Analysis of Relationship between Coagulation Function and Blood Transfusion in Patients with Emergency Rescue / 中国实验血液学杂志

<p><b>OBJECTIVE</b>Through researching preoperative coagulation function in the case of ABO-identical blood insufficient for emergency rescue transfusion according to recommended programs of special emergency rescue transfusion was carried out, the relationship between volume of blood products and coagulation function was analyzed.</p><p><b>METHODS</b>The surgical cases of blood transfusion more than 1 600 ml during operation were collected in our hospitals from Aug 2015 to Dec 2016（n=218）, these cases were divided into the normal coagulation group（Group A） and abnormal coagulation group（Group B）, and the patients of emergency rescue transfusion O type blood group（Group C）. The basic information of cases, the infused volume of red blood cell（RBC）, virus-inactivated frozen plasma（VIFP）, fresh frozen plasma（FFP）, cryoprecipitate（C）and platelets（P）, prothrombin time（PT）, activated partial thromboplastin time（APTT）, fibrinogen（FIB）and international normalized ratio（INR）were analyzed, the relationship between volume of blood transfusion and coagulation function were also analysed. At the same time, the efficiency and safety index were compared before and after transfusion. These indexes, such as hemoglobin（Hb）, indirect bilirubin（IBiL）, direct antiglobulin test（DAT）and irregular antibody were determined at the time-paints of 24 h, 3 d and 7 d after blood transfusion.</p><p><b>RESULTS</b>The differences of age and blood type between group A and B was not statistically significant（P>0.05）. Proportion of A and AB type，transfusion volume of RBC, FFP, C and Plt all were significantly higher in group C （P<0.05）. PT, APTT, FIB and INR in group B and C were significantly different（P<0.05）, which related with the transfusion volume of RBC, FFP and C（P<0.05）. DAT and irregular antibody in every group was all negative before transfusion, No any new irregular antibodies had been detected after transfusion. Hb after blood transfusion was not statistically different before and after transfusion in group C, the IBiL level also was not significantly increased after blood transfusion（P > 0.05）. All those showed that emergency rescue transfusion was safe and effective.</p><p><b>CONCLUSION</b>Preoperative coagulation function is one of factors inflnencing blood products transfusion volume during operation, which also is the basis for evaluating bleeding and blood transfusion. Emergency O type blood and ABO-matched blood transfusions show the same efficiency and safety.</p>

Experimental Study on Neonatal ABO or RhD Compatible Blood Transfusion / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the safety and effectiveness of neonatal ABO or Rh(D) by using compatible blood transfusion through retrospective analysis of data from cases received compatible blood transfusion and type matched blood transfusion.</p><p><b>METHODS</b>The clinical data of 26 cases of neonatal compatible blood transfusion in Chinese Nanchang area from January 2014 to October 2016 were collected, and 26 cases of neonatal type-matched blood transfusion were selected according to ratio of 1:1 cases. The efficiency and safety index of 26 patients compatible blood transfusion were compared with that of type-matched blood transfusion. The efficiency indexes included: patients' basic characteristics, red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Hct), and the safety indexes contain Hb level and indirect bilirubin (IBiL) value before and after blood transfusion, irregular antibody screening, direct antiglobulin test (DAT) results and the adverse reactions of blood transfusion.</p><p><b>RESULTS</b>The age, sex, days of hospitalization between compatible blood transfusion and type matched blood transfusion were not statistically significantly different (P>0.05). The Hb level before transfusion, blood transfusion volume and the increase of Hb, Hct and RBC were not statistically significantly different between two groups (P>0.05). The values of Hb, Hct and RBC in 2 groups significantly increased at the day 1 after blood transfusion (P<0.05). No blood transfusion adverse reaction occurred in 2 groups. The IBiL value significantly decreased in compatible blood transfusion patients at the day 1 after blood transfusion (P<0.05). No new irregular antibodies had been detected after transfusion in all patients, and the others' DAT and screening for irregular antibodies were negative except 22 patients with neonatal hemolysis. The values of Hb and IBiL statistically significantly differenence were not in 12 patients between 1d, 3d, 7d after blood transfusion (P>0.05).</p><p><b>CONCLUSION</b>The efficiency and safety between compatible blood transfusion and type matched blood transfusion are the same in neonatal blood transfusion. Compatible blood transfusion is a safe and effective in clinical blood transfusion.</p>

MicroPET imaging with 2-[18F] fluoropropionic acid in Lewis lung carcinoma-bearing mice / 中华核医学与分子影像杂志

Objective To prepare 2-[18F]fiuoropropionic acid (18F-FPA) and evaluate its biodistribution and imaging capacity in Lewis lung carcinoma-bearing mice.Methods 18F-FPA was prepared by nucleophilic substitution reaction,and its hydrophilicity was analyzed.18F-FPA (7.4-11.1 MBq) was injected into Lewis lung carcinoma-bearing mice via tail vein.MicroPET imaging was performed at 20,80 min after the injection.The biodistribution of 18F-FPA in organs was analyzed.The blocking effects of sodium propionate and dichloroacetate to 18F-FPA were tested in vivo.Data were analyzed by two-sample t test using GraphPad Prism software.Results The synthesis of 18F-FPA took 40 min.18F-FPA had high radiochemical purity (＞99％) and hydrophilicity.18F-FPA was mainly distributed in the carcinoma,the urinary bladder and the caecum.The radioactive uptakes in muscles,brown fat and bones were relatively low.Quantitative analysis showed that the uptake of 18F-FPA in Lewis lung carcinoma from 20 min to 80 min was slightly increased ((17.03±2.87) ％ID/g vs (19.33±2.45) ％ID/g) without significant difference (t=1.100,P＞0.05).Neither sodium propionate nor dichloroacetate could block the uptake of 18F-FPA in Lewis lung carcinoma (t=1.544,0.894;both P＞0.05).Conclusions 18F-FPA can be quickly synthesized and has good physicochemical properties.It can be used as a tracer to visualize Lewis lung carcinoma in mice,and its tumor uptaking can not be blocked by propionate and dichloacetate.18 F-FPA PET has the potential to detect lung cancer noninvasively in clinic.

Imaging Potential and Biodistribution in vivo of 2-18FFluoropropionic Acid in Breast Cancer-bearing Mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the imaging potential and biodistribution in vivo of a novel positron imaging agent,2-[(18)F]fluoropropionic,in breast cancer-bearing mice. Methods: 2-[(18)F]fluoropropionic acid (7.4-11.1 MBq)was injected into the breast cancer-bearing mice via tail vein,followed by micro positron emission tomography at 60 min and 120 min.The radioactivity per volume (Bq/ml) in organs was transferred to percentage injected dose per gram(% ID/g)by Inveon Research software and the biodistribution of 2-[(18)F]fluoropropionic acid in organs was deduce.The same operations were done with (18)F-FDG.</p><p><b>RESULTS</b>2-[(18)F]fluoropropionic acid was mainly distributed in the urinary bladder,intestine,and liver between 60 min to 120 min.The breast cancer at right flank was visualized clearly,and the radioactivity uptake was (13.74±1.97)% ID/g and (14.84±1.06)% ID/g,respectively,at these two time points (P=0.454).The radioactivity uptakes in muscle and brown tissue were relatively low.The radioactivity uptake of (18)F-FDG was (10.27±2.34)% ID/g at the breast cancer 60 min after injection,and radioactivity uptake of the brown fat on the back was obvious.</p><p><b>CONCLUSIONS</b>Positron imaging agent 2-[(18)F]fluoropropionic acid can be used to image breast cancer.It may be applied in the noninvasive imaging of breast cancer in clinical settings.</p>

Preparation of HER2 radioligand 99Tcm-ABH2 and its in vivo imaging in breast cancer-xenografted mice / 中华核医学与分子影像杂志

Objective To prepare 99Tcm-human epidermal growth factor receptor 2 (HER2) affibody (ABH2) and explore its feasibility as an imaging agent in HER2-positive breast cancer.Methods Sodium glucoheptonate and SnCl2 · 2H2O were used to label ABH2 with 99Tcm.The labeling yield and radiochemical purity of 99Tcm-ABH2 were determined.The stability of 99Tcm-ABH2 was tested in PBS and serum.The equilibrium disassociation constant (Kd) of 99Tcm-ABH2 was measured with MBA-MD-361 breast cancer cells.SPECT/CT imaging was carried out at 1.0 h and 4.5 h after injection of 37 MBq 99Tcm-ABH2 in nude mice (n=4) xenografted with MBA-MD-361 breast cancer.The T/NT (liver,brain,lung,heart,bone and muscle) ratios were deduced from SPECT/CT acquired data.For the blocking experiment,200 μg ABH2 was injected intravenously before 99Tcm-ABH2 injection.SPECT/CT imaging was performed in the same way.The T/NT ratios were compared between non-blocked and blocked groups by one-way analysis of variance.Results Fhe labeling yield of 99Tcm-ABH2 was above 99％.99Tcm-ABH2 was substantially stable in PBS and serum;the radiochemical purity was (95.0± 1.0)％ after incubation with serum at 37 ℃ for 6.0 h.The Kd of 99Tcm-ABH2 was 1.7 nmol/L.The radioactive uptake in cancer was visualized at 1.0 h and 4.5 h after injection of 99Tcm-ABH2 in HER2 positive MDA-MB-361 breast cancer.99Tcm-ABH2 was cleared out mainly through urinary system.The ratios of tumor to liver,lung,brain,heart,muscle and bone were 1.81± 0.60,8.95±1.13,20.08±6.12,7.61±0.56,10.62±1.78,11.42±2.07,respectively at 4.5 h after 99Tcm-ABH2 injection.After ABH2 blocking,the corresponding T/NT ratios were 0.60±0.23,3.05± 1.38,5.24±2.17,2.42±1.02,8.16±2.66,2.76±0.48 (F=29.38,P＜0.05) respectively.Conclusion 99Tcm-ABH2 could be synthesized with high purity,and this new agent could be used to image HER2-positive breast cancer specifically.

Expression and affinity purification of recombinant human epidermal growth factor receptor-2 affibody with C-terminal cystein / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prepare the modified ZHER2V2 affibody with amino-terminal HEHEHE sequence and carboxyl-terminal GGGC sequence by gene recombinant expression,which is the basis for invasive HER2 imaging with affibody.</p><p><b>METHODS</b>The encoded affibody gene was optimized by codon preference of E. coli with gene designer software. The N-terminal of affibody was fused with HEHEHE sequence,while the C-terminal was fused with GGGC sequence. The synthetic gene was confirmed by Hind 3 endonuclease restriction and gene sequencing. The human epidermal growth factor receptor-2(HER2)affibody gene was sub-cloned into pET22b(+)plasmid and transformed into competent BL21(DE3)bacteria. The expression of modified affibody was induced with isopropyl Β-D-1-thiogalactopyranoside(IPTG)and identified by SDS-PAGE. The affibody was purified by nickel affinity binding and imidazole elution. The purified affibody was labeled with (68)Ga and its affinity was determined by saturation analysis with HER2-positive cells MDA-MB-361.</p><p><b>RESULTS</b>The affibody gene containing N-terminal HEHEHE and C-terminal GGGC sequences were confirmed by Hind 3 endonuclease restriction and gene sequencing. A newly expressed 8×10(3) protein was expressed from the induced recombinant bacteria identified by SDS-PAGE after sub-cloning HER2 affibody gene into pET22b(+)plasmid,transforming recombinant plasmid into competent BL21(DE3)bacteria and inducing the recombinant bacteria with IPTG. The expressed protein was purified from nickel agarose by 60 mmol/L imidazole eluting. The affinity Kd value of (68)Ga labeled affibody to HER2 positive MDA-MB-361 cells was 1.5 nmol/L.</p><p><b>CONCLUSION</b>The affiibody ZHER2V2 containing N-terminal HEHEHE and C-terminal GGGC was successfully prepared by gene optimization,recombinant expression and affinity purification.</p>

68Ga-labeled HER2 affibody with C-terminal cysteine and its PET imaging / 中华核医学与分子影像杂志

Objective To label the modified human epidermal growth factor receptor 2 (HER2) affibody with 68Ga at cysteine position of carboxyl-terminal Gly-Gly-Gly-Cys (GGGC) sequence,and to image the mice model bearing HER2-expressed breast cancer with microPET.Methods The HER2 affibody with carboxyl-terminal cysteine was produced by gene engineering.The 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid (DOTA) was conjugated to the thiol (SH)-group of cysteine by DOTA-10-maleimidoethylacetamide (MMA-DOTA).The newly eluted 68Ga was labeled to DOTA-HER2 affibody at 90 ℃.The labeled mixture was purified by C18 cartridge and eluted with anhydrous alcohol.The purified 68Ga-labeled affibody was prepared for cell binding analysis and microPET imaging.The HER2-expressed breast cancer cells MBA-MD-361 were used to analyze cell binding and establish cancer model with BALB/c nude mice.68Ga-labeled affibody (3.7 MBq) was administered to 4 nude mice with breast cancer via tail vein for microPET imaging at 20 and 60 min post injection.The mice were sacrificed immediately after imaging in unconscious state and their tissue/organs (tumor,muscle,bone and heart) were removed for weighing and radioactivity counting by γ counter.The tumor to normal organ ratios of radioactivity (tumor to liver,muscle,bone and heart)were calculated.Results The purity of HER2 affibody was more than 95％.The radiochemical purity of 68Ga-labeled HER2 affibody was 97％.The KD value (affinity) of 68Ga-labeled HER2 affibody was 1.5 nmol/L in HER2positive cells.MicroPET imaging showed that 68Ga-labeled HER2 affibody could bind HER2-positive cancer rapidly and excreted mainly through urinary system.The radioactivity ratios of tumor to liver,muscle,bone and heart were 17.4±1.0,35.1±10.9,20.7±6.2 and 20.9±4.0,respectively.Conclusions 68Ga could be labeled to HER2 affibody.The 68Ga-labeled HER2 affibody may be useful for PET imaging of HER2-positive tumor.

Study on RHCE genotyping of Rh blood group in Uygur nationality of Xinjiang in China / 中国实验血液学杂志

This study was aimed to investigate the characteristics of RHCE genotyping of Xinjiang Uygur nationality population in China. Primers for detecting RHCE genes were designed according to the references, 89 Uygur nationality RhD-negative samples, 233 Han nationality RhD-negative samples and 109 Han nationality RhD-positive samples were detected by sequence-specific primer-polymerase chain reaction (SSP-PCR) for RHCE genotyping. All above-mentioned samples were unrelated. The results indicated that RHE/e genotyping results were consistent with the serological test results in the samples of Uygur and Han nationality, regardless of the RhD-negative samples or the RhD-positive samples. The RHC/c genotyping results from 89 RhD-negative samples of Uygur nationality were consistent with serological test results. However, total error of RHC/c genotyping from 233 RhD-negative and 109 RhD-positive samples of Han nationality was 5.05%. In conclusion, this method of RHCE genotyping is suitable for the analysis of the RHE/e genotyping of Uygur nationality, no erroneous RHC/c genotyping of Uygur nationality was found in this study, but this method needs to be further studied.

The measurement of affinity of site-specific labeled annexin V with ~(99m)Tc by calcium titration / 基础医学与临床

Objective To derermine the affinity of site-specific labeled annexin V with ~(99m)Tc to phosphatidylserine (PS)exposed erythrocytes.Methods The annexin V fused with a metal chelating binding site was obtained from Pichia Pastoris culture and methanol induction expression.The annexin V was purified from the culture supernatant crude product by uhrafihration.The annexin V was conjugated with ~(99m)Tc site-specifically through sodium glucoheptonic acid and SnCl_2.The radioactive annexin V was added to determine its affinity to PS exposed erythrocytes.Results The calcium concentration at which half of the protein is bound to cells(EC50)is changed with varying ratio of protein to cells.The affinity was determined at a low protein/cell ratio as 33.4.Conclusion The annexin V recombinantly expressed in Pichia Pastoris shows high affinity to PS exposed erythrocytes.

Application of magnetic resonance contrast agent ultrasmall superparamagnetic iron oxide combined with magnetic transfer imaging in the animal model of experimental allergic encephalopathy / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the sensitivity of magnetic resonance contrast agent ultrasmall superparamagnetic iron oxide (USPIO) enhancement scan in detecting experimental allergic encephalopathy (EAE) lesions and the change of magnetic transfer of USPIO enhancement lesions in the animal model of EAE.</p><p><b>METHODS</b>The routine T1-weighted imaging, T2-weighted imaging, three dimensional T1 magnetic transfer, three dimensional T1 no magnetic transfer, Dimeglumine Gadopentetate injection (Gd-DTPA) enhancement, and USPIO enhancement scan were performed in 11 EAE rats and 10 control rats respectively. The sensitivity of USPIO and Gd-DTPA enhancement in detecting the lesions in EAE rats was calculated. Magnetic transfer ratio (MTR) of USPIO enhancement area for the first time in EAE rats and MTR of the same area of the last scan were calculated respectively. HE and myelin staining of brain tissues were performed.</p><p><b>RESULTS</b>No abnormally enhanced lesions were showed in EAE rats' brain in Gd-DTPA enhancement scan, while abnormally enhanced lesions were showed in 11 EAE rats' brain in USPIO enhancement scan. The MTR value of USPIO enhancement area for the first time was significantly different from MTR of the same area of the last scan in EAE rats (P < 0.05). Inflammation cells and demyelination lesions were found in USPIO enhancement area histopathologically. There were no positive findings in control rats.</p><p><b>CONCLUSIONS</b>The sensitivity of USPIO enhancement scan in detecting EAE lesions was high. Magnetic transfer imaging, together with USPIO enhancement scan, was helpful to determine the features of the EAE lesions.</p>

Application of 99Tc(m)-annexinV in predicting the therapeutic effect of chemical agent on lung cancer / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prepare 99Tcm-annexinV and evaluate its application in the early prediction of therapeutic effect of chemical agent on lung cancer.</p><p><b>METHODS</b>Annexin V was obtained by recombinant pichia pastoris expression, ammonium sulfate precipitation, and size-exclusion chromatography. The purified protein was labeled with 99Tc(m) at the N-terminal site by stannous chloride reduction and purified by desalting. The labeling yield and radiochemical purity of 99Tc(m)-annexinV were determined by instant thin-layer chromatography. The biological activity of 99Tc(m)-annexinV was tested by phosphatidylserine-exposed erythrocytes bound radioactivity counting. The lung cancer mice models were established by inoculating LA795 cells to right flank of 615 mice subcutaneously and tumor tissue transplantation. The biodistribution of 99Tc(m)-annexinV in lung cancer mice models were investigated at 6, 12, 24, and 48 h after cyclophosphamide administration.</p><p><b>RESULTS</b>The annexin V was secreted from pichia pastoris and purified by ammonium sulfate precipitation and size-exclusion chromatography with high yield. The annexin V could be labeled at room temperature with 50.2% radioactivity yield. The radiochemical purity of 99Tc(m)-annexinV reached up to 93.9% with intact biological activity. The biodistribution analysis demonstrated that 99Tc(m)-annexinV was excreted from kidney. The uptake of 99Tc(m)-annexinV at tumor reached maximum 48 h after cyclophosphamide administration while tumor to muscle ratio was 6.34 and tumor to blood ratio was 4.09.</p><p><b>CONCLUSIONS</b>99Tc(m)-annexinV derived from pichia pastoris was successfully prepared. It is useful in predicting the therapeutic effect of chemical agent on lung cancer.</p>

Comparison between genotyping and serological phenotyping in RhCE blood group / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To genotype the RHCE gene of Hans, Xinjiang's Uigurs and Kazakstans in China, and to compare the results of RHCE genotyping with that of RhCc/Ee phenotyping.</p><p><b>METHODS</b>RHCE genes of 98 Hans with RhD positive and 230 Hans, 72 Uigurs and 18 Kazakstans with RhD/RHD negative were genotyped with PCR-sequence specific primer (SSP) technique.</p><p><b>RESULTS</b>The results of RHE/RHe genotyping from samples with RhD positive and negative were in accord with that of phenotyping. It would result in 4.44% error using C-->G polymorphism at nt48 of RHCE gene to genotype RHCE, and 4.05% failure of detection using the 109 bp insertion to detectRHCE gene in Chinese Hans. The results of RHE/RHe genotyping in unrelated 72 Uigurs and 18 Kazakstans with RhD phenotype were consistent with that of phenotyping, and false positive and false negative were not found in genotyping in Uigurs and Kazakstans tested.</p><p><b>CONCLUSION</b>The results of RHE/RHe and RHc genotyping were correct with PCR-SSP and accordant with that of phenotyping. Using the C48G polymorphism in exon 1 of RHCE to genotype RHC gene would result in false positive resulting from RHc mutation at this locus, and using the 109 bp insertion to genotype RHC gene would result in false negative because of the absence of the 109 bp. Therefore it is necessary to genotype RHC gene using more than two polymorphic loci.</p>

Investigation of the characteristics of Rh blood group of Uygur nationality in Xinjiang / 中国实验血液学杂志

The study was to investigate the characteristics of Rh blood group of Uygur nationality in Xinjiang. 1 230 blood samples of Uygur nationality were studied by Rh serological typing, modified antiglobulin test, chloroform/trichloroethylene absorption elution test, upstream, downstream and hybrid Rhesus boxes, 10 exons of D gene, RHD(psi) pseudogene. The results showed that the frequency of RHD negative was 5.8%, and no Del type was found. The further investigation of 72 samples of RhD (-) found that ccee (57.02%) and Ccee (29.08%) phenotype as well as RHD(-)/RHD(-) genotype (94.44%) and complete deletion type of D gene exon (91.12%) were all in high frequency, no RHD(psi) pseudugene was detected. In conclusion, the Rh blood group of Uygurs nationality in Xinjiang possesses both oriental and caucasian Rh characteristics, which enriches the diversity of blood types in chinesenation.

Biological activity of human single-chain antibody against amyloid beta peptide involved in Alzheimer's disease / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the biological activity of recombinant human single-chain antibody against amyloid beta peptide in vitro.</p><p><b>METHODS</b>Human single-chain antibody against amyloid beta peptide was obtained from recombinant bacteria. The antigen-binding activity of this antibody was measured by enzyme-linked immunosorbent assay (ELISA) and competitive ELISA. Human neuroblastoma SH-SY5Y cells were used as cell models to test the protective role of human single-chain antibody against amyloid beta peptide.</p><p><b>RESULTS</b>Recombinant human single-chain antibody was mainly located in the insoluble inclusion bodies of bacteria. The antibody was dissolved by urea and purified by metal affinity chromatography as active form to bind synthetic amyloid beta peptide 40 or amyloid beta peptide 42. The improvement of the survival rates of human neuroblastoma cells was significantly superior in amyloid peptide 42 plus equimolar antibody group than in amyloid peptide 42 group (P < 0.05), and was significantly superior in the amyloid peptide 40 plus equimolar antibody group than in amyloid peptide 40 group (P < 0.01).</p><p><b>CONCLUSION</b>The recombinant human single-chain antibody against beta amyloid peptide 40 from E. coli can partially inhibit the neurotoxicity effect of amyloid beta peptide in vitro.</p>

Screening analysis of irregular antibodies from random donor population in Shaoguan area / 中国实验血液学杂志

The study was purposed to analyze the frequency and distribution of irregular antibodies in Shaoguan area. Screening 15 033 random donor antibodies in Shaoguan area by screening cells, polybrene and antiglobulin tests. The results indicated that the irregular antibodies were found in 42 samples. The frequency of irregular antibodies in female was higher than that in male (P < 0.001), and Rh blood group antibodies such as anti-D, anti-E, and anti-Ec C were common (47.6%). 2 samples of Le antibodies were failed to be found by polybrene test. 2 samples of irregular antibodies with titer 2 were undiscovered by screening test of 10 pooled samples. In conclusion, because of irregular antibodies resulting in hemolytic transfusion reaction, the investigation of frequency and distribution of irregular antibodies is very important for safe transfusion. Antibody screening must be done for female donors, and especially for massive plasma transfusion of patients with severe and dangerous illness and infants so as to ensure safety.

Recombinant expression of mutant human annexin V / 中国医学科学院学报

<p><b>OBJECTIVE</b>To mutate human annexin V gene and transform it to Pichia Pastoris for mutant human annexin V expression, so as to be purified as active annexin V with endogenous metal chelating site.</p><p><b>METHODS</b>The 5' and 3' end of native annexin V gene were mutated by specific primers. The mutant annexin V gene was inserted into pPIC9K and sequenced. The correct plasmid was linearized and transformed into Pichia Pastoris strain GS115 by electroporation. The transformants were selected from MD plates and cultured in BMGY medium and induced with methanol. The culture was centrifuged and the supernatant was analyzed by SDS-PAGE and silver staining. The binding activity of mutant human annexin V from culture supernatant was determined with phosphatidylserine exposed erythrocytes and fluorescein isothiocyanate-annexin V.</p><p><b>RESULTS</b>The 5' end of native human annexin V gene was fused with GCAGGCGGCTGCGGCCAT coding sequence and 3' end 946-948 site TGT was mutated to AGC. Pichia Pastoris transformants secreted proteins of relative molecular mass 36 000 48 h after methanol induction. The concentration of this protein that inhibited 50% of the binding of fluorescein-annexin V was 4nmol/L.</p><p><b>CONCLUSION</b>Highly-active recombinant mutant human annexin V with endogenous metal-chelating sites can be expressed in Pichia Pastoris system.</p>

The Conjugation of Fluorescein Isothiocyanate to Annexin V and its Application / 医学研究杂志

Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in boric buffer and the conjugate annexin V-FITC was purified by ion-exchange chromatography.Sheep red blood cells were treated with Glutaraldehyde to expose membrane PS.The annexin V-FITC binding to PS exposed erythrocytes was investigated by fluorescent microscopy.Results The novel annexin V was purified and concentrated from pichia pastoris culture by ion-exchange chromatography and ammonium sulfate precipitation.After conjugating with FITC,the annexin V was found to bind PS exposed erythrocytes by fluorescent microscopy.Conclusion The annexin V expressed recombinantly in pichia pastoris retains the binding ability to PS exposed erythrocytes and is applicable to apoptosis detection in vitro.

The Preparation Investigation on Human Annexin V Dimmer(diannexin) / 医学研究杂志

Objective To prepare human annexin V dimmer and make chance for its pharmacological research.Methods Pichia pastoris transformed by human annexin V gene was used in this investigation.The engineered yeast was cultured in BMGY media and induced with methanol media BMMY for secreting expression of human annexin V.The final BMMY media supernatant was adopted for preparation of human annexin V dimmer by ammonium sulfate precipitation,molecular sieve separation and ultra-filtration concentration.The purification process was monitored by SDS-PAGE and coomassie brilliant blue staining.Results Human annexin V was obtained after two days induction of pichia pastoris in BMMY media.The human annexin V dimmer was successfully prepared from culture supernant by serial ammonium sulfate precipitation,molecular sieve separation and ultra-filtration concentration.Conclusion We prepare the human annexin V dimmer(diannexin V)successfully.

A comparative research on RHD gene structures of Chinese Han and Uigur population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To research comparatively on the RHD gene structures in unrelated RhD negative individuals of Chinese Uigur and Han population.</p><p><b>METHODS</b>The upstream, downstream, hybrid box and 10 exons of RHD gene were detected with sequence specific primer-PCR technique.</p><p><b>RESULTS</b>The results showed the genotypes of RhD negative individuals to have the significant difference between Chinese Uigur and Han population, that 94.44% Uigur individuals were with RHD(-)/RHD(-) genotype but just 61.40% Han population were with this genotype(94.44% versus 61.40%, P<0.01); 2.78% Uigur individuals were with RHD(+)/RHD(-) genotype but 34.21% Han population were with this genotype(2.78% versus 34.21%, P<0.01). However, there was significantly no RHD(+)/RHD(+) genotype difference between Chinese Uigur and Han population(2.78% versus 4.39%, P>0.05). In 78 cases of RhD negative Chinese Hans with single RHD gene, of which the RHD gene structure showed that 53(67.95%) cases were RHD(1-10) allele(of 53 RHD(1-10) alleles, 14 alleles were unexpressed); 15(19.23%) were RHD-CE(2-9)-D(2) allele; 5(6.41%) cases were RHD-CE(2-7)-D(2) allele; 2(2.56%) were similar to RHD-CE(3-6)D allele; 1(1.28%) case was RHD-CE(5-6)-D allele; and 2(2.56%) were RHD-CE(6)-D or point mutation respectively. Of 2 RhD negative Chinese Uigurs with RhD(-)/RHD(+) genotype, one carried RHD(1-10) allele, another carried RHD-CE(2-9)-D(2) allele.</p><p><b>CONCLUSION</b>The most frequently unexpressed RHD alleles were RHD-CE(2-9)-D(2), RHD(1-10) and RHD-CE(2-7)-D(2) respectively in Chinese Han population who carried single RHD allele with RHD(-) phenotype and RHD(+) genotype. It showed the confluent character of RH gene in Chinese Han and Uigur population that there existed unexpressed RHD-CE(2-9)-D(2) allele in Chinese Uigur nationality, which was infrequent in Chinese Uigur population but frequent in Chinese Han population.</p>

The packaging and purification of recombinant adeno-associated virus loaded with anti-amyloid ? peptide single-chain antibody gene / 基础医学与临床

Objective To produce and purify recombinant adeno-associated virus(AAV) loaded with anti-amyloid ? peptide single-chain antibody gene for gene therapy of Alzheimer's disease.Methods The plasmid pSNAV2.0-Abeta-scFv was used to transform BHK-21 cell by Lipofectamine 2000 for stable package cell line establishment.The helper virus HSV1-rc/?UL2 was used to transfect package cell for anti-amyloid ? peptide single-chain antibody gene loaded recombinant adeno-associated virus production.The chloroform-PEG/NaCl-chloroform extraction and ion-exchange chromatography were employed for recombinant AAV purification.SDS-PAGE and PCR amplification were adopted for purified virus identification.The final virus physical titer was determined by digoxin-labeled DNA probes.The effect of gene therapy was tested with transgenic mice by water maze test.Results The purity of recombinant adeno-associated virus with our target gene reach up to 98% after stable cell package and serial purification.The physical titer of the final virus was 1?1012vg/mL.The latency of treated mice in water maze test were reduced significantly.Conclusion The adeno-associated virus carrying anti-amyloid? peptide single-chain antibody gene was produced by HSV1system and purified.The animal behavior test demonstrated the recombinant AAV waseffective in the gene therapy of Alzheimer's disease.