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Title: [Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice].
Author: Ma TX, Xu YW, Jiang DY.
Journal: Zhonghua Shao Shang Za Zhi; 2020 Mar 20; 36(3):234-243. PubMed ID: 32241050.
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. 目的： 探讨白细胞介素17(IL－17)修饰的小鼠骨髓间充质干细胞(BMSC)对异体小鼠皮肤移植的影响及机制。 方法： (1)取5只BALB/c小鼠(均为雌性，4～8周龄，性别、鼠龄下同)，处死后取股骨、胫骨和肱骨，采用全骨髓密度梯度离心联合贴壁分离法分离纯化培养BMSC，取第3代细胞行形态学观察，该代细胞经成骨、成脂诱导分化鉴定。第4代细胞经干细胞表面标志物鉴定。取第3～6代BMSC，经终质量浓度50 ng/mL小鼠重组IL－17预处理5 d后，行形态学观察。取IL－17预处理的BMSC和未经IL－17预处理BMSC标记碳花青荧光染料(CM－Dil)，行形态学观察并计算标记率。(2)取45只C57BL/6J小鼠，按随机数字表法分为磷酸盐缓冲液(PBS)对照组13只、单纯BMSC组16只、BMSC＋IL－17组16只。在小鼠皮肤移植术前1 d，单纯BMSC组13只小鼠经尾静脉注射5×10(6)个/mL未经CM－Dil标记的BMSC 0.1 mL，另3只小鼠经尾静脉注射5×10(6)个/mL CM－Dil标记的BMSC 0.1 mL；BMSC＋IL－17组13只小鼠经尾静脉注射5×10(6)个/mL未经CM－Dil标记的IL－17预处理的BMSC 0.1 mL，另3只小鼠经尾静脉注射5×10(6)个/mL CM－Dil标记的IL－17预处理的BMSC 0.1 mL；PBS对照组13只小鼠经尾静脉注射0.1 mL PBS。取45只BALB/c小鼠作供体，将前述3组处理后的45只C57BL/6J小鼠作为受体，建立背－背全厚皮移植模型。于移植术后第2天，再次对3组小鼠注射与移植术前1 d一样的等量相应细胞或PBS。于移植术后第7天，分别取单纯BMSC组和BMSC＋IL－17组中注射CM－Dil标记的BMSC和CM－Dil标记的IL－17预处理BMSC的3只小鼠，脱颈处死后采用荧光显微镜观察BMSC的CM－Dil示踪并计数。于移植术后第6天拆除敷料后，从单纯BMSC组注射未经CM－Dil标记的BMSC的13只小鼠和BMSC＋IL－17组注射未经CM－Dil标记的IL－17预处理BMSC的13只小鼠及PBS对照组13只小鼠中选取7只小鼠，记录移植皮片的存活时间。于移植术后第8天，从3组剩余的6只小鼠中分别取3只小鼠进行移植皮片的大体观察，酶联免疫吸附测定法检测小鼠血清中γ干扰素、IL－10、转化生长因子β(TGF－β)的水平，流式细胞仪检测脾脏CD4(＋)CD25(＋)叉头翼状螺旋转录因子p3(Foxp3)(＋)调节性T细胞(Treg)比例，行苏木精－伊红染色观察移植皮片的组织形态。于移植术后第14天，取3组剩余3只小鼠同前行组织形态观察。对数据行独立样本t检验、单因素方差分析、LSD检验。 结果： (1)培养5 d，经IL－17预处理的BMSC与未处理的BMSC形态和大小并无明显差别。(2)CM－Dil标记后，经IL－17预处理的BMSC与未处理的BMSC生长状态均较良好，标记率几乎可达100%。(3)移植术后第7天，单纯BMSC组小鼠皮片及邻近皮下组织CM－Dil标记阳性的BMSC数量为每100倍视野下(6.2±2.6)个，明显少于BMSC＋IL－17组CM－Dil标记阳性的IL－17预处理BMSC的每100倍视野下(15.0±5.3)个(t＝－2.962，P<0.05)。(4)单纯BMSC组和BMSC＋IL－17组小鼠移植皮片的存活时间分别为(13.3±1.2)、(17.0±1.5)d，明显长于PBS对照组的(8.7±0.8)d(P<0.01)，而BMSC＋IL－17组小鼠移植皮片的存活时间明显长于单纯BMSC组(P<0.01)。(5)移植术后第8天，PBS对照组小鼠移植皮片大部分发黑变硬且结痂坏死，单纯BMSC组小鼠移植皮片大部分存活良好，BMSC＋IL－17组小鼠移植皮片均存活良好。(6)移植术后第8天，与PBS对照组相比，单纯BMSC组和BMSC＋IL－17组小鼠的血清中IL－10和TGF－β含量明显升高(P<0.01)，γ干扰素含量明显降低(P<0.01)；与单纯BMSC组相比，BMSC＋IL－17组小鼠的血清中IL－10和TGF－β含量明显升高(P<0.01)，γ干扰素含量明显降低(P<0.01)。(7)移植术后第8天，单纯BMSC组和BMSC＋IL－17组小鼠脾脏CD4(＋)CD25(＋)Foxp3(＋)Treg比例明显高于PBS对照组(P<0.01)，BMSC＋IL－17组小鼠脾脏CD4(＋)CD25(＋)Foxp3(＋)Treg比例明显高于单纯BMSC组(P<0.01)。(8)移植术后第8天，PBS对照组小鼠移植皮片可见大量炎性细胞浸润，表皮、真皮坏死；单纯BMSC组小鼠移植皮片可见局灶性的炎性细胞浸润，仅有轻微的表皮变性；BMSC＋IL－17组小鼠移植皮片皮肤附属器存活完好，同时有血管形成。移植术后第14天，单纯BMSC组小鼠移植皮片可见广泛的炎性细胞浸润，并有较重的表皮变性和局灶性坏死；BMSC＋IL－17组小鼠移植皮片可见局灶性的炎性细胞浸润和轻微表皮变性；PBS对照组小鼠移植皮片已完全坏死。 结论： IL－17可通过提高小鼠BMSC诱导免疫耐受的能力和增强BMSC的归巢能力，最终起到减轻异体皮片移植后免疫排斥反应，延长小鼠移植皮片存活时间的作用。.

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