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.

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.

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.

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.

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 investigate the genetic diagnosis and molecular pathogenesis of four patients with combined deficiency of coagulation factor Ⅴ and Ⅷ and their family members. Methods The APPT, FT, FⅤ: C, FⅧ: C were detected for phenotypic diagnosis. Thrombin generation assay was applied to determine the generation condition of thrombin in patients and healthy controls. Cenomic DNA was extracted from peripheral blood using the TianGen RelaxCene Blood DNA System;amniotic fluid DNA was extracted with phenol-ethyl ether method. The LMAN1 and MCFD2 genes were analyzed by PCR. Gene mutations were detected with nucleotid sequences by using end-labeling dideoxy method. Results The APTT of Proband 1 was significantly prolonged to 88. 2s and her PT was prolonged to 19. 6 s. The combined deficiency was identified with FⅧ (FⅧ: C 24. 2% ) and FV(FⅤ: C 9. 1% ). Proband 2 and 3 were sisters. The coagulation studies revealed that both of them had prolonged APTT (71.6 s and 74.6 s respectively) and PT (22. 1 s and 18. 3 s respectively). The combined deficiency of FⅤ (FⅤ: C 7. 6% and 14. 5% respectively) and FⅧ( FⅧ: C 25% and 19.6% respectively) were identified. Proband 4 was detected to have the prolonged APTT (70.3 s),PT (18.2 s) and the deficiency of FⅤ(FⅤ: C 9. 4% ) and FⅧ (15. 7% ). The remaining phenotype indicators test of the 4 probands were normal. The diagnosis for the 4 probands was combined deficiency of factor Ⅴ and Ⅷ. The proband 1 was detected to have compound heterozygous mutations in LMAN1 gene while having the LMAN1 and MCFD2 direct gene sequencing. One mutation was a small insertion located on exon 8 [ nt912insA (X71661. 1)] that resulted in p. 305frameshiftX20 and her mother was detected to have the same heterozygous mutation on the the locus. The other mutation was located on exon 11: nt1366C > CT ( X71661. 1 ) , p. 456Arg > Stop which was inherited from her father. Amniocyte DNA was detected to have only one heterozygous mutaion [nt1366C > CT (X71661. 1) , 456Arg > Stop] inherited from the father. No mutation in MCFD2 gene was found in proband 1 and her parents. The analysis of the MCFD2 gene in proband 2 and 3 revealed a novel homozygous single base substitution (nt411T>C) in exon 4, which results in the exchange of the amino acid isoleucine by the amino acid threonine at amino acid position 136 (p. Ile136Thr). Sequencing of the whole LMAN1 gene showed that the proband 4 had one homozygous nonsence mutation in the exon 5 of the LMAN1 ( nt615C >T,p. 202 Arg> Stop). All of the 4 probands with combined deficiency of FⅤ and FⅧ showed declined endogenous thrombin potential in the thrombin generation tests. Conclusion The combined deficiency of FⅤ and FⅧ in the proband 1 results from the compound heterozygous mutations ( nt1366C > CT and nt912insA) in LMAN1 gene, which are inherited from her parents respectively. The prenatal genetic investigation for the patient mother with preganency indicates that the fetus is a female carrier with one mutation (nt1366C > CT) inherited from the father. The homozygous missence mutation ( nt411T > C, p. Ile136Thr) in the MCFD2 gene accounts for the proband 2 and 3. The daughter of the proband 2 is a carrier with a heterozygous mutation inherited from her mother. The homozygous nonsence mutation in the LMAN1 gene of the proband 4 results in the deficency of F Ⅴ and FⅧ.

Objective To identify the gene mutations of platelet membrane glycoprotein Ⅱ b,Ⅲa(GPⅡb/Ⅲa)in three Chinese pedigrees with Glanzmann thrombastIlenia.Methods All exons and exonintron boundaries of GP Ⅱ b/Ⅲ a gene were amplified by PCR analysis followed by DNA sequencing.DNA sequencing was used to exclude gene polymorphisms.Results The probands in the three pedigrees had a normal platelet count,coagulation profiles,scattered platelets on the blood film,a prolonged cutaneous bleeding time,and impaired or minimal ex vivo platelet aggregation in response to ADP,thrombin,collagen,adrenaline and arachidonic acid,but normal platelet aggregation in response to ristoeetin.Both FACS and Western blotting demonstrated trace content of αⅡb in the platelets from proband 1 and proband 3,who were classified as type Ⅰ GT,and a small amount of αⅡb in the platelets from proband 2,who was classified as type Ⅱ GT.Compound heterozygous mutations,T2255G(Leu721Arg)and C2671T(Gln860Stop)were identified in proband 1.The proband 2 had homozygous A2334C(Gln747Pro)missense mutation.Nonsense mutations C1750T (Arg584Stop)and 69-79 deletion mutation were identified in proband 3. Conclusions Compound heterozygous mutations T2255G and C2671T of αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 1. Homozygous mutation A2334C of αⅡb gene leads to type Ⅱ Glanzmann thrombasthenia for proband 2. Compound heterozygous mutations C1750T and 69-79del αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 3. T2255G,C1671T and 69-79del aye novel mutations for αⅡb gene.