The neuroprotective role of brain-derived neurotrophic factor for embryonic rat cortical neurons against hypoxia via CREB phosphorylation / 生物医学工程学杂志

Transcription factor cyclic AMP response element-binding protein (CREB) in embryonic cortical neurons is an important modulator of Brain-derived neurotrophic factor (BDNF) induced gene expression. Meanwhile, our early researches have indicated that BDNF possesses neuroprotective role for hypoxic neurons against hypoxia. In order todisclose whether the neuroprotective role of BDNF for embryonic rat cortical neurons against hypoxia is fulfilled via nucleoprotein CREB phosphorylation, we used western blotting method to detect the expression of CREB and phosphorylated CREB in experimental groups (with BDNF) and hypoxic control group (without BDNF) with the time changes of exposure to hypoxia. Results indicated that hypoxia and BDNF both could induce phosphorylation of CREB in embryonic cortical neurons. Phosphorylation of CREB in experimental group (with BDNF) was much higher than that in hypoxic control group at the same time points (P<0.01). The expression of phosphorylated CREB reached the highest level at the first hour after being exposed to hypoxia in experimental groups, then phosphorylated CREB decreased slowly and remained at the level for much longer time in experimental groups than in control group. The total amount of CREB in embryonic cortical neurons at the first 0-3 hours after being exposed to hypoxia in experimental groups were the same as that in hypoxic control group. CREB decreased more quickly in hypoxic control group at 5-6 hours after hypoxia. This in vitro research demonstrates that BDNF plays its neuroprotective role for embryonic rat cortical neurons against hypoxia via CREB phosphorylation.

Isolation, culturing and growth characteristics of mesenchymal stem cells from bone marrow of Rhesus monkey, Macaca mulatta / 生物医学工程学杂志

Mesenchymal stem cells from bone marrow of Rhesus monkey (RhBMSCs) were isolated by density gradient centrifugation, purified by adherence separation, and further identified by phenotype and karyotype analysis. Growth characteristics of RhBMSCs were investigated by observation of cell proliferation and detection of apoptosis. Density gradient centrifugation and adherence separation revealed a simple method to obtain fairly pure RhBMSCs. Fusiform was the most common form of cell under observation, and cell karyotype was normal. Phenotyping assay revealed that the RhBMSCs are highly positive (99.2%) for CD29 when compared with the low positive rates (less than 3.2%) for CD34, CD45 and HLA-DR, which indicated a successful isolation of high purity RhBMSCs and a vigorous activity of cells to proliferate. But the proliferation activity of RhBMSCs gradually decreased following the increasing of cell generations. The methods and results of isolation, expansion, identification and growth characteristics of RhBMSCs were discussed in this paper, which may be helpful to understanding the bone marrow mesenchymal stem cells of human, and may serve as the groundwork for orientated differentiation of RhBMSCs and tissue repairment on experimental animal model of rhesus monkey.

Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli / 生物医学工程学杂志

To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.

Neonatal murine keratinocytes split express CD80/CD86 upon culture / 生物医学工程学杂志

It was previously thought that keratinocytes did not express the CD80 and CD86 which provide the most important costimulatory signals for the antigen-specific T-cell activation. The cultured keratinocytes allografts were initially accepted, but eventually, all grafted donor cells were gradually replaced by recipient cells. The precise mechanisms are not very clear. In this study, neonatal murine keratinocytes were cultured for 7 days, the results of flow cytometry and confocal microscopy showed that CD80 could be detected on keratinocytes, while CD86 could not be detected all the time. RT-PCR analysis confirmed this result. The expression level of the CD80 mRNA amplified significantly from day 1 to day 7, as expression of the control beta-actin, but CD86 was not detected. Mixed Lymphocyte Reaction (MLR) showed that keratinocytes cultured with 10% serum for 7 d stimulated effectively allogeneic rather than syngeneic T cell proliferation. This study demonstrated for the first time that costimulatory molecule CD80 can be expressed on keratinocytes in vitro. These data provided an alternative explanation for the ultimate rejection of allogeneic keratinocytes in which keratinocytes act as antigen-presenting cells.

Transfection of vascular endothelial growth factor gene promotes osteogenesis activities of osteoblasts / 中国组织工程研究

BACKGROUND: Ectogenesis vascular endothelial growth factor (VEGF) could enhance the activity of alkaline phosphatase (AKP) and concentration of cycli adenosine monophosphate (cAMP) fivefolds in cultured osteoblast cell. What' s the effect of ectogenesis VEGF gene transfection on osteoblasts is still by no means clear.OBJECTIVE: To investigate the effect of gene transfection and expression of ectogenesis VEGF on the osteogenesis activities of osteoblast cell.DEDIGN: A completely randomized controlled study.SETTING: Laboratory of Transplantation and Immunity, West China Hospital, Sichuan University MATERIALS: Cranial osteoblasts of newborn two or three-day male SD rat.METHODS: The experiment was conducted in the Laboratory of Transplantation and Immunology of Huaxi Hospital, Sichuan University from April to December 2003 The cranial osteolasts of newborn rat were separated and cultured with enzyme digestion method then were identified by teoblasts cultured in vitro with cation liposomes transfection as gene transations, immunohistochemical staining was performed on VEGF and collagen type I and osteocalcium were detected.collagen I and secretion of osteocalcium of osteoblasts.RESULTS: The concentration of osteocalcium and expression of type I collagen of the 1- 5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group were significantly higher than that in the control group (P ＜0.05).CONCLUSION : It is found in this experiment that the synthesis of collagen I was enhanced obviously after sussceful transfection of pBLAST49-mVEGF plasmid. Compared with the control group, the diffence of intergrated optical density gained by Mias image analysis system was significant( P ＜ 0.05),indicating that pBLAST49-mVEGF plasmid transfection can improve the synthesis of type I collagen and secretion of osteocalcium of osteoblasts.

Effect of multiple-point injection of the mixture of pBLAST49-mVEGF plasmid and liposomes into the subperiosteum on both sides of the fractured bone of rats on fracture healing: Expression of vascular endothelial growth factor at the local part of the fractured bone at different time points through a randomized blank control trial / 中国组织工程研究

BACKGROUND: Research has shown that the vascular endothelial growth factor (VEGF) of the ends of the fractured bone is heavily expressed 72 hours to 3 weeks after the fracture and it is supposed that it has a promoting effect on fracture healing. Inducing angiogenesis through VEGF gene transfection has gradually attracted the attention of the researches.OBJECTIVE: To find an efficient way of exogenous VEGF gene in vivo transfection through injecting the mixture of positive ion liposome transfection agent and plasmid and to study the promoting effect of extra VEGF gene expression on bone fracture healing.DESIGN: A randomly grouping, blank control trial.SETTING: Animal Laboratory of Huaxi Medical Center of Sichuan University MATERIALS: Totally 40 adult male SD rats, weighing 230 to 250 g,were involved. All the animals were randomly divided into the experiment group and the control group with 20 rats in each group.METHODS: The experiment was conducted at the Animal Laboratory of Huaxi Medical Center of Sichuan University from April to December 2003.Altogether 40 rats were involved to establish fractured models of right shaft of femur. Cut the bone in the middle of bone stem, retroplanted a Kirsh' nail with 1 mm diameter through intercondylar part and the fractured bone was fixed. In the experimental group, a mixture of 100 μL of liposome transfection agent and 100 μg of pBLAST49-mVEGF plasmid was injected in multiple points into the subperiosteum of the both sides of the ends of the fractured bone. The same volume of normal saline was injected into the rats in the control group. Then, 2 rats in each group were put to death 3,7,14,28,42,56,70 days after the operation and femoral bone specimen was collected.MAIN OUTCOME MEASURES: Observation of right femoral fractured staining results of VEGF, with the apperance of brown granules as positive.RESULTS: Two rats were selected at 7 time points separately, and altogether 28 rats entered the stage of result analysis. The other 12 rats were fracture at different time points: For the experimental group, 28 days after the operation, cartilage callus appeared and replaced fibrocallus gradually,and the fracture line disappeared. Fifty-six days after the operation, the bone healed completely. For the control group, 28 days after the operation , fibrocallus was observed, and the fracture line was still clear. 56days after the operation, much callus appeared, and the fracture line beof fractured bone was stained with hemotoxylin eosin (HE). In the experiment group, 56 days after the operation, the bone healed completely and trabecular like bones were rebuilt. The bone marrow cavity of the fractured region was open again. In the control group, Fifty-six days after the operation, no mature bone was formed, and the bone marrow cavity was not different time points: The expression in the two group reached to the peak on day 14 and began to decrease on day 28. The expression of VEGF in the experimental group was obviously higher than that in the control group.CONCLUSION: Injection of the mixture of positive ion liposome transfection agent into the subperiosteum of rats is an effective approach for in vivo transfection and pBLAST49-mVEGF gene transfection can effectively facilitate the bone fracture healing of rats.

The influence of IL-3 on the differentiation and development of dendritic cells from mouse bone marrow / 生物医学工程学杂志

In order to compare the effects of GM-CSF + IL-4 with the effects of IL-3 + IL-4 on the differentiation and development of dendritic cells (DCs) from mouse bone marrow, we cultured bone marrow hematopoietic progenitor cell in vitro and examined whether DCs induced by IL-3 + IL-4 (IL-3 DCs) differed from the DCs induced by GM-CSF + IL-4 (GM-CSF DCs) in morphology, yields, phenotype and endocytic activity. Scanning electron microscope, laser scanning confocal microscope (LSCM) and flow cytometer (FCM) were used. The results showed that the DCs induced by IL-3 + IL-4 and the DCs induced by GM-CSF + IL-4 had similar morphology, and the DCs induced by IL-3 + IL-4 expressed high level of major histocompability complex (MHC) class II. Besides, IL-3 DCs had the characteristics of higher yields and better consistency. But CD80 and CD86 were expressed at low levels or absent on IL-3-treated DCs. The capability of uptaking antigen of IL-3 DCs was more powerful than that of GM-CSF DCs. IL-3 could substitute for GM-CSF in culturing DCs from mouse bone marrow cells and could obtain the tolerance DCs that lack costimulatory molecules.

Visualizing living fibroblast on co-cultured denture base resin by green fluorescent protein marker introduced into the cell / 生物医学工程学杂志

Visualizing living cells growing on co-cultured biomaterials is ideal for material biocompatibility evaluation in vitro. In this experiment, mouse fibroblasts L929 were labeled by introducing the gene coding enhanced green fluorescent protein (EGFP) marker into the cells. Morphology as well as proliferation of labeled cells surrounding or on the surface of co-cultured denture base resin slides were observed by use of phase-contrast microscope and fluorescent microscope directly. It was found that residual methyl methacrylate (MMA) in the denture base resin exhibited transient cytotoxicity to fibroblasts and this transient cytotoxicity could be eliminated by pre-extracting the resin with ddH2O for a short time. This fact demonstrated that even slight cytotoxicity of materials could be detected through imaging of living cells near material or material touched. And it was suggested that imaging of living cells co-cultured with biomaterial is helpful to understanding biocompatibility of materials more accurately.

Application of enzyme histochemistry in evaluation of in vitro and in vivo biocompatibility of HA/TCP / 生物医学工程学杂志

In order to explore the possibility of applying enzyme histochemistry in biocompatibility evaluation, we investigated the effect of biomaterials on the activities of intracellular enzymes in this experiment. It was found that there was no obvious difference in morphology between osteoblasts co-cultured with HA/TCP and with Ti-alloy. However, transient down-regulation of NADH, SDH, LDH and CCO of the osteoblasts co-cultured with HA/TCP was detected by enzyme histochemistry, but these enzymes of osteoblasts the co-cultured with Ti-alloy were not down-regulated. It was indicated that something extracted from HA/TCP injured the co-cultured osteoblasts slightly. Similar early acute inflammatory reactions were observed after HA/TCP and Ti-alloy were separately implanted into the dorsal muscle of rabbit. There was also no obvious difference between the tissue response to HA/TCP and that to Ti-alloy. Activities of enzymes in tissues around implanted materials were down-regulated at the early injury period and recovered gradually within 30 days post-operation. But the mild toxicity of extracts from HA/TCP was demonstrated by the fact that the recovery period of HA/TCP group was longer than that of Ti-alloy group. It was proved that enzyme histochemistry is more sensitive than tissue morphology analysis in detecting the cell or tissue responses to biomaterials. Therefore, it is possible to use enzyme histochemistry in biocompatibility evaluation.

The quantity analysis of reverse transcriptase in porcine endogenous retrovirus expressed in Banna minipig inbred / 生物医学工程学杂志

Quantitative RT(reverse transcriptase) assay was established to detect the reverse transcriptase in plasma of thirty-four Chinese Banna minipig inbred in this work. The protocol was given in the RT kit (Roche), using HIV-1 as the positive control of the kit and supernatant of PK-15 as the PERV positive control respectively. The results show that positive reverse transcriptase reaction can be detected in the plasma of the pigs, but the levels are much lower than that of HIV-1 and lower than that of PERV in supernatant of PK-15.

Study on rabbit mesenchymal stem cells differentiation to the adipogenic or osteogenic lineage in vitro / 生物医学工程学杂志

Rabbit bone marrow-derived mesenchymal stem cells(MSCs) are multipotent. We studied the adipogenic and osteogenic differentiation potent using adipogenic supplement (AS) or osteogenic supplement (OS) in vitro. Specific markers of this induced adipogenic and osteogenic lineage were identified. The findings showed that the rabbit MSCs are capable of differentiating into adipogenic and osteogenic lineages spontaneously. On the 21st day, approximately 75% rabbit MSCs were induced to adipogenic or osteogenic cells in medium containing AS or OS, respectively. These results demonstrated that the differentiation of MSCs could be regulated in vitro. The underlying molecular mechanisms of adipogenic or osteogenic differentiation await elucidation.

Bone marrow mesenchymal stem cells: progress in bone/cartilage defect repair / 生物医学工程学杂志

Mesenchymal stem cells (MSC) are thought to be multi-potent cells that have the potential to differentiate into lineages of mesenchymal tissues, including bone, cartilage, tendon, fat, muscle, and marrow stroma during embryo morphogenesis. In recent years, cells that have the characteristics of mesenchymal stem cells were isolated from marrow aspirates of human and a few animals. It was found that these cells retain the characteristics of stem cells in vitro and could be induced to differentiate exclusively into the osteocytic, chondrocytic, myoblastic and adipocytic lineages. It was demonstrated that MSC could heal clinically significant bone and cartilage defects in animal models. The role of MSC in repairing tendon defect was also testified. In addition, for its multi-potential to differentiate into lineages of mesenchymal tissues, MSC could be used as gene vehicle for gene therapy of trauma care.

Osteogenesis of HA/TCP biphasic ceramics implanted into muscle: a long-term study / 生物医学工程学杂志

New bone formation in long-term intramuscle implant of Ca-P biomaterial was investigated in this experiment. After implanting into dog dorsal muscle for 15 months, a thin fibrous membrane that wrapped HA/TCP implant was still observed obviously. Three types of tissues, i.e. mesenchymal tissue, bone and bone marrow, regularly distributed in different pores of implant. Nearly all the pores of implants were occupied by bone. Bone in the pores located in the central region of implant was matured lamellar bone characterized by obvious lacuna and rich bone marrow. However, bone in the peripheral pores was immature woven bone without bone marrow formation. Furthermore, mesenchymal tissues only exist in the peripheral pores and usually were connected with immature woven bone. It was demonstrated that porous HA/TCP has bone inductivity and it could induce new bone formation at non-osseous site. Well-regulated distribution of mesenchymal tissue, bone and bone marrow in the pores suggest bone morphogenesis in the implant must obey a specific space-time program.

Human peripheral blood monocyte derived dendritic cell culture and mature regulation / 生物医学工程学杂志

Mature dendritic cells are potent antigen-presenting cells that initiate primary immune responses, while immature dendritic cells have quite different properties from mature dendritic cells and are tolerance inducer actually. Here we describe the method of using monocyte condition medium to generate dendritic cells of different maturation phases from nonproliferating progenitors in human peripheral blood. The procedure involves two steps. The first step(or priming phase) is to work on a 6-7-day culture of plastic-adherent blood monocyte in medium supplement with GM-CSF and IL-4. The second step (or differentiation phase) requires the exposure to monocyte conditioned medium. Only the dendritic cells generated by the first step are actually immature, with strong immature dendritic cell features such as active endocytosis, the same expression of monocyte marker CD14, and much of the MHC class II still lies within intracellular compartments (MIIC). The second stage dendritic cells have all the features of mature dendritic cell, including a stellate shape, nonadherence to plastic, the expression of dendritic cells restricted marker CD83, and very strong T cell stimulatory function. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. Since progression from immature to mature dendritic cell is entirely dependent on exogenously added growth factor such as monocyte condition medium, the peripheral blood monocyte may help to harness synchronized population of mature and immature dendritic cells for studies or therapies.

TEST OF LEMPHOCYTE TRANSFORMATION,ACTIVATION OF IL-2 AND IL-6FROM SPLEEN T-CELL IN GUINEA PIGS IMMUUIZED WITH RECOMBINANT LEPTOSPIRA GENE VACCINE / 中国人兽共患病学报

To identification the immunization activation of recombinant Leptospira gene vaccine from many-siden MethodsThe guinea pigs were immunized with recombinant Leptospira gene vaccine [plasmid vector pT7-7 was negtive control ,inactivated whole cell vaccine (WCV) was positive control]. Then spleen cells were taken out. Particularity lymphocyte transformation test(LTT),IL-2 and IL-6 activation of these spleen cells were determined by MTT and 3H-TdR respectively. Results1)The Relative transformation index of gene vaccine group was significance higher than pT7-7 group (vaccin group: 2. 19±0. 18, pT7-7 group 1.42±0. 27 ( P＜0. 005 ); 2 ) the activation of IL- 2 and IL- 6 from recombinant gene vaccine group waw significance stronger than pT7-7 group (vaccine group IL-2:34. 8±3.11,IL-6:94. 6±6.03, pT7-7 group IL-2:20. 4±3. 05,IL-6: 67±6.28), (P＜0. 005). Conclusion1) The activation of Th1 and Th2 lymphocyte cell were increased in the guinea pigs with gene vaccine immunized. It suggested the recombinant Leptospira gene vaccine could elicit an extremely strong immunization effect of T-cell cooperate with B-cell and the gene vaccine was equal the WCV(P＞0. 05)but the gene vaccine sideeffect was small and the applying prospect was good.