Objective To isolate and identify the adult neural stem cells from the parenchyma of spinal cord in adult mouse.Methods The parenchymal spinal cord from adult mouse was dissected and dissociated by mechanical trituration.The tissue suspension was cultured in serum-free DMEM/F12 medium supplemented with EGF and B27.The cell colonies generated from a single cell were screened by limited dilution and incubated with BrdU.The cell colonies were transferred into medium with serum to induce differentiation.The cells were identified with antibodies to Nestin,BrdU,MAP2 and GFAP by immunofluorescence staining.Results The cells were cultured for seven days to generate proliferative neurospheres.The majority of cells in these neurospheres expressed Nestin and were differentiated into MAP2-positive cells and GFAP-positive cells in medium containing with fetal bovine serum.Conclusion A significant number of neural stem cells are present in the parenchymal adult mouse spinal cord and can proliferate and also give rise to neurons and glia in vitro.

OBJECTIVETo analyze the surgical treatment result and clinical characteristics of hilar cholangiocarcinoma in order to improve the rate of early diagnosis and radical resection.

METHODSBetween 1986 and 2004,84 hilar cholangiocarcinoma patients underwent surgery, and their data were retrospectively reviewed.

RESULTSAccording to the Bismuth-Corlette staging system, 7 were type I, 18 type II, 22 type II a, 12 type IlI b, 20 type IV and 5 unclassified. 32 patients (38.1%) had had the history of operation for cholelithiasis before or were found to have cholelithiasis simultaneously at the time of diagnosis. The rate of making correct diagnosis by ultrasound, CT and MRCP was 71.4% , 84.0% and 91.4% , respectively. Of these 84 patients, 24 (28.6%) underwent radical resection, 14 (16.7%) palliative resection and 30 (35.7%) only internal or external drainage, while 16 patients was found to have contraindication for any further surgical intervention. The overall operation rate was 81.0% (68/84) with a radical resection rate of 35.3% (24/68). The 1-, 3- and 5-year survival rates was 70.8%, 50.0% and 20.8% in the radical resection group, and 50.0%, 21.4% and 0 in the palliative resection group, respectively. There was a statistically significant difference in the survival between two groups. Whereas in the internal or external drainage group, the 1-, 3- and 5-year survival rates was 20.0% ,10.0% and 0. All of the patients who did not undergo surgical intervention died within one year.

CONCLUSIONCholelithiasis may play an important role in the pathogenesis of hilar cholangiocarcinoma. Early diagnosis and radical resection are two important factors to improve the prognosis of hilar cholangiocarcinoma. Skeletonization of hepatoduodenal ligament with partial liver resection can improve the rate of radical resection for hilar cholangiocarcinoma.

Adenocarcinoma ; diagnosis ; surgery ; Adenocarcinoma, Mucinous ; diagnosis ; surgery ; Adult ; Aged ; Aged, 80 and over ; Bile Duct Neoplasms ; diagnosis ; surgery ; Bile Ducts, Intrahepatic ; Biliary Tract Surgical Procedures ; methods ; Cholangiocarcinoma ; diagnosis ; surgery ; Drainage ; methods ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Survival Analysis

Objective To investigate the treatment effects of operation and nerve growth factor on adult multisegmental cervical spinal cord injury without fracture dislocation.Methods Sixty-eight patients with multiple segmental cervical spinal cord injury without fracture dislocation,admitted to our hospital from January 2004 to May 2011,were chosen in our study; according to the will of the patients,18 patients received conservative treatment (group A),25 patients were treated by posterior single open-door laminoplasty (group B) and the other 25 patients were treated with posterior single open-door laminoplasty combined with nerve growth factor (group C,once daily for 4 weeks); their clinical data were retrospectively analyzed.Follow up was performed at 3,6 and 12 months after the treatments;Japanese Orthopaedic Association (JOA) scale was employed to evaluate the clinical efficacy.Results Three,6,12 months after the treatments,the JOA scale scores were statistically different among the three groups (P＜0.05); the scores in group B and C were significantly higher than those in group A at all time points (P＜0.05); 6 and 12 months after the treatmemts,the JOA scale scores in group C were significantly higher than those in group B (P＜0.05).Conclusion To patients with multisegmental cervical spinal cord injury without fracture dislocation,conservative treatment can make the spinal cord function partially restored,but the effect is limited; exogenous nerve growth factor has good repairing effect on spinal cord injury.

OBJECTIVETo develop a real-time fluorescent PCR protocol suitable for the routine screening of AZFc/DAZ microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients.

METHODSA set of real-time fluorescent PCR was established. Eighty-seven azoospermic and ligozoospermic patients undergoing ICSI in the IVF center and 30 azoospermic men undergoing testicular biopsy in the clinic of urology surgery were screened for AZFc/DAZ microdeletions of Y chromosome.

RESULTSEleven cases (9.4%) of AZFc/DAZ microdeletions were found in 117 cases of azoospermic and oligozoospermic patients by screening of realtime fluorescent PCR. Four cases (6.6%) were found in 61 oligozoospermic patients, and 7 cases (12.5%) were found in 56 azoospermic patients.

CONCLUSIONThe real-time fluorescent PCR protocol presented in this study is an easy and reliable method for detection of AZFc/DAZ microdeletions on the Y chromosome, which yields identical results to those of the multiplex PCR.

Chromosome Deletion ; Chromosomes, Human, Y ; Deleted in Azoospermia 1 Protein ; Fluorescence ; Humans ; Infertility, Male ; genetics ; Male ; Polymerase Chain Reaction ; methods ; RNA-Binding Proteins ; genetics

OBJECTIVETo develop a multiplex PCR protocol, which could be suitable for routine screening of microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients.

METHODSFive multiplex sets were established. Eighty-seven azoospermic and oligozoospermic patients undergoing intracytoplasmic sperm injection (ICSI) in the in vitro fertilization (IVF) center and 30 azoospermic men undergoing testicular biopsy in the clinic of Urology Surgery were screened for microdeletions of Y chromosome.

RESULTSA total of 19 (16.2%) cases of microdeletions were found in 117 azoospermic and oligozoospermic patients by screening of Y chromosome microdeletions. Of these, 11 cases (18.0%) were found in 61 oligozoospermic patients, and 8 cases (14.3%) were found in 56 azoospermic patients.

CONCLUSIONThe multiplex PCR protocol presented in this study is an easy-to-do and reliable method for detecting microdeletions on the Y chromosome. Routine screening of microdeletions on the Y chromosome for azoospermic and oligozoospermic patients is essential.

Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; Female ; Genetic Testing ; methods ; Humans ; Infertility, Male ; diagnosis ; genetics ; Male ; Polymerase Chain Reaction

OBJECTIVETo study the inducible expression of hypoxia-inducible factor 1alpha (HIF-1alpha) on the proliferation and invasion property of HepG2 cells under normoxia in vitro.

METHODSConstructed the HepG2(Tet-on-HIF-1alpha) cell line which could induce the expression of HIF-1alpha by doxycycline; Under normoxia in vitro, MTT assay was used to observe the proliferative and adhesive activity of cells, and the invasive activity was determined by transwell cell culture chamber method.

RESULTSUnder normoxia, the HIF-1alpha mRNA and protein of HepG2(Tet-on-HIF-1alpha) cells could be induced up to (5.899 +/- 2.176) and (2.179 +/- 0.742) folds by doxycycline (1 microg/ml); There were no difference of A(490 nm) between the Dox(+)and Dox(-) group in experiment detecting the proliferation activity (P > 0.05); But in adhesive experiment, the A(490 nm) of Dox (+) group was 0.662 +/- 0.058, higher than the Dox(-) group 0.526 +/- 0.808 (P = 0.008); The invasive cell number of Dox(+) group was 37.611 +/- 8.424, but in the Dox(-) group, the number was 25.333 +/- 8.117 (P < 0.01).

CONCLUSIONSUnder normoxia in vitro, the Tet-on gene regulate system could increase the HIF-1alpha protein by inducing the HIF-1alpha mRNA; HIF-1alpha has no influence with the proliferation activity, but it could enhance the adhesive and invasive properties of HepG2 cells.

Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cell Survival ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; physiology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Neoplasm Invasiveness ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the quantities of monocyte-derived dendritic cell precursors (pDC1) and plasmacytoid dendritic cell precursors (pDC2) in peripheral blood mononuclear cells (PBMC) of severe aplastic anemia (SAA) patients before and after immune suppressive therapy (IST), the ratio of the pDC1 to pDC2, and the expression of co-stimulating molecules (CD80, CD86, CD40) on dendritic cells (DC) and B cells in SAA patients.

METHODSBy means of three color monoclonal antibody labeling technology, the quantities and ratio of pDC1 and pDC2 in PBMC were detected in 26 SAA patients at active phase, 13 at recovery phase and 15 normal controls respectively. The aforementioned parameters of 10 SAA patients were tested before and 2 months after IST. The expression of CD80, CD86 and CD40 on DC and B lymphocytes were detected in 16 SAA patients and 15 normal controls.

RESULTSThe percentages of pDC1 and the ratio of pDC1/pDC2 of controls were (0.41 +/- 0.05)% and 1.58 +/- 0.18 respectively, and those of SAA patients at active phase were (0.67 +/- 0.13)% and 2.70 +/- 0.32 respectively, [pDC1 (P < 0.05); pDC1/ pDC2 ratio (P < 0.01)]. The aforementioned parameters in convalescent SAA patients decreased to (0.43 +/- 0.10)%, and 1.78 +/- 0.36 respectively, being no difference from those of normal controls. The percentages of pDC1 and pDC2 in 10 SAA patients were (0.87 +/- 0.31)%, and (0.35 +/- 0.09)%, before IST, and (0.24 +/- 0.09)%, (0.14 +/- 0.04)%, after IST, being significantly decreased (P < 0.05). The percentages of CD86 expression on DC of controls was (11.97 +/- 4.31)%, and that of SAA patients was (29.84 +/- 3.02) % (P < 0.05). The percentages of CD80, CD40 and CD86 expression on lymphocytes of controls were (2.57 +/- 0.44)%, (7.34 +/- 1.22)% and (1.86 +/- 1.11)%, respectively, and those of SAA patients were (5.17 +/- 0.68)%, (8.85 +/- 2.94)% and (5.98 +/- 0.96)% respectively (P < 0.05, P < 0.01). The percentage of CD86 expression on B lymphocytes in controls was 8.04 +/- 0.66%, and in SAA patients was (20.46 +/- 2.78)%, (P < 0.05).

CONCLUSIONThe pDC subtypes were abnormal and the percentage of pDC1 is increased in SAA patients, which are associated with stage of this disease. DC and B Lymphocytes in SAA patients upregulated expression of costimulatory molecules (CD86) which cause the T lymphocyte abnormally activated.

Adolescent ; Adult ; Anemia, Aplastic ; immunology ; B-Lymphocytes ; immunology ; metabolism ; B7-1 Antigen ; blood ; B7-2 Antigen ; blood ; CD40 Antigens ; blood ; Case-Control Studies ; Child ; Convalescence ; Dendritic Cells ; immunology ; metabolism ; Female ; Flow Cytometry ; Humans ; Male ; Middle Aged

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

OBJECTIVETo investigate the types and therapies of malignancies in renal allograft recipients.

METHODSWe retrospectively analyzed the occurrence, types, and therapies of malignancies in 498 renal allograft recipients who had received operations in Peking Union Medical College Hospital from May 1986 to October 2008.

RESULTSAmong 498 renal allograft recipients, 18 patients (3.6% ) were diagnosed with malignancies, which included bladder cancer (n = 5), renal pyloric cancer or ureteric cancer (n = 4), leukemia or lymphoma (n = 3), hepatic cancer (n = 2), skin cancer, rectum carcinoma, pulmonary carcinoma and thymoma (n = 1 each). Surgical operations were performed in 10 cases, 6 of whom survived with normal renal function and had no rejection of transplanted kidneys. Three patients with bladder cancer and one patient with ureteric cancer experienced recurrences 7 to 15 months after operations; among them one bladder cancer patient died. One hepatic carcinoma patient died of pulmonary metastasis 8 months after operation. One non-Hodgkin's lymphoma patient died 11 months after chemotherapy. Five cases with advanced unresectable malignancies died 8 to 17 months after the diagnosis.

CONCLUSIONSThe incidences of malignancies, especially urological epithelial carcinoma, are high in renal allograft recipients. Radical surgery of the solid malignancies is a preferred option.

Adult ; Aged ; Female ; Humans ; Kidney Transplantation ; Male ; Middle Aged ; Neoplasms ; epidemiology ; therapy ; Postoperative Complications ; epidemiology ; therapy ; Retrospective Studies

OBJECTIVESTo investigate the expression of P120 catenin in pancreatic carcinoma and to explore the association between P120 catenin gene polymorphism at T755G position and pancreatic carcinoma.

METHODSThe expression of P120 catenin in 52 cases of pancreatic carcinoma and normal pancreatic tissues on the mRNA and protein levels were evaluated by RT-PCR and Western Blot methods respectively. P120 catenin gene polymorphism at T755G position of in 52 patients and 60 healthy controls were examined by PCR-restriction fragment length polymorphism (PCR-RFLP) technique.

RESULTSThe mRNA and protein expressions of P120 catenin in pancreatic carcinoma tissues were significantly lower than normal pancreatic tissues (P=0.000, P=0.002). Reduced expression of P120 catenin mRNA was significantly correlated with differentiated (P=0.033), lymph node metastasis (P=0.004), vascular invasion (P=0.022), and pTNM stage (P=0.003). Additionally, there were significant difference of P120 catenin gene polymorphism genotypes and alleles at T755G position between patients and healthy controls (P=0.008, P=0.016). The GG genotype of P120 catenin gene was associated with higher risk of incidence for pancreatic carcinoma compared with the TT genotype (OR=2.765, 95%CI=1.312-3.958).

CONCLUSIONSThe reduced expressions of both P120 catenin mRNA and protein in pancreatic carcinoma suggest its association with pancreatic carcinoma development. Polymorphism of P120 catenin gene at T755G situation might be a risk factor for pancreatic carcinoma, and it may be used to diagnosis and prevent pancreatic carcinoma early.

Case-Control Studies ; Catenins ; genetics ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Pancreatic Neoplasms ; genetics ; metabolism ; Polymorphism, Genetic