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【Objective】 To study the red blood cell substitute preparation method of glutaraldehyde(GDA) and Bis(3, 5-dibromosalicyl) fumarate(DBBF) double polymerized, and also observe its curative effect. 【Methods】 The affecting factors of the crosslink of DBBF and human placental hemoglobin(Hb) were selected, including solution pH, inositol hexaphosphoric acid concentration, and molar ratios of DBBF/Hb. The changes of P50, Hill Co and methemoglobin(MetHb) content during the preparation process were studied and the molar ratio of GDA to DBBF-Hb were determined. The final product was obtained through the steps of ultrafiltration, filtration, and deoxygenation, etc., and the final product was subjected to a study on the curative effect of anti-hemorrhagic shock in rats. 【Results】 Under the condition of pH 8.0, the concentration of inositol hexaphosphoric acid was 10mM/L, the molar ratio of DBBF/hemoglobin was 6∶1, the content of dimer was(22.67±3.28)%, the content of tetramer was (74.64±7.05)%, and its P50 was (25.25±2.31) mmHg. As the molar ratio of DBBF/Hb increased, P50 and MetHb content would gradually increase, while the Hill Co will gradually decrease. The molar ratio of GDA and DBBF-Hb was 7∶1, the content of dimer was (8.23±0.79)%, the content of tetramer was (27.87±3.63)%, the content of super-macromolecule was (1.05±0.31)%. As the molar ratio of GDA and DBBF/Hb increased, MetHb content would increase, while the P50 and Hill Co would gradually decrease. The 24h survival rate of the product in rats with hemorrhagic shock was 83.33%(10/12), which was significantly higher than that in the negative control group[41.67%(5/12)], while similar with the positive control group [91.67%(11/12)]. 【Conclusion】 The red blood cell substitutes with GDA and DBBF double polymerized can effectively reduce the dimer content, which is more conducive to industrial production, and has a positive effect on the treatment of hemorrhagic shock.
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This paper briefly reviewed blood substitutes, focusing on the background, history of research and development(R&D), types, functions, characteristics, the indications that will be mainly targeted in clinical, and their special status and role in the future, especially in the rescue of war/trauma patients using hemoglobin-based oxygen carriers(HBOCs). Additionally, the authors put forward unique insights and suggestions on some key concepts and R&D bottlenecks in the development of HBOCs through combining the authors′ own R&D experience for decades with efforts and lessons given by many scholars in this field at home and abroad, hoping to provide some new inspirations and ideas for colleagues to innovate and develop blood substitutes to benefit the clinical as soon as possible.
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【Objective】 To explore the protective effects of hemoglobin base on oxygen carries (HBOCs) with different oxygen affinity on isolated rat hearts. 【Methods】 Using Langendorff isolated heart perfusion model, 45 adult male SD rats (SPF grade), perfused with 30 min KH solution baseline, were randomly divided into sham operation group and control group: St. Thomas (STS) solution perfusion volume was 3mL/100g body weight; high P50 HBOCs group: [STS + high P50 HBOCs (P50=35.0 mmHg, 2.5 mg/100 g) product] perfusion volume was 3mL/100g body weight; medium P50 HBOCs group: [STS + medium P50HBOCs (P50=26.5.0 mmHg, 2.5 mg/100 g) product] perfusion volume was 3 mL/100 g body weight; low P50 HBOCs group: [STS + low P50 HBOCs (P50=11.0 mmHg, 2.5 mg/100 g) product] perfusion volume was 3mL/100g body weight, and the heart was arrested and placed in a 37℃ water bath to make the heart ischemic for 35 minutes, and then reperfused for 2 hours. The left ventricular development pressure (LVDevP), left ventricular end diastolic pressure (LVEDP), the rate of change of left ventricular pressure (LVPCR) and heart rate (HR) in the rat heart during reperfusion were observed and recorded. 1 min perfusion fluid from each rat in the basic and reperfusion phase was taken, and blood gas analyzer was used to measure the blood gas indexes of rats, and the myocardial injury marker enzymes [cardiac enzyme creatine kinase (CK-MB), lactate dehydrogenase (LDH) and the release of α-hydroxybutyrate dehydrogenase (α-HBDH)] were measured by ELISA kit. 【Results】 The cardiac function and the release of myocardial enzymes in the 5 groups of rats in the basal cardiac perfusion stage were similar (P>0.05). However, in the reperfusion stage, except for the insignificant changes in HR (P>0.05), the heart LVDevP (mmHg) of the three P50 HBOCs groups and the control group were 10.69±3.65 vs 8.50±2.88, 23.26 ±5.62 vs 8.50±2.88, 35.60±3.82 vs 8.50±2.88, LVEDP (mmHg) were 43.34±8.08 vs 54.64±7.42, 39.43±8.30 vs 54.64±7.42, 31.46±4.11 vs 54.64±7.42, dp/dt were 12.09±9.96 vs 6.09±0.98, 25.65±8.87 vs 6.08±0.98, 35.32±9.33 vs 6.09±0.98, -dp/dt were 17.53±11.28 vs 11.39±2.16, 28.80±13.70 vs 11.39±2.16, 43.36±3.83 vs 11.39±2.16, respectively (all P<0.05); the rebound situation and the release of CK-MB, LDH, and α-HBDH in the three P50 HBOCs groups were better than those in the control group (P<0.05). Among the three P50HBOCs products, the low P50HBOCs group had the best cardiac function indexes. The myocardial enzyme indexes of the high, medium and low HBOCs groups were CK-MB (ng/mL): 110.47±4.04, 90.2±2.46, 77.1±3.51; LDH (U/L): 162.23±7.71, 135.13±23.69, 92.20±4.21; a-HBDH (U/L): 228.00±8.03, 172.30±8.99, 131.00±2.02. 【Conclusion】 STS solution containing HBOCs products can improve the function of the reperfused heart at normal temperature ischemia for 35 min and 2 h reperfusion, and reduce heart damage. The STS solution containing low P50 HBOCs has the most obvious protective effect in rat isolated heart perfusion.
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BACKGROUND:Pasteurization is a perfect method for albumin virus inactivation, which may not be required for virus inactivation validation. However, there are no systematical reports concerning virus inactivation of hemoglobin blood substitutes. OBJECTIVE:To explore the effects of pasteurization on the physicochemical properties and biological function of hemoglobin blood substitutes. METHODS:Appropriate cord blood samples were taken fol owed by centrifugation, washing blood, rupture of membranes, stabilizer treatment. In the control group, the samples were placed in 55℃water bath, and when the temperature of hemoglobin solution reached (55±1)℃, a heat treatment began and lasted for 2 hours. In the pasteurization group, the samples were placed in 60℃water bath, and when the temperature of hemoglobin solution reached (60±1)℃, a heat treatment began and lasted for 10 hours. The heating process was under continues nitrogen protection. Then, the hemoglobin solution was placed in ice bath and cooled to below 4℃fol owed by low-speed centrifugation and filtration via microporous membrane, purification and viral inactivation thereby to obtain cord blood hemoglobin. RESULTS AND CONCLUSION:The products in the pasteurization group were al red clear liquid. There was no significant difference between the two groups in the yield, methemoglobin concentration, and oxygen-carrying capacity. The purification of the two groups was more than 98%. Two kinds of purification methods had no effects on the oxygen-carrying capacity of hemoglobin. Therefore, pasteurization method can replace thermosensitive purification method of 55℃, 2 hours. The pasteurization method wil not only ensure the physicochemical and biological properties of hemoglobin, but also achieve the purpose of virus inactivation.
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Aquilaria sinensis callus induced by stem tips were used to establish the suspension cell system. The results showed that the most suitable medium for callus induction and subculture is MS + 2.0 mg x L(-1) NAA + 1.0 mg x L(-1) 6-BA. After 12 times of subculture, the energetic and loose callus, which were appropriate for cell suspension culture, were cultured and shook in liquid medium MS + 2.0 mg x L(-1) NAA + 1.0 mg x L(-1) 6-BA + 500.0 mg x L(-1) casein hydrolysate (CH) to establish the suspension cell system. The growth curve of suspension cells showed a "S" type. At the beginning of the culture, cell density increased slowly; during 4 to 6 days, suspension cells reached logarithmic growth period; during 7 to 12 days, suspension cells were in the platform period; but after 12 days, cell density and activity went down obviously. Agarwood sesquiterpenes were not detected in the suspension cells during the growth period, however, they could be detected in MeJA treated suspension cells. In this study, a stable and active growing suspension cell system was established, which was a proper system to study the mechanism of agarwood sesquiterpene formation, and additionally provided a potential way to generate agarwood sesquiterpenes through application of cell culture.
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Objective To study the optimization process of matching conditions using glutaraldehyde (GDA) as crosslinking agent of hemoglobin based oxygen carriers (HBOCs),to further reduce the average molecular weight and the content of super-weight molecular,and improve the conversion ratio of polymerization.Methods The orthogonal designs were done on the basis of the previous single influencing factor research of human placenta hemoglobin crosslinking GDA.Three factors were selected including molar ratio of GDA to hemoglobin,mass concentration of hemoglobin and the rate of the feeding GDA.Results The molar ratio of GDA to hemoglobin is the most important influencing factor on the molecular weight distribution of polymerized hemoglobin,followed by the mass concentration of hemoglobin and the rate of feeding GDA.When analyzing the impact on the mean molecular weight,there were significant differences between mean molecular weight corresponding to different molar ratios of GDA to hemoglobin (P＜0.05),while there was no statistical significance between mean molecular weight corresponding to different mass concentrations of hemoglobin and the rates of feeding GDA (P＞0.05).When analyzing the impact on the effective conversion ratio,there were significant differences between effective conversion ratios corresponding to different molar ratios of GDA to hemoglobin and different mass concentrations of hemoglobin (P＜0.05),while there were no statistical significances between effective conversion ratios corresponding to different rates of feeding GDA (P＞0.05).When analyzing the impact on the content of super-weight molecular,there were significant differences between content of super-weight molecular corresponding to different molar ratios of GDA to hemoglobin,while there were no statistical significances between content of super-weight molecular corresponding to different mass concentrations of hemoglobin and different rates of feeding GDA.Conclusions The optimal matching conditions of hemoglobin polymerization process were determined by orthogonal designs.
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The present study was aimed to investigate the influence of freeze-drying on the quality of polymerized human placenta hemoglobin (PolyPHb). The PolyPHb solution was freeze-drying under suitable conditions. Hemoglobin concentration, methemoglobin (MetHb) content, UV spectrum, Fe3 content, oxygen-carrying capacity, pH, the average molecular weight and its distribution, circular dichroism, oxygen equilibrium curve and other indicators were measured before and after freeze-drying. The appearance, residual water content, rehydration time of the lyophilized product were also evaluated. The results showed that there was no significant difference on all the indicators measured above, which indicated that freeze-drying process had no effect on the physical and chemical properties of PolyPHb, as well as on its biological activity. Therefore, the properties of PolyPHb were stable during this freeze-drying process and could be preserved after such freeze-drying process.
Subject(s)
Female , Humans , Pregnancy , Blood Substitutes , Freeze Drying , Methods , Hemoglobins , Chemistry , Methemoglobin , Placenta , Chemistry , PolymerizationABSTRACT
Five influencing factors of the polymerization process of glutaraldehyde with hemoglobin were studied in cluding the approach of feeding glutaraldehyde, hemoglobin concentration, the molar ratio of glutaraldehyde and hemoglobin, reaction time and temperature, in order to reduce the average molecular weight and to improve the effective polymerization ratio. The results showed that the optimal process reduced the average molecular weight of hemoglobin based oxygen carriers (HBOCs) from (350.20 +/- 35.45)kD to (158.60 +/- 8.70)kD and improved the effective polymerization ratio from 53.27% +/- 4.95% to 69.50% +/- 3.70%. When the experiments expanded 30 folds, the results of the effective polymerization ratio and the average molecular weight mentioned above could be achieved.
Subject(s)
Female , Humans , Pregnancy , Glutaral , Chemistry , Hemoglobins , Chemistry , Molecular Weight , Oxygen , Chemistry , Placenta , PolymerizationABSTRACT
Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D).The highest regeneration rate (34.6%)was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine (BA).The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant (2n = 12)with only one haploid plant(n = 6).Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture orB.chinense.
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The purpose of this study was to test whether oxygen carriers could decrease tissue injury in a rat model of acute myocardial infarct. The study included 3 groups: SD rats in group II and group III were subjected to permanent occlusion of their left anterior descending coronary arteries; SD rats in group I were subjected to sham-operation. The success of modeling was assartained by ECG. Then the rats were given drug via caudal veins for 2 days. A quantitative evaluation was made with an automatic device for interpretation of cardiac troponin T (cTnT); heart staining was made for the calculation of myocardial infarction size (MIS); and myocardial tissue was taken and subjected to routine pathological hematoxylin-eosin (HE) staining for showing myocardial cell injury. cTnT in the sham-operation group was significantly lower by comparison with that in the model group (P < 0.01), and it was slightly lower in the oxygen carriers group than that in the model group, but there was no statistically significant difference (P = 0.18); MIS was significantly smaller in the sham-operation group than that in the model group (P < 0.01), and it was greater in the model rats than that in the oxygen carriers rats (P < 0.05). HE staining of myocardicum in the oxygen carriers group was significantly better than that in the model group (P < 0.01). The evidence suggested that oxygen carriers increased oxygen supply to ischemic myocardium, reduced the myocardial injury, and thus might offer a novel treatment of myocardial infarction.
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Animals , Male , Rats , Blood Substitutes , Pharmacology , Hemoglobins , Metabolism , Myocardial Infarction , Metabolism , Oxygen , Metabolism , Rats, Sprague-Dawley , Troponin T , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the characteristics of male sterility of Bupleurum chinense and further explore the developmental period and reason of abortion for the male sterile plants.</p><p><b>METHOD</b>The morphological characteristics of B. chinense male sterile and normal plants were investigated and compared. The anther development process and pollen viability of two types of plants were examined by microscopic assay.</p><p><b>RESULT AND CONCLUSION</b>The shapes and sizes of anther and filament were different between the male sterile and the normal plants. For the male sterile plant's, the filament size was no more than 1/2 of that of normal plants and the anthers were shriveled, failed to dehisce and pollinate naturally, and the pollen grains in the anthers had no vitality. Other morphological characteristics were similar between two types of plants. The main reason leading to male sterility of B. chinense was the abnormal development of tapetum cells with two circumstances. The one is that the tapetum cells degraded early during the period of microsporocyte phase to tetrad phase and the other is that the tapetum cells proliferated with delayed degradation in the tetrad to uninucleate phase.</p>
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Bupleurum , Cell Biology , Physiology , Plant Infertility , Pollen , Cell Biology , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To breed new varieties with better uniformity and commercial quality as well as higher saikosaponin contents.</p><p><b>METHOD</b>The excellent germplasm resources were selected from "zhongchai no. 1" population. Single plant method was applied to get better varieties. All the breeding material was investigated according to morphological characters, agronomic characters and the contents of saikosaponin a and saikosaponin d. The experiments of comparative test and varieties regional test were carried out.</p><p><b>RESULT</b>The bred new varieties of "zhongchai No. 2" and "zhongchai No. 3" had better uniformity. The dark brown roots ratios of the two varieties were 83.2%, 89.9%, respectively. The contents of saikosaponins (a + d) of the two varieties reached 1.31%, 1.02%, respectively.</p><p><b>CONCLUSION</b>"zhongchai No. 2" and " zhongchai No. 3" both had the advantages of better uniformity, darker brown roots and higher saikosaponin contents.</p>
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Breeding , Bupleurum , Chemistry , Genetics , Oleanolic Acid , Plant Extracts , SaponinsABSTRACT
Purified hemoglobin was modified with pyridoxal 5-phosphate(PLP) and polymerized with glutaric dialdehyde(GDA) to get the products. By comparison of the physical, chemical and biological properties of different procedures for modification before and after polymerization, there is no significant difference in molecular distribution, methemoglobin(MetHb) concentration, oxygen carrier capacity, P50 and spectra. Furthermore, the procedure of modification after polymerization can save PLP greatly and decrease cost greatly. So the procedure of modification after polymerization is a better way in research and production. The addition of GDA could control the increasing of MetHb. By comparison on the physical, chemical and biological properties of different procedures, there is no significant difference in molecular distribution, MetHb concentration, oxygen carrier capacity and spectra between the procedure of adding GDA before PLP and that after PLP. But the P50 of adding GDA before PLP is much lower than that after PLP. So the procedure of adding GDA after PLP is a better way.
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Humans , Blood Substitutes , Chemical Phenomena , Glutaral , Chemistry , Hemoglobins , Chemistry , Methemoglobin , Chemistry , Pyridoxal Phosphate , ChemistryABSTRACT
Oxygen-carrying substances based on poly-placental hemoglobin were put into freeze-drying. Sucrose was chosen to inhibit the methmoglobin(MetHb) formation. MetHb content, ultravioletes(UV) spectrum, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), High performance liquid chromatography (HPLC) were monitored during freeze-drying. As a result, when the mass ratio of sucrose to protein was above 0.5, MetHb formation was under control;the characteristic absorption of UV spectrum, SDS-PAGE and HPLC showed no visible change. Freeze-dried solids were kept under room temperature and refrigerator for 3 months. As a result, MetHb formation depended upon storage temperature and the mass ratio of sucrose to protein. For group C in which the mass ratio of sucrose to protein was 1.0; there is no marked change in MetHb content, UV spectrum, SDS-PAGE and HPLC after 3 months.
Subject(s)
Female , Humans , Chromatography, High Pressure Liquid , Drug Stability , Drug Storage , Electrophoresis, Polyacrylamide Gel , Freeze Drying , Hemerythrin , Hemoglobins , Chemistry , Methemoglobin , Placenta , Chemistry , Sucrose , PharmacologyABSTRACT
The term "blood substitutes" includes plasma substitutes and blood cell substitutes in the broad sense, but in its narrow sense, it means red blood cell (RBC) substitutes, platelet substitutes and white blood cell (WBC) substitutes. The RBC substitutes includes perfluorocarbon, hemoglobin-based and encapsuled substitutes. The hemoglobin-based substitutes which was widely researched in the world includes human hemoglobin-based, animal hemoglobin-based and gene recombined hemoglobin based substitutes. The function and immunology of WBC is very complicated, so it is rarely used in clinic. Nowadays the platelet substitutes pursued by the researches and developments includes mainly the liposome and collagenic fiber species substitutes.
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Humans , Blood Substitutes , Fluorocarbons , Chemistry , Hemoglobins , Chemistry , Recombinant Proteins , Chemistry , Tissue Engineering , MethodsABSTRACT
This study was intended to establish a method for preparation of purified and viral-inactivated placenta hemoglobin. The optimum preparative condition resulted in the up-grading of purity,recovery and so on. A quality control was also established for the purification of hemoglobin. Compared to present purification methods, this method is easy to operate, needs low investment and running cost, and has the advantage of simultaneous operation for purification and viral-inactivation. The resulted hemoglobin had high purity and recovery, and the physicochemical property measured up to that in international reports. So this method is suitable for preparing purified and viral-inactivated hemoglobin on an adequate scale, and is useful for further development of blood substitutes.
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Humans , Blood Substitutes , Fetal Blood , Chemistry , Virology , Hemoglobins , Virus InactivationABSTRACT
Objective To compare the resuscitation effect of 6% hydroxyethyl starch (HES) of different molecular weight in normal saline on hemorrhagic shock in rats. Methods Hemorrhagic shock model was established in 50 SD rats by 45% hemorrhage,and then they were equally divided into 5 groups. The rats were administered with HES40/,HES130/,HES200/normal saline,Voluven or Haes. Their mean arterial blood pressure (MAP),left intraventricular systolic pressure (LVSP),the maximal change rate of left intraventricular pressure (?dp/dtmax) of hemorrhagic shock rat were observed. Meanwhile,the artery blood gas,the survival time and 24-hour survival rate were also observed. Results Similar hemodynamic parameters values were obtained in the rats treated with our HES and those with Voluven or Haes. HES130 and HES200 prolonged the survival time of shocked animals in comparison with HES40. Among the 3 types of HES,HES200 had the best effect,which is equivalent to Voluven and Haes. Conclusion HES of 3 molecular weights have beneficial effect on hemorrhagic shock,and HES200 exerts best.