Objective: To construct eukaryotic expression vector pSecTag2/HygroB-DAF of human decay accelerating factor (DAF) and transfect NIH/3T3 cells after encapsulated by chitosan. Methods:The human DAF fragments were obtained by PCR form DAF-pGEM-T Easy Vector, cloned into the eukaryotic expression vector pSecTag2/HygroB, and identified by restriction endonuclease’s digestion and DNA sequencing. After the particles of pSecTag2/HygroB-DAF were encapsulated by chitosan, the NIH/3T3 cells were transfected by chitosan-DAF nanoparticles and detected DAF expression by immunohistochemistry stain. Results:The DAF fragment was 1049 bp. Its sequence was as same as DAF cDNA in Genebank. After having been transfected by chitosan-DAF nanoparticles 24 hours, the NIH/3T3 cells showed diffusely positive in cytoplasms by anti-DAF immunohistochemistry. Conclusion:Eukaryotic expression vector of human DAF were constructed successfully and transfected it to NIH/3T3 cells after encapsulated by chitosan.

OBJECTIVETo study the anti-endotoxin of different concentration baicalin.

METHODS6.250 microg/mL, 3.125 microLg/mL, 1.562 microg/mL and 0.781 microg/mL baicalin solutions were mixed with I EU/mL endotoxin, respectively. The mixtures were put into water of (37+/-1) degrees C for 15 min, 30 min and 60 min. The degrading effects were determined by using limulus amebocyte lysate test (LAL test).

RESULTS1) The degrading effect of 6.250 microg/mL, 3.125 microg/mL and 1.562 microg/mL baicalin solution on I EU/mL endotoxin was degraded completely in 15 min, 30 min and 60 min, respectively. 2)The degrading effect of 3.125 microg/mL, 1.562 microg/mL and 0.781 microg/mL baicalin solution on 1 EU/mL endotoxin after these mixtures had been incubated for 15 min. Endotoxin values were (0.155 5 +/- 0.002 8) EU/mL, (0.212 1+/-0.004 9) EU/mL, (0.355 9+/-0.013 9) EU/mL, respectively. These differences among them were statistically significant (P<0.01). 3) The degrading effect of 1.562 microg/mL and 0.781 microg/mL baicalin solution on 1 EU/mL endotoxin after these mixtures had been incubated for 30 min. Endotoxin values were (0.1640+/-0.0025) EU/mL and (0.2094+/-0.004 4) EU/mL, respectively. These differences between them were statistically significant (P<0.01).

CONCLUSIONThe action of anti-endotoxin of baicalin is dose-dependent and time-dependent. The results show that baicalin has the stronger anti-endotoxin effect.

Endotoxins ; Flavonoids ; Limulus Test

AIMTo observe the effect of FAK-related non-Kinase (FRNK) plasmid on hepatic stellate cell (HSC) proliferation stimulated by fibronectin (FN).

METHODSFRNK plasmid was transfected into HSC with transient liposomal transfection. The proteins of FRNK, FAK and p-FAK(Tyr397) were assayed by Western blotting analysis. The proliferation of HSC was evaluated by improved MTT assay, and cell cycle pattern was determined by flow cytometry (FCM).

RESULTS(1) The expression of FRNK protein increased after FRNK transfected HSC, and it was at 48 h that the expression of FRNK protein was the highest (P < 0.01). The protein level of FAK was no significant difference between before FRNK plasmid transfection and after transfection (P > 0.05). The expression of p-FAK(Tyr397) protein was down-regulated after FRNK had been transfected in HSC, (P < 0.01). (2) The HSC proliferation inhibition rates at 12 h, 24 h and 48 h after FRNK transfection were 20.07%, 26.16%, 29.77%, respectively (P < 0.01). (3) Compared with the non-FRNK plasmid group, the FRNK-transfected HSCs almost arrested in G0/G1 phase (71.4 +/- 2.81 vs 48.9 +/- 1.66, P < 0.01).

CONCLUSIONAfter FRNK were transfected successfully in HSCs using lipofectamine, the phosphorylation of FAK was inhibited. The HSC proliferation was restrained in a time-dependent manner and the HSC was arrested in G0/G1 phase.

Cell Line ; Cell Proliferation ; Fibronectins ; Hepatic Stellate Cells ; cytology ; Humans ; Phosphorylation ; Plasmids ; genetics ; Protein-Tyrosine Kinases ; genetics ; Transfection

OBJECTIVETo analyze the mid-term clinical and radiographic results obtained with the Bernese periacetabular osteotomy for the treatment of severe hip dysplasia.

METHODSFrom October 1997 to December 2002 20 hips of 18 patients were classified as having severe acetabular dysplasia (Severin classification Grade IVb). Preoperatively, all patients had hip pain, and sufficient hip joint congruency on functional radiographs. All 20 hips underwent Bernese periacetabular osteotomy. Postoperatively, the hips were assessed radiographically on center edge angle (CE), acetabular roof obliquity and the progression of osteoarthritis. Clinical results and hip function were measured with the Harris hip score at an average of 6.2 years follow-up.

RESULTSComparison of preoperative and follow-up radiographs demonstrated significant improvements in the lateral CE angle, the anterior CE angle, and roof obliquity. The average Harris hip score improved from 78.5 points preoperatively to 91.1 points at the time of the latest follow-up. Fourteen of 18 patients were satisfied with the result of the surgery, and 16 of 20 hips had a good or excellent clinical result. Under-correction occurred in 5 hips.

CONCLUSIONSThe Bernese periacetabular osteotomy is an effective procedure for surgical correction of the severe dysplastic hip. This osteotomy can predictably obtain major reorientation of the acetabulum in all planes. The clinical results in the mid-term follow-up are encouraging.

Acetabulum ; surgery ; Adolescent ; Adult ; Female ; Follow-Up Studies ; Hip Dislocation, Congenital ; surgery ; Humans ; Male ; Osteotomy ; methods ; Treatment Outcome ; Young Adult

OBJECTIVEPatients with typical clinical manifestations of Hemorrhagic fever with renal syndrome (HFRS) are becoming fewer. We conducted analysis on clinical features of HFRS in order to reduce the mistakes in diagnosis.

METHODS64 patients were diagnosed as HFRS during May, 2000 to June, 2006 in our hospital. All the patients' serological tests (HFRS-NP-specific IgM, IgG antibody) by ELISA method were positive. We collected their clinical manifestations and test results. SPSS 12.0 was used in our statistical analysis.

RESULTSAmong the 64 patients, 71.6% of all the cases occurred from Feb. to June. Most of patients were admitted to the hospital with untypical manifestation. Only 30.6% patients appeared headache, lumbago, and pain of orbital cavity. 32.8% patients had obviously signs of injection and hemorrhage. However, there were 90.6% patients with headache and 84.4% patients with nausea or vomit. Hypotensive or oliguric phases were absent in 56.3% patients. There were only 31.3% patients with all five stages. Thrombocytopenia (79.7%) and heavy proteinuria (71.9%) were common. But 54.7% of patients shown normal or even decreased white blood cell count. Only 2/3 of patients had elevated serum creatinine (Cr). Liver involved was common showing as elevated aminotransferase. ALT level was not always parallel to Cr level. There was an opposite trend between them.

CONCLUSIONWe must recognized the untypical manifestations of HFRS. Further study focus on pathogenesis was useful for diagnosis and therapy.

Adolescent ; Adult ; Aged ; Alanine Transaminase ; blood ; Creatinine ; blood ; Female ; Hemorrhagic Fever with Renal Syndrome ; blood ; pathology ; physiopathology ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Male ; Middle Aged

The objective of the study is to find out the effect of shark chondroitin on T lymphocyte subsets in cancer patients. Patients were divided into two groups. One group was treated with chemotherapy alone, and the other group was treated with chemotherapy plus shark chondroitin. Using immunofluorescence technique, T lymphocyte subsets in the peripheral blood were determined in two groups before and after chemotherapy. The results showed that CD4(+)/CD8(+) ratio increased in the patients received shark chondroitin. In the chemotherapy group, CD3(+) had no change, but CD4(+) decreased while CD8(+) increased significantly. The results suggest that shark chondroitin could enhance immune function in cancer patients, especially during chemotherapy.

Adenine ; analogs & derivatives ; pharmacology ; therapeutic use ; Adult ; Drug Resistance, Viral ; Female ; Genes, Viral ; Genotype ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; Humans ; Male ; Middle Aged ; Organophosphonates ; pharmacology ; therapeutic use

BACKGROUNDIntratracheal instillation of blood induces self-repaired acute lung injury. However, the mechanism of repair has been unclear. Heme-oxygenase (HO)-1, which catalyzes heme breakdown, acts as an inducible defense against oxidative stress and plays an important role in inflammation. The objective of this study was to test the role of HO-1 in lung injury caused by intratracheal instillation of red cells.

METHODSForty healthy, male Sprague-Dawley rats were randomly divided into five groups: normal group, saline group, erythrocyte group, erythrocyte+zinc-protoporphyrin (ZnPP, HO-1 inhibitor) group and saline+ZnPP group. At 2 days after intratracheal instillation of red cells, lung tissues and lavage samples were isolated for biochemical determinations and histological measurements.

RESULTSHistological analysis revealed that administration of ZnPP worsened the acute lung injury induced by instilled erythrocytes. HO-1 was over-expressed in the erythrocyte group and in the erythrocyte + ZnPP group. Compared with the erythrocyte + ZnPP group, the levels of total protein, lactate dehydrogenase and tumor necrosis factor-alpha in the lavage were lower (P < 0.01), while the level of interleukin-10 was higher in the erythrocyte group (P < 0.01).

CONCLUSIONHO-1 protects against erythrocyte-induced inflammatory injury in lung.

Animals ; Erythrocytes ; physiology ; Heme Oxygenase (Decyclizing) ; analysis ; physiology ; Interleukin-10 ; analysis ; Lung ; pathology ; Lung Injury ; prevention & control ; Male ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVETo set up a stand for surgical classification of pancreatic duct stone and evaluate the benefits of different management according to the classification.

METHODSRetrospectively analysis the diagnosis and prognosis of different management of 33 cases pancreatic duct stones to establish a new standard of classification and strategy of management of pancreatic duct stone.

RESULTSAccording to the results of imaging examination (B-US, CT, ERCP) and finding during surgery, pancreatic duct stone can be classified into four different types: Type I: The stones mainly located in the head of pancreas. Endoscopic pancreas drainage and remove of stones is the first line choice of treatment. If it fail the Whipple procedure should be applied. Type II, The stones mainly located in the body of pancreas. It can be treated by Pusetow procedure. Type III, The stones mainly located in the tail of pancreas. The resection of the tail of pancreas or combined with spleenectomy was recommended for the management of this type stones. Type IV, The stones can be found from the head to tail of the main duct of pancreas. The Pusetow-Gillesby procedure or dividing of the neck of pancreas removing stones from both ends of pancreatic duct and reconstructed by two ends pancreatic duct-ileostomy in Roux-en-Y fashion are the choice of management.

CONCLUSIONThe invadulaized strategy of the management based upon correct diagnosis and classification play the most important role in the treatment of pancreatic duct stone.

Adult ; Aged ; Calculi ; classification ; diagnosis ; surgery ; Cholangiopancreatography, Endoscopic Retrograde ; Female ; Humans ; Male ; Middle Aged ; Pancreatic Diseases ; classification ; diagnosis ; surgery ; Pancreatic Ducts ; diagnostic imaging ; pathology ; Retrospective Studies ; Tomography, X-Ray Computed ; Ultrasonography

Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM - P2 vector using restriction endonuclease, then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E. coli. Positive recombinant plasmids were selected sequenced and named pPIC9K-P2 and it was linearized by Sal I and Bgl II, then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K - P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS+ MUT+ (Methanol utilization plus) and HIS+ MUT(S) (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris. The multicopy recombinants were named GS115/pPIC9K - P2HIS+ MUT+ and GS115/pPIC9K-P2HIS+ MUT(S), both HIS+ MUT+ and HIS+ MUT(S) were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33 % of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen.
Animals ; Blotting, Western ; Cysticercosis ; diagnosis ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Genome, Fungal ; genetics ; Helminth Proteins ; genetics ; metabolism ; Humans ; Phosphoproteins ; genetics ; metabolism ; Pichia ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; metabolism ; Swine ; Taenia ; metabolism