0.05) in results. The positive rate was 26.99% for these 3 kits, and any two of them could achieve a positive rate of 53.95%. The total positive rate was 80.95%, which was significantly different as compared with non-tuberculosis group(P

Objective To study two new factor Ⅸ mutations Cys82Ser and Ile288Ser in vitro and research the molecular mechanism of haemophilia B. Methods PcDNA3. 1 ( - ) FⅨwt expression plasmid was prepared. The mutated FⅨcDNA expression plasmids, PcDNA3.1 ( - ) FⅨM1 (Cys82Ser) and PcDNA3. 1 ( - ) F Ⅸ M2 (Ile288Ser) were constructed by megaprimer method respectively. Transient expression experiments were performed using HEK293 cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA. PcDNA3. 1 ( - ) was used as a blank control. The expression proteins were detected by ELISA, factor activity assay and flourescence stain. Results The results suggested that the two FⅨ gene mutations did not induce the reduction of the mutant FⅨ mRNA compared with the wild-type FⅨ mRNA. The FⅨ:Ag in culture media and cell lysate of wild type conduct were assigned as 100. 0%. The results of PcDNA3.1 ( - ) FⅨ M1 mutation protein were (27. 1 ± 5. 2)% and (99.4 ±4. 1)% respectively. For PcDNA3. 1( - )FⅨM2, the results were (5.3 ± 1.8)% and (31.7 ±2. 5)% respectively. The FⅨ: C in culture media of wild type conduct was also assigned as 100. 0%. Then the two types of mutant protein were ( 8. 5 ± 3.2 ) % and ＜ 1%, respectively. Immunofluorescence microscopy result suggested that the intensity of perinuclear spot was reduced in cells transfected with PcDNA3.1 ( - ) FⅨM2 while staining for PcDNA3. 1 ( - ) FⅨM1 was predominantely diffuse without perinnclear enhancement. Conclusions These results strongly suggest that the FⅨ Cys82Ser mutation protein is not been correctly folded, by any possibility. The mutation protein has secretion defect. The secretion dysfunction and the protein degradation intracellular are possiblely the molecular pathology of Ile288Ser mutant protein.

Objective To detect the expression of hypoxia-inducible factor 1α (HIF-1α) and bcl-2 ovarian serous carcinoma and their clinical significances. Methods Paraffin specimens including 61 cases of ovarian serous carcinoma and 50 normal ovarian tissues were selected. The expressions of HIF-1α and bcl-2 proteins were detected by immunohistochemical EnVision method and their relationship between them was analyzed. Results The positive rate of HIF-1α and bcl-2 proteins expression in 61 ovarian serous carcinoma was 68.9%and 54.1%, respectively. There was a significant difference between the two groups (χ2=55.381, P< 0.05; χ2= 38.493, P< 0.05). The clinical pathological parameters showed that the positive expression of HIF-1αand bcl-2 proteins were not related with the age (P>0.05). HIF-1αpositive expression was correlated with tumor grades, the state of lymph node metastasis and FIGO stages (χ2=4.931, 25.008, 5.610, P<0.05). Bcl-2 was significantly associated with tumor grades and lymph node metastasis (χ2= 6.956, 33.869, P<0.05), but not with FIGO stages (χ2=3.391, P>0.05). The expression of bcl-2 was positively correlated with HIF-1α in ovarian serous carcinoma (r= 0.304, P= 0.017). Conclusions The expressions of HIF-1α and bcl-2 play a synergic role in the progression of ovarian serous carcinoma. The combined detection of HIF-1αand bcl-2 is effective for patients'prognosis judgment.

Objective: To study the expression of negative costimulatroy molecule B7-H4 in non-small cell lung cancer (NSCLC) tissues and its relationship with the clinical features of NSCLC. Methods: Fifty-two NSCLC specimens from pa-tients who were pathologically diagnosed in our hospital during January 2008 to April 2009 were included in the present study. B7-H4 expression and infiltration of CD3~+ T cells in NSCLC tissues were detected by immunohistochemistry. The correlation between B7-H4 expression, CD3~+ T infiltration, and the clinical features of NSCLC was studied. Results: The positive rate of B7-H4 in 52 NSCLC tissues was 48.08% (25/52), and B7-H4 expression in normal lung tissues was neg-ative or low (P <0.05). B7-H4 expression was positively correlated with the clinical tumor stages and lymph node metas-tasis of NSCLC (P < 0.05), and negatively correlated with tumor infiltration of CD3~+ T cells (P < 0.05), but had no re-lationship with clinicopathologie parameters of NSCLC (P > 0.05). Conclusion: Negative costimulatroy molecule B7-H4 may play important roles in the development of NSCLC. Positive expression of BT-H4 is correlated with the clinical tumor stages and lymph node metastasis of NSCLC, which provides a foundation for diagnosis and therapy of NSCLC.

BACKGROUND: Some researches suggest that placenta is regarded as a new source of mesemchymal stem cells (MSCs). Adherent cells derived from placenta tissue have similar morphological characteristics and surface markers to MSCs; meanwhile, they can differentiate into osteoblasts and nerve cells.OBJECTIVE: To find a new source and way for MSCs separation.DESIGN: Observational study.SETTING: The Third People's Hospital of Wuxi.MATERIALS: Placenta was sourced from cesarean in our department of obstetrics and gynecology and provided confirmed consent from the relatives and ethics committee. The main reagents contained culture medium, sABC kit, DAB staining kit, caprine-anti-rat FITC, recombinant human basic fibroblast growth factor (rh-bFGF), rabbit-anti-human glial fibriliary acidic protein (GFAP).METHODS: The experiment was carried out in the Laboratory of Cells & Molecular Biology, the Third People's Hospital of Wuxi from May 2005 to August 2006. Placenta entity tissue was digested, adherent cells were cultured, morphological characteristics were observed, and growth curves of placenta-derived multipotent cells (PDMCs) in various generations were drawn. On the 1st and 7th days, supernatant was derived from cells in primary culture to measure content of β-glycerophosphate disodium (β-HCG) with chemiluminescence technique. In addition, expression of surface antigen and differentiating potency were detected at the same time. At 24 hours (at phase of nerve cells) and 2 weeks (at phase of osteoblasts) after inducible differentiation, cultured cells were dealt with routine immunocytochemical stain, and then they were observed under routine microscope or fluorescence microscope.MAIN OUTCOME MEASURES: ① Morphological indexes and growth curves of PDMCs; ② measurement of β-HCG in supernatant of PDMCs and expression of antigen of PDMCs with flow cytometer; ③ immunohistochemical analysis of PDMCs during and after inducible differentiation.RESULTS: ① Primary culture of PDMCs: After digestion of placenta tissue, a few of adherent cells were obtained and gradually formed thin and flat monolayer cells two weeks later. The monolayer cells grew like whirlpool or cluster. With the increase of cell density, soma was slenderer and slenderer and like fibroblast. ② Growth curves of PDMCs: Growth latency ranged from 2 to 8 days after cell inoculation. During this period, adherence was observed gradually but not obviously amplified. Eight days later, cells entered log growth phase. During this period, proliferation was active and cell process surrounding extended under phase contrast microscope. A lot of MSCs were observed in division phase between two nuclei. In addition, density was increased and cells connected to each other. Within 11-14 days, growth curve gradually entered platform phase. MSCs covered the bottom of bottle, cells slowly expanded, and primary culture stopped. ③ Measurement of β-HCG: Expression of β-HCG was not detected in supernatant at two time points. ④ Characteristics of surface antigen of PDMCs: PDMCs could express CD29, CD44 and CD105, but not CD34, CD45,CD19 and CD106. ⑤ Inducible differentiation of PDMCs: At 24 hours after inducing to nerve cells, form of PDMCs obviously changed, soma rebounded, refraction of nucleus was partially reinforced, structure was similar to dendrite and axis-cylinder, and positive neuro-specific enolase and GFAP were observed after staining.CONCLUSION:Placenta tissue contains PDMCs whose morphological function is similar to MSCs; in addition, placenta is regarded as an effective source of MSCs.

In order to better fulfill the tasks of research,to turn out more quality papers,to produce outstanding results,and to further strengthen management and supervision of scientific research,the"quantitative economic management of scientific research quotas" was established in the hospital.Applying of the measure in scientific research management in the past eight years it was shown that the desired results were achieved,the academic advancement and the personnel growth were greatly promoted.

Objective To identify the clinical phenotypic diagnosis and gene mutation detection of two kindreds with PS deficiency. MethodsPS: A was measured by chromogenic substrate method;TPS:Ag, FPS: Ag levels were measured by ELISA method; PS gene(PROS1 gene)was detected by amplifying 15 exons and flanking intron sequences from the propositus with PCR method. PCR products were purified and directly sequenced. Results For propositus 1,PS: A was 48.6% ,TPS: Ag was 136 mg/L, FPS : Ag was 41 mg/L, PROSI gene exon 2 was in c. Heterozygous base substitutions was detected in C121T locus, which led to Arg-1Cys (R-1C) heterozygous roissense mutation encoded in PS proteins. For propositus 2, PS: A was 29.2%, TPS: Ag was 83 mg/L, FPS: Ag was 26 mg/L, PROSI gene exon 14 was in c. Heterozygous base substitutions was identified in CI687T locus, in which Gln.522Stop heterozygous nonsense mutation was encoded in PS proteins. Conclusions c. C121T is a novel mutation locus detected in PROS1 gene. This heterozygous mutation could lead to type Ⅱ PS hereditary deficiency, while c. C1687T heterozygous mutation could bring about type Ⅰ PS hereditary deficiency.

0bjective To make genetic and prenatal diagnosis of a female with Haemophilia A.Methotis The FⅧ:C.BT and VWF were detected to make phenotypic diagnosis.LD-PCR was adopted for screening the intron 22 inversion and PCR was adopted for the screening the intron 1 inversion.The coding and boundary sequences of FⅧgene were analyzed by PCR and DNA equencing.Eight combined polymorphie markers(Amelo,F8IVS13,CA22,DXS15,DXS9901,G6PD,DXS1073 and DXS1108)were applied for linkage analysis of the family by multiplex fluorescent PCR.The polymorphism of DXS52 (ST14)was analyzed by PCR and electrophoresis. Assessment of X inactivation was performed using an Hpa II-polymerase chain eaction (PCR)assay for the X-inked human androgen receptor gene(HUMARA). Results The female HA patient showed severe FW deficiency(FⅧ:C 2.1%)and other phenotypie tests were normal.Her family members showed normal in all tests.The female proposita was found to be a carrier of FW gene intron 22 inversion.But her family members as well as her etus showed negative results.Except this inversion,no other mutation Wag found then.The female inherited two X chromosomes from both her parents' and her fetus inherited the maternally derived X chromosome from the female proposita according to the linkage analysis.Furthermore.X-inactivation paRern of the female was unbalanced and her aternally derived X chromosome Wag inaetived mostly while the majority of her paternal derived one kept active.Conclusions The severe haemophilia A in the proposita resulted from the de novo Ⅷ intron 22 inversion which most probably arose in the paternal germ line.Associated with a skewing pattern of inactivation of the maternally derived X chromosome.Her etus is normal female.

Objective To investigate function of Arg327Ile (R327I) and Arg327Ala(R327A) FⅨ mutants and to study the molecular pathogenesis of haemophilia B(HB) caused by R3271 mutation.Methods Hygromycin-resistant cell line was screened and the secretion of FⅨ antigen into the medium was measured by ELISA.The cell line with appropriate expression levels of F Ⅸ antigen was selected for culture.Recombinant F Ⅸ (rF Ⅸ ) was purified from concentrated medium by two step methods of Q-Sepharose Fast Flow and anion exchange chromatography.The concentration and purity of rF Ⅸ were determined by ELISA and SDS-PAGE,respectively.The activation of wild-type ( WT),R327I and R327A of rFⅨ by FⅦa/TF/Ca2+ or FⅪa/Ca2+ was identified by Western blot in different time periods.The FⅨa and FⅧa complex formed by interaction with different concentrations of FⅧa was used to activate F X,the apparent dissociation constant (Kd) for FⅧa binding was calculated by the kinetic results.The kinetic data of the activation of FX by WT,R327I and R327A FⅨa with or without FⅧa were calculated.Results The amount of WT,R327I and R327A rFⅨ were 450,210,64 μg,and the purity of rFⅨ was confirmed by SDSPAGE.Both R3271 and R327A could be normally activated by FⅧa/TF/Ca2+ or FⅪa/Ca2+.Kd for FⅧa binding showed that the binding capacities of R327I and R327A were 4 and 5 times lower than WT,respectively.The catalytic efficiencies of R327I and R327A F Ⅸ a for F X were 6 and 8 times lower with FⅧa,and 3 and 7.4 times lower without F Ⅷ a,respectively.Conclusions R327I and R327A rF Ⅸ mutants impair their binding to the FⅧa.The site on R327 contributes to FⅧa binding.It is partly related to the activation of FX.The low FⅧa binding to R327I FⅨa may cause HB.

OBJECTIVE:To evaluate the management of the free anti-TB drugs in He'nan province,and to analyze ex-periences and find out the gap.METHODS:Based on the requirements of the Chinese TB Control Program-Free anti-TB drug management manual,a questionnaire comprised of 17 items was developed and 20 TB drug storerooms at city or county level were randomly sampled for on the spot investigation.RESULTS:Of the total drug storerooms investigated,75% had yearly drug demand plan,but only 35% was up to the standard in inventory control,95% had no expired drug,90% had inventory/supply vouchers and detailed inventory records,only 25% achieved conformity between records and physical counts.The condition of drug storerooms and the storage of drugs were unable to meet the requirement.CONCLUSION:Tuberculosis Control Agency at different levels haven't paid due attention to the management of free anti-TB drugs.The personnel in this agency should raise their drug management responsibility from aspects of saving public belongings and ensuring patients' medication quality.