SUMMARY Objective:To investigate the influence of cryoprotectants and cooling rates in vitrificationmethod on the spindles of rabbit M Ⅱ oocytes.Methods:Rabbit oocytes were verified by using cryoloopwith ethylene glycol(EG)singly or EG combined with dimethyl sulphoxide(DMSO)as cryoprotectants,and cooled by taking oocytes directly into liquid nitrogen or by vitrification machine.After frozen rabbit o-ocytes thawed,the microtubulin and chromosome of the spindles were fixation and stained by immunofluo-rescent method.Confocal microscope was used to reveal spindle configuration.Results:In the two proto-cols of single EG used and EG combined with DMSO,the spindles were severely injured.But in protocolof EG combined with DMSO and at ultra-rapid cooling rate,the normal configuration of spindle rate ofthawed rabbit oobytes was similar to that of the control group.Conclusion:The protocol of EG combinedwith DMSO as cryoprotectants and with extremely high cooling rate by vitrification machine can producethe best effect on conservation of spindle configuration in vitrification of rabbit oocytes.

BackgroundIt is important to measure the corneal curvature, anterior chamber depth (ACD) and axial length accurately for calculating IOL power. The interchange outcomes from different measuring methods and apparatus will cause unreliable IOL power. ObjectiveThe present study was to compare the differences of corneal curvature, anterior chamber depth (ACD) measured by IOLMaster and Orbscan Ⅱbefore and after laser in situ keratomileusis(LASIK) and further compare the axial length measured by IOLMaster and A-ultrasound. Methods One hundred and thirty eyes from 65 consecutive myopic patients before LASIK and 56 eyes of 28 cases with 1-month follow-up duration after LASIK in Henan Eye Institute were enrolled in this study. The K value, ACD between IOLMaster and Orbscan Ⅱ as well as results of axial length between IOLMaster and A-ultrasound were compared by using paired t test. The agreements of the measured values among IOLMaster, Orbscan Ⅱ and A-ultrasound were evaluated using Bland-Altman plot. ResultsBefore LASIK,the K value measured by IOLMaster,Orbscan Ⅱ were ( 43.32 ± 1.52 ) D and ( 42.99 ± 1.45 ) D respectively with the difference value of( 0. 33 ±0. 03 ) D, showing a significant difference(t=10. 380,P=0.000) and a positive relation between them(r=0.971,P=0.000). After LASIK,the K value measured by IOLMaster, Orbscan Ⅱwere(39. 02±2. 14) D and ( 38.91 ±2. 04) D with the difference value (0. 12±0. 33 ) D, presenting a significant differences between them (t =2.715, P =0.009). Bland-Altman plots indicated the disagreement in K value and uninterchangeable. Before LASIK, the ACD measured by IOLMaster,Orbscan Ⅱ and A-ultrasound were ( 3.72 ± 0. 22 ) mm, ( 3.69 ±0. 22 ) mm and ( 3.75± 0.27 )mm respectively and no significant differences were found between them (P ＞ 0. 05 ). Axial length measured by IOLMaster significantly prolonged in comparison with A-ultrasound(25.59± 1. 01 mm vs 25.22±0.99 mm ) , and the difference was( -0. 37 ±0. 30 ) mm, showing significant difference ( t =- 14. 098, P =0. 000 ) and positive correlation ( r =0. 954, P =0. 000 ). Axial length values measured by IOLMaster were ( 25.54 ± 1.05 ) mm in preoperation and ( 25.48 ± 1.01 ) mm in postoperation with the difference (0.052±0. 412)mm, showing statistically insignificant difference between them (t=0. 946,P=0. 348). ConclusionsKeratometries measured by IOLMaster,Orbscan Ⅱ are much more different. Therefore,these two methods are not recommended to use interchangely. ACD measured by IOLMaster,Orbscan Ⅱ and A ultrasound are proved to obtain the similar results and is clinically interchange. Axial length measured by IOLMaster is longer than that measured by A-ultrasound.

Objective To investigate the effects of separate and direct bee sting punctures at acupoints on ESR and RF in rheumatoid arthritis. Method Seventy-two patients with rheumatoid arthritis were randomized to observation and control groups, 36 cases each. The observation group received separate bee sting puncture at acupoints and the control group, direct bee sting puncture at acupoints. In both groups, treatment was given once every other day, three times a week, one week as a course, for two courses. ESR and RF were measured in the two groups before treatment and at one and two weeks after. Result ESR and RF changed significantly in both groups after treatment compared with before (P<0.05). The effects of the two treatments on rheumatoid arthritis-related ESR and RF were equal and there was no statistically significant difference between the two groups (P>0.05). Conclusion Both separate and direct bee sting punctures at acupoints can reduce ESR and RF in rheumatoid arthritis. Separate bee sting puncture at acupoints is easy for the patients to accept.

OBJECTIVETo establish two-dimensional electrophoresis profiles from human laryngeal squamous cell carcinoma tissue and paired normal tumor-adjacent mucosa epithelia tissue, and to identify differential expression proteins.

METHODSThe total proteins of human laryngeal squamous carcinoma tissue and paired normal tumor-adjacent mucosa epithelia tissue were separated by immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differential expression proteins were analyzed using image analysis software, then identified using mass spectrometry and database searching.

RESULTSWell-resolved, reproducible 2-DE patterns of laryngeal squamous cell carcinoma and adjacent normal mucosa epithelial were obtained. Differential protein spots were defined as spots in 2-DE gels. Thirteen proteins were preliminarily identified, naming which 10 proteins were upregulated in laryngeal cancer tissue. Such as cofilin-1, nuclear body protein SP140, GRP94, HSP 90, GSTP1-1, superoxide dismutase [Mn], cyclophilin A, proteasome activator complex subunit 2, apolipoprotein A-I precursor, CaM-like protein and so on. There were 3 proteins downregulated in laryngeal cancer tissue, which were fatty acid-binding protein, calgranulin A and calgranulin B.

CONCLUSIONSThirteen proteins which are associated with the tumorigenesis of the laryngeal squamous cell carcinoma were characterized. These extensive protein variations indicate that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. It is better for understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis-for the rational designs of diagnostic and therapeutic methods.

Aged ; Carcinoma, Squamous Cell ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Proteomics ; methods

OBJECTIVETo observe the effect of acupoint injection by astragalus injection on local SIgA and pathomorphologial changes in rats with chronic pelvic inflammatory disease (CPID).

METHOD50 female Wistar rats were randomly devided into 6 groups, in which CPID model was made except the normal group and sham operation group. The astragalus injection group and the 0.9% NaCl injection group were treated by acupoint injection in Guanyuan (RN4) and Zusanli (ST36). The group was fed Qianjinpian solution into stomach. The histopathologic changes of rats' uterus of each group were observed and SIgA in vagina flushing was detected.

RESULTThe model group showed inflammatory changes, and astragalus injection group and Qianjinpian group showed little histopathologic changes. The levels of SIgA in astragalus injection group were significantly higher than those in other groups, but that in the model group was the lowest.

CONCLUSIONThe deficiency of local SIgA lead to repeatedly attack of CPID. The treatment of acupoint injection by astragalus injection can improve the excretion of SIgA, reinforce the local immunity, and prevent the repeatedly attack.

Acupuncture Points ; Animals ; Astragalus membranaceus ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Immunoglobulin A, Secretory ; blood ; Injections ; Pelvic Inflammatory Disease ; blood ; pathology ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Wistar ; Uterus ; pathology

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONHighly-active recombinant mutant human annexin V with endogenous metal-chelating sites can be expressed in Pichia Pastoris system.

Annexin A5 ; genetics ; Base Sequence ; Humans ; Molecular Sequence Data ; Mutation ; Pichia ; genetics ; RNA, Messenger ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo study long-term behavioral and ultrastructural alterations in a hypoxic-ischemic brain damage (HIBD) model of neonatal rats.

METHODSSixty seven-day-old Sprague-Dawley rats were randomly subjected to unilateral carotid artery ligation followed by hypoxic exposure (HIBD group) or sham operation (n=30 each). A battery of behavioral tests, including Morris water maze test and sensorimotor tests, were performed at a postnatal age of 5 weeks. Nissl staining was used for counting neurons. Transmission electron microscopy was used for observing synapse structures and measuring the thickness of the postsynaptic density area and the length of the postsynaptic active area. The correlations of histological changes with the results of behavioral tests were evaluated.

RESULTSThe HIBD group showed a significantly longer escape latency (P<0.05) and a lower frequency of original platform crossing (P<0.05) in the Morris water maze test compared with the sham operation group. The sensorimotor function test showed that the sensorimotor function in the HIBD group was worse than in the sham operation group. Nissl staining showed that the number of neurons in the HIBD group was significantly reduced (P<0.01) compared with the sham operation group. Transmission electron microscopy showed that synapses were significantly reduced in number, and that the thickness of the postsynaptic density area and the length of the postsynaptic active area were reduced in the HIBD group. The thickness of the postsynaptic density area was negatively correlated with escape latency in the Morris water maze test (r=-0.861, P<0.01), and also negatively correlated with the total score of sensorimotor function tests (r=-0.758, P<0.05) in the HIBD group.

CONCLUSIONSHypoxia ischemia can lead to neuron loss and ultrastructure damage, resulting in long-term deficit of behavioral functions in neonatal rats.

Animals ; Animals, Newborn ; Brain ; pathology ; ultrastructure ; Female ; Hypoxia-Ischemia, Brain ; pathology ; psychology ; Male ; Maze Learning ; Microscopy, Electron, Transmission ; Rats ; Rats, Sprague-Dawley ; Reaction Time

Objective To explore the nursing measures of patients complicated with esophago-tracheal fistula treated by the implantation of self-expansion membrane-covered esophageal stent. Methods Observed postoperative complications of 62 patients with advanced carcinoma of middle third of esophagus after the implantation surgery of self-expansion membrane-covered esophageal stent under direct vision of gastroscopy.Results The incidence of postoperative complications was higher in patients complicated with esophago-tracheal fistula than in the others. Conclusions The enhancement of postoperative nursing measures, especially the instruction of cough, could decrease the incidence of postoperative complications effectively.

Objective To investigate the effect of change in neurotrophins level in the bronchial asthma rats' lung on airway hyperresponsiveness and airway neural plasticity. Methods A total of forty male SD rats were randomly divided into 4 groups with 10 rats in each group: control group,asthmatic group, NGF+BDNF prevention group and anti-NGF+anti-BDNF prevention group. The asthmatic model was established by inhalation and injection of ovalbumin. The airway responsiveness was measured after 8 weeks.The bronchial inflammation was assessed by HE staining,and nerve growth factor and brain derived neurotrophic factor expressions of left lung were assayed by the immunohistochemistry staining.Then the expressions of synaptophysin and neurofilament were detected by RT-PCR. Results Compared with the control group, the lung tissue of the asthma group and NGF+BDNF prevention group had more infiltrating inflammatory cells; The expressions of NGF and BDNF were higher in the asthma group and NGF+BDNF prevention group than those in the control group and anti-NGF+anti-BDNF prevention group (P＜0.05),and significantly higher in the NGF+BDNF prevention group than those in the asthma group (P＜0.05).Both the airway responsiveness and the levels of SYN mRNA and NF mRNA in the lung tissues were significantly higher in the asthma group and NGF+BDNF prevention group than those in the control group (P＜0.05).In asthma group,the expressions of NGF and BDNF were positively related to the expressions of SYN (r=0.889,P＜0.05; r=0.985,P＜0.05)and NF(r=0.956,P＜0.05; r=0.927,P＜0.05),and also positively related to the airway hyperresponsiveness (r=0.938,P＜0.05; r=0.906,P＜0.05). Conclusion NGF and BDNF might be involved in rat airway bronchial neural plasticity changes,which resulted in the airway hyperresponsiveness.

OBJECTIVETo establish a method for detecting the polymorphism of methylenetetrahydrofolate reductase gene (MTHFR).

METHODSThe MTHFR was amplified, and the amplified products were detected by denaturing high performance liquid chromatography (DHPLC), and the amplified MTHFR was confirmed by sequencing and restriction enzyme digesting.

RESULTSA total of 334 individuals of Han people in southern China were recruited in our study, and their polymorphisms of MTHFR were detected. The accurate rate of the DHPLC method, that was very sensitive with 100% detection rate available, was over 99%. The frequencies of CC, CT and TT genotypes were 56.9%, 38.3% and 4.8% individually, and the frequencies of T and C alleles were 23.95% and 76.05% individually.

CONCLUSIONThe DHPLC method can detect polymorphism of MTHFR rapidly, effectively and economically. And there is the existence of different MTHFR polymorphisms in area and race.

Adult ; Aged ; Aged, 80 and over ; Alleles ; China ; ethnology ; Chromatography, High Pressure Liquid ; methods ; DNA Mutational Analysis ; Female ; Humans ; Male ; Methylenetetrahydrofolate Dehydrogenase (NAD+) ; genetics ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Middle Aged ; Nucleic Acid Amplification Techniques ; Polymorphism, Genetic