Objective This is a feasibility study of rabbit meniscus regeneration evaluated with the use of autologous bone marrow derived mesenchymal stem cells(MSCs) and collagen glycosaminoglycan(GAG). Methods Autologous MSCs were prepared from the rabbit proximal tibial bone marrow and stimulated in vitro to start fibrocartilage differentiation lineage by bFGF and TGF ? 1. Then, collagen GAG templates enriched with these MSCs were implanted in vivo to the menisci excised rabbit knees as the substitute for the excised meniscus. After 3, 6, 12, 24 weeks postoperatively, the implants were evaluated by the gross, histological and ultrastructural observations. Results The MSCs enriched collagen GAG implants underwent inflammation, degradation, MSCs division and remodeling stages in vivo, and consequently formed a meniscus like fibrocartilage tissue. Special staining and electronic microscope observation proved that the regenerated fibrocartilage were chondrocyte like fibrochondrocytes; in contrast, the results of the control group showed that both collagen GAG implants without MSCs and no substitute had limited regenerating tissue, further evaluations by histological and electronic microscope showed no evidence of fibrochondrocytes, and hence these regenerated tissue were fibrous rather than fibrocartilaginous. Conclusion The inducing of rabbit meniscus regeneration by the autologous bone marrow derived MSCs and porous collagen GAG template is proved to be feasible in this study. However, further studies to improve the biomaterial design, to evaluate the biomechanical properties of the regenerated tissue and to ensure clinical safety etc are needed prior to its clinical application.

Objective: To synthesize the collagen-GAG template and to evaluate its feasibility to be used as the MSCs vehicle for meniscal tissue engineering. Methods: The collagen-GAG template was synthesized from rat tail type Ⅰ collagen and GAG using Yannas method. Then the post-stimulated MSCs by bFGF and TGF-β1 were added in. The MSCs-enriched collagen sponges were cultured in vitro， two weeks later the histological and ultrastructure detection was performed. Results: The histological and ultrastructure of the collagen-GAG template remained intact after 2 weeks' culture, and the MSCs in it remained viable. Conclusion: The collagen-GAG template synthesized in this experiment is suitable for the meniscal tissue engineering reconstruction as the vehicle for MSCs seed cells.

The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34%, 0.025 < P < 0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42%, significantly lower than those of the cells transfected with chkl sense chain (54.64%, 0.005 < P < 0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.
*Apoptosis/radiation effects ; Cell Cycle/radiation effects ; HL-60 Cells ; Oligonucleotides, Antisense/*genetics ; Protein Kinases/*genetics ; Protein Kinases/metabolism ; Radiation Tolerance/*genetics ; Transfection

AIM: In this study, CD147 antibody was used to carry out targeted modification of nanoparticles for protein kinase Cε (PKCε)-siRNA gene therapy to target lung cancer cells.The inhibitory effects of the nanoparticles on the proliferation and invasion of the lung cancer cells were observed.METHODS: The magnetic nanoparticles targeting CD147 protein were assembled as gene vector.The expression of CD147 in the lung cancer cells was observed under laser scanning confocal microscope.The cells were divided into CP group, CN group and LP group as the experimental groups. Targeted nanoparticles were used as CA group.Non-transfected cells were used as control group.The cell transfection was carried out with 250 ng plasmids/well in 6-well plate.The effect of nanocontrast agent on the cell endocytosis was observed under laser scanning confocal microscope.The mRNA expression of PKCε was detected by RT-qPCR.The protein expres-sion of Ki67, MMP3, PKCε, Wnt1 and GAPDH was determined by Western blot.The cell proliferation ability was detec-ted with colony formation assay.The cell invasion ability was detected by Transwell method.RESULTS: The expression of
CD147 protein in the human lung cancer A549 cells was confirmed by immunofluorescence staining.The endocytosis of siRNA into the A549 cells in CP group was observed with the highest efficiency as compared with CN group and LP group. The relative mRNA expression of PKCε in the A549 cells of CP group, CN group, LP group and CA group were (9.76 ± 0.18)%, (98.51 ±0.32)%, (99.17 ±0.16)% and (99.68 ±0.11)%, respectively.The difference between CP group and control group was statistically significant (P <0.05).No significant difference among CN group, LP group and control group was observed.The protein expression of PKCε, Ki-67, MMP3 and Wnt1 in CP group was significantly reduced, and the protein expression levels among CN group, LP group and control group had no significant difference.The colony number in CP group was significantly smaller than that in control group (P <0.05).The effective colony numbers in CN group, LP group and CA group had no significant difference as compared with control group.The number of the invading cells in CP group was significantly less than that in control group (P <0.05).The numbers of the invading cells in CN group, LP group and CA group had no significant difference as compared with control group.CONCLUSION: Nanogene vector targe-ting CD147 can carry PKCε-siRNA to conduct gene therapy efficiently on the lung cancer cells to achieve effective inhibito-ry effects on the proliferation and invasion of the lung cancer cells.

Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good anti-tumor and anti-metastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in I-III phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.

The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34 %,0.025＜P＜0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42 %, significantly lower than those of the cells transfected with chk1 sense chain (54.64 %, 0. 005＜P＜0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.

Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good anti-tumor and anti-metastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in I-III phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.
Adenoviridae ; genetics ; Animals ; Cell Line ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Virus Replication ; genetics

Objective To explore the effect of galangin(Gal)on inflammation in hepatitis B rats.Methods The rats were randomly divided into control group,hepatitis B group,Gal-L and Gal-H groups,positive drug lamivudine group and Gal-H+AMPK inhibitor(compound C)group with 12 in each.After modeling,medication treatment was performed once a day for 8 weeks.The level of alanine aminotransferase(ALT),total bilirubin(TBIL)and aspartate aminotransferase(AST)in the serum of rats were detected.HE staining microscopy was ap-plied to detect pathological changes in liver tissue.TUNEL staining microscopy was applied to detect cell apoptosis in liver tissue.Chromatin immuno-precipitation was applied to detect HBV viral load in liver tissue.ELISA was ap-plied to detect the levels of monocyte chemotactic protein-1(MCP-1),interleukin-12(IL-12),and tumor necrosis factor-α(TNF-α)in liver tissue.Western blot was applied to detect the expression of caspase-3,Bcl-2 associated X protein(Bax),p-AMPK and SIRT1 proteins in liver tissue.Results Compared with hepatitis B group,the liver damage of rats in the Gal-L group,Gal-H group and lamivudine group was alleviated;The level of serum AST,TBIL,and ALT,apoptosis rate,HBV viral load and the level of MCP-1,IL-12,TNF-α as well as the expression of caspase-3 and Bax proteins in liver tissue were all reduced.The expression of p-AMPK and SIRT1 proteins in liv-er tissue increased(P＜0.05).Compound C baffled inhibitory effects of high-dose Gal on inflammation,cell apopto-sis,and HBV viral load in liver tissue of hepatitis B rats.Conclusions The mechanism of Gal in inhibiting inflam-mation,cell apoptosis and HBV virus replication in hepatitis B rats is potentially attributed to up-regulation of the AMPK/SIRT1 pathway.