1.The experimental study of cryopreserved neural stem cells transplanting on Wistar rat spinal cord injury
Jun CHEN ; Xueli ZHANG ; Han JIANG ; Rui LIU ; Xiaoli ZHENG ; Chengrui TIAN
Chinese Journal of Laboratory Medicine 2008;31(10):1182-1185
Objective To observe the therapeutic effect of spinal cord injury(SCI)by transplantation cryopreserved neural stem ceils(NSCs).Methods 45 Wistar rats were randomly divided into three groups.with 15 rats in each group.Transplantations were carried out two days after SCI,group A:0.9% saline water,group B:NSCs,group C:cryopreserved NSCs.After the transplantation,immunohistochemical and tissue incision were appeared to detect the survival and differentiation of the transplanted ceils in nivo,and the methods of BBB and oblique plate were used to estimate the recovery of function.Results Group B and C all had active NSCs except group A.The injuried spinal cord A[(3.9±0.1)mm2],B[(1.2±0.3)mm2]and C[(1.1±0.3)mm2],there was an variance in the three groups (F=423.949,P<0.01),BBB test:group A[(2.0±0.5)mark],B[(16.4±0.8)mark]and C [(16.0±1.4)mark],there was an variance in the three groups(F=970.157,P<0.01),the oblique plate test:group A[(31.3±2.9)degree],B[(46.8±2.1)degree]and C[(46.5.4-2.4)degree],there was an variance in the three groups(F=151.099,P<0.01),the tests demonstrate there were no variance in group B and C(all P>0.05),there was no variance in living rate between the cryopreserved NSCs group [(55.9±5.2)%]and no eryopreserved[(65.1±3.4)%,t=3.334,P>0.05].Conclusions Cryopreserved NSCs keep the ability of reproducdon and differentiation.NSCs is a kind of valuable cell resource for the therapy of SCI.
2.The dynamic changes of plasma yon willebrand factor and P-selectin in the finger replanted patients and their clinical significance
Lanfen PENG ; Dongsheng CHEN ; Wenjin FU ; Jincai LUO ; Guangzhong XIE ; Changqin YE ; Zhihong HUANG ; Huihua TANG
Chinese Journal of Laboratory Medicine 2008;31(10):1157-1160
Objective To explore the dynamic changes of von willebrand factor(VWF)and Pseleetin in the finger-replanted patients,and the relationship between the prognosis of the surgery and hypercoagulability.Methods From December 2004 to December 2006,eishty finger-replanted patients were recruited to our study.with 40 healthy volunteers as controls.Plasma VWF and P-selectin were detected by enzyme-linked immunosorbent assay(EUSA)in both controls and patients before or after replantation.Results The VWF and P-selectin levels had significant differences between the replantations and controls(F=14.76,11.76,P<0.01).The VWF levels in the patients of 1,4,8,16 hours after replantation were(1 715±493),(1 396±549),(1 266±504),(1 163±436)U/L respectively,all markedly higher than the controls(P<0.01).The P-selectin levels in patients of 1,4,8,16,24 hours after operation were(14.7±2.6),(12.5±3.0),(11.8±3.2),(11.1±3.0)、(10.5±2.6)μg/L,significanfly higher than the controls(P<0.01).The VWF levels in patients of pre-replantion and the 1,4,8,16,24,48,72 hours after replantation were(854±209),(1 535±389),(1 177±407),(1 040±283),(958±216),(829±193),(777±151),(713±137)U/L in successful group,and were(1 202±164),(2 333±243),(2 146±161),(2 039±244),(1 865±170),(1 645±283),(1 427±331),(1 188±262)U/L in unsuccessful groups.They were all significantly different at the same test-time points between two groups(t=4.44,5.12,6.10,8.43,10.17,8.85,5.10.4.61,P<0.05).The P-selectin levels in patients of 1,4,8,16,24,48,72 hours after replantation were(13.9±2.5),(11.2±2.0),(10.2±1.6),(9.6±1.2),(9.2±0.9),(9.5±0.6),(9.3±0.4)μg/L in successful group,and(17.2±1.0),(16.9±1.0),(17.0±1.3),(16.1±1.1),(14.9±1.5),(13.8±1.4),(12.8±1.2)μg/L in unsuccessful group.Significant difference existed at the same testtime points between two groups again(t=5.22.9.91,10.35,12.79,9.46.9.45,9.33,P<0.01).After replantation,both VWF and P-selectin were rapidly elevated and went to the summit 4 hours later,then declined to pre-replantation level about 24 to 48 hours later after replantation.Conclusions VWF and P-selectin were associated with the hypercoagulability.Dynamic monitoring VWF and p-selectin may be useful in determining the existence of hypercoagulability and the therapy of anti-coagulability.
3.Quantitative analysis of fetal RhD genotyping with fetal DNA from RhD-negative maternal plasma
Xuedong WANG ; Baolong WANG ; Shulai YE ; Lanfang WANG ; Yanqiu LIAO ; Jianjun SHEN ; Guangming JIANG ; Zuojun SHEN
Chinese Journal of Laboratory Medicine 2008;31(10):1147-1152
Objective To explore the feasibility of fluorescence quantitative PCR(FQ-PCR)in prenatal diagnosis of the fetal RhD genotyping using free DNA from RhD-negative pregnant women.Methods The fetal RhD gene was amplified from 78 RhD-negative pregnant women with single fetus maternal plasma (gestation from 11 to 40 weeks).Rhe existence of fetal DNA was confirmed by amplification ofnine different polymorphic short tandem repeat loci(STR)and sex-determining region Y chromosome(SRY)gene.Exon5,7,10 and intron 4 were amplified by real-time polymerase chain reaction with TaqMan probe.The results of fetal RhD genotyping were evaluated retrospectively by the serologic analysis of infant cord blood.Results Among the 78 specimens,the SRY positive signals were detected from samples of 41 and were all identified male fetal through 8ex observation after newborn infants delivered from the women enrolled.The mean concentration of SRY gene reached(214.7±120.9)eopies/ml.RhD genotyping results of 70 cases were in complete concordance with the resets through serological detection of fetal cord blood after delivery.In addition,5 cases were false-positive.3 cages were considered inconclusive.The coincidence rate was 90%(70/78).From 5 false-positive cases,4 cases were identified as RhDel phenotype by detecting RHDl227A allele gene.The final accuracy rate of FQ-PCR was 95%(74/78)in the fetal RhD genotyping.Conclusion FQ-PCR analysis for noninvasive prenatal of fetal RhD genotyping could be useful in prevention and diagnosis of hemolytic disease of newborn.
4.Identification of plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in Enterobacteriaceae
Rong ZHANG ; Jiachang CAI ; Hongwei ZHOU ; Gongxiang CHEN
Chinese Journal of Laboratory Medicine 2008;31(10):1134-1141
Objective To investigate the molecular epidemiology and mechanism of earbapenem resistance of Serratia marcescens,Klebsiella pneumoniae and Escherichia coli isolates from intensive care units(ICUs).Methods Twenty-one S.marcescens,ten K.pneumoniae and one E.coli isolates with carbapenem resistance or reduced carbapenem susceptibility were recovered from two ICUs in our hospital from April 2006 to Febmary 2007.Pulsed-field gel electrophoresis(PFGE)and enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR)were performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined bv agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained bv using an alkalinelysis technique and was digested by various endonucleases.Elimination of plasmid from S.marcesceus isolates were performed by repeated SDS treatment.The crude β-lactamase extracts of original isolates and E.coli transconjugants were subjected to isoelectric focusing(IEF);Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.Results ERIC-PCR indicated that all S.marcescens isolates belonged to a clonal strain.PFGE indicated that ten K. pneumoniae isolates were indistinguishable or closely related to each other.The MICs of imipenem and meropenero for all isolates were 2 to 8 μg/ml except K.pneumoniae K10(128 and 256 μg/ml).Conjugation studies with E.coli(EC600)resulted in the transfer of reduced carbapenem susceptibility from original isolates(MICs:from≤0.125 μg/ml to 1-2μg/ml).IEF,PCR and DNA sequence analysis confirmed that S.marcescens isolates produced KPC-2(pI of 6.7)and a β-lactamase(pI 6.5).k pneumoniae isolates produced TEM-1(pI 5.4),KPC-2,CTX-M-14(pI 7.9),and a β-lactamase(pI 7.3).E.coli El produced KPC-2,CTX-M-15(pI 9.0),and a β-laetamase(pI 7.3).Only a KPC-2 was detected in E.coli transeonjugants.Plasmid restricfion analysis using EcoR Ⅰ,Hind Ⅲ,and Bcu Ⅰ showed identical restrietion patterns among all E.coli transconjugants.SDS-PAGE and ompK 35/36 gene sequence analysis of OMPs revealed that K.pneumoniae K10 failed to express OmpK36 because of insertional inactivation by an insertion of ISEcp1.Conclusions Carbapenem-non-susceptible S.marcescens.K.pneumoniae and E.coli were epidemic in two ICUs in our hospital.Resistance or reduced susceptibility to carbapenems in these strains is mainly due to production of KPC-2.Presence of KPC-2 combined with porin deftciency result in high-level carbaoenem resistance in K.pneumoniae.The game blaKPC2-encoding plasmid spreads among the three different genera.
5.Objectives and significance of stipulation for"regulation on national management of HIV laboratory testing"
Chinese Journal of Laboratory Medicine 2008;31(10):1091-1094
Laboratory testing for HIV infection is the most basic diagnosis evidence for HIV infection and case with AIDS.However,HIV testing is not only professional issue but also quite psychologically different from other tests for infectious diseases.It requires that the HIV test need reach up nearly both 100% of the sensitivity and specificity.If false positivity occurs,it may cause personal,familial and social problems,even result in extremist behavior such as action of suicide.If false negativity occur,it may lead not to receive medical intervention prompdy and to increase infection for society due to blood and sexual transmission modes.As of December 2007,there were 6 918 HIV screening labs and 202 HIV confirmatory labs,which has been approved in China.Therefore,the purpose of"the regulation on national management of HIV laboratory testing"(Management)is to require the management of HIV laboratory testing work legally and to ensure the testing as a technical protection measure for priority prevention and control of AIDS in the country.This paper presents the development and formulation of the Management,and also describes people's concern and understand some key points to carry out the Management in hospital laboratory departments currently.
6.The interference of common methods in determination creatinine
Chinese Journal of Laboratory Medicine 2001;24(2):102-104
Objective To investigate the interference of common methods in determination of creatinine. Methods Standardized with Fuller′s earth, Jaffe′s method, enzymatic method (ultraviolet), enzymatic method (colour), enzymatic method (electrode) were compared and evaluated. Results In eight interference materials, Jaffe′s method was positively affected by pyruvic acid (≥0.186 mmol/L), α-ketoglutaric acid(≥0.0615 g/L), ascorbic acid(≥0.82 mmol/L) and negatively affected by bilirubin(≥165.5 μmol/L). Enzymatic method (ultraviolet) was not interfered. Enzymatic method (color) was positively affected by creatine(≥0.21 mmol/L) and negatively interfered by dopamine(≥0.28 mmol/L). At last, enzymatic method (electrode) was positively interfered by creatine(≥0.62 mmol/L). Conclusion Enzymatic method(ultraviolet) is the best in the above methods.
7.Effects of different inner face of human venous blood container on platelet activation
Yingchun ZHOU ; Xifeng TANG ; Mingfei XU
Chinese Journal of Laboratory Medicine 2008;31(8):937-941
Objective To study the effects of different inner face status of human venous blood container on platelet activation. Methods The plastic( polyethene terephthalate,PET) and glass tubes were coated with polyalkyleneoxide modified polydimethylsiloxane(L722). The contact angles of L722-coated glass and L722- coated PET tubes, glass tubes, PET tubes, silane coupling agent-coated glass tubes and polypropylene (PP) tubes were analyzed respectively. The blood were drawn into above tubes, and then incubated in a roller bottle at room temperature for 10-60 mira The marker of activated platelete, CD62p, was detected by flow cytometry(FCM). Results The inner face contact angle of the blood collection tubes with different material and surface treatment reflected platelet activation to a certain extent, but was not linear. The percent age of CD62p positive platelets in L722-modified PET tubes reduced from (37.4 ±14. 8) % to(21.9 ± 12. 4) %. The platelet activation by glass tubes was (54.5 ± 18.6 ) %, markedly more than PET tubes. While membrane formed, the platelet activation by glass tubes decreased remarkably, and the percentage of CD62p positive platelet in silane coupling agent-coated glass tubes were ( 28. 3 ± 8.2 ) %,markedly less than that of the L722-coated glass tubes. The platelet activation by PET-based material ectally modified with L722 was obviously less than L722-coated glass tubes and the percentage of CD62p positive platelet in silane coupling agent-coated glass tubes were ( 41.5 ± 15.9 ) % and ( 22. 0 ± 12. 8 ) %,respectively. The time course of platelet activation by different tubes showed that the platelet activation by L722-coated PET tubes and polypropylene tubes in 60 rain was not significantly different from the results in30 mitt Conclusions The diverse surface energy status induced by different material and surface treatment of blood collection tubes have obvious effects on the activation of platelets. The silicic oil surface treatment can effectively improve the blood compatibility of blood collection tubes. CD62p detectod by FCM is a sensitive marker for the evaluation of the activation of platelets induced by the material of blood collection tubes. It is of importance in the establishment of surface treatment model of blood container and screening out the material of clinical application.
8.Expression and preliminary use of human sperm-specific lactate dehydrogenase in Escherichia coli
Na XIN ; Ping CHEN ; Lianghu HUANG ; Xiangdong TU ; Yushui WU ; Fenghua LAN
Chinese Journal of Laboratory Medicine 2008;31(8):933-936
Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.
9.Construction of lentiviral vectors of shRNA targeting human APRIL gene
Feng WANG ; Lin CHEN ; Jianguo SHAO ; Zhenbiao MAO
Chinese Journal of Laboratory Medicine 2008;31(8):919-923
Objective To construct small hairpin RNA(shRNA) lentiviral vectors targeting human a proliferation-inducing ligand(APRIL) gene and detect the titer of virus and suitable multiplicity of infection (MOI) after 293T cells were infected by the lentival vectors. Methods Three RNA interference targeting sequences of APRIL gene were screened including shAPRIL1210, shAPRIL1754 and shAPRIL1604. Both sense and antisense Oligo DNA of the targeting sequences were synthesized and cloned into the pGCL-GFP vector, respectively. The resulting lentiviral vectors containing shAPRIL were named LV-shAPRIL1210, LV-shAPRIL1754, LV-shAPRILI604. Then they were confirmed by PCR and DNA sequencing. 293T cells were co-transfected with LV-shAPRIL, pHelper 1.0 and pHelper 2. 0 to product lentivirus, respectively. The titer of virus and suitable MOI were tested according to the expression level of GFP in the 293T cells. Results PCR analysis and DNA sequencing confirmed that three shAPRIL DNA were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5 × 107, 6 × 107 and 4 × 107(transduction units )TU/ml, respectively, and the suitable MOI was 5. Conclusions Three shRNA lentiviral vectors targeting human APRIL gene have been successfully constructed, which lays a foundation for future studying APRIL's gene silencing in related target cells.
10.Evaluation of aldosterone-renin ratio in the diagnosis for primary aldosteroulsm
Mei ZHANG ; Mingjie HUANG ; Lin ZHANG ; Xia LI ; Zhongyun XIONG ; Deying HE ; Zhenmei AN
Chinese Journal of Laboratory Medicine 2008;31(8):903-907
Objective To evaluate the diagnostic value of the aldosterone-renin ratio (ARR) for primary aldosteronism (PA). Methods Serum aldosteronos ( ALD ) and plasma renin activities (PRA)among 44 subjects with primary aldosteronism, 9 subjects with phecchromocytoma, 8 subjects with nonfunctional adrenal tumors, 12 subjects with Cushing syndrome, 4 subjects with stenosis of renal artery and 13 subjects with primary hypertension were retrospectively reviewed. ARR was calculated. The receiver operating characteristics (ROC) curves for every index were used to evaluate diagnostic value. Results The area under the curve(AUC) in the ROC curve of ALD in a supine position was 0. 947, the cut-off value of diagnosis of PA. The AUC for the ROC curve of ALD in upright position was 0. 889, the cut-off value of ALD diagnosis of PA. The AUC for the ROC curve of ARR in a supine position was 0. 978, the cut-off value of diagnosis of PA. The AUC for the ROC curve of ARR in upright position was 0. 981, the cut-off value of specificity. If ARR was combined with ALD in upright position was used, the diagnostic value was better than either index. When ALD > 275 ng/L and the AUC for the ROC curve in upright position was 0. 989,specificity. Conclusions The diagnostic value of ARR in diagnosis of primary aldosteronism is higher than ALD and PRA. ARR in upright position is better than that in supine position, especially when combined with ALD > 275 ng/L in upright position.