ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of local delivery of delta12-prostaglandinJ2-loaded poly (lactic-co-glycolic acid) (Δ(12)-PGJ2-NC) on growth factors expression and bone formation.</p><p><b>METHODS</b>Δ(12)-PGJ2-NC was prepared by the emulsion solvent diffusion method. The physical and chemical properties of the nanoparticles were evaluated by particle size analysis, transmission electron microscopy, drug-loading ratio and the in vitro release study. Then standardized transcortical defect (5.0 mm × 1.5 mm) was conducted in the femur of 48 male Wistar rats which were randomly divided into four groups (n = 12), S, K, F, and N. Thirty microliter of saline (S), unloaded nanoparticles (K), Δ(12)-PGJ2 (F) and Δ(12)-PGJ2-NC(N) in a collagen vehicle were delivered inside a titanium chamber fixed over the defect. Then, four subgroups were randomly divided in each group named as D3, D7, D14, and D28 (n = 3) according to the days 3, 7, 14, and 28 after the surgery. At days 3, 7, 14, and 28, the mRNA expression of the bone morphogenetic protein-6 (BMP-6), platelet-derived growth factor-B (PDGF-B) in defect aera was analyzed by real time quantitive-polymerase blotting. HE staining was employed to reveal new bone formation in weeks 2 and 4.</p><p><b>RESULTS</b>Δ(12)-PGJ2-NC appeared opalescent white and remained relatively stable, with an average particle size of (135.2 ± 0.85) nm. The images from transmission electron microscopy showed that Δ(12)-PGJ2-NC was spherical in shape and homogeneously distributed. The encapsulation efficiency of Δ(12)-PGJ2 with the poly (lactic-co-glycolic acid) (PLGA) nanocapsules was about 92%. The in vitro release of Δ(12)-PGJ2-NC at 37 °C showed a sustained fashion and the average accumulated amount was 30%, 52%, 77%, 91%, and 98% respectively, at 0.5, 1, 2, 4 and 6 h. Compared with the animals treated with saline, after dose of 100 mg/L Δ(12)-PGJ2 and Δ(12)-PGJ2-NC apllication, the mRNA expression level of BMP-6, PDGF-B increased significantly (P < 0.05, P < 0.001). The protein expression of BMP-6, Ephrin-B2 also was up-regulated. Histomorphometry revealed that new bone formation increased at the same dose of 100 mg/L. But the unloaded nanoparticles did not have the same effect (P > 0.05).</p><p><b>CONCLUSIONS</b>A stable Δ(12)-PGJ2 loaded nanoparticle was successfully prepared. Δ(12)-PGJ2-NC may upregulate the expression of BMP-6, PDGF-B and Ephrin-B2, and promote new bone formation in bone defect area.</p>
Subject(s)
Animals , Male , Rats , Bone Morphogenetic Protein 6 , Genetics , Metabolism , Bone Regeneration , Ephrin-B2 , Genetics , Metabolism , Femur , General Surgery , Lactic Acid , Pharmacokinetics , Pharmacology , Nanocapsules , Nanoparticles , Particle Size , Polyglycolic Acid , Pharmacokinetics , Pharmacology , Prostaglandin D2 , Pharmacokinetics , Pharmacology , RNA, Messenger , Metabolism , Random Allocation , Rats, Wistar , Receptor, Platelet-Derived Growth Factor beta , Genetics , Metabolism , Time Factors , Up-RegulationABSTRACT
Objective Aim to set up the method of glycoprotein B genotype of human cytomegalovirus (HCMV) analysis by using nested polymerase chain reaction(nPCR) and restriction fragment length polymorphism(RFLP). Methods Eleven blood samples and twenty-three urine samples were obtained from thirty-four HCMV-infected patients. A fragment of gB gene was amplified by nPCR. HCMV gB genotyping was carried out by RFLP, and the amplified DNA fragments were verified by DNA sequencing. Results Of the 34 patients, gB type Ⅰ was found in 13 patients, gB type Ⅱ in 12 patients, gB type Ⅲ in 9 patients, and none had the gB type Ⅳ sequence. The similarities of PCR products of HCMV gB Ⅰ, Ⅱ and Ⅲ amplified compared with the sequences of prototype strains in GenBank were 98.1%～99.6%, 98.9%～100%, 97.3%～98.9%. Conclusions The nPCR assay developed in this study was sensitive and specific for detection of HCMV, and RFLP analysis of HCMV gB genotype was definite and reliable.