Influence and its mechanism of allogeneic dermal papilla cells on the wound healing of full-thickness skin defects in mice
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摘要:
目的 探索异体毛乳头细胞（DPC）对小鼠全层皮肤缺损创面愈合的影响及其机制。 方法 该研究为实验研究。利用显微解剖结合胶原酶消化法自5只6周龄雄性C57BL/6J小鼠触须毛囊中提取DPC并成功鉴定。将18只8周龄雄性C57BL/6J小鼠按照随机数字表法分为磷酸盐缓冲液（PBS）组及DPC组（每组9只小鼠）,在所有小鼠背部创建全层皮肤缺损创面模型。分别于伤后2、4、6 d,通过创面周围皮下注射给予DPC组小鼠含1×106个第4代DPC的细胞悬液100 μL、PBS组小鼠等体积的PBS。伤后3、7、10、14 d,观察2组小鼠创面愈合情况及毛发生长情况并测量创面剩余面积；另测量2组小鼠伤后14 d创面毛发覆盖面积。伤后14 d,收集2组小鼠创面组织标本,行苏木精-伊红染色观察新生毛囊情况并计数,行Masson染色观察真皮层胶原沉积情况并测量胶原沉积面积,采用免疫荧光法检测Wnt/β连环蛋白信号通路相关分子β连环蛋白、淋巴增强结合因子1（Lef1）的蛋白表达,分别采用蛋白质印迹法和实时荧光定量反转录PCR法检测β连环蛋白、Lef1的蛋白和mRNA表达。各实验样本数均为3。 结果 与PBS组相比,DPC组小鼠伤后各时间点创面再上皮化速度加快,且在伤后10、14 d可见更多毛发生长。伤后7、10、14 d,DPC组小鼠创面剩余面积分别为（13.92±2.90）、（3.69±1.78）、（1.09±0.14）mm2,分别明显小于PBS组的（26.19±2.06）、（10.84±3.59）、（6.75±2.11）mm2（t值分别为5.85、3.09、4.63,P值均<0.05）。伤后14 d,DPC组小鼠创面毛发覆盖面积为（62±7）mm2,明显大于PBS组的（35±6）mm2（t=2.89,P<0.05）。伤后14 d,与PBS组比较,DPC组小鼠创面组织中新生毛囊数量明显增多（t=5.43,P<0.05）,真皮层胶原沉积面积明显缩小（t=3.56,P<0.05）。伤后14 d,免疫荧光法和蛋白质印迹法检测均显示,DPC组小鼠创面组织中β连环蛋白（t值分别为5.49、4.25,P值均<0.05）和Lef1（t值分别为7.50、11.54,P值均<0.05）的蛋白表达均明显高于PBS组；DPC组小鼠创面组织中β连环蛋白和Lef1的mRNA表达均明显高于PBS组（t值分别为7.68、9.67,P<0.05）。 结论 DPC通过激活Wnt/β连环蛋白信号通路加快小鼠全层皮肤缺损创面再上皮化,并促进创面愈合过程中的毛囊再生。 -
关键词:
- 创伤和损伤 /
- 伤口愈合 /
- 淋巴样增强子结合因子1 /
- 胶原 /
- 毛囊再生 /
- 毛乳头细胞 /
- Wnt/β连环蛋白信号通路
Abstract:Objective To explore the influence and its mechanism of allogeneic dermal papilla cells (DPCs) on the wound healing of full-thickness skin defects in mice. Methods This study was an experimental study. DPCs were isolated from the whisker follicles of five 6-week-old male C57BL/6J mice by combining microdissection with collagenase digestion and were successfully identified. Eighteen 8-week-old male C57BL/6J mice were divided into phosphate buffer solution (PBS) group and DPC group according to the random number table, with 9 mice in each group, and the full-thickness skin defect wound model was created on the back of all mice. On day 2, 4, and 6 after injury, the mice in DPC group were administered 100 μL of cell suspension containing 1×106 DPCs of the 4th passage by subcutaneous injection around the wound, and the mice in PBS group was administered an equal volume of PBS. On day 3, 7, 10, and 14 after injury, the wound healing and hair growth of mice in two groups were observed, and the residual wound area was measured, and the hair coverage area on the wound of mice in two groups was measured on day 14 after injury. On day 14 after injury, the wound tissue samples of mice in two groups were collected. Hematoxylin-eosin staining was performed to observe the condition of newborn hair follicles and the number was counted, Masson staining was performed to observe the collagen deposition in the dermis and the collagen deposition area was measured, the immunofluorescence method was used to detect the protein expressions of Wnt/β-catenin signaling pathway related molecules β-catenin and lymphoid enhancer binding factor 1 (Lef1), and Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction were used to detect the protein and mRNA expressions of β-catenin and Lef1, respectively. The number of samples in each experiment was 3. Results Compared with those in PBS group, the mice in DPC group had accelerated wound re-epithelialization at each time point after injury, and more hair growth on day 10 and 14 after injury. On day 7, 10, and 14 after injury, the residual wound areas of mice in DPC group were (13.92±2.90), (3.69±1.78), and (1.09±0.14) mm2, respectively, which were significantly smaller than (26.19±2.06), (10.84±3.59), and (6.75±2.11) mm2 in PBS group, respectively (with t values of 5.85, 3.09, and 4.63, respectively, P values all <0.05). On day 14 after injury, the hair coverage area on the wound of mice in DPC group was (62±7) mm2, which was significantly larger than (35±6) mm2 in PBS group (t=2.89, P<0.05). On day 14 after injury, compared with those in PBS group, the number of newborn hair follicles in the wound tissue of mice in DPC group was significantly increased (t=5.43, P<0.05), and the dermal collagen deposition area was significantly reduced (t=3.56, P<0.05). On day 14 after injury, both the immunofluorescence method and the Western blotting detection showed that the protein expressions of β-catenin (with t values of 5.49 and 4.25, respectively, P values all <0.05) and Lef1 (with t values of 7.50 and 11.54, respectively, P values all <0.05) in the wound tissue of mice in DPC group were significantly higher than those in PBS group; the mRNA expressions of β-catenin and Lef1 in the wound tissue of mice in DPC group were significantly higher than those in PBS group (with t values of 7.68 and 9.67, respectively, P<0.05). Conclusions DPCs can accelerate the re-epithelialization of full-thickness skin defect wounds in mice by activating Wnt/β-catenin signaling pathway and promote hair follicle regeneration during the process of wound healing. -
参考文献
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图 1 小鼠毛乳头细胞的生长及标志物表达情况。1A.毛乳头组织培养7 d后,细胞呈短梭形,围绕组织块密集生长 倒置荧光显微镜×40；1B.成脂诱导培养2周后,细胞内可见脂滴（红色）形成 油红O×100；1C.成骨诱导培养3周后,细胞内可见钙结节（红色）形成 茜素红×100；1D.细胞中碱性磷酸酶（灰色）表达为阳性 对硝基苯磷酸二钠×40；1E、1F、1G、1H.分别为细胞中多能蛋白聚糖（红色）、CD133（红色）、β连环蛋白（绿色）及碱性磷酸酶（红色）表达情况,均为阳性 花青素3-异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚×100
图 2 2组全层皮肤缺损小鼠伤后各时间点创面愈合及毛发生长情况。2A、2B、2C.分别为PBS组伤后3、7、14 d创面形态,创面逐渐愈合,并可见新生毛发生长；2D、2E、2F.分别为DPC组伤后3、7、14 d创面形态,与图2A、2B、2C相比,图2D、2E、2F创面面积均更小,且图2F可见更多毛发生长
注：分别于伤后2、4、6 d,通过创面周围皮下注射给予毛乳头细胞（DPC）组小鼠DPC、磷酸盐缓冲液（PBS）组小鼠PBS；各图中圆环为防止创面快速收缩的硅胶环
图 3 2组全层皮肤缺损小鼠伤后14 d创面组织中新生毛囊及胶原沉积情况。3A、3B.分别为PBS组、DPC组新生毛囊情况,图3B中新生毛囊的数量明显多于图3A 苏木精-伊红×10；3C、3D.分别为PBS组、DPC组胶原沉积情况,图3C真皮层新生胶原的含量明显多于图3D Masson×10；3E、3F.分别为图3A、3B中方框中图形的放大图,图3E中无新生毛囊结构形成,而图3F中可见新生毛囊结构形成 苏木精-伊红×100；3G、3H.分别为图3C、3D中方框中图形的放大图,图3G中胶原排列致密、紊乱,图3H中胶原排列疏松 Masson ×100
注：分别于伤后2、4、6 d,通过创面周围皮下注射给予毛乳头细胞（DPC）组小鼠DPC、磷酸盐缓冲液（PBS）组小鼠PBS
图 4 2组全层皮肤缺损小鼠伤后14 d创面组织中β连环蛋白与Lef1的蛋白表达情况。4A、4B.分别为PBS组、DPC组β连环蛋白荧光表达情况,图4B中β连环蛋白荧光强度明显强于图4A Alexa Fluor 594-4',6-二脒基-2-苯基吲哚×100；4C、4D.分别为PBS组、DPC组Lef1荧光表达情况,图4D中Lef1荧光强度明显强于图4C Alexa Fluor 488-4',6-二脒基-2-苯基吲哚×100
注：分别于伤后2、4、6 d,通过创面周围皮下注射给予毛乳头细胞（DPC）组小鼠DPC、磷酸盐缓冲液（PBS）组小鼠PBS；Lef1为淋巴增强结合因子1；Lef1阳性染色为绿色,β连环蛋白阳性染色为红色,细胞核阳性染色为蓝色
图 5 蛋白质印迹法检测的2组全层皮肤缺损小鼠伤后14 d创面组织中β连环蛋白与Lef1的蛋白表达
注：分别于伤后2、4、6 d,通过创面周围皮下注射给予毛乳头细胞（DPC）组小鼠DPC、磷酸盐缓冲液（PBS）组小鼠PBS；Lef1为淋巴增强结合因子1,GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3指示PBS组3个样本,4、5、6指示DPC组3个样本
Table 1. 采用实时荧光定量反转录PCR法检测细胞因子mRNA表达的引物序列与产物大小
引物名称 引物序列（5'→3'） 产物大小（bp） β连环蛋白 上游：GTGGTCATCGCCACCTTAATA 101 下游：TTCTCGCTGTTGGAGTTCAG 淋巴增强结合因子1 上游：TCTGCTGACATTTGCCTCTAC 103 下游：GCTGGTGGATGGCTCTTATATT 3-磷酸甘油醛脱氢酶 上游：GGTGAAGGTCGGTGTGAACG 179 下游：CTCGCTCCTGGAAGATGGTG 
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