The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.

Adult ; Aortic Valve ; microbiology ; pathology ; Autopsy ; Brain ; microbiology ; pathology ; Endocarditis, Bacterial ; complications ; microbiology ; pathology ; Female ; Heart Ventricles ; microbiology ; pathology ; Humans ; Mitral Valve ; pathology ; Sepsis ; complications ; microbiology ; pathology ; Substance Abuse, Intravenous ; complications ; microbiology ; pathology ; Young Adult

OBJECTIVETo observe the effects of co-transfection of platelet derived growth factor antisense oligodeoxynucleotides (PDGF-AODN) and tissue-type plasminogen activator (tPA) gene on inhibition of intimal proliferation of auto-transplantion artery.

METHODSOne hundred male New Zealand rabbits were randomly divided into four groups (25 in each): Group A (control group), Group B (PDGF-AODN transfection group), Group C (tPA gene transfection group) and Group D (PDGF-AODN and tPA co-transfection group). The left and right external iliac arteries were transplanted reciprocally. The transplanted arteries were respectively soaked in PDGF-AODN, pBudCE4.1/tPA and PDGF-AODN plus pBudCE4.1/tPA solution about 15 minute before transplantation. The rabbits were sacrificed at 3d, 1w, 2w, 4w and 8w after operation. The specimens were harvested for pathologic examination, electron microscopy, chromogenic substrate test, 3H-TdR incorporation test and immunohistochemical staining.

RESULTThe scan electron microscopy showed that there were a few thrombocytes on vas-wall of Group C and D without thrombus, whereas there were abundant thrombocytes and thrombus forming on vas-wall of Group A and B. The intimal area, stenosis ratio of transplanted artery, 3H-TdR incorporation,the number of PDGF positive cell in Group D were significantly less than those in Group A (P<0.01),Group B and Group C (both P<0.05). The activity of tPA gene products in transplanted vas-wall of Group D was significantly higher than that of Group A (P<0.01).

CONCLUSIONLocal co-transfection of PDGF-AODN and tPA gene can effectively inhibit the proliferation of vascular smooth muscle cells, hyperplasia of intima and restenosis of transplanted artery.

Animals ; Endothelium, Vascular ; pathology ; Graft Occlusion, Vascular ; prevention & control ; Hyperplasia ; prevention & control ; Iliac Artery ; transplantation ; Male ; Oligodeoxyribonucleotides, Antisense ; biosynthesis ; genetics ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Rabbits ; Random Allocation ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; Transfection ; Tunica Intima ; pathology

OBJECTIVETo study the effect of platelet-derived growth factor (PDGF) and proliferating cell nuclear antigen (PCNA) antisense oligodeoxynucleotides (AODN) together on inhibiting the proliferation of the stenosis of transplanted vascular.

METHODSThe left and right external iliac arteries (length 1.0 cm) of rabbits were transplanted reciprocally. The transplanted vascular were respectively soaked in liposomes, PDGF-AODN, PCNA-AODN and PDGF-AODN adding PCNA-AODN solution about 20 minute, the vascular anastomotic were sutured by 8/0 suture of soaked in AODN solution. Four weeks later, the specimens were harvested for microscopy. The pathological morphology of transplanted vascular were observed under microscope (HE). The intimal thickness and area, stenosis ratio(%) of transplanted vascular were calculate and analysed statistically among group by computer system. The number of positive cells of PDGF's mRNA in transplanted vascular wall were counted with in situ hybridization histo-cytochemistry and the number of positive cells of PCNA's protein in transplanted vascular wall were counted by S-P immunochemistry.

RESULTSThe intimal thickness and area, stenosis ratio of transplanted vascular, the number of PDGF and PCNA positive cell in PDGF-AODN adding PCNA-AODN group were significantly lower than those in other group (P < 0.01), and that were lower evidently than PDGF-AODN group and PCNA-AODN group.

CONCLUSIONPDGF and PCNA antisense oligodeoxynucleotides together could significantly inhibit the proliferation of vascular smooth muscle cell and stenosis of transplanted vascular.

Animals ; Graft Occlusion, Vascular ; pathology ; prevention & control ; Iliac Artery ; pathology ; transplantation ; Male ; Oligonucleotides, Antisense ; genetics ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Rabbits ; Transfection ; Transplantation, Homologous

OBJECTIVETo observe the effects of local co-transfection vascular endothelial growth factor165 (VEGF165) and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia and restenosis in rabbits artery after operation injury and possible mechanisms.

METHODSMicrology operation injury was used to establish the model of intimal injury of right external iliac artery in rabbits. To select 120 male New Zealand rabbits and were randomly divided into 3 groups (n = 40, in each group): Group A (physiological brine control group), Group B (pBudCE4.1 group), Group C (pBudCE4.1/VEGF165-tPA group). The vas-wall of micrology operation injury were infused respectively physiological brine, pBudCE4.1 and pBudCE4.1/VEGF165-tPA transfection solution by micro-injector. Each group were divided into 5 subgroups (n = 8, in each subgroup) randomly according to the sacrifice times (2 d, 1 week, 2 week, 4 week and 8 week after operation). The injured vascular specimen were harvested for pathology test, electric microscopy study, reverse transcription-PCR examining and immunochemistry detecting.

RESULTSThe intimal area and narrow ratio of vases in Group C at every time point after operation were significantly lessened than that in Group A and Group B (P < 0.01). The narrow ratio of vases in Group C at 8 week after operation were decreased respectively by 57.9% and 59.0% than that in Group A and B. The expression of VEGF165 mRNA in Group C were increased significantly than that in Group A and B at every time point after operation (P < 0.01), the expression reached the peak at 1 week and continued to 4 week after operation. Immunohistochemical identified that tPA positive cell in Group C were significantly increased than that in Group A and B (P < 0.01) at every time point after operation.

CONCLUSIONLocal co-transfection VEGF165 and tPA genes could restrain intimal hyperplasia and restenosis of vas, which lay a foundation for future multi-gene therapy of vascular intimal hyperplasia.

Animals ; Arteries ; pathology ; Endothelial Cells ; cytology ; Hyperplasia ; prevention & control ; In Vitro Techniques ; Male ; Myocytes, Smooth Muscle ; cytology ; Plasmids ; Rabbits ; Random Allocation ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; Transfection ; Tunica Intima ; pathology ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo study the clinicopathologic features, immunohistochemical findings, diagnosis and differential diagnosis of atypical teratoid/rhabdoid tumors (AT/RT) of central nervous system in childhood.

METHODSThe clinicopathologic data, morphologic features and immunophenotypes were reviewed in 6 cases of AT/RT. EnVision method was applied. Antibodies include cytokeratin (CK), epithelial membrane antigen (EMA), vimentin, smooth muscle actin (SMA), muscle specific actin (MSA), glial fibrinary acid protein (GFAP), desmin, placental alkaline phosphatase (PLAP) and INI1.

RESULTSFive of the six cases of AT/RT occurred in infancy and early childhood. Histologically, the predominant component was rhabdoid cells. Cytoplasmic inclusions were present in all cases. Primitive neuroectodermal tumor (PNET) component was also identified in 5 of the 6 cases studied. Immunohistochemically, the tumor cells were positive for cytokeratin, epithelial membrane antigen and vimentin. The staining for INI1, desmin and PLAP was negative. Smooth muscle actin was expressed in 2 cases and glial fibrillary acidic protein in 5 cases. The proliferative index as demonstrated by Ki-67 staining was high.

CONCLUSIONSAT/RT is not a particularly uncommon malignancy in childhood. The histologic hallmark is the presence of rhabdoid cells with cytoplasmic inclusions. The tumor cells are positive for cytokeratin, epithelial membrane antigen and vimentin, and negative for INI1. Differential diagnosis includes PNET, medulloblastoma and medullomyoblastoma.

Brain Neoplasms ; metabolism ; pathology ; surgery ; Child, Preschool ; Diagnosis, Differential ; Female ; Humans ; Infant ; Keratins ; metabolism ; Male ; Medulloblastoma ; metabolism ; pathology ; Mucin-1 ; metabolism ; Neuroectodermal Tumors, Primitive ; metabolism ; pathology ; Rhabdoid Tumor ; metabolism ; pathology ; surgery ; Teratoma ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

Endotoxemia ; metabolism ; pathology ; HMGB1 Protein ; Humans ; Liver ; metabolism ; pathology

UNLABELLEDOBJECTIVE To observe the clinical efficacy by Qingying Huoxue Decoction (QHD) combined ursodeoxycholic acid (UDCA) in treating patients with early and mid-term primary biliary cirrhosis (PBC). METHODS Totally 78 patients were randomly assigned to the treatment group and the control group, 39 in each group. All patients received basic treatment and took UDCA (at the daily dose of 13-15 mg/kg). Patients in the treatment group took QHD, one dose per day. The treatment course for all was 6 weeks. Clinical efficacy, gamma-glutamyl transferase (γ-GGT), alkaline phospatase (ALP), TBIL, alanine aminotransferase (ALT), and aspartate transaminase (AST) were observed before and after treatment. RESULTS Totally 21 (53. 8%) patients obtained complete response in the treatment group, with statistical difference when compared with that of the control group (11 cases, 30. 8%). Levels of GGT, ALP, ALT, AST, and TBIL decreased in the two groups after treatment (P < 0.01). Levels of ALP, GGT, and TBIL were obviously lower in the treatment group than in the control group (P < 0.05).

CONCLUSIONSQHD combined UDCA in treating early and mid-term PBC patients was superior to the effect of using UDCA alone. It also could improve patients' liver function.

Alanine Transaminase ; metabolism ; Aspartate Aminotransferases ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Liver Cirrhosis, Biliary ; drug therapy ; Ursodeoxycholic Acid ; therapeutic use ; gamma-Glutamyltransferase ; metabolism

OBJECTIVETo study the distribution of mutations of UDP-glucuronosyltransferase 1A1 (UGT1A1) gene and its relationship with hyperbilirubinemia among neonates with hyperbilirubinemia of Guangxi Heiyi Zhuang nationality.

METHODSTotal genomic DNA was extracted from the blood of 100 neonates with hyperbilirubinemia (case group) and 100 neonates without hyperbilirubinemia (control group), all of whom were selected from Guangxi Heiyi Zhuang population. TATA box and all exons of UGT1A1 gene were amplified by PCR and directly sequenced.

RESULTS(TA)7 insertion mutation in TATA box, G71R missense mutation in exon 1, and 4 single nucleotide polymorphisms (SNPs) (rs199539868, rs114982090, rs1042640 and rs8330) in exon 5 were observed. The allele frequency of G71R mutation in the case group was significantly higher than that in the control group (P<0.01). There were no significant differences in the genotype distribution and allele frequency of TATA box mutation and SNPs (rs1042640 and rs8330) between the two groups (P>0.05). The logistic regression analysis showed that the odds ratios (95% confidence intervals) of UGT1A1 TATA box mutation, G71R mutation, and SNPs (rs1042640 and rs8330) associated with the development of neonatal hyperbilirubinemia were 0.846 (0.440, 1.629), 3.932 (1.745, 8.858), 0.899 (0.364, 2.222), respectively.

CONCLUSIONS(TA)7 insertion mutation and G71R missense mutation of UGT1A1 gene are common mutation types in neonates with hyperbilirubinemia of Guangxi Heiyi Zhuang nationality. Four SNPs (rs199539868, rs114982090, rs1042640, and rs8330) was first reported in China. UGT1A1 G71R missense mutation is a risk factor for hyperbilirubinemia in neonates of Guangxi Heiyi Zhuang nationality.

China ; ethnology ; Glucuronosyltransferase ; genetics ; Humans ; Hyperbilirubinemia, Neonatal ; genetics ; Infant, Newborn ; Logistic Models ; Mutation ; Polymorphism, Single Nucleotide ; TATA Box

The study was to explore the telomerase activity and the expression of hTERT, c-myc and bcl-2 mRNA during terminal differentiation of HL-60 cells induced by all trans-retinoic acid (ATRA) and to study the possible molecular mechanism. By use of the model of differentiated HL-60 cells induced by ATRA, the telomerase activity was determined by TRAP-PCR-ELISA and the expression of hTERT, c-myc, bcl-2 mRNA was detected by RT-PCR in differentiated HL-60 cells. The results showed that during differentiation of HL-60 cells, the telomerase activity was decreased, the expression of hTERT, c-myc and bcl-2 mRNA was downregulated, and the downregulation of hTERT occurred prior to suppression of telomerase activity. It is concluded that the telomerase activity is related to decrease expression of hTERT, c-myc and bcl-2 mRNA during HL-60 cell differentiation induced by ATRA.
Cell Differentiation ; DNA-Binding Proteins ; Gene Expression Regulation, Neoplastic ; Genes, bcl-2 ; Genes, myc ; HL-60 Cells ; Humans ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism ; Tretinoin ; pharmacology