Demonstrate

In iGEM 2016, we, SYSU-MEDICINE, not only confirmed our devices (BBa_K1993008 and BBa_K1993009) at molecular level, but also on animal models (IBD and DTH). We tried our best to improve them to acclimatize real conditions.

IBD

IBD (Inflammatory bowel diseases) including Crohn’s disease (CD) and ulcerative colitis (UC), are caused by the multiple factors such as genetic susceptibility, breakdown of mucosal immune tolerance, and self-immune activation to gut microbiota. MSC therapy utilizing immunosuppressive and differentiation properties of MSCs have been tested in clinical trials for both luminal and fistulizing forms of IBD. In our project, MSCs engineered with the gene of CXCR4 and marking proteins were used and evaluated.

Figure 4.1.1
1. Grouping

IBD models with 42 BALB/c mice were established and divided into 5 groups：
(1) 10 mice were included in this experiment. After sensitization, 6 mice were injected with MSCsCXCR4.
(2) 10 mice were included in this experiment. After sensitization, 6 mice were injected with MSCseGFP.
(3) 10 mice were included in this experiment. After sensitization, 6 mice were injected with saline.
(4) 6 mice were treated with alcohol for the enema as the alcohol control.
(5) 6 mice without any further treatment were as the normal control group.

2. Evaluation

To evaluate how MSCs contribute to relief of IBD, colon length, DAI score, survival rate, the concentration of the cytokines and HE staining of each group were recorded. All the data we harvested indicated that MSCs group had better performance than control group.

- We established murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-colitis (IBD animal model). After injecting with MSCsCXCR4, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.

Figure 4.1.2.1	Figure 4.1.2.2
Figure 4.1.2.3	Figure 4.1.2.4

- Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCsCXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.

Figure 4.1.3
- Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSCCXCR4 group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSCCXCR4 group.

Figure 4.1.4.1	Figure 4.1.4.2
Figure 4.1.4.3	Figure 4.1.4.4

- Under IVIS Spectrum, MSCsCXCR4 exhibited enhanced capacities for targeted migration to the bowels in inflammatory condition in vivo.

Figure 4.1.5

DTH

DTH (delayed type hypersensitivity) is an experimental model for human allergic contact dermatitis (ACD), one of the prevalent skin diseases worldwide. The unique immunomodulatory functions of MSCs on various types of immune cells may render them as a novel approach to desensitize allergic diseases. In our project, MSCs engineered with the gene of CXCR5 and marking proteins were used and evaluated.

Figure 4.2.1
BBa_K1993008
1. Grouping
In our design, DTH model with 28 BALB/c mice were established and divided into 4 groups：
(1) 8 mice were included in this experiment. After sensitization, 6 mice were injected with MSCsCXCR5.
(2) 8 mice were included in this experiment with treatment of sensitization and injection of MSCseGFP to the caudal veins.
(3) 6 mice were included in this experiment with treatment of sensitization and injection of PBS to the caudal veins.
(4) 6 mice containing in this group will receive identical volume of acetone/olive oil solution application to the backs and ears. PBS were injected to the caudal veins.

2. Evaluation
Ear samples were measured and harvested for qPCR, HE staining or immuno-fluorescence, which showed better curative effect of engineered MSCs.

- MSCsCXCR5 infusion dramatically ameliorated DTH. MSCsCXCR5 displayed better treatment efficacy than DTH+MSCeGFP group, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSCsCXCR5 significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection.

Figure 4.2.2.1

Figure 4.2.2.2

- Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of the anti-inflammatory cytokines (IL-10), elevated in DTH+MSCsCXCR5 group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSCsCXCR5 group.

Figure 4.2.3

- The inflamed ears were collected and subjected to in situ immunofluorescence staining. Our results revealed that MSCsCXCR5 highly accumulating in inflamed ears displayed better chemotaxis than almost undetectable MSCseGFP.

Figure 4.2.4