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Objective:A new type of bio-ink and polycaprolactone (PCL) were used to construct an integrated osteochon-dral composite tissue block by multi-nozzle 3D bioprinter. And the repair results to osteochondral defects were evaluated.Methods:In freeze-drying group: Freeze-dried composite scaffold made by silk fibroin (SF) and β-tricalcium phosphate was used to repair osteochondral defects, as control. In the 3D printing group: PCL was used to printed a hollow multi-layer cylinder frame by 3D biological printer. Extracellular matrix, SF and bone marrow mesenchymal stem cells were used as chondral bio-ink. Then, chon-dral bio-ink was used to print tissue-engineered cartilage on top of PCL frame. Before implantation of cartilage defect, autogenous cancellous bone was filled in PCL frame, then the tissue-engineered osteochondral composite was used to repair osteochondral defects. In mosaic group: Autologous osteochondral transplantation was performed. The repair results of the above three groups were compared by histological score, biochemical analysis and biomechanical test to evaluate the effect of repairing rabbit cartilage defects.Results:The compression modulus of neo-cartilage in the 3D print group 2.56±0.30 MPa was close to that of the mosaic group 2.51±0.13 MPa ( P>0.05), and significantly higher than that of freeze-dried group 1.37±0.14 MPa ( F=11.058, P<0.05). The sGAG contents in the 3D print group 14.49±0.7 μg/mg was close to that of the mosaic group 14.98±0.81 μg/mg ( P>0.05), and significantly higher than that of freeze-dried group 8.72±0.73 μg/mg ( F=20.973, P<0.05). However, there was no significant difference in collagen content between the three groups ( P>0.05). The results of ICRS cartilage repair histology score showed that the scores of the 3D print group were close to those of the mosaic group in the matrix, cell distribution, cell viability and subchondral bone ( P>0.05), and were significantly higher than those of freeze-dried group in the surface and cartilage mineralization scores ( F=19.544, P<0.05). Conclusion:Using the new bio-ink to make bone cartilage composite scaffold by 3D bio printing can simplify the construction of tissue-engineered bone cartilage composite tissue in vitro, and can repair cartilage defects in vivo.
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Objective:To repair the articular cartilage defects of animal models with cartilage tissue block made by multi-nozzle three-dimensional bio-printer and observe its effect.Methods:Bio-ink was made by adding silk fibroin, polyethylene glycol and bone mesenchymal stem cells into extracellular matrix (ECM) solution. Rheological properties of biological ink were evaluated by rheometer, the protein secondary structure of biological ink was identified by Fourier transform infrared spectroscopy, and a tissue engineering scaffold with thickness of 2mm and diameter of 6mm was printed by using a pressure sprinkler loaded with cartilage biological ink. The compression modulus of tissue engineering scaffold was measured by tension machine. The degradation rate of each scaffold was evaluated by dry weight loss method, and the viability and proliferation of cells on the scaffold were evaluated by CCK-8 and live&dead cell staining. The differentiation of cellular cartilage on the scaffold was evaluated by real-time fluorescence quantitative PCR. The scaffold was embedded into the defect area of animal articular cartilage to repair articular cartilage defect according to the principle of autogenous cartilage transplantation. The effect of cartilage repair after 3 months was evaluated by histological staining and biochemical detection.Results:We found that all biological inks showed the flow characteristics of shear thinning. The absorption peak of biological ink amide I region containing silk fibroin moved to 1 623 cm -1. With the increase of silk fibroin content, the mechanical strength and degradability of biological ink were improved, and the compression modulus of 10% and 15% printing stand reached 19.96±5.66 kpa and 26.87±10.68 kpa, respectively. All biological inks had no obvious cytotoxicity. Real-time quantitative PCR showed that when the content of silk fibroin reached 10%-15%, the bone marrow mesenchymal stem cells in the tissue mass had stronger ability to differentiate into cartilage. In vivo studies showed that after 3 months, the sGAG/DNA content of 10% and 15% silk fibroin scaffolds reached 0.25±0.01 μg/ng and 0.24±0.02 μg/ng, respectively, and the collagen/DNA content reached 17.71±0.83 ng/ng and 16.69±2.39 ng/ng, respectively. Tissue engineered cartilage printed with high concentration silk fibroin can better repair articular cartilage defects. Conclusion:TThe chondrogenic differentiation and extracellular matrix (collagen and glycosaminoglycan) secretion of BMSCs were superior to those of the other two scaffolds when the content of silk fibroin reaches 10%-15%. The changes of chondrogenic differentiation ability and extracellular matrix secretion of stem cells from different scaffolds, as well as the repair effect on articular cartilage defects are caused by the differences of mechanical properties of scaffolds, which can be produced by the changes of silk fibroin concentration.
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Objective To investigate the effects and its possible mechanisms of tiopronin (TIP) on interleukin-2 (IL-2) immunotherapy of human leukemia KG-1 cells transplanted in nude mice.Methods KG-1 cells (1 x 107/ml) in logarithmic growth phase were injected subcutaneously into the groin of the left hind leg of the 45 5-week-old nude mice.When the subcutaneous tumor diameter was about 8 mm,nude mice were randomly divided into three groups (n =15):Control group (intraperitoneal injection of phosphate buffer),IL-2 group (hypodermic injection of IL-2),IL-2 + TIP group (hypodermic injection of IL-2 and intraperitoneal injection of TIP).The therapeutic effect of TIP combined with IL-2 on human leukemia KG-1 cells transplanted in nude mice was observed.The number of nature killer (NK) cells in peripheral blood of nude mice was detected by flow cytometry.Nitrate reductase assay was used to detect reactive nitric metabolite (RNM) levels in peripheral blood of nude mice.Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-β (TNF-β) and interferon-γ (IFN-γ) in peripheral blood of nude mice.Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay was used to analyze apoptosis.Results Both IL-2 and IL-2 + TIP could inhibit the growth of transplanted tumor.Compared with IL-2 group [(54.32 ± 4.32) ％],the tumor inhibition rate of IL-2 + TIP group was (90.15 ± 3.75)％,and its inhibition of tumor growth was more obvious (t =11.893,P ＜ 0.001).The tumor weights of Control group,IL-2 group and IL-2 + TIP group were (0.95 ± 0.05)g,(0.58 ± 0.03)g and (0.27 ± 0.07)g,and there was statistically significant difference among the three groups (F =52.716,P ＜ 0.001).Compared with IL-2 group,the tumor weight of IL-2 + TIP group was significantly reduced (P =0.008).The number of NK cells in IL-2 + TIP group was (0.658 ±0.157)/L,which was significantly higher than (0.452 ±0.124)/L of IL-2 group (P =0.021).The concentration of RNM in IL-2 + TIP group was (42.92 ± 4.68)μmol/ml,which was significantly lower than (163.38 ± 5.49)μmol/ml in IL-2 group (P =0.007).The concentrations of TNF-β and IFN-γin IL-2 + TIP group were (247.68 ± 8.24) pg/ml and (185.61 ±7.58) pg/ml,which were significantly higher than (97.48 ± 7.28)pg/ml (P =0.021) and (70.62 ± 8.47)pg/ml (P =0.015) in IL-2 group.The apoptotic rate of tumor cells in IL-2 + TIP group was (47.38±4.25)％,which was significantly higher than (21.41 ±2.79)％ in IL-2 group (P ＜0.001).Conclusion TIP can increase the sensitivity of leukemia cells to IL-2 immunothe-rapy by removing RNM,promoting NK cells activity and increasing NK cells-induced tumor cell apoptosis.
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Objective To investigate the effect of Tiopronin (TIP) on interleukin (IL)-2 immunotherapy of human leukemia KG-1 cells and its possible mechanism.Methods KG-1 ceils in logarithmic growth phase were randomly divided into KG-1 + IL-2 group and KG-1 + IL-2 + TIP group.Methyl thiazolyl tetrazolium (MTI) assay and colony formation assay were used to detect the sensitivity and proliferation of KG-1 cells.The changes of reactive nitric metabolites (RNM) were detected with nitrate reductase method.The production of tumor necrosis factor (TNF)-3 and interferon (IFN)-γ,was detected with enzyme linked immunosorbent assay (ELISA).The expression of CD3ξ was detected with Western blot and real time polymerase chain reaction (RT-PCR).Results IL-2 and IL-2 + TIP could inhibit the growth of KG-1 cells.The inhibitory rate of KG-1 + IL-2 + TIP group was significantly higher than that of KG-1 + IL-2 group,and the sensitivity of KG-1 cells to IL-2 was 6.2 times higher.Both IL-2 and IL-2 + TIP group inhibited the colony formation of KG-1 cells.Compared to KG-1 + IL-2 group,KG-1 + IL-2 + TIP group inhibited the colony formation of KG-1 cells by 3.5 times.The RNM production of KG-1 + IL-2 group was (158.26 ± 3.82) μmol/ml,which was significantly higher than (45.18 ± 4.29) μ mol/ml of KG-1 + IL-2 + TIP group (P ＜ 0.05).The levels of TNF-β and IFN-γin KG-1 + IL-2 + TIP group were (253.28 ± 7.84) pg/ml and (181.25 ±6.41) pg/ml,which was significantly higher than (98.45 ±6.43) pg/ml and (68.74 ±8.26) pg/ml of KG-1 +IL-2 group (P＜0.05).The expression of CD3ξ in KG-1 +IL-2 +TIP group was significantly higher than that in KG-1 + IL-2 group.Conclusions Tiopronin can promote NK/T cell activity and increase the sensitivity of leukemia KG-1 cells to IL-2 by eliminating reactive nitrogen metabolites.
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Objective To investigate the effect of E1A gene on the radiosensitivity of human nasopharyngeal carcinoma cells and its possible mechanism. Methods The E1A gene was transfected into nasopharyngeal carcinoma CNE-2R cells by adenovirus vector. The expression of E1A gene was detected by RT-PCR. Untransfected CNE-2R cells(PBS group)and CNE-2R cells transfected with empty vector Ad-β-gal(Ad-β-gal group)and E1A(Ad-E1A group)were given 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy 6 MV X-ray irradiation. The changes in radiosensitivity of CNE-2R cells were determined by colony-forming assay. Flow cytometry was used to analyze cell apoptosis in each group. The expression of NF-κB, CK2α, Bcl-2, and cleaved caspase-3 was measured by Western blot. Results RT-PCR confirmed that the E1A gene was transfected into CNE-2R cells and stably expressed. The Ad-E1A group had a significantly lower plating efficiency than the PBS group and the Ad-β-gal group(P<0.05). The Ad-E1A group had significantly lower cell survival rate at 2 Gy irradiation than the PBS group and the Ad-β-gal group(0.217 vs. 0.602, P<0.05;0.217 vs. 0.585, P<0.05). The Ad-E1A group had a significantly higher α/β value than the PBS group and the Ad-β-gal group(24.680 vs. 5.268, P<0.05;24.680 vs. 5.132, P<0.05). Flow cytometry results showed that irradiation alone could promote the apoptosis of CNE-2R cells,when combined with E1A gene,the apoptosis rate was significantly increased(P<0.05). Western blot results showed that E1A gene down-regulated the expression of NF-κB/p65,CK2α,and Bcl-2 and up-regulated the expression of cleaved caspase-3. Conclusions E1A gene can enhance the radiosensitivity of nasopharyngeal carcinoma cells by inhibiting the expression of CK2 to block the NF-κB signaling pathway and promoting cell apoptosis.
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Objective:To establish neonatal ?-ray irradiated injury model of diffuse cortical dysplasias(DCD)in rats to observe the features of pathology,and discuss the mechamism of pathology and epileprogenesis in this model.Methods:?-ray-induced model of DCD was established in rat.We observed the cerebral cortex change with naked eyes and under a light microscope,and assessed the hippocampal mossy fiber sprouting after HE staining,Nissl staining and Timm's histochemical method.The comparisons between every two groups were performed with Nemenyi test.Results:The rats had seizure by chance,but most of these rats displayed more active action,exiting and so on.EEG showed frequent spontaneous sharp waves,or spikes from the frontal or the hippocampal neocortical structures.Neonatal ?-ray irradiated injury could result in DCDs in cerebral cortex and mossy fiber sprouting in hippocampi CA3 area bilaterally(nonparametric Kruskal-Wallis H test and Nemenyi test,P