Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Icariine is a flavonoid isolated from a traditional Chinese medicine Epimedium pubescens and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis. But the mechanism is unclear. The aim of the study was to research the effect of Icariine on the proliferation and differentiation of human osteoblasts.</p><p><b>METHODS</b>Human osteoblasts were obtained by inducing human marrow mesenchymal stem cells (hMSCs) directionally and were cultured in the presence of various concentrations of Icariine. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of Icariine on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of calcified nodules were assayed to observe the effect on cell differentiation. The expression of bone morphogenetic protein 2 (BMP-2) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Icariine (20 microg/ml) increased significantly the proliferation of human osteoblasts. And, Icariine (10 microg/ml and 20 microg/ml) increased the activity of ALP and the amount of calcified nodules of human osteoblasts significantly (P < 0.05). BMP-2 mRNA synthesis was elevated significantly in response to Icariine (20 microg/ml).</p><p><b>CONCLUSIONS</b>Icariine has a direct stimulatory effect on the proliferation and differentiation of cultured human osteoblast cells in vitro, which may be mediated by increasing production of BMP-2 in osteoblasts.</p>

SCT-OGP fusion protein expression and its potential role as osteoporosis therapy / 生物工程学报

Osteoporosis is a disease of bone metabolic disorder, the incidence of which increases sharply in old people. Calcitonin (CT) is a peptide hormone containing 32 amino acid that can inhibit osteoclasts activity. Osteogenic Growth Peptide (OGP) is a peptide hormone with 14 amino acid. It is an autocrine mitogen for osteoblastic and firbroblastic cells which has anabolic activiy. Six SCT and OGP DNA segments were chemically synthesized and ligated into a yeast expression vector pPIC9. The recombinant plasmid was transformed into pichia pastoris GS115. Finally, we got two stable SCT-OGP high expression clones after screening. Purifed protein can stimulate the proliferation of osteoblastic and fibroblastic cells, and also stimulate serum ALP activity and decrease serum calcium level using mice as animal models, demonstrating its potential role in oteoporosis therapy.

Advances in the Research of Long Life Recombinant Protein Drug / 中国生物工程杂志

The recombinant protein drug usually has a short half-life after intravenous (IV) or subcutaneous (SC) administration. The methods of prolonging the half-life of recombinant protein drug in common use are mainly based on three principles: 1 Amplifying the molecule weight of protein drug; 2 Making use of drug balance in the blood; 3 Reducing Immunogenicity. Three methods were focused on: Analog construction, PEGylation and Albumin fusion technology. The characteristics, half-life and immunogenicity problem of their products in the market and under development are summarized.

Expression in Pichia pastoris and properties of human serum albumin-interferon alpha2b chimera / 生物工程学报

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.

Clinical characteristics and treatment of flexion-distraction stage I injuries in subaxial cervical spine / 中华外科杂志

<p><b>OBJECTIVE</b>To study the clinical characteristics and treatment of flexion-distraction stage I injuries in subaxial cervical spine.</p><p><b>METHODS</b>Twelve cases of flexion-distraction stage I injuries with delayed symptoms, admitted in our hospital between January 1995 and December 2004, were studied retrospectively. In acute phase, all of 12 cases had neck pain and limited neck movements, neurological deficits were found in 6 of 12 cases. Eight cases had a correct diagnosis, and 2 cases had a error diagnosis, 2 cases missed. All cases were satisfactory by the primary conservative treatment. After 274 days average asymptomatic intervals, all of 12 cases had recurrence of neck pain, delayed neurological deficits were found in 10. MRI showed that all of 12 cases were unstable injuries.</p><p><b>RESULTS</b>All of the 12 patients were treated operatively. Decompression, fusion and fixation were performed by anterior approach in 9 cases, and by combined anterior and posterior approach in 3 cases. The average follow-up period was 33.1 months. Neck pain had great recovery in all cases, 10 cases with neurological deficits, 7 returned normal. Radiographic evidences of intervertebral bony fusion and good cervical alignment were observed in all of 12 cases.</p><p><b>CONCLUSIONS</b>Flexion-distraction stage I injuries is often caused by ligament and disc injuries, and often missed with subtle symptoms and radiographic changes. Inadequate primary treatment options are often due to failure to recognize the instability, and maybe result in delayed injuries. MRI is helpful for the early accurate evaluation of spinal stability. Unstable injury require early surgical treatment. The anterior approach operation is recommended to most of these patients with acute and old injuries. Combined anterior and posterior approach operation should be considered in these patients who have old injuries with stiff kyphosis.</p>

The effect of early diagnosis of recurrence and revision after resection of primary spine tumors / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the effect of early diagnosis of recurrence and early revision after resection of primary spine tumors.</p><p><b>METHODS</b>From March 1989 to September 2005, the relate clinic data of 55 patients with giant cell tumors, osteoblastomas, chondrosarcomas and chordomas in spine in big piecemeal and current fashion was analysed.</p><p><b>RESULTS</b>In 55 cases, 43 patients were followed up and had complete materials. The follow-up time ranged from 1.6 to 16.5 years, averagely 5.8 years. Thirty-four patients followed up regularly, and 12 were found recurrent, in which one C(1) giant cell tumor was found extensively large 3 months after initial surgery and was undertaken palliatory curet. The other eleven lesions were small and re-operated with wide margin. As a result, six patients lived without tumors during the 1 approximately 9.5 years follow-up, one patient gave up revision when found recurred again for economic reason, another four patients recurred repeatedly, but they persisted in regular follow-up and took revision surgeries whenever the recurred lesion were found. As a result, 3 of them lived without tumor and the other one died of other disease without sign of recurrence. In contrast, there were another nine patients who came to follow up until they had symptoms and were confirmed recurrent extensively. Two of them were excised radically for the tumors located in the relatively easily exposed segments of spine and lived without tumor now. While the other seven patients only received palliatory curet and all died of tumors.</p><p><b>CONCLUSIONS</b>Regular follow-up, early diagnosis of recurrence and early revision need to be regarded as part of radical excision and are very important of surgical treatment of spinal tumors, which can prolong the patients' survival time.</p>

Surgical factors underlying the recurrence of primary spine tumor / 中华外科杂志

<p><b>OBJECTIVE</b>To find the causes of the spinal primary tumors recurrence in surgical technique.</p><p><b>METHODS</b>From 1989 to 2002, 38 cases of primary spine tumors, including giant cell tumor, osteoblastoma, chondrosarcoma and chordoma with wide resection via a piece-meal fashion. By adopting a retrospective method, the present study investigated the clinical and imaging materials of pre- and post-operation period and those in follow-up.</p><p><b>RESULTS</b>The study included 18 cases of giant cell tumor, 6 osteoblastoma, 6 chondrosarcoma and 8 chordoma. In all cases, 63% of tumors were in cervical and cervicothoracic (C(7)-T(2)) spine; 29% in thoracolumbar (T(4)-L(5)) spine and 8% in sacrum. Tumors involved in multi-segment were 34%. And 71% patients had the tumor in the body and the arch simultaneously. And 71% of tumors formed paraspinal masses, 42% in both sides. The tumors invaded the channel in 58% of the cases. The compartment were invaded in 79% patients. Finally, 32 patients were followed up, from 1.0 approximately 14.9 years, 5.1 years average. Seventeen patients recurred after the surgery, the recurrence rate was 53%. The recurrence rate of giant cell tumor was 35%, osteoblastoma 50%, chondrosarcoma 75%, chordoma 100%. The recurrence rate of tumor in cervical and cervicothoracic spine was 63%, thoracic-lumbar 33%, sacrum 67%. The recurrence rate of multi-segment tumors was 80%, and that of single segment 41%. The recurrence rate of the tumors involving in vertebral body alone or involving the arch simultaneously reached 75% and 55% respectively; the recurrent rate in the arch alone was 33%. The recurrence rate of the tumors involving in vertebral body ranged in different segments. Those growing in cervical and cervicothoracic spine reached 73%; those growing in thoracolumbar spine was only 25%. The recurrence of the tumors without soft masses was 20%, those of single-sided soft masses was 45% and those of double-sided 91%. Among the 17 recurrent patients, 83% of the tumors were in the cervical and cervicothoracic spine. Those extending to the upper cervical and cervicothoracic amounted to 58%. All the 17 recurrent patients had body lesion and paraspinal soft masses.</p><p><b>CONCLUSIONS</b>During the primary spinal tumor operation, that failure to get adequate exposure and full division is thought to be the cause of recurrence. So the precise design before surgery and adequate exposure of the tumor in the surgery is the guarantee of wide excision.</p>

Enhancement of immune responses to hepatitis B DNA vaccine by superantigen SEA in mice / 生物工程学报

To investigate the adjuvant effect of plasmid DNA encoding superantigen SEA (D227A) (pmSEA) on immune responses induced by HBV DNA vaccine containing HBV preS2 and S antigen in BABL/c (H-2d). BALB/c mice were immunized intramuscular injection with HBV DNA vaccine (pHBVS2S) mixed with or without pmSEA plasmid. Antibodies againat HBV PreS2 and S antigen in the sera were accessed by Anti-HBs ELISA, and the HBsAg specific cytotoxic T lymphocytes (CTLs) activity was determined by 5 Chromium Release Assay. The HBs peptide-specific IFN-gamma secreting T cells were detected by ELISPOT. Anti-HBs antibody titers and CTLs activity in mice immunized with pmSEA + pHBVS2S group were significant higher (P < 0.05) than pHBVS2S DNA vaccine group. The ratio of IgG1/IgG2a (0.282) was apparently different from the group immunized with peptide (10). Mice immunized with HBV DNA vaccine plus adjuvant produce higher titer of IgG1 and IgG2a antibodies against HBV S antigen 1.36 and 1.73 time higher than that without adjuvant respectively. HBs peptide--specific IFN-gamma secreting T cells increased 2 - 3 times by the pmSEA adjuvant, compared to DNA vaccine group. HBV DNA vaccine (pHBVS2S) induces humoral and cellular immuno-responses in BALB/c mice, and the responses could be significantly boasted by the plasmid encoding mSEA. Therefore the pmSEA was a potential adjuvant for DNA vaccines.

Effects of Epimedium pubescens icariine on proliferation and differentiation of human osteoblasts / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of epimedium pubescens icariine on the proliferation and differentiation of human osteoblasts.</p><p><b>METHOD</b>Human osteoblasts were obtained by inducting human marrow mesenchymal stem cells (hMSCs) directionally. MTT was used to observe the proliferation and activity of ALP was assayed to observe the differentiation of the third passage human osteoblasts cultured in vitro. The expression of BMP-2 mRNA was checked by RT-PCR.</p><p><b>RESULT</b>Epimedium pubescens icariine at the dose of 20 microg x mL(-1) increased greatly the proliferation and differentiation of human osteoblasts and promoted the expression of BMP-2 mRNA.</p><p><b>CONCLUSION</b>Epimedium pubescens icariine enhances significantly the proliferation and differentiation of human osteoblasts, which may be mediated by increasing the expression of BMP-2 mRNA.</p>

The efficacy of allogenic strut bone graft and instrumentation for anterior cervical fusion following subtotal corpectomy and decompression in cervical myelopathy / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of allogenic strut bone graft and instrumentation for anterior cervical fusion following subtotal corpectomy and decompression in cervical myelopathy.</p><p><b>METHODS</b>Thirty-five patients with cervical myelopathy were treated by the procedure of allogenic strut bone graft and instrumentation for anterior cervical fusion following subtotal corpectomy and decompression. The preoperative average JOA scale score was 8.7 point (Range 4-15).</p><p><b>RESULTS</b>Sixty-nine vertebral were corpectomized and 104 levels were decompressed and fused with an average of 3 levels. Among the cases, 1 vertebrae was corpectomized in 7 cases, 2 vertebra in 22 cases, 3 vertebra in 6 cases. There were no surgery-related complications. The patients were followed up from 11-37 months, with an average of 17.4 months. No plate breakage, screw loose, graft infection, lysis and absorption was discovered. The fusion rate was 100%, the average time of fusion was 9.3 months (range from 6-15 months). The postoperative average JOA scale score was 14.8 point (range 7-17), the recovery ratio was 73.5% and the excellent and good results was 82.8%.</p><p><b>CONCLUSIONS</b>The use of allogenic strut bone graft and instrumentation for anterior cervical fusion following subtotal corpectomy and decompression in cervical myelopathy may not only simplify surgical procedure and decrease injuries and complications, but also the fusion is satisfactory and reliable.</p>

Diagnosis and treatment of esophageal fistula in anterior cervical spine surgery / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the causes, diagnosis, treatment and prevention of esophagocutaneous fistula in anterior cervical spine surgery.</p><p><b>METHODS</b>Thirteen cases with esophagocutaneous fistula in anterior cervical spine surgery were studied.</p><p><b>RESULTS</b>The causes includes: (1) During the operation, esophagus was oppressed by a clasp for so long time that made a pressure necrosis of the esophagus; (2) Esophagus was injured by loose plates and screws; (3) Loose bone grafts oppressed esophagus; (4) Esophagus was injured by operative appliance in the operation; (5) Esophagus was oppressed by the plate.</p><p><b>DIAGNOSIS</b>After anterior cervical spine surgery if patients had a high fever, sore throat, swelling incision, and food sediment was found in the incision, esophagocutaneous fistula should be considered. The final diagnosis could be done by esophageal radiography.</p><p><b>TREATMENT</b>Fasting cure, nasogastric tube and wound drainage should be used; When the inflammation ended, patients should undergo operation of closure of the esophageal fistula.</p><p><b>CONCLUSIONS</b>The esophagocutaneous fistula in anterior cervical spine surgery has several causes mentioned above. We should take precautionary measures to avoid the complication, and use appropriate treatment to cure when it happens.</p>

Evaluation of neck moving ability after anterior cervical fusion / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the relationship of adjacent segment degeneration and neck symptom after anterior cervical fusion.</p><p><b>METHODS</b>Making follow-up to 66 cases who had accepted anterior cervical fusion for 1 - 16 years, average 10.5 years. Observe the mobilization and gliding degree of adjacent segment in flexion-extension X-ray film, neck symptom, and the relationship of them. In 59 of these patients, the mobilization of entire cervical spine and non-fusion segment was observed before/after operation.</p><p><b>RESULTS</b>The incidence of adjacent segment instability in follow-up term was 72.7%, and 40.9% of all patients had significant neck symptom. The rate of significant neck symptom in patients who had instability was 48%, whereas which in patients without instability was 18.8%, and the difference was statistically significant (P < 0.05). The difference of mild instability and severe instability was not statistically significant (P > 0.1). The mobilization of entire cervical spine decreased obviously (P < 0.001). The mobilization of adjacent segment increased obviously (P < 0.01), and which of non-adjacent segment had no change (P > 0.05).</p><p><b>CONCLUSION</b>Majority of patients who have accepted anterior cervical fusion possessed instability of adjacent segment, but many of them have no symptoms. Adjacent segment instability is one of the reasons that induce neck symptoms.</p>

Functional expression of DNA binding domain of Zif268 in Escherichia coli / 生物工程学报

As the ubiquitous nucleic acids recognizing motif, Zinc finger protein play important role in regulation of gene expression. The study of recognization specific will greatly facilitate understanding the delicate interaction of Zinc finger protein and DNA. By the choice of expression vector, the induction and culture conditions, the DNA binding domain of Zif268 was expressed in Escherichia coli partly solubly. The gel mobility shift assay shows that purified DNA binding domain can bind its natural target sequence specifically, which indicates the DNA binding domain remains its DNA binding activity in Escherichia coli. The functional expression of DNA binding domain of Zif268 will greatly facilitate the development of in vivo genetic selection assay for the study of Zinc fingers-DNA interaction.

Induction of protective immunity in rhesus monkey by inoculation with recombinant fusion protein of cholera toxin B subunit-multivalent epitopes of Plasmodium falciparum / 生物工程学报

Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi.

Study of recombinant stem cell factor / 生物工程学报

Stem cell factor is an important hematopoietic growth factor. In this study, the human stem cell factor was produced by recombinant E. coli, and the structure and biological activity of the recombinant stem cell factor(rhSCF) was studied. It was indicated that the rhSCF was a uncovalent dimer in phosphate buffer,and had the correct mass spectra, mass peptides spectra, composition of amino acid, N-terminal sequernce, C-terminal sequence and intrachain disulfide linkages, rhSCF alone or synergy with rhG-CSF could mobilze hematopoietic progenitors to blood in monkey.

High expression and characterization of human parathyroid hormone in Escherichia coli / 生物工程学报

Human parathyroid hormone (hPTH) was highly expressed in Escherichia coli by inserted the synthesized whole hPTH cDNA into the vectors pBV220 and pET22b. After expression and disruption, the purified product was acquired through cation exchange chromatography and reverse phase chromatography. From the results of N-terminal sequencing and MALDI-TOF-MS analysis the recombiant prtein was indentified as intact hPTH. In in vitro Bioassays the recombinant hPTH stimulated adenylate cyclase as the standard did. In ovariectomized rats the recombinant hPTH markedly increased the femoral bone mass and bone mineral density.

Over-expression in Escherichia coli and purification of nucleocaspid and membrane protein of SARS coronavirus / 生物工程学报

Genes encoding nucleocaspid (N) and membrane (M) protein of SARS coronavirus were obtained by RT-PCR and were cloned into expression vector pET22b and pBV222. DNA sequencing showed that the genes cloned from a patient in Beijing were identical to the gene sequences from reported Toronto strain. The genes were over-expressed in E. coli either as inclusion body or as soluble form. The recombinant proteins were purified by ion-exchange, or ion-exchange followed by metal chelate affinity chromatography. The recombinant N protein was demonstrated highly antigenic and could be employed as antigen to detect SARS antibodies in ELISA system for SARS diagnosis.

Gene cloning, soluble expression and activity analysis of rSEA / 生物工程学报

The superantigen,such as staphylococcal enterotoxins, had been identified as possible anti-cancer molecules in many reports. In this paper, we cloned the entA gene encoding Staphylococcal enterotoxin A from the genomic DNA of Staphylococcus aureus(ATCC13565) by PCR, the sequence cloned was accordance with that reported in Genebank. The entA gene could be expressed effectively after inserted into plasmid pET-22b( + ), The rSEA was expressed as inclusion bodies when induced by IPTG at 37 degrees C and became soluble after induced at low temperature, the soluble part is about 55% of total rSEA products. Only one band was detected by western-blotting in expression product of BL-21 (DE3) with pET-SEA. The soluble rSEA was purified by Ni2+ chelating sepharose column. No other protein except rSEA was seen in SDS-PAGE gel stained by both Coomassie brilliant blue and silver salt, which showed that the rSEA was purified effectively. Homology modeling of rSEA determined the structure change was conducted, which indicated there was no apparent structure change between rSEA and native SEA. This result was also confirmed by proliferation assay of PBMC, for the rSEA could induced proliferation of PBMC as effectively as native SEA. The increasing anti-tumor activity of rSEA was also detected after the spleen cell activated in vivo by rSEA, which was accordance with others reports. This work paved the way for the further study of anti-cancer with rSEA.

Expression plasmid-host strain using chromosome-plasmid balanced lethal system based on the Escherichia coli thyA / 生物工程学报

To construct a vector for DNA vaccine and protein expression by using chromosome-plasmid balanced lethal system which was based on the thyA+ gene/deltathyA Escherichia coli. The thyA genes from Escherichia coli and Vibrio cholerae were amplified by polymerase chain reaction and cloned into pCDNA3 by replacing ampilicilin resistant gene. Multiple cloning sites, the prokaryotic replicon, CMV promoter and the boving growth hormone polyA signal were also included in the vectors. Two new non-antibiotic recombinant plasmids renamed as pcDNATE and pcDNATC which had the nutritional marker as thyA were constructed and were transformed respectively into the deltathyA derivative of E. coli K-12 strain DY330-TI, then two chromosome-plasmid balanced systems for E. coli based on the thyA were developed. To test the efficiency and stability of the newly constructed chromosome-plasmid balanced lethal system, a reporter gene--red fluorescent protein (DsRed2) gene was cloned into pcDNATE, pcDNATC and expressed as fusion to the c-myc. The two recombinant plasmids, pcDNATE-DsRed2, pcDNATC-DsRed2, were transfected into HEK293 solely and DsRed2-myc was detected by the fluorescence microscope assay and western-blot. Meanwhile, the loss of recombinant plasmids were not seen in cultures without thymidine after 20 generations. The chromosomal-plasmid balanced lethal system is proved to be an effective vector system for the expression of target genes and share the same stability with the antibiotic-resistant plasmid vector system. It holds great potential in gene vaccine vector because obviating the weakpoints of the drug resistance marker during application.

Prophylactic and therapeutic vaccines against Alzheimer's disease / 生物工程学报

Beta-amyloid(Abeta) immunization as vaccines has now become a promising approach for the prevention and treatment of Alzheimer's disease (AD)after its debut in 1999. Transgenic mouse models of AD that develop age-dependent Abeta deposition, damage to the neuropil, and behavioral deficits have enabled researchers to test if the approach can influence these AD-like pathologic changes in their brains. Active immunization with different forms of A beta and protocols have been shown to decrease brain Abeta deposition and improve cognitive performance in these mice models in the following studies. Although the phase II clinical trials of active immunization with Abeta(AN1792)were halted last year due to the occurrence of CNS inflammation in a small subset of patients, researchers found that strong humoral responses can be induced by the vaccination. Furthermore, the active immunization also brings an almost complete clearance of Abeta from much of the cerebral cortex. Abeta specific antibodies are believed to cross blood-brain barrier by minimal destroy of vascular wall where amyloid depositions exist. Three possible mechanisms on removal of Abeta deposition from brain have also been reviewed. Still some problems should be clarified before this strategy could be applied for clinical therapy. Whether vaccination will improve the cognitive decline in AD patients will depend upon clinical assessments, which was vital to destiny of the approach.