Objective To investigate the efficacy of continuous blood purification ( CBP) in the treatment of severe sepsis, and explore the related immune regulatory mechanisms. Methods Forty-eight patients with severe sepsis were randomly divided into a control group ( n =23) and a CBP group ( n =25) .CD4 + CD25 + regulatory T cells ( Treg% ) in peripheral blood and APACHEⅡ score were measured dynamically before treatment and 12, 24, 36, 48, 60, 72 hours after treatment. Meanwhile the length of ICUstay, duration of mechanical ventilation, and 28 day mortality were determined. Results Compared with the control group, the length of ICU stay, ventilator time, incidence of multiple organ failure, and mortality decreased significantly in the CBP group ( P lt; 0. 05) . And CBP also decreased Treg% and APACHEⅡ score significantly. There was a positive correlation between Treg% and APACHEⅡ score ( r =0. 804, P lt;0. 01) .Conclusion Early CBP treatment can reduce Treg%, improve cellular immunity and improve the prognosis of sepsis.
Objective To investigate the value of continuous blood purification （CBP）in early treatment of patients with ARDSexp （ARDS caused by extrapulmonary causes），especially in reducing inflammation mediators and extravascular lung water （EVLW）.Methods According the hospital admission sequence，the patients with APACHEⅡ scores from 15 to 20 and PaO2/FiO2 from 100 to 200 were recruited.The ARDSexp patients were divide into an intervention group treated with CBP （Mode：CVVHDF，rate of displacement liquid and dialysate：1.5 L/h，rate of blood：100-200 mL/h，and the time of CBP：72 hours），and a control group without CBP treatment. The NICO and PICCO monitoring data and the survival rates were recorded and analyzed using the SPSS software. Results The mortality rate of the intervention group was lower than that of the control group （6.3% vs. 36.8%，P=0.032）. In the 72 h monitoring dada of NICO and PICCO，the time of improving PCBF，Pm，Cdyn，VCO2，MValv，Pm，PIP，Raw，RSBI，Vd/Vt，and PaO2/FiO2 of the intervention group was severer than those in the control group，and the severety was also more than that of control group which was was significantly different at 72 h（Plt;0.05）. In the PICCO data，the time of decreasing EVWL and PVPI was shorter than the control group，and the decreasing extent was more than the control group，with significant difference at 72 h. But the changes of Apm，CI，and CVP were not significant （Pgt;0.05）. Conclusions In treatment of ARDSexp patients，CBP therapy can induce the PCBC and EVLW，improve pulmonary compliance and MValv，and reduce the mortality rate，while doesn’t influence heart function and the stability of circulation.
Objective The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mell itus. To set up an improved method of the isolation and purification of rat islets, which can obtain highpurity,high-yield, and high-viabil ity islets. Methods Ten healthy and adult male SD rats, weighing 250-300 g were used asorgan donors. Collagenase V was perfused into pancreas via pancreatic duct. Pancreas was digested with collagenase in water bath at 38℃ about 15 minutes, islet purification was performed using two techniques: with Ficoll 400 density gradient (group A), and Ficoll-Paque™ PLUS (group B). Dithizone (DTZ) was util ized for identifying islets, counting islets equivalent quantity (IEQ) and islets’ purity. Trypan blue staining was used to detect the viabil ity of islets. Islets of group B was encapsulated with alginate/poly-L-lysine/alginate (APA). Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insul in release test. Results DTZ staining showed that islets shape were round, ell ipse and irregular with a clear edge and a diameter range of 50-300 μm. The IEQ values were 338.04 ± 76.61 and 834.80 ± 54.00 in groups A and B, respectively, showing significant difference (P lt; 0.05). The purities were 88.31% ± 2.67% and 95.63% ± 1.96% in groups A and B, respectively, showing no significant difference (P gt; 0.05). The activities of islets were 67.40% ± 5.15% and 86.05% ± 2.52% in groups A and B, showing significant difference (P lt; 0.05). Islet APA microcapsules had round shape, unified size, and its diameter was between 1.5 and 2.0 mm. Each microcapsule was encapsulated of 1 to 3 islets. The result of insul in release assay was that the concentrations of insul in secretion with islets of microencapsulated and nonmicroencapsulated were (5.53 ± 1.64) ng/ mL and (4.76 ± 0.26) ng/mL in low glucose, and its concentrations of insul in secretion in high glucose were (11.95 ± 2.07) ng/ mL and (14.34 ± 3.18) ng/mL. Stimulated insul in secretion in high glucose was 2 times more than that in low glucose (P lt; 0.05), but there was no significant difference (P gt; 0.05) in the stimulation index between group A (2.16 ± 0.30) and group B (3.01 ± 0.59). Conclusion The method of islets isolation and purification using Ficoll-Paque™ PLUS own the virtues of more convenient, high islet yield, and high islet purity. Both microencapsulated and nonmicroencapsulated islets show high-viabil ity while culture in vitro.
Objective Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root gangl ion (DRG) tissue culture on β3-tubul in positive axon. Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and non neuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of β3-tubul in positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 μmol/L), and cytrarabine (Ara-C, 0, 10, and 20 μmol/L). Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P lt; 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P lt; 0.05). In PDL/LN gruop, there was no significant difference (P gt; 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P gt; 0.05); the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of β3-tubul in positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P lt; 0.05). The highest number of β3-tubul in positive axon distal end was obtained at 5% concentration level of FBS (P lt; 0.05), but showing no significant differences in β3-tubul in positive axon length among three levels (P gt; 0.05). Both the most of β3-tubul in positive axon distal ends and the longest β3-tubul in positive axon average length were obtained at 0 μmol/L concentration level of 5-Fu, showing significant differences between 0 μmol/L level and 20, 40 μmol/L levels (P lt; 0.05). A similar result of β3-tubul in positive axon distal end was got at the 0 μmol/L level and 10 μmol/L level of Ara-C, which was significantly higher than that of 20 μmol/L level (Plt; 0.05). Conclusion? A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 μmol/L Ara-C are recommended in β3-tubul in positive axon research.
Objective To review the general approaches in isolation and purification of pancreatic islets and progress in several aspects. Methods The latest l iterature concerning acquisition of pancreatic islets was reviewed and analyzed interms of the choice of pancreatic islet donors, the digestion and isolation of pancreas, the purification of islet and the assay of outcome. Results The profile of the isolation and purification depends on the selection of reagents and methods of operation in every step and l inkup between every step. Conclusion Pancreatic islet transplantation is the most effective method to treat type 1 diabetes, the problem of inadequate sources of pancreatic islets could be resolved by the optimal process and the establ ishment of standardized operation.
To set up an economic and effective method for islet isolation from rat, and thereby prove a laboratory protocol of animal model for cl inical islet transplantation. Methods Twenty-five adult male SD rats weighing 230-380 g were used as organ donor. In each of 5 repeated experiments, pancreatic islets of 5 animals were isolated by intraductal infusion of compound sodium chloride injection (CSCI), and subsequently, digested with low concentration (0.5 mg/mL)of collagenase V solution. Islet purification was performed by using a discontinuous density gradient centrifugation thatwas prepared with 27.0%, 23.0%, 20.5% and 11.0% of Ficoll 400. Islet yield and purity were determined by dithizon (DTZ)stain, and propidium iodide (PI)/fluorescein diacetate (FDA) double stain was used to check viabil ity of islets. The endocrine secretory function was assessed by insul in secretion in either low (2.8 mmol/L) or high (25.0 mmol/L) glucose incubation after 3 days of culture in RPMI1640 media. Results Average islet digestion time of 5 experiments was (13.8 ± 1.6) min. Before purification, average isolated number was (5 626 ± 422) islets, and the number was significantly reduced to (2 914 ± 485) islets after purification (P lt; 0.01). The average recovery rate was 51.6% ± 6.0%, and the average yield was (583 ± 97) islets/pancreas. The average purity and viabil ity of islets were 90.2% ± 3.4% and 81.6% ± 7.0%, respectively. After 3 days of culture, insul in secretion of the islets was (116.1 ± 17.4) EU/L in high glucose incubation, which was significantly higher than that of low glucose environment [(39.7 ± 7.5) EU/L, P lt; 0.01)]. The average insul in stimulation index was 3.0 ± 0.4. Conclusion The islet isolation with the CSCI solution and digestion with low concentration of collagenase V decrease experimental cost and also have a beneficial effect on islet recovery and their function.
bjective To separate the SO-Rb50 cells antigen corresponding to the monoclonal antibody of anti-retinoblastoma. Methods The antigen corresponding to the monoclonal antibody of anti-retinoblastoma was separated elementarily by ion-exchange chromatography, and was identified by dot-blotting using the monoclonal antibody of anti-retinoblastoma. The target protein band of the antigen was separated in light of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. Results A special unmixed band of SO-Rb50 cells antigen was separated with the relative molecular weight of 83×103.Conclusion The antigen corresponding to the monoclonal antibody of anti-retinoblastoma could be separated from SO-Rb50cells.(Chin J Ocul Fundus Dis，2003，19：152-155)
In this article, the first time use of continuous veno-venous hemofiltration(CVVH) combined with toxic absorption for the treatment of a crush syndrome patient injured in earthquake is described. Correct therapeutic regimen, close observation and prescription condition are crucial for the success of management. The evidence for the application of this method is also discussed in detail.
Alpha-glycerophosphate oxidase (α-GPO) from Enterococcus casseliflavus was successfully isolated and purified by using polyethylene glycol (PEG)/(NH4)2SO4 aqueous two-phase system (ATPS). The results showed that the chosen PEG/(NH4)2SO4 ATPS could be affected by PEG molecular weight, pH, concentration of PEG and (NH4)2SO4, and inorganic salt as well as additional amount of crude enzyme. After evaluating these influencing factors, the final optimum purification strategy was formed by 16.5% (m/m) PEG2000, 13.2% (m/m) (NH4)2SO4, pH 7.5 and 30% (m/m) additive crude enzyme, respectively. The NaCl was a negative influencing factor which would lead to lower purification fold and activity recovery. These conditions eventually resulted in the activity recovery of 89% (m/m), distribution coefficient of 1.2 and purification fold of 7.0.
To obtain recombinant human β2-microglobulin (rhβ2M) with properties of good solubility and high purity from E.coli, prokaryotic expression conditions were optimized and protein purification was performed in this study. After testing the effect of different IPTG concentrations, temperatures and induction times on the production of rhβ2M, the optimum expression conditions were determined, i.e. joining IPTG to final concentration being 0.8 mmol/L and inducing time 6 h and at temperature of 25℃. Under the optimum induction conditions, the ratio of soluble rhβ2M to soluble bacterial protein was 63.7%. After purified by Ni Sepharose 6 Fast Flow, the purity of rhβ2M achieved a greater value of 95%. Western blot analysis revealed that rhβ2M possessed the antigen property that specifically interacted with anti-β2M antibody.