The method evaluation of culturing df-1 to proliferate canine distemper virus in mink with cephodex microcarrier

As an acute and highly lethal infectious disease, there is no specific therapeutic drug for canine distemper (CD). Although the process of large-scale production of canine distemper virus (CDV) vaccine of mink has been greatly improved, there are still many deficiencies to be perfected. As one of the most promising technologies for large-scale vaccine production, microcarrier suspension culture technology needs to be further improved. In this study, the application effect of the new Cephodex microcarrier in CDV culture was evaluated to establish a set of technical process for DF-1 cell high-density growth and CDV efficient proliferation. To perfect the large-scale CDV production process, Cephodex was used to suspension culture DF-1 cells for proliferating CDV. In a shake flasks culture system, the optimal culture conditions were established by optimizing culture temperature, virus inoculation and harvest time. Therefore, mink CD vaccine high-efficiency production was laid on the preliminarily established technology of CDV microcarrier suspension culture. The cell density could reach over 3×106 cells/mL after 72 h cultured with Cephodex microcarrier at 37°C. Proliferated at 35°C, the CDV titer after 72 h was about 100.5 TCID50/0.1ml higher than that at 33°C and 37°C. These results show that the Cephodex microcarrier could be used for large-scale culture of DF-1 cells and efficient proliferation of CDV

The role of N-acetyltransferase 8 in mesenchymal stem cell-based therapy for liver ischemia/reperfusion injury in rats.

To evaluate the impact of mesenchymal stem cells (MSCs) against hepatic I/R injury and explore the role of N-acetyltransferase 8 (NAT8) in the process.We investigated the potential of injected MSCs systemically via the tail vein in healing injuried liver of the SD rat model of 70% hepatic I/R injury by measuring the biochemical and pathologic alterations. Subsequently, we evaluated the expression levels of NAT8 by western blotting in vivo. Concurrently, hydrogen peroxide (H2O2)-induced apoptosis in the human normal liver cell line L02 was performed in vitro to evaluate the protective effects of MSC conditioned medium (MSC-CM) on L02 cells. In addition, we downregulated and upregulated NAT8 expression in L02 cells and induced apoptosis by using H2O2 to study the protective role of NAT8.MSCs implantation led to a significant reduced liver enzyme levels, an advanced protection in the histopathological findings of the acutely injured liver and a significantly lower percentage of TUNEL-positive cells, which were increased after I/R injury. In vitro assays, MSC-CM inhibited hepatocyte apoptosis induced by H2O2. Moreover, overexpression or downregulation of NAT8 prevented or aggravated hepatocyte apoptosis induced by H2O2, respectively.MSC transplantation provides support to the I/R-injured liver by inhibiting hepatocellular apoptosis and stimulating NAT8 regeneration

NAT8 participation in MSC-mediated hepatic regeneration after I/R damage.

<p>A. Immunohistochemical staining of liver specimens is shown, in which antibodies to NAT8 were used. B. Western blot analysis shows that the NAT8 level was decreased after I/R injury, and MSC treatment resulted in the restoration of the protein level at 6 and 24 h. C. The quantified histogram of Western blot images. *P<0.05, **P<0.01.</p

Effects of pcDNA3.1-NAT8, NAT8-siRNA673, and NAT8-siRNA173 on NAT8 expression.

<p>A. Western blot analysis shows that pcDNA3.1-NAT8 significantly upregulated NAT8 expression. On the other hand, NAT8-siRNA673 and NAT8-siRNA173 could significantly downregulate NAT8 expression in the L02 cells respectively compared to scrambled siRNA, of which, NAT8-siRNA173 showed more significant RNA inhibiting effect. B. The quantified histogram of Western blot images. *P<0.05, **P<0.01.</p

L02 cells with NAT8 overexpression exhibited an increased capacity to resist H₂O₂-induced apoptosis.

<p>(A and C) Typical effect of NAT8 overexpression and downregulation on H₂O₂-induced apoptosis of L02 cells. (B and D) the percentage of apoptotic L02 cells among the total cells. All values were expressed as mean ± SEM of five independent experiments. **P<0.01, ***P<0.001.</p

MSCs promoted the regeneration of the liver in rat with I/R injury.

<p>A. Appearance examination of livers from the three groups. B. The weight of livers from the three groups. The data are represented as mean ± SEM. n = 6 independent livers. *P<0.05, **P<0.01. C. H&E staining of the liver sections from the three groups. Low magnification (bottom left), scale bars represent 1mm; High magnification (upper right), scale bars represent 100 µm. D. The mean pathological score of the liver sections. All values were expressed as mean ± SEM. n = 5 independent sections. **P<0.01.</p

Distribution of DAPI-positive MSCs in the tissues of rat.

<p>The frozen sections of lung (A), kidney (B), liver (C), and spleen (D) were observed by fluorescence microscopy. Scale bars represent 100 µm.</p

Characterization of UC-derived MSCs.

<p>A. Bright-field image. B. H&E staining image. C. Immunophenotype of MSCs at passage 5 by flow cytometry. Scale bars represent 200 µm.</p

MSCs reduced the apoptosis of liver in rat with I/R injury.

<p>A. TUNEL staining of the liver sections from the three groups. Scale bars represent 100 µm. The arrows show TUNEL-positive hepatocyte nuclei. B. Quantification of TUNEL-positive hepatocyte nuclei. n = 5 independent sections. **P<0.01. C. The expression of apoptosis-associated proteins determined by Western blot analysis. D and E. The quantified histogram of Western blot images. *P<0.05, **P<0.01, ***P<0.001.</p