Aim DNA vaccinating BALB/c mice with the constructed recombinant plasmid, pcDNA3, containing ROP1 gene from Toxoplasma gondii to observe the effect on the production of the cytokines, IFN- γ、 IL - 2 , and NO in the immunized mice. Methods Large-scale preparation of plasmid DNA by alkaline lysis,the DNA were injected through muscles of left leg in each mouse at the dosage of 100μg. A booster vaccine was given at the same dosage after two weeks. Control groups were injected with pcDNA3 blank plasmid and normal saline respectively. After 30,50 and 70 days of the booster injection, following tests were carried out 3 times separately :the serum IFN-γand IL-2 were detected by sandwich ELISA;the NO was detected by enzyme assay. Results The 3 times detected results of IFN-γγ、IL-2 and NO were significant higher in the vaccinated group than that of in control groups and the contents were increased with the vaccinated time prolonged. Conclusion The IFN - γγ、 IL - 2 and NO were produced by vaccinating BALB/c mice with the recombinant plasmid, pcROP1.

Objective Patients with knee osteoarthritis undergoing total knee arthroplasty may have a different degree of pain during the perioperative period. This will not only bring a lot of adverse effects to the patients,and directly affect the early postoperative functional exercise and rehabilitation of the knee joint. At present,the commonly methods used to relieve the pain after TKA are:patient con-trolled Intravenous analgesia(PCIA)、patient controlled epidural analgesia( PCEA)、continuous femoral nerve block analgesia(CFNB)、joint peripheral injection analgesia and some methods without using medi-cine. In this paper,the analgesia methods used during the perioperative period of TKA and the latest de-velopment are reviewed.

Objective　To construct the eukaryotic expression recombinant plasmid, pcIFN-γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3-rhoptry protein 1 （pc-ROP1） combined with pcIFN-γ against Toxoplasma gondii (T. gondii) infection in mice. Methods　A fragment of the IFN-γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion. pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100*!μg, and a booster vaccination was given at the same dosage after two weeks. Control groups were injected with pcDNA3 blank plasmid or normal saline. At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC-ELISA for the determination of IFN-γ, IL-2 and IL-10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera. Results　The recombinant plasmid, pcIFN-γ was constructed. The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN-γ, IL-2 and NO in mice injected with pcROP1 and pcIFN-γ were higher than in those injected with pcROP1 alone. There was no difference in IgG antibody levels between the two groups. Conclusion　The genetic adjuvant, pcIFN-γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection.

BACKGROUND:In recent years, repair of articular cartilage injury has become an important field in basic medical research. Because injured articular cartilage is difficult to repair, the repair of articular cartilage injury has become a difficult hotspot.

BACKGROUND:Mesenchymal stem cels, underin vivo orin vitro specific induction conditions, can differentiate into the cartilage, muscle, tendons and so on. Clinical trials concerning mesenchymal stem cels mainly include tissue repair (such as bone, cartilage and joint repair) and treatment of heart, liver, spinal cord injury and nervous system diseases.
OBJECTIVE:To compare the biological characteristics of mesenchymal stem cels from different sources.
METHODS: PubMed and CNKI databases were retrieved for articles related to sources of mesenchymal stem cels and biological characteristics of mesenchymal stem cels published from 1987 to 2015. The retrieved articles were summarized and analyzed in the folowing aspects: cel surface marker, proliferation, differentiation, migration, and function, so as to explore the merits and demerits of mesenchymal stem cels from different sources.
RESULTS AND CONCLUSION:A difference in the proliferation ability and surface markers is found between different sources of mesenchymal stem cels. Immunological competence of mesenchymal stem cels from different sources may be correlated with their activation status, species differences, tissue sources and culture conditions, resulting the immunological competence of mesenchymal stem cels from different sources is not exact the same. In-depth understanding of the factors and mechanisms by which influence the migration of mesenchymal stem cels from different sources can enhance the migration ability of different sources of mesenchymal stem cels, and increase their efficiency in wound healing, tissue repair and regeneration treatment.

BACKGROUND:Osteoarthritis is a joint disease that primarily affects the cartilage. With the changes of the extracelular matrix, chondrocytes appear to have apoptosis. Vascular endothelial growth factor plays an important role in promoting endothelial cel division and proliferation and inducing angiogenesis. Hypoxia-inducible factor is a celular transcription factor and produces different reactions due to the oxygen content. Vascular endothelial growth factor and hypoxia-inducible factor are focused on inhibiting chondrocyte apoptosis. OBJECTIVE:To elaborate the effects of vascular endothelial growth factor and hypoxia-inducible factors on chondrocyte apoptosis. METHODS: Recent literatures related to chondrocyte apoptosis were summarized and analyzed. During the process of osteoarthritis, changes in vascular endothelial growth factors in chondrocytes and regulatory effects of vascular endothelial growth factor and hypoxia-inducible factor on chondrocyte apoptosis were elaborated.

BACKGROUND:Articular cartilage repair has been a difficulty in the clinical setting, which is mainly treated with autologous or al ogeneic osteochondral grafts, and cartilage periosteum or periosteum grafts. However, the limited source, secondary lesion and immunological rejection force some researchers to search for a novel treatment strategy, cartilage tissue engineering, that is of great significance for cartilage regeneration and repair. OBJECTIVE:To investigate the tissue-engineered scaffolds for the repair of articular cartilage defects. METHODS:The first author searched the PubMed and WanFang databases for the articles addressing tissue-engineered cartilage for articular cartilage defects published between 1991 and 2015 using the keywords“articular cartilage defect, scaffold, tissue engineered cartilage”in English and Chinese, respectively. The irrelative and repetitive literatures were excluded. RESULTS AND CONCLUSION:Final y 48 eligible literatures were enrol ed based on the inclusion and exclusion criteria. Cartilage tissue engineering possesses the advantages of control ability, little damage to tissue itself, and biological repair of injured cartilage. Tissue-engineered scaffold material is a critical factor in tissue engineering construction;therefore, it should hold biodegradability and histocompatibility. The commonly used scaffold materials include natural macromolecule materials (col agen, silk fibroin and chitosan), and synthetic polymer materials (polylactic acid and tricalcium phosphate). It is necessary to prepare composite scaffolds with high bioactivity integrate advantages of each material. The tissue engineering is bound to be a hotspot in the field of articular cartilage repair.

Objective　To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods　Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed. Results　The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion　pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

OBJECTIVETo characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis.

METHODSMice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally.

RESULTSSignificant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival.

CONCLUSIONHumoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

Animals ; Antibodies, Protozoan ; blood ; Antigens, Protozoan ; Cytokines ; biosynthesis ; Female ; Immunization ; Immunoglobulin G ; blood ; classification ; Interleukin-2 ; genetics ; Mice ; Protozoan Proteins ; genetics ; Protozoan Vaccines ; immunology ; Toxoplasma ; immunology ; Toxoplasmosis, Animal ; prevention & control ; Vaccines, DNA ; immunology

OBJECTIVETo evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii (T. gondii) surface antigen1 (SAG1) and rhoptry protein 1 (ROP1) in mice as a hybrid gene.

METHODSTruncated SAG1 and ROP1 DNA fragments were amplified using polymerase chain reaction (PCR) and inserted into pEGFP-N3 vector to construct recombinant plasmid pSAG1-ROP1. NIH3T3 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10 micro g plasmid DNA entrapped in liposome. Four weeks after the final booster injection, blood samples were collected and subjected to enzyme-linked immuno sorbent assay (ELISA) to investigate humoral and cell-mediated immune responses. Reversal transcript-polymerase chain reaction (RT-PCR) was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. Dot-blot hybridization was employed in order to detect whether or not the injected DNA was incorporated into the genomic DNA of the immunized mice.

RESULTSGreen fluorescence was observed in pSAG1-ROP1-transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1-ROP1 was between 58 kDa and 75 kDa. No expression was observed in blank control plasmid-transfected cells. The sera of immunized mice exhibited antibodies to T. gondii tachyzoites and primarily interferon-gamma and interlukin-2. RT-PCR showed that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post the first injection. Dot-blot hybridization revealed that the presence of foreign DNA in the splenocytes and peripheral blood leukocytes was transient and that no foreign DNA had inserted into the genomic DNA of mice immunized with pSAG1-ROP1.

CONCLUSIONSImmunization with a liposome-encapsulated DNA construct encoding the T. gondii SAG1 and ROP1 can induce humoral and cell-mediated immune responses.

Animals ; Antibody Formation ; Antigens, Protozoan ; genetics ; DNA ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Vectors ; Immunity, Cellular ; Immunization ; Liposomes ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Protozoan Proteins ; genetics ; Toxoplasma ; genetics ; immunology ; Transfection