[ ABSTRACT] AIM:To investigate the biological characteristics of newborn rabbit tracheal chondrocytes in vitro. METHODS:Newborn rabbit tracheal chondrocytes were obtained by the method of enzyme digestion, and then cultured in monolayer in vitro.Morphological and growth observations were performed under inverted phase contrast microscope.The ultrastructures of the cells were observed under scanning electron microscope and transmission electron microscope.The bi-ological characteristics of secreted extracellular matrix components were detected by real-time PCR, immunocytochemistry staining and toluidine blue staining.RESULTS: Newborn rabbit tracheal chondrocytes isolated and cultured in vitro showed short triangular or irregular shapes, and adherent growth very well.The ultrastructures of the cells showed pore and abundant cytoplasm and organelles, with a lot of protein secretions in the cells.The chondrocytes expressed the mRNA of collagen I, collagen II and proteoglycans, mainly collagen II and proteoglycans.Immunocytochemistry staining showed col-lagen II and SOX9 positive, and collagen I weakly positive.Toluidine blue staining was also positive.CONCLUSION:Enzyme digestion and monolayer culture are suitable method to obtain newborn rabbit tracheal chondrocytes.These cells, secreting extracellular matrix components, are able to be selected as seed cells for tissue engineering of trachea in vitro, and used to study the therapeutic method for neonatal rabbit tracheal stenosis.

Objective To investigate the quality of concentrated platelet with less WBC by improved white membrane method,so as to provide the basis for preparation of the concentrated platelet in the future.Methods Testing the quality indicators of 56 cases of concentrated platelet with less white blood cells by improved white membrane method,and comparing the quality indicators of 56 cases of collected platelet at random by machine.Testing the expression rate of platelet membrane surface glycoprotein molecules CD62P and PAC-1 before and after filtering in different storage time,and comparing the expression rate of 37 cases of collected platelets at random by machine.Results The differences of quality indicators between concentrated platelet with less white blood cells by improved white membrane methods and collected platelet at random by machine were not statistically significant (P＞0.05),such as volume,platelet content,Ph,amount of RBC mixed within.But The differences of amount of WBC mixed with were statistically significant (P＜ 0.01),both of sterility tests were negative.The differences of the expression rate of platelet membrane surface glycoprotein molecules CD62P and PAC-1 during storage period within 3 days were not statistically significant (P＞0.05).Conclusion The various quality indicators of concentrated platelet with less white blood cells by improved white membrane methods attain mixed concentrated platelet and platelet national standards,and in vitro platelet activity kept well.Platelet special leucocyte filter does not cause obvious platelet activation and damage within three days of storage period of concentrated platelet in filter processing.Collected platelet by machine is being activated and damaged continuously when kept in platelet oscillation device 22±2℃ for 3 days,but activation and damage does not significantly change the platelet activity in vitro.

BACKGROUND: Bone marrow mesenchymal stem cells are important seeded cells for construction of tissue-engineered trachea, but there is no special surface marker. Therefore, identification of bone marrow mesenchymal stem cells is mostly based on morphology, phenotype antigen and the function of differentiation. OBJECTIVE: To explore the feasibility of the tracheal chondrogenic differentiation of bone marrow mesenchymal stem cells under a special condition through isolation, cultivation and identification of bone marrow mesenchymal stem cells. METHODS: Rabbit bone marrow was acquired in the sterile environment to isolate and culture bone marrow mesenchymal stem cells to passage 2 by bone marrow adherence and screening method. Flow cytometry identified the phenotype CD44, CD45 of bone marrow mesenchymal stem cells at passages 1 and 2. Rabbit tracheal samples were acquired in the sterile environment, the tracheal chondrocytes were isolated and cultured by enzyme digestion, and toluidine blue staining was used to detect aggrecan. Bone marrow mesenchymal stem cells were co-cultured with tracheal chondrocytes by Transwel and transforming growth factor β1. Cel morphology was detected under an inverted microscope. Real-time quantitative PCR and toluidine blue staining detected the extracel ular matrix components, such as type Ⅱ col agen and aggrecan.RESULTS AND CONCLUSION: After isolation and culture, cells were spindle and irregular in morphology, and passaged cells thrived that were gathered into a fish-like colony growth. For passage 1 bone marrow mesenchymal stem cells, the positive rates of phenotype antigen CD44 and CD45 were respectively 96.97% and 13.72%; for passage 2 cells, the positive rates of phenotype antigen CD44 and CD45 were 99.11% and 8.54%, respectively. Tracheal chondrocytes were positive for toluidine blue staining. The morphology of induced bone marrow mesenchymal stem cells changed from long fusiform to triangular or irregular shape, indicating the chondrocytes expressed type Ⅱ col agen and aggrecan, and toluidine blue staining was positive. These results showed bone marrow adherence and screening method could acquire bone marrow mesenchymal stem cells, and the purity of passage 2 cells is higher. Under a special condition, bone marrow mesenchymal stem cells have the potential of chondrogenic differentiation, and can be selected as seed cells for construction of tissue-engineered trachea.

OBJECTIVETo evaluate the clinical effects of fine root canal preparation with rotary ProTaper.

METHODSNinety-two teeth among 92 patients were treated with root canal therapy which root canals prepared by rotary ProTaper with Glyde gel (contains EDTA) and lateral condensation, including 66 teeth that 141 root canals were fine,17 teeth being root canal therapy and one or more than root canal were omitted,6 maxillary first molar containing second mesiobuccal canal(MB2), 3 maxillary second molar MB2.

RESULTSOne hundred and thirty five in 141 fine root canals among 66 teeth were successfully prepared by rotary ProTaper, the prepared successful ratio was 95.74%,X-ray showed that obturator had smooth taper. Two palatal root canals of maxillary first molar were underfilling, 4 root canals appeared ledge at apical one third. Seventeen omitted root canals were successfully prepared and obturated. Five in 9 MB2 were successfully prepared and obturated to root-apex, four in 9 were not to end. No instrument separated in all patients.

CONCLUSIONThe preparation of fine root canals with rotary ProTaper and EDTA is an effective and safe and limited. Nevertheless, the preventive importance of ledge and perforation should be emphasized.

Adult ; Aged ; Dental Instruments ; Female ; Humans ; Male ; Middle Aged ; Root Canal Preparation ; instrumentation

Background Erythropoietin (EPO) has a protective effect on retinal neurons in many retinal diseases,but regarding the effect of EPO on apoptosis of retinal photoreceptor cells in retinal detachment (RD) is uncompletely clear.Objective This study was to investigate the protective effect of endogenous EPO on photoreceptors in a rat model of RD and explore its possible mechanism.Methods Seventy-two Sprague- Dawley (SD) rats were randomly assigned to control group,RD group,RD+PBS group,RD+erythropoietin soluble receptor (EPOsR) 2, 20, 200ng groups with 12 rats for each group.1.4% hyaluronic acid was slowly injected into the subretinal space to induce RD in rats,and PBS or 2,20 or 200ng EPOsR was then injected into the vitreous space.On day 3 after RD,apoptotic photoreceptors were detected using transferase-mediated dUTP nickend labeling (TUNEL),and caspase-3 activity was assessed by Western-blot and immunofluorescence staining.On day 14 after RD,retinal histopathologic examination was carried out and outer nuclear layer (ONL) thickness was measured under the light microscope.The use of animals complied with the Statement of Association for Research in Vision and Ophthalmology. Results Apoptotic photoreceptors were seen in ONL of rats of the RD group.Apoptotic photoreceptors were gradually increased with the elevation of EPOsR dose in the vitreous cavity.Western blot and immunofluorescence consistently showed that the gray scale of caspase-3 activity was 0.15±0.04,0.49±0.03,0.50±0.07,0.63±0.03,0.69±0.04 and 0.83±0.04 in the normal group,RD group,RD +PBS group,RD+EPOsR 2,20,200ng groups respectively with statistically significant differences (F=76.016;P=0.000),and caspase-3 activity was considerably stronger in the RD+EPOsR 200ng group than the other groups (P＜0.01).On day 14 after RD,the ONL thicknesses in the normal control group,RD group,RD+PBS group,RD+EPOsR 2,20,200ng groups were (47.39±3.39)μm,(33.96±3.54)μm,(31.83±5.21)μm,(31.40±2.63)μm,(24.99±2.06)μm and (19.30±3.71)μm,showing significant differences among these groups (F=44.733,P=0.000).ONL thicknesses the groups treated with different doses of EPOsR were markedly thinner than that of the RD group and RD +PBS group (P＜0.01).Conclusion EPOsR induces apoptosis of retinal cells and enhances the activity of caspase-3 in a dose-dependent manner.Endogenous EPO can protect photoreceptors against anoxia-mediated damage in RD eyes through decreasing caspase-3 activity and inhibiting apoptosis.

Objective To investigate the correlation between the indications,findings,interventions of fibrobronchoscopy(FB) in neonates and their correlative diseases with neonatal FB results and clinical data.Methods Retrospective case series of 243 consecutive patients of 28 days old or younger were investigated underwent FB for the first time from January 2010 to December 2014,at a tertiary care hospital.The common indications for FB and detection rate of respiratory tract diseases were collected.If the findings of FB had significant associations with premature birth and other diseases were analyzed.Associations between interventions and basic illnesses were also analyzed.Results Of the 243 patients undergoing 275 procedures of FB,201 cases were boys(73.1％).The age of FB was (13.34 ± 9.76) days and the weight was (3.08 ± 0.68) kg.Forty-five cases were premature infants (16.4％).A total of 254 procedures were found to have congenital diseases (92.4％),and 177 cases of them had congenital heart diseases (CHD) (64.4％).Common indications for FB were dyspnea(140 cases,50.9％),tachypnea(82 cases,29.8％),and stridor(71 cases,25.8％).A total of 188 upper airway lesions were found and the most common findings were laryngomalacia(56 cases,20.4％) and vocal cord paralysis(bilateral/unilateral,50 cases,18.2％).A total of 315 lower airway lesions were found and the most common findings were airway mucosal inflammation (98 cases,35.6％),trachea and main bronchial stenosis (73 cases,26.5 ％).A total of 21 cases (7.6％) underwent supraglottoplasty during or after FB,while 17 cases (6.2％) underwent tracheal dilation and 10 cases (3.6％) underwent tracheotomy.Compared with non-CHD neonates,neonates with CHD were statistically significantly less likely to have congenital lesions statistically,such as laryngomalacia(15.8％ vs.28.6％,P =0.012),bilateral vocal cord lesions(6.2％ vs.21.4％,P =0.000) and congenital laryngeal dysplasia(0 vs.7.1％,P =0.001).The tracheotomy(0 vs.10.2％,P =0.000) and supraglottoplasty(2.3％ vs.17.3％,P =0.000) were more rare.Nevertheless,they were more likely to have secondary lesions such as the left main bronchial stenosis caused by extrinsic compression (23.7％ vs.1.0％,P =0.000),abnormal bronchial anatomy(9.6％ vs.2.0％,P =0.018),left vocal cord paralysis(9.0％ vs.1.0％,P =0.008) and airway mucosal inflammation(41.8％ vs.24.5％,P =0.004).The tracheostenosis and main broncial stenosis (37.3％ vs.7.1％,P =0.000) with long-term intubation(78.5％ vs.58.2％,P =0.000) were more common.There was no significant difference between term neonates and premature infants in the detection rate of respiratory tract diseases (P ＞ 0.05),tracheotomy (0 vs.4.3 ％,P =0.322),supraglottoplasty (13.3 ％ vs.6.5 ％,P =0.205) or long-term i ntubation (80.0％ vs.69.6％,P =0.157).Complications caused by procedure were rare and mild.Conclusions FB can detect whether the neonates with dyspnea,tachypnea and stridor have laryngomalacia,vocal cord paralysis,airway mucous edema,tracheal and main bronchial stenosis and other signs,and FB may play an important role in diagnosis,treatment and prognosis evaluation of neonatal respiratory diseases.

OBJECTIVETo evaluate the kinesis of cellular and humoral immune responses to different kinds of recombinant hepatitis B(rHB) vaccines in the immunized mice.

METHODSAt serial time points, the levels of IFN-gamma and IL-2 secreted by spleens mononuclear cells (MNC) of the vaccinated mice were detected by enzyme-linked immunospot methods (ELISPOT) after stimulation in vitro with HBsAg MHC class I peptide S28-39 or HBsAg. The lymphocytotoxicity of the immunized mice were also detected (CTL) by a specific lysis assay and the levels of anti-HBs were measured by the Abbott IMX kit.

RESULTSThe peak values of IFN-gamma and IL-2 in vaccinated mice were detected by ELISPOT, 10 - 14 days after immunization. The CTL and the level of IFN-gamma induced by rHB vaccine derived from yeast cells (Hansenula polymorpha) (rHP vaccine) were significantly higher than the other two vaccines (P < 0.05). The maximum lysis of CTL appeared in the vaccinated mice on day 10 after immunization, with the percentage of 39.8%. The levels of IL-2 induced by rHP vaccine were significantly higher than the other two vaccines (P < 0.05). However, the IL-2 levels in the rSC (saccharomyces cerevisiae) vaccine group were higher as compared with the rCHO vaccine group at day 7 and day 14 (7 d t = 4.595, P = 0.001 < 0.05; 14 d t = 5.721, P = 0.000 < 0.05) after immunization. The cellular immune response to the rHP vaccine was the strongest while it was the lowest to the rCHO vaccine at day 7 after immunization. The sero-positive rates and the titers of anti-HBs in the vaccinated mice increased with time after vaccination. The titers of anti-HBs in the rCHO vaccine group at day 7 were similar to the rSC vaccine group, but significantly higher than that of the rHP vaccine group (P = 0.044 < 0.05). The anti-HBs titers of the rCHO vaccine group at day 14 were significantly higher as compared to the rSC (P = 0.012 < 0.05) and rHP (P = 0.009 < 0.05) vaccine groups.

CONCLUSIONThe immune responses induced by the three kinds of rHB vaccines were different in their patterns and levels. According to the intensity of early cellular immune response, the two yeast HB vaccines were superior to the rCHO vaccine, especially to the rHP vaccine. In contrast, the rCHO vaccine induced early seroconversion and high levels of anti-HBs.

Animals ; Antibodies, Viral ; blood ; Cytotoxicity, Immunologic ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Hepatitis B Vaccines ; classification ; immunology ; Immunity, Cellular ; Immunity, Humoral ; Interferon-gamma ; immunology ; Interleukin-2 ; immunology ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, Synthetic ; classification ; immunology

UNLABELLEDObjective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).

METHODSThis retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.

RESULTSNo significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.

CONCLUSIONRescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.

Acrosome ; pathology ; Acrosome Reaction ; DNA Fragmentation ; Female ; Fertilization ; Fertilization in Vitro ; Humans ; Infertility ; Male ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

OBJECTIVETo study on the dynamics of peripheral blood lymphocytes and their subpopulations in patients with severe acute respiratory syndrome.

METHODSUsing flow cytometry, the absolute numbers of peripheral blood lymphocytes and their subpopulations in 240 SARS patients (696 specimens) and 51 individuals as controls, were counted and compared.

RESULTSThe absolute numbers of peripheral blood lymphocytes and their subpopulations (CD45, CD3, CD4, CD8) were 1298 +/- 785, 897 +/- 606, 510 +/- 372, 362 +/- 263/mm(3), respectively, significantly lower in SARS patients as compared to the normal controls (2024 +/- 423, 1391 +/- 289, 795 +/- 129, 551 +/- 183/mm(3)). Of SARS patients, severe group (1095 +/- 740, 740 +/- 562, 419 +/- 346, 304 +/- 244/mm(3)) had lower counts than that of mild group (1404 +/- 788, 991 +/- 612, 564 +/- 378, 396 +/- 267/mm(3)), and in group with deaths (587 +/- 493, 369 +/- 371, 204 +/- 191, 150 +/- 130/mm(3)) was lower than that of recovery group (1355 +/- 776, 948 +/- 603, 539 +/- 375, 382 +/- 263/mm(3)). There were significant differences (P < 0.01) for CD45, CD3, CD4, CD8, but with no significant difference (P > 0.05) for CD4/CD8 ratio between severe and mild, recovery and death groups. The lymphocytes and their subpopulations (CD45, CD3, CD4, CD8) declined in the 1st week and to the lowest level (977 +/- 579, 641 +/- 466, 360 +/- 275, 270 +/- 216/mm(3)) in the 2nd week. Then the lymphocytes and their subpopulations gradually increased during the recovery of the disease.

CONCLUSIONThe absolute numbers of peripheral blood lymphocytes and their subpopulations in SARS patients might be used as one of the methods for diagnosis on the severity and prognosis of the disease.

Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; blood ; Female ; Flow Cytometry ; Humans ; Lymphocytes ; classification ; immunology ; metabolism ; Male ; Middle Aged ; Severe Acute Respiratory Syndrome ; blood ; Time Factors

OBJECTIVETo analyze the detection rate and gene distribution characteristic of deletional α-thalassemia in Guangxi area, and to provide theoretic basis for thalassemia gene diagnosis and genetic counseling.

METHODSThe regular gene diagnosis of 3 types of α-thal (-- (SEA),- α(3.7),- α(4.2)) was performed by gap-PCR, multiple ligation probe and gene sequencing for globin α or β were used to detect those samples whose genotype and phenotype were not consistent. And the distribution characteristic of α-thalassemia gene in Guangxi area was then analyzed.

RESULTSOut of 51 191 suspected thalassemia patients, there were 19 853 cases of deletional a-thalassemia, accounted for 39.9% in total positive rate, including 19 780 cases of regular types(--(SEA), - α(3.7), - α(4.2)), 61 cases of Thailand-type deletion, 9 cases of triplet type (Hong Kong) (ααα(HK)), 1 case of 21.9 kb deletion type and 2 cases of 809 bp deletion type.

CONCLUSIONTypes of deletional a-thalassemia were complex and accounted for large proportion in Guangxi area. Special gene diagnoses were needed for those couples whose genotype and phenotype were not consistent, in order to provide reliable basis for genetic counseling and prenatal diagnosis.

China ; Genotype ; Humans ; Phenotype ; Polymerase Chain Reaction ; Sequence Deletion ; alpha-Thalassemia ; genetics