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ABSTRACT: The osteoinduction of porous biphasic calcium phosphate ceramics (BCP) has been widely reported and documented, but little research has been performed on rodent animals, e.g., mice. In this study, we report osteoinduction in a mouse model. Thirty mice were divided into two groups. BCP materials (Sample A) and control ceramics (Sample B) were implanted into the leg muscle, respectively. Five mice in each group were killed at 15, 30, and 45 d after surgery. Sample A and Sample B were harvested and used for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC) staining, and Alizarin Red S staining to check bone formation in the biomaterials. Histological analysis showed that no bone tissue was formed 15 d after implantation (0/5) in either of the two groups. Newly-formed bone tissues were observed in Sample A at 30 d (5/5) and 45 d (5/5) after implantation; the average amounts of newly-formed bone tissues were approximately 5.2% and 8.6%, respectively. However, we did not see any bone tissue in Sample B until 45 d after implantation. Bone-related molecular makers such as bone morphogenesis protein-2 (BMP-2), collagen type I, and osteopontin were detected by IHC staining in Sample A 30 d after implantation. In addition, the newly-formed bone was also confirmed by Alizarin Red S staining. Because this is the report of osteoinduction in the rodent animal on which all the biotechnologies were available, our results may contribute to further mechanism research.
Journal of Zhejiang University SCIENCE B 07/2011; 12(7):582-90. · 1.10 Impact Factor
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ABSTRACT: To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells.
A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models.
The IC(50) of MG2B for cancer cells (10-15 μmol/L) was at least 10 times lower than the IC(50) of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC(50) ranging from 10 to 15 μmol/L, which was about 6-10 times lower than the IC(50) for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively.
The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.
Acta Pharmacologica Sinica 01/2011; 32(1):79-88. · 1.95 Impact Factor
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ABSTRACT: A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis. The PY-1 strain showed strong ability against many common plant fungal pathogens in vitro. The antibiotics produced by this strain were stable in neutral and basic conditions, and not sensitive to high temperature. From the culture broth of PY-1 strain, five antifungal compounds were isolated by acidic precipitation, methanol extraction, gel filtration and reverse-phase HPLC. Advanced identification was performed by mass spectrometry and nuclear magnetic resonance spectroscopy. These five antifungal compounds were proved to be the isomers of iturin A: A2, A3, A4, A6 and A7. In fast atom bombardment mass spectrometry/mass spectrometry collision-induced dissociation spectra, fragmentation ions from two prior linear acylium ions were observed, and the prior ion, Tyr-Asn-Gln-Pro-Asn-Ser-betaAA-Asn-CO+, was first reported.
Acta Biochimica et Biophysica Sinica 05/2006; 38(4):233-40. · 1.38 Impact Factor
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ABSTRACT: A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.
Protein Expression and Purification 05/2006; 46(2):495-502. · 1.59 Impact Factor
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ABSTRACT: Abstract A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis. The PY-1 strain showed strong ability against many common plant fungal pathogens in vitro. The antibiotics produced by this strain were stable in neutral and basic conditions, and not sensitive to high temperature. From the culture broth of PY-1 strain, five antifungal compounds were isolated by acidic precipitation, methanol extraction, gel filtration and reverse-phase HPLC. Advanced identification was performed by mass spectrometry and nuclear magnetic resonance spectroscopy. These five antifungal compounds were proved to be the isomers of iturin A: A2, A3, A4, A6 and A7. In fast atom bombardment mass spectrometry/mass spectrometry collision-induced dissociation spectra, fragmentation ions from two prior linear acylium ions were observed, and the prior ion, Tyr-Asn-Gln-Pro-Asn-Ser-βAA-Asn-CO+, was first reported.Edited by Ming-Hua XU
Acta Biochimica et Biophysica Sinica 04/2006; 38(4):233 - 240. · 1.38 Impact Factor
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ABSTRACT: To investigate the antibacterial activity and the cytotoxicity of polypeptide dhvar4.
Solid-phase synthesized dhvar4 was manufactured by MeiLian Biotech Co. Ltd.; we evaluated its antibacterial effect, using the growth curve of bacteria after the bacteria being treated with different concentration of dhvar4. The cytotoxicity of dhvar4 on L-02, ECV-304, 293, 3T3 cells was investigated by MTT assay, and human erythrocytes were subimitted to hemolytic assay.
dhvar4 inhibited the growth of Enterococcus faecium 36418 and Staphylococcus aureus BAA42 only at the concentration of 4 microg/ml. The viability of mammalian cells remained 68%-92% even when the concentration of dhvar4 reached 150 microg/ml. The LD50 (median lethal dose) was determined to be higher than 150 microg/ml. And very low hemolytic activity of dhvar4 was detected even at the highest protein concentration tested (180 microg/ml).
dhvar4 possesses antibacterial activity and has little cytotoxicity to mammalian cells. These results suggest that dhvar4 has great potential in the development of
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 06/2005; 36(3):308-11.
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ABSTRACT: To isolate and characterize mesenchymal stem cells (MSCs) derived from bone marrow of Banna minipig inbred line (BMI).
BMI-MSCs was isolated from bone marrow by density gradient centrifugation and cultured in DMEM (containing 15% bovine serum) at 37 degrees C with humidified 5% CO2. These cultured stem cells were characterized in clonal growth, expression of specific markers and capability of differentiation.
Mesenchymal stem cells were proliferative and could be expanded rapidly in vitro. Clonal growth of these cells can be observed when small amount of cells was inoculated. These cells were SH2, SH3, SH4, SB10 and SB21 positive. And it was proved that these cells possess osteo-differentiation ability, up-regulated alkaline phosphatase expression and calcium secretion after osteosupplement was added into the media for several days.
Mesenchymal stem cells derived from bone marrow of BMI possess the general characters of stem cell.
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 10/2002; 16(5):354-8.
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ABSTRACT: To investigate the influence of different dose levels of hydroxyapatite/tricalcium phosphate (HA/TCP) on the proliferation and alkaline phosphatase (ALP) activity of rabbit osteoblasts.
Three different dose levels of HA/TCP (10%, 40%, 70%) were co-cultivated with rabbit osteoblasts respectively. The proliferation and ALP expression capacity of osteoblasts were examined with MTT method and enzyme histochemistry once every 24 hours until 5 days. Three control groups of other materials were treated and examined in the same way: rabbit osteoblasts as normal control; polyvinylchloride as positive control; titanium alloy as negative control.
There was remarkable time-effect relationship in the proliferation of osteoblasts. Ten percent HA/TCP did not affect osteoblasts growth while 40% HA/TCP could slow the cell growth rate down though time-effect relationship still existed. The proliferation of osteoblasts stagnated when co-cultivated with 70% HA/TCP. On the other hand, 10% HA/TCP could cause reversible damage on ALP activity of osteoblasts, whereas when the dose was 40%, and the cultivation lasted 6 days the damage was irreversible. Three different dose levels of titanium alloy (10%, 40%, 70%) had no effect on the proliferation or ALP activity of osteoblasts.
Dosage is an important factor affecting the biocompatibility evaluation of biomaterial. It suggests that dose choosing should be more specified upon each individual biomaterial. It also indicates that ALP may be a good supplementary index of the cell compatibility of material.
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 04/2002; 16(2):134-8.
