miR-31 promotes the proliferation and migration of bone marrow mesenchymal stem cells北大核心

BACKGROUND: Bone marrow mesenchymal stem cells have been widely used in the clinical treatment of neurodegenerative-related diseases, but their efficacy is reduced by low cell survival and migration rates at the site of injury. OBJECTIVE: To investigate whether miR-31 can enhance the migration and proliferation of bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells from C57BL/6 mice were cultured and identified, and the cells were divided into control, miR-31 agomir, and miR-31 antagomir groups. Bone marrow mesenchymal stem cells at passage 3 were inoculated in six-well plates (1×105/well). When the cells reached 50%-80% fusion, miR-31 agomir and miR-31 antagomir were added to the six-well plates after dilution with serum-free DMEM/F12. After 24 hours of transfection, the proliferation level of cells was analyzed by CCK-8 assay as well as the migration ability of cells was analyzed by Transwell assay. Protein expression of matrix metalloproteinase 2 and CXC chemokine receptor 4 was detected by western blot assay. RESULTS AND CONCLUSION: (1) miR-31 was successfully transfected with bone marrow mesenchymal stem cells without transfection reagents and emitted red fluorescence. (2) After transfection, the proliferation ability of cells in the miR-31 agomir group was enhanced compared with the control group, which increased proportionally with time (P < 0.05). Compared with the control group, miR-31 promoted the migratory ability of bone marrow mesenchymal stem cells in the miR-31 agomir group (P < 0.05) and also upregulated protein expression of matrix metalloproteinase 2 and CXC chemokine receptor 4 (P < 0.05). (3) The results indicated that miR-31 could improve the proliferation ability of bone marrow mesenchymal stem cells and promote the migration of bone marrow mesenchymal stem cells, which provides a basic study for efficient targeting of mesenchymal stem cell migration to the site of injury. © 2023, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro北大核心

BACKGROUND: Atorvastatin has a cardiovascular protective effect that significantly improves endothelial function and promotes the mobilization, migration, and differentiation of endothelial progenitor cells. However, the screening of atorvastatin concentration for in vitro cell culture is not well documented. OBJECTIVE: To investigate the effects of different concentrations of atorvastatin on rat bone marrow-derived EPCs growth characteristics. METHODS: Bone marrow mononuclear cells from Sprague-Dawley rats were induced in selective culture fluid to culture EPCs. Immunofluorescence staining was used to identify cell surface markers. Harvested EPCs were divided into control group and atorvastatin groups with four different concentrations (0.01, 0.1, 1, and 10 µmol/L) for culture. The growth and proliferation of EPCs were observed under light microscope and MTT assay. Flow cytometry was used to detect apoptosis in EPCs. Nitric oxide and endothelial nitric oxide synthase levels in the culture fluid were measured by nitrate reductase method. RESULTS AND CONCLUSION: The number of cells tended to increase in the control and atorvastatin groups, and it was highest in the 1 µmol/L atorvastatin group. The cell number in the 10 µmol/L atorvastatin group began to decrease at 7 days of culture. Among the five groups, the apoptotic rate of cells was lowest in the 1 µmol/L atorvastatin group and highest in the 10 µmol/L atorvastatin group. The levels of nitric oxide and endothelial nitric oxide synthase were significantly higher in the 0.01, 0.1 and 1.0 µmol/L atorvastatin groups compared with the control group (P < 0.01), but lower in the 10 µmol/L atorvastatin group compare with the other groups (P < 0.01). Overall, atorvastatin can promote the proliferation of endothelial progenitor cells and reduce apoptosis by increasing the production of endothelial nitric oxide synthase and nitric oxide, and 1 µmol/L atorvastatin is most suitable for the EPCs culture. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Phenylephrine effects on the morphology of osteoblasts and expression of nicotinamide phosphoribosyltransferase under oxidative stress北大核心

BACKGROUND: Phenylephrine has been proved to exert a protective effect on radiant-induced salivary gland and epithelial cell injuries, but its effect on hydrogen peroxide (H2O2)-induced oxidative stress in osteoblasts are not fully understood. OBJECTIVE: To explore the effect of phenylephrine on H2O2-induced oxidative stress in osteoblasts, and to explore the mechanism underlying the regulation by the expression level of nicotinamide phosphoribosyltransferase (Nampt). METHODS: Primary osteoblasts were cultured and randomly divided into four groups: blank control group, H2O2 group, phenylephrine group, and combination group (0.5 hour pretreatment of 1×10-5 mol/L phenylephrine, and then given 300 µmol/L H2O2). The morphology of osteoblasts was observed at different time points. Osteoblasts were collected after 24-hour culture, and total RNA and protein were then extracted to detect the mRNA and protein expression levels of Nampt by RT-PCR and western blot assay, respectively. RESULTS AND CONCLUSION: Compared with the blank control group, reduced osteoblasts and evident cell shrinks were observed in the H2O2 group, while the number of osteoblasts significantly increased in the combined group compared with the H2O2 group at 12, 24 and 48 hours of culture. RT-PCR results showed that the mRNA level of Nampt in the H2O2 group was reduced by 31.23% of that in the blank control group, while the mRNA level of Nampt in the combination group was dramatically increased by 206.20% of that in the H2O2 group at 24 hours of culture (both P < 0.05). Furthermore, western blot assay findings revealed that the protein level of Nampt in the H2O2 group was reduced by 67.98% of that in the blank control group, while the protein level of Nampt in the combination group was increased by 152.25% of that in the H2O2 group at 24 hours of culture (both P < 0.05). Our results indicate that phenylephrine can alleviate the shrink and atrophy of osteoblasts caused by H2O2, thereby exerting protective effect by up-regulating the mRNA and protein levels of Nampt that may be a regulatory gene. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Application and characteristics of bone graft materials in the treatment of spinal tuberculosis北大核心

BACKGROUND: Thorough removal of local necrotic lesions and one-stage use of bone repair materials can significantly promote local bony fusion, avoid recurrence of tuberculosis in the middle and long terms and reconstruct spinal stability in the surgical treatment of spinal tuberculosis. OBJECTIVE: To review the application of bone graft materials in the treatment of spinal tuberculosis. METHODS: The first author searched the articles related to bone graft materials of spinal tuberculosis in Bailian, CNKI, and Natures databases published from 2001 to 2020. The priority was the articles published recently or in authoritative journals. The search keywords were “bone graft materials, bone tissue engineering; spinal tuberculosis; titanium mesh; autogenous bone” in Chinese and English. RESULTS AND CONCLUSION: At present, bone graft materials have been widely used in clinic, but each has its own disadvantages. For example, the amount of autologous bone is limited, and the transplantation of autologous bone will cause bleeding and potential complications of donor site; allogeneic bone will lead to delayed healing and infection; titanium mesh has the problems of postoperative subsidence and kyphosis correction angle loss; the organic polymer materials such as polylactic acid and polymethyl methacrylate are lack of bone induction performance. Although Ca/P-based ceramic materials can be used as carrier materials of antituberculosis drugs, their biomechanical properties cannot fully meet the clinical needs. In view of the shortcomings of the above materials, it is necessary to find a composite bone tissue engineering material, which can meet the requirements of good biocompatibility, mechanical properties, degradation properties, osteogenic activity, and drug release performance. © 2022, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Proliferation and differentiation of periosteum cells induced by icariin北大核心

BACKGROUND: Icariin has a broad prospect for promoting cell proliferation. Differentiation direction of periosteal cells is uncertain, but the cells are easy to be induced by ultrasound, oxygen or bone morphogenetic protein 7 (BMP7). Periosteal cells have been applied in bone tissue engineering; however, icariin effects on the proliferation and differentiation of periosteal cells is little reported. OBJECTIVE: To investigate the effect of icariin on the proliferation and differentiation of human periosteal cells, thus providing theoretical basis for icariin applied in bone tissue engineering. METHODS: The human periosteum was obtained and the primary cells were isolated in vitro. After culture and expansion, periosteal cells were cultured in 24-well plates, and induced by 0.001, 0.01 and 0.1 mg/L icariin and 50 µg/L BMP7, respectively. The corresponding avsorbance values of different groups were detected. The levels of alkaline phosphatase and calcium nodules in periosteal cells were measured at 1, 3, 5 and 7 days, and the mRNA levels of osteocalcin, osteopontin and Runx2 were detected at 3, 5 and 7 days. RESULTS AND CONCLUSION: The periosteal cells proliferated well after induction with icariin, and could proliferate well in different concentrations of icariin and the positive control group (P < 0.05). Compared with the control group, the periosteal cells induced by icariin were able to produce more alkaline phosphatase and calcium nodules (P < 0.05). The mRNA expression of osteocalcin, osteopontin and Runx2 in periosteal cells could be up-regulated by icariin (P < 0.05). These findings imply that icariin can promote proliferation and differentiate of periosteal cells into osteoblasts, and it can be used as an inducer for the preparation of seed cells in bone tissue engineering. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Role of ABCG2 gene in proliferation and invasion of colorectal cancer stem cells北大核心

BACKGROUND: ABCG2 transporter can mediate multidrug resistance. ABCG2 overexpression can enhance the tolerance of stem cells to chemotherapeutic drugs. However, the mechanism of ABCG2 gene in the proliferation and invasion of colorectal cancer stem cells has not been confirmed. OBJECTIVE: To investigate the role of ABCG2 gene in the proliferation and invasion of colorectal cancer stem cells. METHODS: Colorectal cancer cell lines HCT116 were routinely isolated and cultured, and were randomly divided into four groups. CD133 positive cells were idted by immunomagnetic beads method, and were transfected with ABCG2-siRNA and ABCG2 overexpression plasmids to construct colorectal cancer stem cell modell with ABCG2 low expression and overexpression, and were set as low expression group and ovvrexpression groups, respectively. The remaining HCT116 cells were set as normal and smpty plasmid transfected control groups. The proliferation and invasion of colorectal cancer stem cell in different modell were determined by MTT assay and Transwell assay, respectively. Expressions of matrix metalloproteinase 9 (IMMP-9) mRNA snd protein were detected by enzyme linked immunosorbent sssay (ELISA) and polymerase chain reaction (PCR), respectively. RESULTS AND CONCLUSION: (1) Flow cytometry results showed that: CD133 positive cells in colorectal cancer cell line HCT116 accounted for 2.4%, while increased to 94.51 % of the cell line after being sorted by immunomagnetic beads, suggesting that isolated and cultured cell were colorectal cancer stem cell. (2) MTT assy showed no significant difference between the four groups of cell prior to the cell transfection (P > 0.05). However, MTT value in the low expression group was significantly lower than that in the overexpression group after cell transfection (P < 0.05). (3) Results from the Transwell assay showed that the cells in the low expression groups did not have the ability of migration; on the contrary, in the overexpression group, the number of cells crossing the basement membrane was relatively increased and the ability of cell migration and invasion was enhanced significantly (P <0.05). (4) The IVIMP-9 protein level was lower in the low expression group than in the overexpression group (P < 0.05). Similar results were yielded in the PCR detection. To conclude, down-regulation of ABCG2 protein expression can inhibit colorectal cancer stem cell proliferation and invasion. ABCG2 gene can change the viability of colorectal cancer stem cell by regulating the IMMP-9 expression. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Repair of rabbit radial bone defects using bone morphogenetic protein-2 combined with 3D porous silk fibroin/β-tricalcium phosphate hybrid scaffolds

Our study aimed to investigate the effect of bone morphogenetic protein-2 (BMP-2) bound to silk fibroin and β-tricalcium phosphate (SF/β-TCP) hybrid on the healing of critical-size radial defects in rabbits. A 15-mm critical-size defect was induced at mid-diaphysis in the left radius of 20 New Zealand white rabbits (average age, 3.5 months; weight, 2.5–3.0 kg). The animals were randomized into Group 1 (SF/β-TCP combined with BMP-2), Group 2 (SF/β-TCP alone), and Group 3 (nothing implanted). Radiographs were obtained every 2 weeks and euthanasia was performed after 8 weeks for visual, radiological, micro-computed tomography (micro-CT), and histological studies. Eight weeks after implantation (SF/β-TCP combined with BMP-2), radiographs showed that new bone formed on the surface of the implant and had bridged the defect in Group 1. Micro-CT imaging also confirmed the formation of new bone around the implant, and the newly formed bone was quantified. Histological examination revealed newly formed bone in the implanted area. Meanwhile, there was no formation of new bone in Group 3. Among the groups, most active formation of new bones was found in Group 1, while there was no difference between Group 2 and Group 3. Based on these results, we concluded that BMP-2-SF/β-TCP showed significant improvement in healing of critical-size defects. Therefore, the combination of BMP-2 and SF/β-TCP would be useful in the field of bone tissue engineering.     

Application of engineered exosomes in bone repair and regeneration北大核心

BACKGROUND: Bone defect caused by disease or accident is very common in clinic. The current main treatment method for bone defect repair is autologous bone grafting. Autologous bone grafting may cause complications, such as secondary infections and scars. The other treatments, such as allogeneic bone transplantation, may have risks of immune rejection and disease transmission. Furthermore, safety considerations and the performance of biomaterials and cell-based treatment require further clarification. In recent years, engineered exosomes have shown good application potential in bone repair and bone regeneration, and have become a current research hotspot. OBJECTIVE: To review the research progress of engineering exosomes in promoting bone defect repair and bone regeneration. METHODS: The PubMed, CNKI and Wanfang databases were searched for relevant articles with the key words of “exosome; engineered exosomes; bone repair; bone regeneration” in English and Chinese, respectively. The search time was from January 1980 to September 2020. Articles that were not related to the purpose of the research and repetitive articles were excluded, and 56 articles that met the criteria were finally included for review. RESULTS AND CONCLUSION: The engineered exosomes can not only promote bone differentiation of osteoblast-related cells and new blood vessel formation, but also target tissues and cells. A large number of experiments have proven that the engineered exosomes for repairing bone defect and promoting bone regeneration is a feasible strategy. © 2022, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways

We investigated whether the hyperbaric oxygen (O₂) could promote the proliferation of growth-arrested osteoblasts in vitro and the mechanisms involved in this process. Osteoblasts were exposed to different combinations of saturation and pressure of O₂ and evaluated at 3 and 7 days. Control cells were cultured under ambient O₂ and normal pressure [1 atmosphere (ATA)]; high-pressure group cells were treated with high pressure (2.5 ATA) twice daily; high-O₂ group cells were treated with a high concentration O₂ (50% O₂) twice daily; and high pressure plus high-O₂ group cells were treated with high pressure (2.5 ATA) and a high concentration O₂ (50% O₂) twice daily. Hyperbaric O₂ significantly promoted osteoblast proliferation and cell cycle progression after 3 days of treatment. Hyperbaric O₂ treatment stimulated significantly increased mRNA expression of fibroblast growth factor (FGF)-2 as well as protein expression levels of Akt, p70^S6K, phosphorylated ERK, nuclear factor (NF)-κB, protein kinase C (PKC)α, and phosphorylated c-Jun N-terminal kinase (JNK). Our findings indicate that high pressure and high O₂ saturation stimulates growth-arrested osteoblasts to proliferate. These findings suggest that the proliferative effects of hyperbaric O₂ on osteoblasts may contribute to the recruitment of osteoblasts at the fracture site. The FGF-2/MEK/ERK 1/2/Akt/p70^S6K/NF-κB and PKC/JNK pathways may be involved in mediating this process.     

Research hotspot of biological scaffold materials promoting osteogenic differentiation of bone marrow mesenchymal stem cells北大核心

BACKGROUND: At present, a single biological scaffold material is difficult to meet the osteogenic needs of bone tissue engineering, and bone marrow mesenchymal stem cells have excellent osteogenic characteristics. Composite scaffolds and scaffolds combined with growth factors have better osteogenic ability. It is a research hotspot at present. OBJECTIVE: To review different biological scaffolds and their modified scaffolds to promote the osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: The related articles published in CNKI, Wanfang, VIP, PubMed and Embase databases from January 2014 to July 2020 were searched by the first author with the keywords of “bone marrow mesenchymal stem cells, scaffolds, osteogenic differentiation, hydroxyapatite, collagen, chitosan” in English and Chinese. Finally, 69 articles were selected. RESULTS AND CONCLUSION: The rapid development of bone tissue engineering can effectively solve the problem of bone defect repair. Seed cells and biological scaffold materials are the core of bone tissue engineering. Bone marrow mesenchymal stem cells have excellent osteogenic differentiation ability and are widely used in bone tissue engineering. The combination of different scaffold materials, the use of advanced preparation technology, or the surface modification of scaffolds and the addition of growth factors can fully combine the advantages of various biological scaffold materials, induce the osteogenic differentiation of bone marrow mesenchymal stem cells and the formation of scaffold blood vessels, and achieve the purpose of repairing bone defects, and is the research focus of bone tissue engineering. © 2022, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Research and application of the reamer-irrigator-aspirator北大核心

BACKGROUND: Large volume of bone can be harvested by the reamer-irrigation-aspirator (RIA), which can be used for autologous bone graft, because there are many stromal stem cells and a variety of osteogenesis factors in the harvested bone. OBJECTIVE: To introduce the application progress of the RIA. METHODS: PubMed database was retrieved by the first author for RIA-related articles published from January 2000 to May 2017 using the keyword of " reamer-irrigator-aspirator”. A total of 87 articles were searched initially and finally 38 articles met the inclusion criteria. RESULTS AND CONCLUSION: Compared with harvesting bone tissues from the iliac bone, the RIA can harvest more autologous bone tissues with more bone marrow stromal stem cells. Moreover, the osteogenic effect of the harvested autologous bone is better and there are fewer complications. Therefore, the RIA has been widely used as a method of harvesting non-structural autologous bone tissues. Removal of intramedullary lesions by the RIA is used in the treatment of long bone infection, osteomyelitis and bone tumors, as well as in the removal of intramedullary cement. RIA was originally used as a special reaming during intramedullary nailing for femoral shaft fractures, which could reduce high pressure and high temperature so as to decrease the risks of fat embolism and osteonecrosis. To conclude, the RIA can achieve satisfactory outcomes and result in few complications, but the clinical use is still limited by high cost, frequent fluoroscopy, bleeding and limited indications, as well as risks for iatrogenic fractures and perforation. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

HCMV-encoded UL128 enhances TNF-α and IL-6 expression and promotes PBMC proliferation through the MAPK/ERK pathway in vitro

Cytomegalovirus (CMV) infection enhances expression of several cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-8, to the benefit of virus replication and dissemination. However, the stimulus for certain cytokine production remains unclear. CMV encodes a series of proteins that alter and/or mimic functions of leukocyte migration, activation, and cytokine responses. Our study revealed that human CMV (HCMV)-encoded UL128 protein, which contains signal peptides and has similar amino acid sequences to the CC chemokine, recruits monocytes as human β chemokine (microphage inflammatory protein 1α). Using RNA interference technology, we constructed an HCMV (UL128?/UL128?)-infected tissue cell (MRC-5) and peripheral blood mononuclear cell (PBMC) co-culture system. We measured 6 cytokine levels (IL-2, IL-4, IL-6, IL-10, TNF-α, and interferon-γ [IFN-γ]) in the supernatant, and found significantly elevated IL-6 and elevated TNF-α levels in the HCMV UL128?-infected group. Conversely, we observed decreased levels in the UL128-knockout supernatant. PBMCs presented with UL128 (50?ng/mL) demonstrated better cell viability than the UL128-absent group. Finally, the MAPK/ERK pathway was found to be involved in UL128 induction of cell proliferation. Selective induction of cytokine expression indicates that HCMV-encoded UL128 is a potent inducer of several inflammatory mediators.     

In vitro study of the proliferation and growth of human bone marrow cells on apatite–wollastonite-2M glass ceramics

This study concerns the preparation and in vitro characterization of an apatite–wollastonite-2M bioactive glass ceramic which is intended to be used for the regeneration of hard tissue (i.e. in dental and craniomaxillofacial surgery). This bioglass ceramic has been obtained by appropriate thermal treatment through the devitrification (crystallization) of a glass with a stoichiometric eutectic composition within the Ca₃(PO₄)₂–CaSiO₃ binary system. Crack-free specimens of the bioglass ceramic were immersed in human bone marrow cell cultures for 3, 7, 14 and 21 days, in order to study biocompatibility. Cell morphology, proliferation and colonization were assessed by scanning electron microscopy and confocal laser scanning microscopy. A total protein content assay was used to evaluate the viability and proliferation of cultured bone marrow cells. The results showed that the cells were able to adhere and proliferate on the designed material due to the essentiality of silicon and calcium as accessory factors for cell activity stimulation.     

Dispersion degree of a small-dose bone cement北大核心

BACKGROUND: During the percutaneous vertebroplasty, the optimal dose of bone cement that can bring favorable cement dispersion and remodel the biomechanical balance of the fractured vertebrae remains controversial. OBJECTIVE: To investigate the dispersion degree of small dose of bone cement in vertebroplasty. METHODS: In this experiment, 18 sheep selected with the same condition were randomly divided into three groups (group A, group B, group C), 6 in each group. A model of thoracolumbar vertebral compression fracture (T12, L1, L2) was made in each sheep. The injected volume of bone cement in groups A, B, C was 15%, 20%, 25% of the average volume of adjacent vertebral bodies, respectively. Postoperative CT images were used to evaluate the bone cement dispersion. Dispersion degree of bone cement among the three groups was compared by the Kruskal-Wallis test. RESULTS AND CONCLUSION: There was no statistical difference in the dispersion degree of bone cement among the three groups, and the excellent and good rate of dispersion was over 80%. To conclude, the optimal dose of bone cement injected into the fractured vertebra is 15% of the average volume of adjacent vertebral bodies, which can achieve good dispersion degree and restore the biomechanical stability of the vertebral body. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Effect of α-fetoprotein on the count of bone marrow and splenic stromal precursor cells and proliferation of their cultural descendants

The efficiency of cloning of stromal precursor cell increased more than 2-fold in splenic cultures and more than 3-fold in bone marrow cultures 24 h after injection of Profetal preparation to mice in vivo. The number of nucleated cells did not change in the bone marrow and slightly increased in the spleen. Addition of Profetal in vitro 2-fold decreased the efficiency of stromal precursor cells colony formation in mouse splenic cultures and dose-dependently decreased this process in bone marrow cultures derived from these animals, the maximum (5-fold) inhibitory effect was observed in a dose of 50 μg/ml. Addition of Profetal to cultured human bone marrow fibroblasts did not change the content of stromal fibroblasts in cultures. These data indicate the possibility of indirect effect of α-fetoprotein on the number of stromal precursor cell in hemopoietic and lymphoid organs. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 1, pp. 30–33, January, 2007     

MiR-150-5p inhibits the proliferation and promoted apoptosis of pancreatic cancer cells北大核心CSCD

Objective: To investigate the role of miR-150-5p in cell proliferation and apoptosis in human pancreatic cancer cell lines. Methods: The expression of miR-150-5p in pancreatic cancer was detected by real time qPCR analysis in 11 pairs of pancreatic cancer tissue and matched adjacent normal tissue samples and in 4 pancreatic cancer cell lines. PANC-1, MIA PaCa-2, BxPC-3 and AsPC-1 cells were transfected with chemically synthesized MiR-150-5p mimics, and CCK-8 assays was then performed to assess cellular functions. To fully understand the mechanisms by which miR-150-5p exerted its function, cell cycle analysis was performed on MIA PaCa-2 and PANC-1 cells 48 hours after transfection, by incubating with propidium iodide (PI) and subsequently analyzed by fluorescence-activated cell sorting (FACS). Apoptosis assay was performed on MIA PaCa-2 and PANC-1 cell lines 24 hours after transfection using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) and analyzed by FACS. Results: The expression of miR-150-5p was consistently lower in the pancreatic cancer tissues than in normal tissues,and the miR-150-5p was also down-regulated in pancreatic cancer cell lines (P < 0. 05). MiR-150-5p mimics transfection significantly raised the expression level of miR-150-5p mRNA in PANC-1 and MIA PaCa-2 (P <0. 01). The CCK-8 proliferation assay showed that cell growth was reduced in 4 pancreatic cancer cell lines (AsPC-1, BxPC-3, MIA PaCa-2, PANC-1) of miR-150-5p transfected cells compared with NC-transfected cells. The inhibition rates were 50. 7%,48. 6%,30. 8% and 42. 3%, respectively (P <0. 01). The apoptotic rate was increased in cells transfected with miR-150-5p mimics (P < 0. 01). The cell cycle analysis in MIA PaCa-2 indicated that miR-150-5p treatment induced cell cycle arrest in G1 phase with a significant increase in the percentage of cells in G, phase (P < 0. 01), and a reduction of the S-phase cell population in MIA PaCa-2 and PANC-1 (P<0.01). Conclusions: MiR-150-5p is down-regulated in pancreatic cancer. Overexpression of miR-150-5p inhibits cell proliferation, blocked the cell cycle, but promotes cell apoptosis in pancreatic cancer cells.     

Constructing nanosized cell-type tissue-engineered bone for repair of mandibular bone defects北大核心

BACKGROUND: Nanosized cell-type tissue-engineered bone is a good scaffold material possessing the merits of stem cells and nanomaterials to fabricate bone and soft tissue formation. OBJECTIVE:To explore the method of constructing nano-sized cell-type tissue-engineered bone and to explore its application in the repair of mandibular bone defects. METHODS: One New Zealand white rabbit was taken to isolate bone marrow stromal stem cells by centrifugation. Then, the cells were induced to differentiate into osteoblasts. Osteoblasts (3x108/L, 10 µL) were inoculated into the prepared nano-phase hydroxyapatite/collagen composite to produce the nano-sized cell-type tissue-engineered bone. Another 20 New Zealand white rabbits were taken to make a unilateral puncture-type bone defect model of 15 mmx8 mm. These model rabbits were thereafter randomized into control and artificial bone groups (n=10 per group), followed by no intervention and implantation of nano-sized cell-type tissue-engineered bone, respectively. Repair effects were compared between the two groups. RESULTS AND CONCLUSION: (1) Under the inverted microscope, osteoblasts grew along the material in each group, and the number of cells increased with the prolongation of the culture time. (2) Under the scanning electron microscope, a large number of spindle- or polygon-shaped adherent cells grew well on the surface of the tissue-engineered bone. (3) The defect in the artificial bone group was lessened at 4 weeks after implantation and disappeared at 8 weeks after implantation, and there was no clear boundary with the surrounding tissue. In the control group, the defect size changed little at 4 weeks and reduced at 8 weeks after implantation, and a clear boundary with the surrounding tissue was observed. (4) Bone density, trabecular thickness and trabecular number were significantly higher in the artificial bone group than the control group at 4 and 8 weeks after implantation (P < 0.05). (5) At 4 weeks after implantation, many new bones at the defect site were detected in the artificial bone group, and a large number of mature bone cells were visible at 8 weeks. In the control group, a few osteoblasts were found at the defect site with low bone maturation at 4 and 8 weeks after implantation. These findings suggest that the implantation of nanosized cell-type tissue-engineered bone into the defect site can considerably promote defect healing and achieve ideal repair effects. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.