Zhu Fei-bin, Liu De-wu, Zhang Hong-yan, Xu Jun-ci, Peng Yan, Zhong Qing-ling, Li Yong-tie
Department of Burns, Nanchang University, Nanchang, China.
Zhonghua Shao Shang Za Zhi. 2012 Feb;28(1):25-31.
To observe the effect of sensory neuropeptide substance P combined with epidermal stem cells (ESC) on wound healing and nerve regeneration in diabetic rats.
ESC that had been isolated from SD rats were identified and cultured in vitro, and they were inoculated onto nourishing layer of amniotic membrane to construct amniotic membrane-ESC. Four full-thickness skin wounds were produced on the back of each of 48 diabetic rats. The resulted 192 wounds were randomly divided into ESC + substance P group, ESC group, substance P group, and control group according to the lottery method, with 48 wounds in each group. Wounds in ESC + substance P group and ESC group were transplanted with amniotic membrane-ESC, and those in substance P group and control group were transplanted with amniotic membrane. After transplantation, 250 µL substance P in the concentration of 1 × 10(-7) mol/L was injected around and into the middle of the wounds in ESC + substance P group and substance P group, 2 times a day, and continued for 4 days, while 250 µL PBS solution was injected in the above-mentioned position in ESC group and control group as control, 2 times a day, and continued for 4 days. On post injury day (PID) 4, 7, 10, 14, 17, and 23, the wound healing rate (with 8 wounds at each time point) was observed and determined, and changes in wound tissue structure were observed with HE staining. On PID 4, 7, and 10, collagen distribution in wound tissue was observed with Masson staining, and type I and type III collagen deposition in wound tissue was respectively observed after immunohistochemical staining. The distribution of protein gene product 9.5 (PGP 9.5) and regeneration of substance P positive nerve fibers in wound tissue were observed with immunohistochemical staining on PID 14 and 23. Data were processed with one-way analysis of variance and t test.
(1) The wound healing rate in ESC + substance P group reached 100.0% on PID 14, which was obviously earlier than that in ESC group, substance P group, and control group, healing was respectively observed on PID 17, 17, and 23. The wound healing quality in ESC + substance P group was better than that in the other three groups as shown by HE staining. (2) On PID 10, collagen that was darkly stained and widely distributed was observed in wound tissue of ESC + substance P group and substance P group, while collagen in the other two groups was lightly stained and narrowly distributed. Deposition quantity of type I collagen gradually increased, and that of type III collagen gradually decreased in the wounds of each group over time. On PID 4, 7, and 10, distribution amount of type I collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.72, 118.21, 26.71, P values all below 0.01) and control group (with t value respectively 44.37, 22.76, 30.32, P values all below 0.01), while there was no significance between ESC + substance P group and substance P group. On PID 4, 7, and 10, distribution amount of type III collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.27, 28.68, 14.51, P values all below 0.01) and control group (with t value respectively 35.68, 22.52, 22.24, P values all below 0.01). (3) A large amount of PGP 9.5 and regeneration of substance P positive nerve fibers, and some peripheral nerve fibers in deep wound extending to epidermis were observed in wound tissue of ESC + substance P group and substance P group. A small amount of PGP 9.5 and regeneration of substance P positive nerve fibers without peripheral nerve fibers extending to epidermis were observed in deep wound tissue of ESC group and control group. On PID 14, 23, ratios of area of PGP 9.5 positive nerve fiber in the wounds of ESC + substance P group were (3.86 ± 0.25)% and (7.03 ± 0.28)%, and they were significantly higher than those of ESC group [(1.48 ± 0.30)%, (3.01 ± 0.43)%, with t value respectively 23.95, 30.27, P values all below 0.01] and control group [(1.46 ± 0.23)%, (2.84 ± 0.29)%, with t value respectively 27.35, 40.32, P values all below 0.01]. On PID 14, 23, ratios of substance P positive nerve fiber area in the wounds of ESC + substance P group were (2.01 ± 0.14)% and (1.19 ± 0.11)%, which were obviously higher than those of ESC group [(0.85 ± 0.17)%, (1.34 ± 0.21)%, with t value respectively 20.50, 2.60, P < 0.05 or P < 0.01] and control group [(0.74 ± 0.15)%, (1.30 ± 0.17)%, with t value respectively 23.98, 2.41, P < 0.05 or P < 0.01].
Joint application of substance P and ESC can effectively promote healing of wound and nerve regeneration in diabetic rats.
观察感觉神经肽P物质联合表皮干细胞（ESC）对糖尿病大鼠伤口愈合及神经再生的影响。
从SD大鼠分离ESC并进行体外鉴定和培养，接种于羊膜滋养层构建羊膜-ESC。48只糖尿病大鼠背部各制作4个全层皮肤伤口。将192个伤口按抽签法随机分为ESC + P物质组、ESC组、P物质组和对照组，每组48个伤口。ESC + P物质组和ESC组伤口移植羊膜-ESC，P物质组和对照组伤口移植羊膜。移植后，ESC + P物质组和P物质组伤口周围及中央注射浓度为1×10(-7) mol/L的P物质250 μL，每日2次，持续4天，ESC组和对照组在上述位置注射250 μL PBS溶液作为对照，每日2次，持续4天。在伤后第4、7、10、14、17和23天，观察并测定伤口愈合率（每个时间点8个伤口），HE染色观察伤口组织结构变化。在伤后第4、7和10天，Masson染色观察伤口组织中胶原分布，免疫组织化学染色分别观察伤口组织中Ⅰ型和Ⅲ型胶原沉积。在伤后第14和23天，免疫组织化学染色观察伤口组织中蛋白基因产物9.5（PGP 9.5）分布及P物质阳性神经纤维再生情况。数据采用单因素方差分析和t检验处理。
（1）ESC + P物质组伤后第14天伤口愈合率达100.0%，明显早于ESC组、P物质组和对照组，后三组分别在伤后第17、17和23天观察到愈合。HE染色显示ESC + P物质组伤口愈合质量优于其他三组。（2）伤后第10天，ESC + P物质组和P物质组伤口组织中胶原染色深且分布广泛，其他两组胶原染色浅且分布窄。随着时间推移，每组伤口中Ⅰ型胶原沉积量逐渐增加，Ⅲ型胶原沉积量逐渐减少。在伤后第4、7和10天，ESC + P物质组伤口组织中Ⅰ型胶原分布量明显高于ESC组（t值分别为32.72、118.21、26.71，P值均<0.01）和对照组（t值分别为44.37、22.76、30.32，P值均<0.01），而ESC + P物质组与P物质组之间无显著性差异。在伤后第4、7和10天，ESC + P物质组伤口组织中Ⅲ型胶原分布量明显高于ESC组（t值分别为32.27、28.68、14.51，P值均<0.01）和对照组（t值分别为35.68、22.52、22.24，P值均<0.01）。（3）ESC + P物质组和P物质组伤口组织中观察到大量PGP 9.5和P物质阳性神经纤维再生，部分深部伤口外周神经纤维延伸至表皮。ESC组和对照组深部伤口组织中观察到少量PGP 9.5和P物质阳性神经纤维再生，无外周神经纤维延伸至表皮。在伤后第14、23天，ESC + P物质组伤口中PGP 9.5阳性神经纤维面积比例分别为(3.86±0.25)%和(7.03±0.28)%，明显高于ESC组[(1.48±0.30)%，(3.01±0.43)%，t值分别为23.95、30.27，P值均<0.01]和对照组[(1.46±0.23)%，(2.84±0.29)%，t值分别为27.35、40.32，P值均<0.01]。在伤后第14、23天，ESC + P物质组伤口中P物质阳性神经纤维面积比例分别为(2.01±0.14)%和(1.19±0.11)%，明显高于ESC组[(0.85±0.17)%，(1.34±0.21)%，t值分别为20.50、2.60，P<0.05或P<0.01]和对照组[(0.74±0.15)%，(1.30±0.17)%，t值分别为23.98、2.41，P<0.05或P<0.01]。
P物质与ESC联合应用可有效促进糖尿病大鼠伤口愈合及神经再生。
