Effects of porcine urinary bladder matrix on motility and polarization of bone marrow-derived macrophages in mice
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摘要:
目的 探讨猪膀胱脱细胞基质（UBM）对小鼠骨髓源巨噬细胞的运动和极化情况的影响，为临床创面修复中支架的合理选用提供依据。 方法 采用实验研究方法。采用扫描电子显微镜观察猪UBM和可吸收敷料的微观结构。采用聚丙烯酰胺凝胶电泳观察2种支架浸提液的蛋白质分布。取6只6~8周龄雄性C57BL/6J小鼠（小鼠周龄、性别、品系下同）并提取骨髓源细胞后诱导原代巨噬细胞并鉴定。取3批次巨噬细胞，均分为猪UBM浸提液组和可吸收敷料浸提液组，加入含有相应浸提液的DMEM/F12培养基培养，行划痕试验检测并计算划痕后1、3、7 d的细胞迁移率；行Transwell实验检测培养12、24 h的细胞迁移数量；培养24 h，采用流式细胞仪检测CD206或CD86阳性细胞百分比。以上细胞实验样本数均为3。取12只小鼠，于每只小鼠背部左右两侧各制作1个切口，左侧切口纳入猪UBM组、右侧切口纳入可吸收敷料组，分别植入相应支架。术后7、14 d，行苏木精-伊红染色观测支架中浸润的炎症细胞数量，采用免疫组织化学法观测支架中F4/80、转化生长因子β1（TGF-β1）、血管内皮生长因子（VEGF）和基质金属蛋白酶9（MMP-9）阳性细胞数量和Ⅰ型胶原蛋白沉积情况，采用免疫荧光染色检测F4/80、CD86、CD206阳性细胞百分比。以上动物实验样本数均为6。对数据行析因设计方差分析、重复测量方差分析、独立样本t检验。 结果 猪UBM的一面为致密的基底膜结构，另一面为含有纤维结构的多孔固有层。可吸收敷料的两面均呈三维多孔结构。在（50~70）×103的分子量区间，猪UBM浸提液的泳道出现多条非Ⅰ型胶原蛋白条带，可吸收敷料浸提液的泳道中未出现明显条带。经鉴定，小鼠骨髓源细胞已被成功诱导为巨噬细胞。划痕后1、3、7 d，猪UBM浸提液组细胞迁移率均明显高于可吸收敷料浸提液组（t值分别为15.31、19.76、20.58，P<0.05）。培养12、24 h，猪UBM浸提液组细胞迁移数量均明显多于可吸收敷料浸提液组（t值分别为12.20、33.26，P<0.05）。培养24 h，猪UBM浸提液组细胞中CD86阳性细胞百分比为（1.27±0.19）%，明显低于可吸收敷料浸提液组的（7.34±0.14）%（t=17.03，P<0.05）；猪UBM浸提液组细胞中CD206阳性细胞百分比为（73.4±0.7）%，明显高于可吸收敷料浸提液组的（32.2±0.5）%（t=119.10，P<0.05）。术后7、14 d，猪UBM组支架中浸润的炎症细胞数量均明显多于可吸收敷料组（t值分别为6.58、10.70，P<0.05）；猪UBM组支架中F4/80、TGF-β1、VEGF和MMP-9阳性细胞数量均明显多于可吸收敷料组（t值分别为46.11、40.69、13.90、14.15，19.79、32.93、12.16、13.21，P<0.05）；猪UBM组支架中Ⅰ型胶原蛋白沉积较可吸收敷料组更显著；猪UBM组支架中CD206阳性细胞百分比均明显高于可吸收敷料组（t值分别为5.05、4.13，P<0.05），CD86阳性细胞百分比均明显低于可吸收敷料组（t值分别为20.90、19.64，P<0.05），猪UBM组支架中可见更多的M2型巨噬细胞、可吸收敷料组支架中可见更多的M1型巨噬细胞。 结论 猪UBM可增强小鼠巨噬细胞运动，诱导其发生M2型极化和发挥旁分泌功能，营造含有TGF-β1等生长因子和MMP-9组织重塑分子的微环境，促进组织新生和细胞外基质重塑。 Abstract:Objective To explore the effects of porcine urinary bladder matrix (UBM) on the motility and polarization of bone marrow-derived macrophages in mice, so as to provide evidence for the rational selection of stent in clinical wound repair. Methods The method of experimental research was used. The microstructure of porcine UBM and absorbable dressing was observed under scanning electron microscope. Polyacrylamide gel electrophoresis was used to observe the protein distribution of the two stent extracts. The primary macrophages were induced from bone marrow-derived cells isolated from six 6-8-week-old male C57BL/6J mice (mouse age, sex, and strain, the same below) and identified. Three batches of macrophages were divided into porcine UBM extract group and absorbable dressing extract group. The cells in each group were cultured with Dulbecco's modified Eagle medium/F12 medium containing the corresponding extracts. The cell migration rate was detected and calculated on 1, 3, and 7 d after scratching by scratch test. The number of migrated cells at 12 and 24 h of culture was detected by Transwell experiment. The percentages of CD206 and CD86 positive cells at 24 h of culture was detected by flow cytometer. The numbers of sample in the above cell experiments were all 3. An incision was prepared on the left and right back of twelve mice, respectively. The left incision of each mouse was included in porcine UBM group and the right incision was included in absorbable dressing group, and the corresponding stents were implanted into the incisions respectively. On post operation day (POD) 7 and 14, the number of inflammatory cells infiltrated in the stent was detected by hematoxylin-eosin staining; the number of F4/80, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and matrix metalloprotein-9 (MMP-9) positive cells and type Ⅰ collagen deposition in stents were observed by immunohistochemistry; the percentages of F4/80, CD86, and CD206 positive cells were observed by immunofluorescence staining. The numbers of sample in the above animal experiments were all 6. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, and independent sample t test. Results Porcine UBM has a dense basement membrane structure on one side and porous propria containing a fibrous structures on the other. Both sides of the absorbable dressing had three-dimensional porous structure. In the molecular weight range of (50-70)×103, multiple non-type Ⅰ collagen bands appeared in the lanes of porcine UBM extract, while no obvious bands appeared in the lanes of absorbable dressing extract. It had been identified that mouse bone marrow-derived cells had been successfully induced into macrophages. The cell migration rates in porcine UBM extract group were significantly higher than those in absorbable dressing extract group on 1, 3, and 7 d after scratching (with t values of 15.31, 19.76, and 20.58, respectively, P<0.05). The numbers of migrated cells in porcine UBM extract group were significantly more than those in absorbable dressing extract group at 12 and 24 h of culture (with t values of 12.20 and 33.26, respectively, P<0.05). At 24 h of culture, the percentage of CD86 positive cells in porcine UBM extract group ((1.27±0.19)%) was significantly lower than (7.34±0.14)% in absorbable dressing extract group (t=17.03, P<0.05);the percentage of CD206 positive cells in porcine UBM extract group was (73.4±0.7)%, significantly higher than (32.2±0.5)% in absorbable dressing extract group (t=119.10, P<0.05). On POD 7 and 14, the numbers of inflammatory cells infiltrated in the stents in porcine UBM group was significantly more than those in absorbable dressing group (with t values of 6.58 and 10.70, respectively, P<0.05). On POD 7 and 14, the numbers of F4/80, TGF-β1, VEGF, and MMP-9 positive cells in the stents in porcine UBM group were significantly more than those in absorbable dressing group (with t values of 46.11, 40.69, 13.90, 14.15, 19.79, 32.93, 12.16, and 13.21, respectively, P<0.05); type Ⅰ collagen deposition in the stents in porcine UBM group was more pronounced than that in absorbable dressing group; the percentages of CD206 positive cells in the stents in porcine UBM group were significantly higher than those in absorbable dressing group (with t values of 5.05 and 4.13, respectively, P<0.05), while the percentages of CD86 positive cells were significantly lower than those in absorbable dressing group (with t values of 20.90 and 19.64, respectively, P<0.05), and more M2-type macrophages were seen in the stents in porcine UBM group and more M1-type macrophages were seen in the stents in absorbable dressing group. Conclusions Porcine UBM can enhance macrophage motility, induce M2 polarization and paracrine function, create a microenvironment containing growth factors such as TGF-β1 and MMP-9 tissue remodeling molecules, and promote tissue regeneration and extracellular matrix remodeling in mice. -
Key words:
- Macrophages /
- Cell movement /
- Wound repair /
- Porcine urinary bladder matrix /
- Scaffold remodeling /
- Tissue renewal
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1 可吸收敷料及猪膀胱脱细胞基质（UBM）支架的微观结构。1A、1B.分别为可吸收敷料的上、下表面，均呈三维多孔结构 扫描电子显微镜×200；1C.猪UBM上表面呈致密的基底膜结构 扫描电子显微镜×200；1D.猪UBM下表面呈含有纤维结构的多孔固有层 扫描电子显微镜×200；1E、1F.分别为图1A、1B中方框处放大图，显示可吸收敷料清晰的孔状结构 扫描电子显微镜×1 000；1G、1H.分别为图1C、1D中方框处放大图，显示猪UBM清晰的基底膜结构和纤维结构 扫描电子显微镜×1 000
2 2组小鼠巨噬细胞划痕后各时间点剩余划痕面积 倒置相差显微镜×40。2A、2B、2C、2D.分别为可吸收敷料浸提液组划痕后0（即刻）、1、3、7 d；2E、2F、2G、2H.分别为猪膀胱脱细胞基质浸提液组划痕后0、1、3、7 d，其中图2F、2G、2H剩余划痕面积分别明显小于图2B、2C、2D
3 Transwell实验观察培养各时间点2组小鼠巨噬细胞的迁移情况 结晶紫×100。3A、3B.分别为可吸收敷料浸提液组、猪膀胱脱细胞基质（UBM）浸提液组培养12 h迁移至Transwell小室下层的细胞情况，图3B细胞数量明显多于图3A；3C、3D.分别为可吸收敷料浸提液组、猪UBM浸提液组培养24 h迁移至Transwell小室下层的细胞情况，图3D细胞数量明显多于图3C
4 2组小鼠创面植入支架术后各时间点组织学观察。4A.术后7 d，可吸收敷料组支架底部聚集少量炎症细胞 苏木精-伊红×100；4B.术后7 d，猪膀胱脱细胞基质（UBM）组支架内部出现大量炎症细胞，较图4A明显增多 苏木精-伊红×100；4C.术后14 d，可吸收敷料组支架内部炎症细胞增多 苏木精-伊红×100；4D.术后14 d，猪UBM组支架内部炎症细胞广泛分布，较图4C明显增多 苏木精-伊红×100；4E、4F、4G、4H.分别为图4A、4B、4C、4D中方框处放大图，与放大前相比，可以看到更明显的炎症细胞浸润 苏木精-伊红×400
注：炎症细胞细胞核呈深蓝色
5 2组小鼠创面植入支架术后各时间点F4/80、TGF-β1、VEGF、MMP-9和Ⅰ型胶原蛋白阳性表达（呈棕色） 二氨基联苯胺-苏木精×400。5A、5B、5C、5D.分别为术后7 d可吸收敷料组、术后7 d猪UBM组、术后14 d可吸收敷料组、术后14 d猪UBM组F4/80阳性表达情况，图5B、5D的F4/80阳性细胞数分别明显多于图5A、5C；5E、5F、5G、5H.分别为术后7 d可吸收敷料组、术后7 d猪UBM组、术后14 d可吸收敷料组、术后14 d猪UBM组TGF-β1阳性表达情况，图5F、5H的TGF-β1阳性细胞数分别明显多于图5E、5G；5I、5J、5K、5L.分别为术后7 d可吸收敷料组、术后7 d猪UBM组、术后14 d可吸收敷料组、术后14 d猪UBM组VEGF阳性表达情况，图5J、5L的VEGF阳性细胞数分别明显多于图5I、5K；5M、5N、5O、5P.分别为术后7 d可吸收敷料组、术后7 d猪UBM组、术后14 d可吸收敷料组、术后14 d猪UBM组MMP-9阳性表达情况，图5N、5P的MMP-9阳性细胞数分别明显多于图5M、5O；5Q、5R、5S、5T.分别为术后7 d可吸收敷料组、术后7 d猪UBM组、术后14 d可吸收敷料组、术后14 d猪UBM组Ⅰ型胶原蛋白阳性表达情况，图5R、5T的Ⅰ型胶原蛋白沉积分别较图5Q、5S明显
注：TGF-β1为转化生长因子β1，VEGF为血管内皮生长因子，MMP-9为基质金属蛋白酶9，UBM为膀胱脱细胞基质
6 2组小鼠创面植入支架术后各时间点巨噬细胞的分布和极化 异硫氰酸荧光素-二苯并环辛炔-4'，6-二脒基-2-苯基吲哚×200。6A.术后7 d，猪UBM组支架底层出现大量巨噬细胞；6B.术后7 d，可吸收敷料组支架底部聚集少量巨噬细胞；6C.术后14 d，猪UBM组支架未见明显M1型巨噬细胞；6D.术后14 d，可吸收敷料组支架内部出现少量M1型巨噬细胞；6E.术后7 d，猪UBM组支架底层见少量M2型巨噬细胞；6F.术后7 d，可吸收敷料组支架未见明显M2型巨噬细胞；6G.术后14 d，猪UBM组支架内部散在分布M2型巨噬细胞；6H.术后14 d，可吸收敷料组支架内部未见明显M2型巨噬细胞
注：UBM为膀胱脱细胞基质；各图中右上小图为细胞核染色（蓝色）图、右中小图为F4/80染色(绿色，表示巨噬细胞)图、右下小图为CD86或CD206染色（樱红色）图，各图中左侧大图为右侧3张小图的合成图（F4/80与CD86或CD206的双染呈白色，分别表示巨噬细胞发生M1型或M2型极化）；各图中虚线表示肉膜与材料的分界；图6A~6D为加入F4/80与CD86双抗后的染色，图6E~6H为加入F4/80与CD206双抗后的染色
表1 2组小鼠巨噬细胞划痕后各时间点细胞迁移率比较（%，
组别 样本数 1 d 3 d 7 d 可吸收敷料浸提液组 3 13.6±2.4 19.5±1.5 29.7±2.0 猪膀胱脱细胞基质浸提液组 3 44.9±2.5 59.9±0.9 71.7±0.5 t值 15.31 19.76 20.58 P值 <0.001 <0.001 <0.001 注：处理因素主效应，F=110.80，P<0.001；时间因素主效应，F=1 032.00，P<0.001；两者交互作用，F=8.04，P=0.006 
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表2 Transwell实验观测培养各时间点2组小鼠巨噬细胞的迁移数量比较（个，
组别 样本数 12 h 24 h 可吸收敷料浸提液组 3 16.0±1.3 26.0±2.7 猪膀胱脱细胞基质浸提液组 3 38.0±2.5 86.0±2.1 t值 12.20 33.26 P值 <0.001 <0.001 注：于100倍视野下计数；处理因素主效应，F=516.90，P<0.001；时间因素主效应，F=1 033.00，P<0.001；两者交互作用，F=221.90，P<0.001
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表3 2组小鼠植入支架术后各时间点浸润的炎症细胞数量比较（个，
组别 样本数 7 d 14 d 可吸收敷料组 6 17.0±5.3 40.2±4.8 猪膀胱脱细胞基质组 6 41.8±2.2 77.5±6.5 t值 6.58 10.70 P值 <0.001 <0.001 注：于400倍视野下计数；处理因素主效应，F=132.60，P<0.001；时间因素主效应，F=146.50，P<0.001；两者交互作用，F=5.96，P=0.033
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表4 2组小鼠植入支架术后各时间点F4/80、TGF-β1、VEGF和MMP-9阳性细胞数量比较（个，
组别 样本数 F4/80 TGF-β1 VEGF MMP-9 可吸收敷料组 6 7 d 12.3±1.1 10.2±0.9 14.4±1.3 17.7±2.4 14 d 11.3±1.8 18.7±1.1 14.0±1.5 19.7±1.1 猪膀胱脱细胞基质组 6 7 d 50.0±1.0 48.7±1.6 26.0±0.5 36.5±1.5 14 d 30.0±0.7 49.8±1.4 26.3±1.8 37.2±1.8 t1值 46.11 40.69 13.90 14.15 P1值 <0.001 <0.001 <0.001 <0.001 t2值 19.79 32.93 12.16 13.21 P2值 <0.001 <0.001 <0.001 <0.001 注：于400倍视野下计数；TGF-β1为转化生长因子β1，VEGF为血管内皮生长因子，MMP-9为基质金属蛋白酶9；F4/80、TGF-β1、VEGF、MMP-9处理因素主效应，F值分别为283.30、50.64、0.01、2.14，P值分别为<0.001、<0.001、0.943、0.175；时间因素主效应，F值分别为2 038.00、2 710.00、331.70、374.40，P值均<0.001；两者交互作用，F值分别为231.90、20.17、0.34、0.44，P值分别为<0.001、<0.001、0.571、0.522；t1值、P1值，t2值、P2值分别为组间术后7、14 d各指标两两比较所得
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表5 2组小鼠植入支架术后各时间点CD206和CD86阳性细胞百分比比较（%，
组别 样本数 CD206 CD86 可吸收敷料组 6 7 d 41.2±10.1 80.2±7.9 14 d 56.0±8.9 72.1±2.5 猪膀胱脱细胞基质组 6 7 d 72.0±6.1 8.3±1.5 14 d 81.2±1.7 4.5±0.8 t1值 5.05 20.90 P1值 0.002 <0.001 t2值 4.13 19.64 P2值 0.007 <0.001 注：CD206、CD86处理因素主效应，F值分别为7.73、5.98，P值分别为0.024、0.040；时间因素主效应，F值分别为42.16、821.90，P值均<0.001；两者交互作用，F值分别为0.42、0.79，P值分别为0.543、0.399；t1值、P1值，t2值、P2值分别为组间术后7、14 d各指标两两比较所得
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