Radiographic diagnosis of distal fibula avulsion fractures: Comparison of ankle X-ray and three-dimensional reconstruction of CT / 北京大学学报(医学版)

OBJECTIVE@#To investigate the difference in sensitivity between X-ray and three-dimensional reconstruction of computed tomography (3D-CT) for the diagnosis of distal fibular avulsion fracture, and the radiographic presentation of the ossicle.@*METHODS@#From January to October 2018, 92 patients with distal fibular avulsion fracture were visited for surgical treatment in Department of Sports Medicine, Peking University Third Hospital, and 60 cases were finally enrolled according to the inclusion and exclusion criteria. Intraoperative detection was regarded as the gold standard, and the diagnostic sensitivity of preoperative ankle X-ray and 3D-CT for the distal fibular avulsion fractures was statistically determined. The ossicle maximum diameter as well as the degree of its displacement were also measured. On 3D-CT, the distance from the ossicle center point to the anterior fibular tuberosity (a), the distance to the fibular tip (b), and the a/b value was used to present the ossicle displacement.@*RESULTS@#Among the 60 patients, 36 and the 52 patients were correctly detected by X-ray and 3D-CT, respectively, and the sensitivities was 60.0% and 86.7%, respectively (P=0.004). The mean diameter of the ossicle on X-ray and 3D-CT was (9.2±3.9) mm and (10.5±3.2) mm, respectively. The mean distance from the ossicle center to the anterior fibular tuberosity (a) was (17.5±3.6) mm and the mean distance to the fibular tip (b) was (17.4±4.8) mm, with mean a/b values of 1.1±0.7. The intraclass correlation coefficients (ICC) for each measurement ranged from 0.891-0.998 with a high degree of consistency.@*CONCLUSION@#Compared with X-ray, 3D-CT has higher sensitivity in diagnosing distal fibular avulsion fractures, can help clinicians evaluate ossicle's location and choose surgical methods, and is recommended to be performed in patients with suspected distal fibula avulsion fractures in clinical practice.

Molecular Pathway of Psoralidin-Induced Apoptosis in HepG2 Cell Line / 中国结合医学杂志

OBJECTIVE@#To test the role of psoralidin in human liver cancer HepG2 cells in vitro.@*METHODS@#Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexin V then sorted by flow cytometry. Protein expressions of caspase-3, caspase-8, caspase-9, Bax, Bid, Bcl-2, Bcl-xL and p53 were examined by western blot while activity of caspase-3, -8 and -9 were also determined.@*RESULTS@#Psoralidin reduces cell viability greatly in a time dependent manner (64%, 40%, 21%, 12% at 2, 6, 24 and 48 h treatment with 64 μmol/L psoralidin respectively) and up-regulates activities of caspase-3, -8 and -9 in a concentration dependent manner (between 4 to 64 μmol/L). Psoralidin also increases the expression of pro-apoptosis genes Bax, Bid and p53 while decreases the expression of pro-survival genes Bcl-2 and Bcl-xL, both in a concentration dependent manner between 4 and 64 μmol/L (P<0.05 at 16 and 64 μmol/L). Caspase-3 inhibitor (Ac-DEVD-CHO at concentrations between 10 to 20 μmol/L), p53 inhibitor (pifithrin-α at 5 μmol/L) and cyclosporin A can attenuate the apoptotic effect of psoralidin.@*CONCLUSION@#The cytotoxic role of psoralidin might work through both intrinsic and extrinsic apoptotic pathway.

Therapeutic effect of ticagrelor combined low molecular weight heparin on ACS and its influence on serum inflammatory factors and MACE / 心血管康复医学杂志

Objective : To explore therapeutic effect of ticagrelor combined low molecular weight heparin (LMWH ) on acute coronary syndrome (ACS) and its influence on serum levels of IL-17 ,MMP-9 ,soluble intercellular adhesion molecule (sICAM)-1 and major adverse cardiovascular events(MACE).Methods :A total of 102 ACS patients hospitalized in our hospital from Feb 2016 to Oct 2017 were selected ,randomly and equally divided into atorvastatin group and ticagrelor group ,both groups received corresponding treatment based on routine treatment and LMWH for two weeks .Therapeutic effect ,serum levels of hsCRP ,IL-17 ,MMP-9 ,sICAM-1 ,LVEF ,LVEDd ,LVESd ,peak early diastolic transmitral flow velocity/peak late diastolic transmitral flow velocity (E/A) changes and incidence rate of MACE were compared between two groups.Results :Total effective rate of ticagrelor group was significantly higher than that of atorvastatin group (94.12% vs.80.39%,P＝0.038).Compared with before treatment ,there were significant reductions in serum levels of hsCRP ,IL-17 ,MMP-9 and sICAM-1 ,LVEDd ,LVESd ,and significant rise in LVEF and E/A in two groups after treat-ment , P＜0.01 all.Compared with atorvastatin group ,there were significant reductions in serum levels of hsCRP [(2.96 ± 0.72)mmol/L vs.(1.46 ± 0.41)mmol/L] ,IL-17 [(25.36 ± 4.27)ng/L vs.(19.58 ± 4.46) ng/L] ,MMP-9 [(58.75 ± 10.75)g/L vs.(35.16 ± 9.63)g/L] ,sICAM-1 [(174.53 ± 28.36)ng/ml vs.(124.38 ± 27.34)ng/ml] ,LVESd[(48.01 ± 4.31)mm vs.(39.72 ± 4.16)mm] ,LVEDd[(57.37 ± 5.98)mm vs.(46.51 ± 5.36)mm] and significant rise in LVEF [(45.42 ± 5.68)% vs.(54.33 ± 6.39)%] and E/A[(1.38 ± 0.29) vs.(1.53 ± 0.31)] in ticagrelor group after treatment , P＜0.05 or ＜0.01. Incidence rate of MACE in ticagrelor group was significantly lower than that in atorvastatin group (7.84% vs.23.53%, P＝0.029).Conclusion :Ticagrelor combined LMWH can significantly improve therapeutic effect , lower serum levels of inflammatory factors ,improve cardiac function and reduce incidence rate of MACE in ACS patients .

Relationships between Dietary Diversity and Nutritional Status in Elderly Inpatient / 肠外与肠内营养

Objective: To identify the relationship between Dietary Diversity and Nutritional Status in Elderly Inpatient. Methods: 136 elderly inpatients were selected in a tertiary hospital in Yunnan Provience. Food frequency questionnaire was used to collect dietary information and DDS9 was calculated; We used Mini Nutritional Assessment (MNA) to evaluate nutritional status, then analyzed the effect of Dietary Diversity on nutritional status in elderly inpatients. Results:① Patients at risk of malnutrition and with confirmed malnutrition in participants were 46. 3％, 33. 8％,, respectively. ② The incidence of insufficient diversity, moderate diversity and adequate diversity were 19. 9％, 77. 2％ and 2. 9％, respectively; the total scores of dietary diversity were (4. 68 ± 1. 27). However, apart from vegetables, the rest 8 kinds of food all showed lower scores of intra-group variety; while the consumption of beans, nuts and seafood was seriously inadequate. ③ The incidence of malnutrition risk and confirmed malnutrition in insufficient-diversity group and moderate-diversity group were 5. 109 times and 1. 094 times of adequacy group (P＜ 0. 05). Conclusion: Dietary diversity is strongly associated with putritional status in elderly inpatient. Improvement of dietary diversity status is potentially beneficial in the prevention of malnutrition.

Antibacterial and anti-biofilm activity of the lipid extract from Mantidis ootheca on Pseudomonas aeruginosa / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The aim of this study is to assess the antibacterial and anti-biofilm properties of the lipid extract from Mantidis ootheca against the gentamycin resistant Pseudomonas aeruginosa. The chemical composition of the lipid extract and its relative proportion were determined using the technique of gas chromatography coupled with mass spectrometry (GC-MS). Antibacterial susceptibility tests were performed using a disc diffusion assay and the minimum inhibition concentration (MIC) was determined by way of the agar dilution method. The anti-biofilm test was carried out with crystal violet staining and scanning electron microscopy (SEM). There were 16 compounds detected, and the most abundant components were sesquiterpenoids, monoterpenes, and trace aromatic compounds. The MIC for P. aeruginosa was 4 mg/ml and the eradication effect on preformed biofilms was established and compared with a ciprofloxacin control. The results of our study indicated that a lipid extract from M. ootheca could be used as a topical and antibacterial agent with anti-biofilm activity in the future.

Research on the effect and mechanism of polypeptide extract from scorpion venom combined with 5-fluorouracil on vasculogenic mimicry of H22 hepatoma / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the inhibition effects of polypeptide extract from scorpion venom (PESV) combined 5-fluorouracil (5-Fu) on vasculogenic mimicry (VM) of H2 hepatoma carcinoma cells in mice and its possible mechanisms.</p><p><b>METHODS</b>The H22 carcinoma cell suspension was subcutaneously inoculated into 60 Kunming mice. Then tumor-bearing mice were randomly divided into three groups, i.e., the control group, the 5-Fu group, and the combination group (PESV +5-Fu), 20 in each group. The tumor volume was measured once every other day after 14 successive days of intervention. Then the tumor volume growth curve was drawn, and the tumor inhibitory rate was calculated. The morphological changes of the tumor tissue were observed by HE staining. The VM density of each tumor tissue were detected by immunohistochemical assay and periodic acid-schiff stain (PAS). The protein expression levels of hypoxia inducible factor-la (HIF-la) and matrix metalloproteinase-2 (MMP-2) were detected using immunohistochemical assay. The gray value was semi-quantitatively analyzed using LeicaQwinV3 Image Analysis Software.</p><p><b>RESULTS</b>The growth of H22 hepatoma transplantation tumor was inhibited more obviously in the combination group and the 5-Fu group than in the control group (P <0.05). There was statistical difference in the tumor weight and the tumor volume between the combination group and the 5-Fu group (P <0.05). Immunohistochemical assay and PAS showed that the VM density was obviously lower in the combination group than in the control group and the 5-Fu group (P <0.01). Compared with the control group, the protein expressions of HIF-la and MMP-2 significantly decreased in the combination group (P <0.01).</p><p><b>CONCLUSIONS</b>PESV combined 5-Fu could inhibit the generation of VM of H22 hepatoma transplantation tumor in mice. Its mechanisms might be associated with inhibiting the expressions of HIF-lalpha and MMP-2 in the microenvironment of tumors.</p>

Mechanism of polypeptide from scorpion venom increasing inhibition of 5-fluorouacil on H22 hepatoma / 中草药

Objective: To explore the mechanism of polypeptide from scorpion venom (PSV) on increasing the inhibition of 5-fluorouacil (5-Fu) on H22 hepatoma. Methods: H22 hepatoma model was established through sc inoculating H22 cells suspension into 40 mice in right armpits. Then tumor-bearing mice were divided into five groups randomly on the day 7 after inoculating: model, PSV (20 mg/kg), 5-Fu (15 mg/kg), low-dose PSV (5 mg/kg) + 5-Fu, and high-dose PSV (20 mg/kg) + 5-Fu groups, 10 mice in each group, continuously admininstered for 21 d. Tumor volumes were measured once every other day, then the curves of tumor growth were drawn and the tumor inhibitory rate was calculated; CD34 was used to mark capillary; Immunohistochemical assay was used to detect the protein expression changes of nuclear factor-kappa B (NF-κB), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF). Results: Compared with the model group, the growth of H22 hepatoma transplantation tumor in the combinational groups was obviously inhibited (P < 0.01); The microvessel density (MVD) and the protein expression of NF-κB, MMP-9, and VEGF in the two PSV groups were all decreased significantly (P < 0.01); Compared with 5-Fu group, the MVD and the protein expression of NF-κB, MMP-9, and VEGF in the high-dose PSV + 5-Fu group were also decreased (P < 0.01), while there was no significant difference between the low-dose PSV + 5-Fu group. Conclusion: PSV may increase the inhibition of 5-Fu on H22 hepatoma through blocking NF-κB pathway and reducing the expression of MMP-9 and VGF, which is beneficial to the inhibition of angiogenesis.

Post-stroke flaccid limb dysfunction treated with the comprehensive therapy of acupuncture, Chinese herbal medicine and rehabilitation: muti-center randomized controlled trial / 中国针灸

<p><b>OBJECTIVE</b>To evaluate the clinical value of the comprehensive therapy of acupuncture, Chinese herbal medicine and rehabilitation in the treatment of post-stroke flaccid limb dysfunction.</p><p><b>METHODS</b>The four-center, single-blind, randomized and controlled research method was adopted, 240 qualified subjects were randomized into a comprehensive therapy group, an acupuncture group, a rehabilitation group and a Chinese herbal medicine group, 60 cases in each one, at the ratio of 1 1. In the comprehensive therapy group, the comprehensive therapy of acupuncture, Chinese herbal medicine and rehabilitation was applied. The acupuncture therapy included the scale acupuncture at middle line of vertex, lateral line 1 of vertex, lateral line 2 of vertex, etc. with the single reinforcing and reducing technique by the speed of needle insertion and withdrawal, and the body acupuncture therapy at the acupoints on the antagonistic muscles with the reinforcing and reducing technique by the needle rotation. The Chinese herbal medicine therapy included No. 1 stroke formula for the cases of liver and kidney yin deficiency and the upward disturbance of wind yang, No. 2 stroke formula for qi deficiency and blood stagnation, and the stagnation in meridians and No. 3 stroke formula for the interaction of phlegm and stasis and blockage of meridians according to the pattern/syndrome differentiation. The rehabilitation therapy focused on the promotion technique by putting the healthy limb. The simple acupuncture, rehabilitation and Chinese herbal medicine therapies as the comprehensive therapy group were applied in the acupuncture group, rehabilitation group and Chinese herbal medicine group separately. The Chinese medicine symptom, the limb motor function, the daily life activity, fainting needle reaction, allergic reaction and the others were taken as indices to evaluate the efficacy and safety of the treatment.</p><p><b>RESULTS</b>(1) The results of the four indices named the Chinese medicine symptom, the limb motor function, the limb balance function, the daily life activity were all improved significantly after treatment as compared with those before treatment in four groups (all P < 0.01). (2) Concerning to the improvement degrees, the improvements of the above four indices in the comprehensive therapy group were more significant than those in the other three groups (P < 0.01, P < 0.05). The improvement in Chinese medicine symptom in the acupuncture group and the Chinese herbal medicine group were more significant than that in the rehabilitation group (both P < 0.05). The improvement of the upper limb motor function in the acupuncture group was more significant than that in the rehabilitation group and the Chinese herbal medicine group separately (both P < 0.05).</p><p><b>CONCLUSION</b>The comprehensive therapeutic program of acupuncture, Chinese herbal medicine and rehabilitation is safe and effective in the treatment of post-stroke flaccid limb dysfunction. It is more advantageous in efficacy as compared with any simple therapy.</p>

Experssion of human FGFR2IIIc and its S252W mutant in MDA-MB-231 breast cancer cells with adenovirus vectors / 生物工程学报

To study the functions of human Fibroblast growth factor receptor 2IIIc (FGFR2IIIc) gene in cancer cells, breast cancer cells MDA-MB-231 were infected by recombinant adenoviruses containing FGFR2IIIc and its S252W mutant, respectively. FGFR2IIIc gene was amplified from an existing plasmid and its S252W mutant was obtained by overlapping extension PCR. These two genes were separately cloned into the adenoviral shuttle plasmid pAdTrack-CMV, confivmed by DNA sequencing linearized, and co-transformed into Escherichia coli BJ-5183 with the adenoviral vector pAdEasy-1. The resulting recombinant expression vectors Ad-FGFR2IIIc and Ad-FGFR2IIIcS252W were linearized and transfected into HEK293A cells to get adenoviral particles. GFP was used to verify the gene expression. The recombinant adenoviral particles were harvested, titrated, and then infected MDA-MB-231 cells. The expression of FGFR2IIIc and its S252W mutant were examined by RT-PCR and Western blotting, and the effect of these recombinant adenoviruses on MDA-MB-231 cell proliferation was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. The results showed the recombinant adenoviral particles could infect MDA-MB-231 cells and express the target proteins. MTT showed that both FGFR2IIIc and its S252W mutant inhibited MDA-MB-231 cell proliferation, but the mutant was more effective. Flow cytometry showed that both FGFR2IIIc and its S252W mutant arrested MDA-MB-231 cell cycle at G0/G1 phase, resulting in low cell proliferation.

Preparation and identification of recombinant maxadilan / 生物工程学报

To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentration of serum glucose.

Expression and characterization of VPAC2 in CHO cells / 生物工程学报

VPAC2 is a co-receptor of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) and mediates multiple bio-functions. In order to construct the CHO line expressing VPAC2 stably, pcDNA-VPAC2 was used to transfect CHO cells. The positive clones were selected by G418 and the clone VPAC2-CHO with high sensitivity to PACAP38 was picked out by its ability to promoting the concentration of cAMP. RT-PCR, Western blot and Immunofluorescenece assay were used to identify the express of VPACS. Binding competition with VPAC2 agonist and the bioactivity of mediating the ligand to promote the concentration of cAMP showed that VPAC2 was expressed effectively in VPAC2-CHO. The results of Scatchard analysis revealed that VAPC2-CHO expressed a receptor density of (1.1 +/- 0.2) pmol/mg protein, respectively, with Kd values of (0.55 +/- 0.10) nmol/L for PACAP38 used as a tracer. The construction of CHO cells expressing VPAC2 specially and functionally lays a foundation not only for the further research on the characters and functions of VPAC2 but also for the screening and characterization of novel agonists of antagonists for VPAC2.

High and stable expression of an analog of human basic fibroblast growth factor in Escherichia coli / 中国病理生理杂志

AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET- 3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3)plysS. After induction by IPTG, the analog was obtained and analyzed by SDS - PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein' s bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E. coli.

Controlled study on treatment of cervical spondylopathy of the nerve root type with acupuncture, moxibustion and massage as main / 中国针灸

<p><b>OBJECTIVE</b>To probe into clinical value of comprehensive program of acupuncture, moxibustion and massage as main for treatment of cervical spondylopathy of the nerve root type.</p><p><b>METHODS</b>Five centers, single blind, randomized controlled method were used, 660 cases were divided into a treatment group of 317 cases and a control group of 311 cases. They were treated respectively with comprehensive program of acupuncture, moxibustion and massage as main, and comprehensive program of physical therapy as main. Establish syndrome detection scale and multiply dimensional effect assessment indexes, and evaluate the therapeutic effects and safety.</p><p><b>RESULTS</b>The cured rate, the cured-markedly effective rate were 42.9%, 64.4% in the treatment group, respectively, better than 16.7%, 36.3% in the control group (P<0.01); after treatment of 2 weeks, clinical symptoms improved in the both groups, but the treatment group was better than the control group in the improvement degrees of neck-shoulder-limb pain, neck rigidity, abnormality of cervical anteflexion, etc. (P<0.01 or P<0.05); the treatment group was shorter than the control group in the time of producing the effect and therapeutic course (P<0.01).</p><p><b>CONCLUSION</b>Comprehensive program of acupuncture, moxibustion and massage as main is safe and effective for treatment of cervical spondylopathy, with a better therapeutic effect compared with the comprehensive program of physical therapy.</p>

Preparation and Identification of Recombinant Vasoactive Intestinal Peptide / 中国生物工程杂志

In order to prepare the recombinant vasoactive intestinal peptide (VIP) using intein mediated rapid purification system,the cDNA encoding the recombinant VIP was designed and synthesized according to the preference of E.coli,and then was cloned into the expression vector PTWIN. The recombinant plasmid PTWIN-VIP was transformed into expression host E.coli strain ER2566.The fusion protein consisting of the recombinant VIP,intein and chitin binding domain was expressed and purified by chitin affinity chromatography. The target peptide was released from the fusion protein by changing the temperature and the pH of the cleavage buffer. The molecular weight of the recombinant VIP was determined by the mass spectrometry and the results was conformity with the theoretical value. The preliminary bioactivity assay indicated that the recombinant VIP decreased the serum resistin levels significantly in LPS-induced acute inflammation. The preparation and the characterization of anti-inflammatory effects of the recombinant VIP layed the foundation for its further application.

Cloning and Expression of the New Gene Recombinant RMBAY Against Type-2 Diabetes and Its Production Optimization / 中国生物工程杂志

Constructing prokaryotic expression vector pKY-RMBAY by gene recombination and research its optimizing productive conditions.By PCR technology synthesizing the gene of the RMBAY with preference codon of E.coli and the RMBAY gene was inserted into high efficiency expression vector pKYB-MCS.Expressed fusion proteins in E.coli ER2566 were purified with Chitin-Beads column.Fusion proteins binding on Chitin-Beads was cut on N-terminus of intein due to the induction of ?-mercaptoethanol and the target peptide RMBAY was released.The RMBAY was identified by mass spectrum.Experiment results showed RMBAY can be high efficiently expressed in E.coli ER2566,with optimizing productive conditions the yield of the RMBAY may be 6.7mg/L fermentation product and its purity is greater than 98%.The molecular weight of RMBAY is 3.887 kDa by mass spectrum and that accords with its theory value.

Strong Expression of Recombinant Human Morphogenetic Protein-4 in Escherichia coli and its Bioassay in vivo / 中国生物工程杂志

Objective:To produce rhBMP-4 with bioactivity in E.coli. Methods: The full-length human BMP-4 gene was mutated by PCR without changes in amino acid sequence, then the synthesized gene was cloned into plasmid pET-3c, transducted into BL21(DE)plysS, and induced by adding IPTG to a final concentration of 1.0 mmol/L. The protein product was purified using ion-exchange chromatography method and then renaturated, bioactivity was checked by C2C12 differentiation in vitro and mouse ectopic bone formation in vivo. Results: A 438 bp gene fragment encoding mature peptide of hBMP-4 was cloned , the protein product was mostly in the form of inclusion body, after renaturation, the engineering protein shows better bioactivity. Conclusion:The mutant strategy can enhance the expression of bioactive rhBMP-4 in E.coli expression system.

On-column refolding and purification of human EGF receptor L2 domain inclusion body overexpressed in Escherichia coli / 生物工程学报

The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2. The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21 (DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni2+ -NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni2+ -NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.

Expression, purification and refolding of recombinant human bone morphogenetic protein- 2 in Escherichia coli / 中国病理生理杂志

AIM: To get high biological activity of recombinant human bone morphogenetic protein -2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2,expressed in Escherichia coli, was washed by Triton X- 100 and further purified by DEAE chromatography.The inclusion bodies were resolved in 8 mol/L urea, and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one - step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP - 2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP - 2 was expressed in Escherichia coli in a non - active aggregated form of inclusion bodies using a temperature - inducible expression system. The high - purified rhBMP - 2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP - 2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one - step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP - 2 dimers and monomers. The purified rhBMP - 2 dimers showed the higher biological activity than the commercial rhBMP - 2. CONCLUSIONS: The method achieved the refolding of rhBMP - 2 would be applied to the whole TGF - β superfamily because the BMP - 2 belongs to the superfamily. Meanwhile, the inexpensive,high yield rhBMP - 2 is suitable for clinic application.

Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The aim of this study was to explore the protective effect of basic fibroblast growth factor (bFGF) on brain injury following global ischemia reperfusion and its mechanisms. Brain injury following global ischemia was induced by four vessels occlusion and systemic hypotension. Twenty-four rabbits were randomized into three groups: group A, only dissection of vessels; group B, intravenous infusion of normal saline after reperfusion for 6 h; group C, 30 microg/kg bFGF injected intravenously at the onset of reperfusion, then infused with 10 microg/(kg.h) for 6 h. Serum neuron specific enolase (NSE), S-100B, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-8 (IL-8) were measured before ischemia, 30 min after ischemia, 0.5, 1, 3, 6 h after reperfusion. Brain water content was determined and cerebral histopathological damages were compared. NSE and S-100B were increased 1 h after reperfusion and reached their peaks 6 h after reperfusion, but were much higher in group B than those in group C 3, 6 h after reperfusion. In groups B and C, TNF-alpha was increased after ischemia and IL-1 and IL-8 were increased significantly 0.5 h after reperfusion, then reached their peaks 6 h, 3 h, 6 h after reperfusion respectively. TNF-alpha and IL-8 at the time points of 1 h and 3 h and IL-1 at 3 h and 6 h in group C were correspondingly lower than those in group B. These indices in group A were nearly unchanged. There were less severe cerebral histopathological damages in group C compared with group B, but no difference in brain water content. It could be concluded that bFGF alleviates brain injury following global ischemia and reperfusion by down-regulating expression of inflammatory factors and inhibiting their activities.

Protective effect of ligustrazine on insulin resistance after local cerebral ischemia of rat / 中国中药杂志

<p><b>OBJECTIVE</b>To study the protective effect of Ligustrazine on IR after local cerebral ischemia of rat.</p><p><b>METHOD</b>Models of rat IR after local cerebral ischemia were prepared by electrocagulation of the middle cerebral artery, and changes of serum insulin, tomer necrosis factor-alpha (TNF-alpha), plasma endothelin-1 (ET-1), nitric oxide (NO) and nitric oxide synthase (NOS) were observed 2 weeks after the ischemia.</p><p><b>RESULT</b>Ligustrazine could significantly reduce serum insulin (P < 0.01), the content of plasma ET-1 (P < 0.01) and serum TNF-alpha (P < 0.01), the activity of brain tissue NO and NOS (P < 0.01). The drug also increased insulin sensitivity indexes (ISI).</p><p><b>CONCLUSION</b>The protective effects of Ligustrazion on IR cerebral ischemia may be related to decreasing ET-1 content in plasma, TNF-alpha content in serum, NO content and NOS activities in tissue.</p>