation with alanyl-glutamine dipeptide on insulin resistance and outcome in patients with chronic obstructive pulmonary disease (COPD) and respiratory failure. Methods A prospective, randomized, open and controlled trial was conducted. Patients with COPD and respiratory failure were recruited between Jan 2005 to Feb 2006 and randomly assigned to a trial group (n=14) with glutamine dipeptide supplmented parenteral nutrition and a control group (n=16) with isocaloric, isonitrogenic parenteral nutrition. On the third day and fifth day of nutrition treatment, blood glucose was clamped at level of 4.4 to 6.1 mmol/L by intravenously bumped insulin. Blood gas, blood glucose level, insulin dosage were recorded everyday. The outcomes were mortality, length of stay (LOS) in hospital and in ICU, mechanical ventilation times and the costs of ICU and hospital.Results Thirty patients successfully completed the trial. There was no difference in blood gas between two groups, but PaO2 increased gradually. Compared with control group, blood glucose level had trend to decrease in trial group. The average insul in consumption decreased significantly in trial group on the fifth day. There was no statistical difference between two groups in mortality, length of stay in hospital and the costs of hospital. But compared with control group, length of stay in ICU and mechanical ventilation days had trend to decrease in trial group. Conclusion Alanyl-glutamine dipeptide do not improve pulmonary function of patients with COPD and respiratory failure. However, alanyl-glutamine dipeptide attenuated insul in resistance and stabilized blood glucose. This trial does not confirm alanyl-glutamine di peptide can improve outcome in critically ill patients with COPD and respiratory failure between two groups in mortality at the end of 30 days, length of stay in hospital and the costs of hospital. But the length of stay in ICU and the duration of mechanical ventilation does decrease, but not significantly, in the trial group.
Objective To evaluate the value of preoperative B-type natriuretic peptide （BNP） level in predicting new onset atrial fibrillation （AF） in patients after coronary artery bypass grafting （CABG）. Methods We electronically searched PubMed，EMbase，Cochrane library，CNKI and VIP databases from the establishment of those databases to November 2012. Evaluation standard of diagnostic tests was used to identify and screen literatures which investigated correlations between preoperative BNP levels and new onset AF of patients after CABG. Quality Assessment of Diagnostic Accuracy Studies （QUADAS） was used to evaluate study quality of included literatures. RevMan 5.0 was used for heterogeneity test. Meta-Disc 1.4 software was used for meta-analysis. Included studies were weighted and then combined. Sensitivity，specificity，diag- nostic odds ratio （DOR），positive likelihood ratio，negative likelihood ratio and corresponding 95% confidence interval（95% CI）were calculated. Summary receiver operating characteristic （SROC） curve was drawn，and the area under the SROC curve （AUC） was analyzed. Results A total of 236 studies were identi?ed，and 5 studies met the eligibility criteria including 802 patients for analysis. There were 228 patients with postoperative new onset AF，and 574 patients without postoperative AF. The quality of the included literature was relatively high. DOR of preoperative elevated BNP level with postoperative new onset AF was 4.15 with 95% CI 2.90 to 5.95. Pooled sensitivity was 0.78 with 95% CI 0.72 to 0.83，pooled specificity was 0.58 with 95% CI 0.54 to 0.58，pooled positive likelihood ratio was 1.91 with 95% CI 1.42 to 1.56，pooled negative likelihood ratio was 0.42 with 95% CI 0.32 to 0.54，and the AUC of SROC was 0.79 （Q=0.72）. Conclusion Preoperative elevated BNP level is significantly correlated with new onset AF after CABG，is a powerful predictor of postoperative AF，and can be used to predict new onset AF after CABG to a certain extent of reliability.
To investigate the origin and releasing relation of motilin (MTL), vasoactive intestinal peptide (VIP) and somatostatin (SS) in guinea pig bile as well as its effects during gallstone formation. Guinea pig were divided into three groups: control group (50 animals), on normal diet; lithogenic group (70 animals), fed with lowprotein low fat; and recovering group (50 animals), fed with lowprotein low fat and recovering normal food after the experiment of gallstone formation. MTL, VIP and SS in the bile gallbladder tissue and portal vein plasma of the normal control group were measured with radioimmunoassay. Meanwhile the changes of the gut peptides in the bile and the bile components from different groups were also compared. Results: In control group the levels of MTL, VIP and SS in the bile were higher than those in the plasma, but, obviously lower than those in the tissues, the concentration relationship between in the bile and in the tissue was a positive correlation. In contrast to the control group, MTL concentration decreased but VIP and SS increased in the bile of the lithogenic group, the physicochemical nature of the bile also became lithogenic. In the recovering group the bile also became lithogenic, but, the concentration of those peptides and the nature of the bile all got normal. Conclusion: MTL, VIP and SS in guinea pig bile originate mainly form the gallbladder wall tissues. Food components affect the levels of the gut peptides in bile, which promote the bile lithogenic changes and gallstone formation.
Objective To investigate the significance of sensory neuropeptides [calcitonin gene related peptide (CGRP) and substance P (SP)] in steroid-induced avascular necrosis of the femoral head (ANFH) by using a rabbit model. Methods Fifty-five adult female Japanese White rabbits (weighing 3 kg and aging 24 months) were randomly divided into experimental group (n=45) and control group (n=10). The rabbits in experimental group received a single intramuscularinjection of methylprednisolone at a dose of 4 mg/kg and then were sacrificed after 3 days (n=15), 1 week (n=15), and 2 weeks (n=15) of injection. The rabbits in control group were fed without any treatment. The necrosis of the femoral head was observed. And the expressions of the monoclonal antibodies CGRP and SP were observed with immunohistochemical staining. Also, the integrated absorbance (IA) value of the positive area was calculated. Results All rabbits survived to the end of the experiment. There was no necrosis of the bone or bone marrow in experimental group at 3 days; whereas ANFH was observed in 5 rabbits at 1 week (33%) and in 8 rabbits at 2 weeks (53%). There were significant differences in the rate of ANFH between 1 week, 2 weeks and 3 days (P lt; 0.05); but there was no significant difference between 1 week and 2 weeks (P gt; 0.05). The intensity of CGRP immunoreactivity increased and reached the peak at 1 week, and then decreased at 2 weeks in experimental group. The IA value of CGRP in experimental group at 1 week was significantly higher than that of control group and that of experimental group at 3 days (P lt; 0.05). The IA value of CGRP in experimental group at 2 weeks was significantly lower than those at 3 days and 1 week (P lt; 0.05). The intensity of SP immunoreactivity decreased and reached the lowest at 1 week, and then increased. The IA value of SP in experimental group at 1 week was significantly lower than that of control group and that of experimental group at 2 weeks (P lt; 0.05). Conclusion The sensory neuropeptides may be affected by the steroid, which may play a key role in the process of steroid-induced ANFH by imbalance of bone metabol ism, disturbance of the microcirculation of bone, and disorder of the protective pain-transmission.
Objective To investigate the value and significance of the changes of plasma level of brain natriuretic peptide（BNP） in evaluating ventricle performance of functional single ventricle after total cavopulmonary connection （TCPC）. Methods We studied 11 patients with functional single ventricle undergone TCPC procedure after 2.1 years, who were followed-up at our ward between April 2004 and November 2004, 7 of them were males and 4 of them were females （TCPC group）. The clinical heart function of patients was scored according to the modified scoring system described by Ross. We obtained 3ml blood samples from the extremital vein of all subjects. Blood was collected into chilled tubes containing EDTA and aprotinin （4.5mg and 1 500u/ml blood, respectively）. The blood samples were promptly centrifuged （-4℃, 3 000r/min for 10 min） and the plasma was separated. BNP concentration was determined using immunoradiometric assay kits. Magnetic resonance imaging （MRI） examination was undertaken in 6 patients of TCPC group to analyse the relative factors with the change of BNP. Control group included 9 healthy children. Results （1） Median plasma BNP level for TCPC group and control group was 400pg/ ml （IQR200-690） and 110 pg/ml （IQR90-190）, respectively. There was a significant difference in plasma BNP between them （P=0. 003）. （2） The results of the index of heart function of TCPC group determined by MRI were 65. 76±8. 65 ml/m2 in end-diastolic volume index, 31. 90±6. 36ml/m2 in end-systolic volume index, 39.09±11.76ml/m2 in stroke volume index, 0. 52± 0. 06 in ejection fraction（EF）, 2.38±0.58L/min·m2 in cardiac index （CI）, 103.49±21.57g/m2 in mass index and 1.57±0.24 in mass/EDV. （3） The plasma BNP level for TCPC group was significant correlation with operation ages（r=0.632, P=0.041 ）. There was no correlation between plasma BNP level with EF, CI, score of Ross, gender, ages, percutaneous oxygen saturation（SpO2） and the type of dominant ventricle, respectively. Conclusions Raised concentration of plasma BNP in patients 2 years after undertaken TCPC procedure indicates that nervous-endosecretory system is still under stress condition. This pattern suggests that neurohormonal activation is primarily related to the altered postoperative physiology. the significance of BNP in patients of functional single ventricle after undertaken TCPC is different from tat in biventricular physiology patiens. The plasma BNP level could not be correctly evaluated the cardiac function after TCPC operation.
ObjectiveTo investigate the effect and mechanism of sleeve gastrectomy (SG) on reducing blood glucose level. MethodsThirty GK rats were randomly divided into SG group, sham operation (SO) group, pair-fed (PF) group, and blank control (BC) group. The changes of weight, fasting blood glucose, glucose tolerance (oral glucose tolerance test, OGTT), insulin tolerance (insulin tolerance test, ITT), plasma insulin, ghrelin, and glucagon like peptide-1 (GLP-1) were monitored before and 24 weeks after operation respectively. ResultsFrom the 4th week after operation, weight gain in SG group and PF group began to decrease significantly compared with SO group (Plt;0.01). From the 2nd week after operation, fasting blood glucose level in SG group was lower than that in SO, PF, and BC groups (Plt;005), and the glucose tolerance in SG group obviously improved compared with preoperation and the other 3 groups (Plt;0.01). On the 6th week after operation, the insulin sensitivity in SG group obviously improved compared with SO group (Plt;0.05, Plt;0.01). There was no significant difference of insulin level between SG group and SO group (Pgt;0.05), ghrelin level significantly decreased (Plt;0.01) while GLP-1 level significantly increased (Plt;0.01) in SG group compared with SO group during 2-24 weeks after operation. ConclusionsThe effect of SG on reducing blood glucose is definite. SG can directly lower blood glucose independent with weight loss. Postoperative decreased ghrelin level and increased GLP-1 level may be its primary mechanism.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To explore the effects of calcitonin gene-related peptide (CGRP) on the migration of bone marrow mesenchymal stem cells (BMSCs) and vascular endothel ial growth factor (VEGF) expression in vitro. Methods TheBMSCs were isolated from Sprague Dawley rats using whole bone marrow adherence method. At 1, 2, and 3 weeks after culture, the expressions of CGRP receptor (CGRPR) was detected by Western blot. The BMSCs were treated with CGRP at concentration 1 × 10-8 mol/L (experimental group) and did not treated (control group), and the efficacy of BMSCs migration was analyzed by Transwell chamber assay after 72 hours; at 1, 3, 5, and 7 days, the mRNA expressions of vascular cell adhesion molecule 1 (VCAM-1) were detected by real-time fluorescent quantitative PCR; the protein expressions of VEGF were examined using immunohistochemistry and Western blot. Results CGRPR expressed stably in the cultured BMSCs and reached the peak at 2 weeks. CGRP had a significantly enhanced role in promoting cell migration. The number of cell migration was (3.20 ± 1.77) cells/HP in experimental group and (1.11 ± 0.49) cells/HP in control group, showing significant difference (t=4.230, P=0.001). In experimental group, the expressions of VCAM-1 mRNA increased with time and reached the peak at 7 days. There were significant differences in the expressions of VCAM-1 mRNA between control group and experimental group at 3, 5, and 7 days (P lt; 0.05). Immunocytochemistry results showed positive DAB staining for VEGF at 5 and 7 days in experimental group. Western blot results showed that the protein expressions of VEGF increased significantly at 5 and 7 days in experimental group when compared with control group (P lt; 0.05), which was signfiantly higher at 5 days than at 7 days in experimental group (P lt; 0.05). Conclusion CGRP can promote the migration of BMSCs and stimulate the protein expression of VEGF, which may plays an important role in regulating bone metabol ism by increasing angiogenesis.