Zhu D Z, Wang Y H, Wang R, Fu X B
Key Laboratory of Wound Repair and Regeneration of PLA, the PLA General Hospital, Beijing 100048, China.
Department of General Surgery, Handan First Hospital of Hebei Province, Handan 056002, China.
Zhonghua Shao Shang Za Zhi. 2020 Mar 20;36(3):187-194. doi: 10.3760/cma.j.cn501120-20200105-00005.
To explore the effects and molecular mechanism of tumor necrosis factor α (TNF-α) on differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment. (1) Five 6-8 week-old female C57BL/6 mice were used, with one 1 cm(2) deep partial-thickness to full-thickness scald wound being created on the back of each mouse with a scald apparatus. One day after injury, the full-thickness skin tissue of the wound was taken, and the concentration of TNF-α in the tissue was detected by enzyme-linked immunosorbent assay. (2) Gelatin in the mass of 0.9 g and 0.3 g sodium alginate were mixed and then dissolved in 30 mL phosphate buffer solution to make hydrogel. Full-thickness skin of the planta of 10 male and female one day newborn C57BL/6 mice was ground into dermal homogenate. The mesenchymal stem cells were isolated from femur and tibia of 10 male and female C57BL/6 mice born for 7 days and cultured. A final density of 1.5×10(5) cells/mL of bioink was made of mixture of 8 mL pre-warmed hydrogel, 1 mL mouse foot dermal homogenate, and 1 mL the second or third passage of mesenchymal stem cell suspensions. The three-dimensional bioprinter was used to print 12 cylindrical blocks arranged in a crisscross pattern in petri dish. The printed blocks were cross-linked with 25 g/L calcium chloride solution for 10 min and then cultured for 12 hours by adding a medium for mesenchymal stem cells. Subsequently, the printed blocks were divided into blank control group and TNF-α treatment group according to the random number table, with 6 plates and 6 blocks in each group. Both groups of printed blocks were cultured with fresh sweat gland induction medium, and a final mass concentration of 20 ng/mL TNF-α was added into the medium of TNF-α treatment group. After 6 hours of culture, the mRNA expression of pluripotency marker Nanog in the mesenchymal stem cells of two plates of each group was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Nanog in the mesenchymal stem cells of one plate of each group was detected by Western blotting, both with triplicate samples. After 14 days of culture, the mRNA expression of sweat gland cell markers cytokeratin 14 (CK14), CK18, sodium potassium adenosine triphosphatase protein a1 (ATP1a1), and aquaporin 5 (AQP5) was detected by real-time fluorescent quantitative RT-PCR in the mesenchymal stem cells of 2 plates of each group (=3), and the protein expression distribution of CK14, CK18, ATP1a1, and AQP5 of the mesenchymal stem cells in one plate of each group was detected by immunofluorescence staining. Data were statistically analyzed with independent sample test. (1) One day after injury, the mass concentration of TNF-α in the scald wound tissue of mouse was (19±3) ng/mL. (2) After 6 hours of culture, the mRNA and protein expression levels of Nanog in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.39±0.04 and 0.36±0.03, respectively, which were significantly lower than 1.00±0.05 and 1.00±0.07 of blank control group (=16.51, 14.56, <0.01). (3) After 14 days of culture, the mRNA expression levels of CK18, CK14, ATP1a1, and AQP5 in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.38±0.03, 0.42±0.11, 0.23±0.06, and 0.25±0.03, respectively, which were significantly less than 1.00±0.03, 1.00±0.05, 1.00±0.05, 1.00±0.07 of blank control group (=25.31, 8.31, 17.07, 17.06, <0.01). (4) After 14 days of culture, the CK18, CK14, ATP1a1, and AQP5 protein were widely distributed in the cytoplasm of mesenchymal stem cells in printed blocks of blank control group, while the distribution of CK18, CK14, ATP1a1, and AQP5 protein in the cytoplasm of mesenchymal stem cells in printed blocks of TNF-α treatment group were significantly reduced in comparison. Exogenous TNF-α inhibits the directional differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment, which may be related to the inhibition of the expression of Nanog mRNA and protein by TNF-α that subsequently results in the down-regulation of multi-directional differentiation potential of mesenchymal stem cells.
探讨肿瘤坏死因子α（TNF-α）在三维环境中对小鼠间充质干细胞向汗腺细胞分化的影响及分子机制。（1）选用5只6-8周龄雌性C57BL/6小鼠，用烫伤仪在每只小鼠背部造成1处面积为1 cm²、深度从浅Ⅱ度至Ⅲ度的烫伤创面。伤后1天，取创面全层皮肤组织，采用酶联免疫吸附测定法检测组织中TNF-α浓度。（2）将0.9 g明胶与0.3 g海藻酸钠混合，然后溶于30 mL磷酸盐缓冲液中制成水凝胶。取10只1日龄新生C57BL/6雌雄小鼠足底的全层皮肤研磨制成真皮匀浆。从10只7日龄C57BL/6雌雄小鼠的股骨和胫骨中分离出间充质干细胞并进行培养。将8 mL预热的水凝胶、1 mL小鼠足底真皮匀浆和1 mL第二代或第三代间充质干细胞悬液混合制成终密度为1.5×10⁵个细胞/mL的生物墨水。使用三维生物打印机在培养皿中打印出呈交错排列的12个圆柱形块体。将打印好的块体用25 g/L氯化钙溶液交联10分钟，然后加入间充质干细胞培养基培养12小时。随后，根据随机数字表将打印好的块体分为空白对照组和TNF-α处理组，每组6个培养皿、6个块体。两组打印块体均用新鲜的汗腺诱导培养基培养，TNF-α处理组培养基中加入终质量浓度为20 ng/mL的TNF-α。培养6小时后，采用实时荧光定量逆转录聚合酶链反应（RT-PCR）检测每组2个培养皿中间充质干细胞多能性标志物Nanog的mRNA表达，采用蛋白质免疫印迹法检测每组1个培养皿中间充质干细胞Nanog的蛋白质表达，均设3个重复样本。培养14天后，采用实时荧光定量RT-PCR检测每组2个培养皿（n = 3）中间充质干细胞汗腺细胞标志物细胞角蛋白14（CK14）、CK18、钠钾三磷酸腺苷酶蛋白α1（ATP1a1）和水通道蛋白5（AQP5）的mRNA表达，采用免疫荧光染色检测每组1个培养皿中间充质干细胞CK14、CK18、ATP1a1和AQP5的蛋白质表达分布。数据采用独立样本t检验进行统计学分析。（1）伤后1天，小鼠烫伤创面组织中TNF-α的质量浓度为（19±3）ng/mL。（2）培养6小时后，TNF-α处理组打印块体中间充质干细胞Nanog的mRNA和蛋白质表达水平分别为0.39±0.04和0.36±0.03，显著低于空白对照组的1.00±0.05和1.00±0.07（t = 16.51，14.56，P < 0.01）。（3）培养14天后，TNF-α处理组打印块体中间充质干细胞CK18、CK14、ATP1a1和AQP5的mRNA表达水平分别为0.38±0.03、0.42±0.11、0.23±0.06和0.25±0.03，显著低于空白对照组的1.00±0.03、1.00±0.05、1.00±0.05和1.00±0.07（t = 25.31，8.31，17.07，17.06，P < 0.01）。（4）培养14天后，空白对照组打印块体中间充质干细胞的CK18、CK14、ATP1a1和AQP5蛋白在细胞质中广泛分布，而TNF-α处理组打印块体中间充质干细胞细胞质中CK18、CK14、ATP1a1和AQP5蛋白的分布相比显著减少。外源性TNF-α抑制三维环境中小鼠间充质干细胞向汗腺细胞的定向分化，这可能与TNF-α抑制Nanog mRNA和蛋白质表达，进而导致间充质干细胞多向分化潜能下调有关。
