Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice
10.3760/cma.j.cn501120-20190510-00232
- VernacularTitle:白细胞介素17修饰小鼠骨髓间充质干细胞对异体小鼠皮肤移植排斥反应的影响及机制
- Author:
Tengxiao MA
1
;
Yingwei XU
;
Duyin JIANG
Author Information
1. 河南省人民医院整形外科，郑州 450000
- Keywords:
Interleukin-17;
Mesenchymal stem cells;
Immune tolerance;
Skin transplantation
- From:
Chinese Journal of Burns
2020;36(3):234-243
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects and mechanism of interleukin-17 (IL-17)-modified mouse bone marrow mesenchymal stem cells (BMSCs) on the allogeneic skin transplantation in mice.Methods:(1) The femur, tibia, and humerus were isolated from five BALB/c mice (all female, aged 4 to 8 weeks, the same gender and age below) after sacrifice. BMSCs were isolated, purified, and cultured by whole bone marrow density gradient centrifugation combined with adherent separation method. The third passage of cells was used for morphological observation and identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of the expression of stem cell surface markers. The third to sixth passages of BMSCs were pretreated with mouse recombinant IL-17 at a final mass concentration of 50 ng/mL for 5 days, and then were harvested for morphological observation. After being labeled with carbocyanine fluorescent dye (CM-Dil), IL-17-pretreated BMSCs and IL-17-unpretreated BMSCs were obtained for morphological observation and the labeling rates were calculated. (2) Forty-five C57BL/6J mice were divided into phosphate buffer solution (PBS) control group ( n=13), BMSCs alone group ( n=16), and BMSCs+ IL-17 group ( n=16) according to the random number table. One day before the skin transplantation of mice, 0.1 mL BMSCs (5×10 6 cells/mL) without CM-Dil labeling were injected to the 13 mice in BMSCs alone group through the tail vein, and 0.1 mL BMSCs (5×10 6 cells/mL) labeled with CM-Dil were injected to the other 3 mice in BMSCs alone group through the tail vein. IL-17-pretreated BMSCs (5×10 6 cells/mL) without CM-Dil labeling in the volume of 0.1 mL were injected to the 13 mice in BMSCs+ IL-17 group through the tail vein, and 0.1 mL IL-17-pretreated BMSCs (5×10 6 cells/mL) labeled with CM-Dil were injected to the other 3 mice in BMSCs+ IL-17 group through the tail vein. PBS in the volume of 0.1 mL was injected to the 13 mice in PBS control group through the tail vein. Forty-five BALB/c mice were used as donors, and forty-five treated C57BL/6J mice in the 3 groups were used as recipients to establish a back-to-back full-thickness skin transplantation model. On the 2nd day after transplantation, the same number of corresponding cells and the equal amount of PBS were injected to the recipient mice of each group again. On the 7th day after transplantation, three mice injected with CM-Dil-labeled BMSCs in BMSCs alone group and three mice injected with CM-Dil-labeled IL-17-pretreated BMSCs in BMSCs+ IL-17 group were sacrificed by cervical dislocation to track the CM-Dil-labeled BMSCs by fluorescence microscope, which was counted. After the dressing removal on the 6th day post transplantation, 7 mice were selected respectively from 13 mice in BMSCs alone group injected with BMSCs without CM-Dil-labeling, 13 mice in BMSCs+ IL-17 group injected with IL-17-pretreated BMSCs without CM-Dil-labeling, and 13 mice in PBS control group, respectively, to record the skin graft survival time. On the 8th day post transplantation, three of the remaining six mice in the three groups were taken for general observation of the grafted skin, serum levels of interferon-γ, IL-10, and transforming growth factor β (TGF-β) by enzyme-linked immunosorbent assay method, the percentage of CD4 + CD25 + forkhead/winged helix transcription factor p3 (Foxp3) + regulatory T cells (Tregs) in spleen by flow cytometer, and the histopathological observation of the grafted skin by hematoxylin eosin staining. The rest three mice in each group were also taken for histopathological observation as above on the 14th day post transplantation. Data were statistically analysed with independent sample t test, one-way analysis of variance, and least significant difference test. Results:(1) There were no significant differences in the morphology and size between IL-17-pretreated BMSCs and IL-17-unpretreated BMSCs on culture day 5. (2) After CM-Dil labeling, BMSCs and IL-17-pretreated BMSCs grew well, and the labeling rate was almost 100%. (3) On the 7th day post transplantation, there were 6.2±2.6 CM-Dil-labeled BMSCs per 100 fold visual field in the skin and adjacent subcutaneous tissue of mice in BMSCs alone group, which were significantly fewer than the 15.0±5.3 CM-Dil-labeled IL-17-pretreated BMSCs per 100 fold visual field in BMSCs+ IL-17 group ( t=-2.962, P<0.05). (4) The skin graft survival time of mice in BMSCs alone group and BMSCs+ IL-17 group was (13.3±1.2) and (17.0±1.5) days respectively, significantly longer than (8.7±0.8) days in PBS control group ( P<0.01), and the skin graft survival time of mice in BMSCs+ IL-17 group was significantly longer than that in BMSCs alone group ( P<0.01). (5) On the 8th day post transplantation, most of the skin grafts of mice in PBS control group was black, scabby, and necrotic. Most of the skin grafts of mice in BMSCs alone group survived well, while all the skin grafts of mice in BMSCs+ IL-17 group survived well. (6) On the 8th day post transplantation, compared with those of PBS control group, the serum levels of IL-10 and TGF-β of mice in BMSCs alone group and BMSCs+ IL-17 group were significantly higher ( P<0.01), and the serum level of interferon-γ was significantly lower ( P<0.01). Compared with those of BMSCs alone group, the serum levels of IL-10 and TGF-β of mice in BMSCs+ IL-17 group were significantly higher ( P<0.01), and the serum level of interferon-γ was significantly lower ( P<0.01). (7) On the 8th day post transplantation, the percentages of CD4 + CD25 + Foxp3 + Treg in spleen of mice in BMSCs alone group and BMSCs+ IL-17 group were significantly higher than the percentage of PBS control group ( P<0.01), and the percentage of CD4 + CD25 + Foxp3 + Treg in spleen of mice in BMSCs+ IL-17 group was significantly higher than that of BMSCs alone group ( P<0.01). (8) On the 8th day post transplantation, infiltration of a large number of inflammatory cells and necrosis of epidermis and dermis were found in the skin grafts of mice in PBS control group; focal infiltration of inflammatory cells and slight epidermal degeneration were found in the skin grafts of mice in BMSCs alone group; the skin appendages of the skin grafts of mice in BMSCs+ IL-17 group survived well with angiogenesis. On the 14th day post transplantation, the skin grafts of mice in BMSCs alone group showed extensive infiltration of inflammatory cells, severe epidermal degeneration and focal necrosis; the skin grafts of mice in BMSCs+ IL-17 group showed focal infiltration of inflammatory cells and slight epidermal degeneration; the skin grafts of mice in PBS control group were completely necrotic. Conclusions:IL-17 can reduce the immune rejection in allogeneic skin grafting and prolong the survival time of mouse skin grafts by improving mice BMSCs′ capabilities to induce immune tolerance and enhancing the homing ability of BMSCs.
