Therapeutics
Mouse 1 (control)
Phase
Mouse 4
Mouse 5 (1)
Mouse 5 (2)
DAPI
Anti-insulin
Merge
Figure 3: Immunofluorescent analysis of liver tissues of mice treated with control protein or with Pdx1-PTD, Ngn3-PTD and MafA-PTD proteins. Mice 1, 2 and 3 were from the control group, and mice 4, 5 and 6 were treated with the three proteins. The first row of panels are bright phase images, the second are fluorescent images with DAPI staining, and the third are fluorescent images with insulin antibody staining. Each image on the fourth row of panels is the merge image of the three images above. Each column of panels are for the same field
Mouse 1 (control)
Phase
Mouse 5 (1)
Mouse 5 (2)
Mouse 6
DAPI
PTD (1mg/kg), and MafA-PTD (1mg/kg) were injected into each mouse by intraperitoneal (IP) in treatment group (Mouse-4, Mouse-5 and Mouse- 6) and BSA (1mg/kg) was injected into each mouse in the control group (Mouse-1, Mouse-2 and Mouse-3). Injections were repeated every day for seven days. Mice of both treatment and control group were sacrificed on day 10, three days after completion of injections. The mouse liver and pan- creas were washed with 1X PBS and fixed by 4% paraformaldehyde for overnight. Then the liver and pancreatic tissues were processed by standard Paraffin Embedding protocol. The Tissue sections, 5-micro in thickness, were prepared routinely with histology microtomes and mounted on standard histology glass slides. The wax in tissues was dis- solved by xylene during processing of tissue sec- tions. Tissue sectioning and histologic and immunohistochemical staining were performed using routine methods. For indirect fluorescent- antibody (IFA) assay, the slides were blocked with 0.05% Tween 20 (TBST) and 3% BSA for one hour at RT and were incubated with mouse anti- insulin antibody (Invitrogen) at 4°C overnight. The slides were washed three times with PBS for 15 minutes at RT and incubated with fluorescein isothiocyanate (FITC) conjugated swine anti- mouse antibody (KPL) for two hours at RT. The same concentration of Mouse IgG was used as iso- type control. Anti DAPI antibody was added to slides as a nuclear marker (Figures 3 and 4). The results showed that the treatment group had more insulin-producing cells in livers compared with the control group (Figure 3). There are some insulin positive cells in the liver of control animals (mouse 1, Figure 3). This observation is not sur- prising since the same kind of leakiness in mice has been reported in the literature before13. In the liv- ers of protein-treated mice, clusters of insulin-pos- itive cells emerged, many of which were concen- trated in the periportal areas.
Anti-insulin Merge
In the pancreas of mice treated with the three transducible proteins, clusters of insulin-producing cells were observed beside the normal insulin- intensive islets. Different from the native islets, these clusters of insulin-producing cells are more mosaic and diffusive (Figure 4).
Conclusions
Figure 4: Immunofluorescent analysis of pancreatic tissues of mice treated with control protein or with Pdx1-PTD, Ngn3-PTD and MafA-PTD proteins. Mice 1, 2 and 3 were from the control group, and mice 4, 5, and 6 were treated with the three proteins. The first row of panels are bright phase images, the second are fluorescent images with DAPI staining, and the third are fluorescent images with insulin antibody staining. Each image on the fourth row of panels is the merge image of the three images above. Each column of panels are for the same field
88
IVR potentially combines the advantages of drug- based and cell-based therapies while avoiding the shortcomings of both. Since the modality of IVR therapeutics are proteins, small molecules or RNAs, their risk profiles and cost structures of development and commercialisation are similar to
Drug Discovery World Summer 2011
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