Haemophilia is caused by lack of coagulation factor VIII or IX in patients′blood with inadequate hemostasis.Currently recombinant coagulation factor VII(rFVII)produced in different cells is used against clini-cal bleeding of haemophilia patients.To enhance the production and activity of rFVII;some eukaryotic cells such as baby hamster kidney(BHK);Chinese hamster ovary(CHO);insect cell and fish embryo;were used to express rFVII.Meanwhile;the effect of functional gene on the activity of rFVII and the limitation of rFVII production caused by post-translational modification were investigated by different methods.The role of rFVII in hemostasis;synthesis of rFVII in different eukaryotic cells and impact on production of post-translational modification are reviewed in this article.

AIM: To construct subtractive libraries in association with heat adaptation differential expressed genes. METHODS: The experiment was carried out with heat adapted rat model and normal temperature control. mRNA was extracted from liver tissue and reverse-transcripted into cDNA. After cut and ligation, suppression subtractive hybridization was executed with each group of cDNA as tester and the other one as driver to construct subtractive libraries. Specificity of the libraries was evaluated by approach of comparing G3PDH gene PCR products with template from the library and unsubtractive sample. Reliability of the libraries was evaluated by primarily isolation and screening. RESULTS: PCR results confirmed that G3PDH concentration was greatly reduced compared with control group, which suggested that specificity of the libraries was high. 300 target segments were isolated from the library, 27 of them were verified to be differential expressed genes, which suggested that the libraries were reliable and efficient. CONCLUSION: This study founded the basis of further investigation on heat adaptation mechanism by approach of constructing subtractive libraries in association with heat adaptation differential expressed genes.

Objective To establish a method of reverse hybridization to detect five subfamilies of low risk Human Papillomaviruses(HPV6,11,42,43 and 44)and eighteen subfamilies of high risk HPV (HPV16,18,31,33,35,39,45,51,52,53,56,58,59,66,68,73,83 and MM4)in one reaction.Methods Special probes for twenty-three HPV subfamilies were fixed on nylon membrane bars,biotin labeled general primers mediated polymerase chain reaction(GP-PCR)were applied in HPV DNA amplification.PCR amplified DNA fragments were reversely hybridized with special probes that were fixed on the membranes. All samples(136)detected by reverse hybridization method were paralleled with the methods of Hybridization Capture Ⅱ(HC-Ⅱ)and sequencing.Results Positive rate of the 136 samples detected by reverse hybridization was 41.9%,while HC-Ⅱ 42.6% and sequencing 40.4%.Reverse hybridization detection indicated coherence with the other two methods(Kappa 0.8644 and 0.9089,respectively).While sequencing was lab standard for DNA test,the sensitivity was 96.36%,specificity was 95.06%,accuracy was 95.59%.Conclusions Method of reverse hybridization is adaptable to 23 kinds of HPV subfamilies, which can confirm the exactly subfamilies of HPV infection.This method is adaptable in clinical detection of HPV,with high sensitivity,high specificity,simply and convenient operation and the results are easily to be read.

Three bactreiophages of Pseudomonas aeruginosa were isolated from sewage and named as PaP1, PaP2 and PaP3. All belong to double-strand DNA phages, their genome is about 47kb, 34kb and 24kb respectively. The titre (pfu/mL) of three phages is respectively 109, 1011 and 1011, PaP1 is lytic phage, both PaP2 and PaP3 are lysogenic. Under electron microscope, All show icosahedral heads with diameter of 70nm, 55nm and 65nm respectively. PaPl belongs taxonomically to Myoviridae, and both of PaP2 and PaP3 belong to Pedoviridae. The phage-re-sistance and substitution phenomenon of the resistant flora for the sensitive were observed, and the mutation frequence of Pseudomonas aeruginosa resistant to the phage is about 1.4 ? 10-7 ~ 7.9 ?10-7 determined by end-point -titer method.

Chronic osteomyelitis is one of the most common disorder in clinic. In recent years due to diabetes, peripheral vascular disease and trauma induced disease increased, the prevalence rate increased. With the development of magnetic resonance imaging and CT imaging technology, it greatly improved the accuracy of clinical diagnosis of chronic osteomyclitis and ability to describe the infection characteristics, and provide a reliable basis for clinical treatment. The current research on chronic osteomyelitis mainly concentrated on the aspects of imaging applications and ways of using antibiotic optimization control inflammation, defect restoration and reconstruction of blood supply and treatment. But the best time to the antibiotic therapy and the use of program is still uncertain, for after debridement, bone grafting time and defect repair function of fast recovery still need further research.
Anti-Bacterial Agents ; therapeutic use ; Chronic Disease ; Humans ; Osteomyelitis ; diagnosis ; therapy

Aim To screen antibodies of novel purinoceptor as a marker for further study of the purinoceptor. Method BALB/c mice were immunized for 4 times with rat aortic endothelial cell. Then the phage display system was used to construct a single-chain Fv fragment (ScFv) cDNA library from the total RNA of immunized mice. The characteristics of novel purinoceptor not existing on vascular smooth muscle cell but on aortic endothelium were used to enrich the aortic endothelium specific antibodies. Induced with IPTG, these antibodies were secreted into the periplasm of E. coli. The functional experiment of novel purinoceptor named organ bath experiment was used to screen out the positive ScFv from the soluble expressed antibodies. Immunohistochemistry experiment was used for positive ScFv identification. Results The total mouse anti-rat endothelium lgG is 1 ∶16 000. 8?106 mouse anti-rat endothelium ScFv cDNA library was successfully constructed. After 4 times of rat endothelium and rat smooth muscle cells screening, 2 500 ScFv cDNA binding membrane of aortic endothelium was enriched. After 4 times of functional screening, a phage-ScFv named B inhibiting the adenosine induced NO dependent construction by 83.4%?21.6% was selected from the expressed antibodies. Immunohistochemistry experiment showed that ScFv-B combined with aortic endothelium specifically and functional experiment showed that ScFv-B did not have any effect on adenosine induced ileum contraction, indicating that ScFv-B specifically binding to the novel purinoceptor. Conclusions ScFv-B binding specifically to the novel purinoceptor was selected by phage display technique and functional screening experiment which provide a good marker for further study of the novel purinoceptor.

Objective To analyze the diagnosis and treatment of pancreatic pseudocyst. Method This study included 46 pancreatic pseudocyst cases, 7 received conservative therapy, 12 received internal drainage, 9 received external drainage, 5 received sequential internal and external drainage and 13 received partial pancreatectomy. Result Cases receiving conservative therapy recovered well without recurrence, one case receiving internal drainage suffered from postoperative pancreatic fistula, one receiving external drainage was complicated with pancreatic fistula and recurrence developed in another 2 cases, one case undergoing partial pancreatectomy was complicated with postoperative pancreatic fistula. Conclusion Pancreatic pseudocyst should be managed individually according the course and patients' clinical condition.

To improve the efficiency of halophilic actinobacteria screening and carry out the rapid selection of targeted strains, we tested 34 strains of Streptomonospora by PCR-single strand conformation polymorphism analysis (PCR-SSCP) based on genus-specific primers for the PCR identification. This approach employs PCR with two pairs of primers located in the 16S rRNA sequence flanking two variable region, then build clustering tree according as SSCP data. Synchronously, we sequenced all the 16S rRNA partial sequences for these strains to verify them. The results showed that the PCR-SSCP analysis was an efficient, easy-to-handle and economic method for rapid selection of halophilic actinobacteria resources.

In this study, plate transparent circle by Davis method was introduce firstly screening ?-Rhamnosidase high-yield strain. The spore-sprouted Aspergillus niger 8-hour were mutagenized by ethyl methane sulphonate and pre-screened via transparent circle. 11% mutants yield 40% higher of ?-rhamnosidase than the original strain. A high-yield strain, T-226 with the highest ?-rhamnosidase activity of 373.4 U/mL was finally selected from these potential high-yield mutants after rescreened by shake flask fermentation twice. When the T-226 strain was fermented in 5 L bioreactor, the enzyme activity could reach to 631.9 U/mL after 84 h. Thus, the established screening method is highly efficient to isolate ?-rhamnosidase high-yield mutant of A. niger.

Objective To investigate the effects of different degrees of hemodilution with the artificial plasma substitutes most commonly used in clinical practice on coagulation in vitro. Methods Ten male ASA physical status I volunteers aged (28.8?1.6) yr and weighing (66?8) kg were enrolled. Venous blood samples obtained from each volunteer were diluted with 10% HES (200 000/0.4-0.55), 4 % succinylated gelatin (gelofusine GEL), 3.5 % polygeline (Haemaccel HAE) and lactated Ringer's solution (RL) by 33% [blood: diluent(B:D) =2:1], 50% (B:D=1:1) and 66% (B:D=1:2). Coagulation of the undiluted blood (control) and diluted blood was measured with sonoclot coagulation and platelet function analyzer (SCT) and by routine coagulation tests. The parameters measured included activated clotting time (ACT), clot rate, platelet function (PF), Hct, platelet count (Pit), plasma fibrinogen concentration (Fig) and activated partial thromboplastin time (APTT) . Results (1) At 33 % dilution ACT was significantly shortened in all the four groups as compared with control value; at 50 % dilution ACT was significantly shorter than the control in RL, HES and HAE groups; at 66 % dilution ACT was significantly prolonged in HES group. (2) Clot rate was significantly decreased at 33 % dilution only in HES group but was significantly decreased in all the four groups at 50 % and 66 % dilution. (3) PF decreased significantly only in GEL group at 33 % dilution but was significantly decreased in GEL and HES groups at 50 % and 66 % dilution. (4) With increasing dilution Hct, Pit and Fig gradually decreased and APTT was prolonged. Conclusion Coagulation changes are closely related to the degrees of dilution. At 33 % dilution, the three artificial plasma substitutes tested do not significantly affect hemostasis. At 50 % and 66 % dilution coagulation is badly impaired, but there are differences among the substitutes used for dilution. HAE impairs oagulation least as it contains higher calcium