ABSTRACT
Objective::To investigate the role and mechanism of Testudinis Carapax et Plastrum aqueous extract in promoting osteogenic differentiation of mouse preosteoblast cell line(MC3T3-E1) by regulating nuclear transcription factor-κB(NF-κB) inflammation microenvironment. Method::MC3T3-E1 cells were cultured in vitro, and osteogenic induction (OI) was performed. Testudinis Carapax et Plastrum was prepared and treated the cells. Cells were devided into control group, osteogenic induction group and Testudinis Carapax et Plastrum (20 mg·L-1)with osteogenic induction group. The proliferation of MC3T3-E1 was detected by cell counting kit-8 (CCK-8), and the optimum concentration of intervention was determined. MC3T3-E1 differentiation and osteogenic mineralization were assayed using alkaline phosphatase (ALP) and Alizarin red staining (ARS), respectively. The expressions of NF-κB p65, NF-κB p105, interleukin-6(IL-6), ALP and Collagen-Ⅰ(COL-Ⅰ) mRNA were detected by Real-time PCR. Result::The results of CCK-8 showed that the proliferation of MC3T3-E1 did not change statistically with time, but it showed an upward trend, while the proliferation at 20 mg·L-1 was more obvious than other groups. The ALP and ARS showed that the positive staining rate of osteogenic induction group and Testudinis Carapax et Plastrum with osteogenic induction group were higher than control group.Real-time PCR results showed that on the 7th day in culture, the expression of NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01), and the expression of ALP and COL-Ⅰ mRNA was significantly upregulated(P<0.05), on the 14th day, the expression of NF-κB p65, NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01). The expression of ALP and COL-Ⅰ mRNA was significantly increased (P<0.05, P<0.01). Conclusion::Testudinis Carapax et Plastrum aqueous extract can promote osteogenic differentiation of MC3T3-E1 via a mechanism associated with the regulation of inhibition of NF-κB inflammatory microenvironment.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.</p><p><b>METHODS</b>Recombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.</p><p><b>RESULTS</b>The tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.</p><p><b>CONCLUSION</b>Immunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.</p>