P物质与表皮干细胞联用对糖尿病大鼠创面愈合与神经再生的影响

Effect of substance P combined with epidermal stem cells on wound healing and nerve regeneration in rats with diabetes mellitus

目的 观察感觉神经肽P物质与表皮干细胞（ESC）联合应用对糖尿病大鼠创面愈合与神经再生的作用. 方法 分离培养SD大鼠ESC（经鉴定）,接种于羊膜滋养层上构建羊膜-ESC备用.选择48只糖尿病模型大鼠,每只背部制作4个全层皮肤缺损创面.按随机抽签法将此192个创面分为ESC＋P物质组、ESC组、P物质组、对照组,每组48个创面.ESC＋P物质组和ESC组创面均移植羊膜-ESC,P物质组、对照组创面移植羊膜.移植后,ESC＋P物质组、P物质组在创周及创面中央注射1×10-7 mol/L的P物质250μL,ESC组、对照组在创周及创面中央注射PBS 250μL作对照,各组每日注射2次,连用4d.于大鼠伤后4、7、10、14、17、23 d,观察并计算创面愈合率（每时相点8个创面）,HE染色观察创面组织结构改变.伤后4、7、10 d,行Masson染色观察创面组织总胶原分布,免疫组织化学染色观察Ⅰ、Ⅲ型胶原沉积量.伤后14、23 d,用免疫组织化学染色法观察创面组织中蛋白基因产物9.5（PGP 9.5）及P物质阳性神经纤维分布情况.对数据行单因素方差分析和t检验. 结果（1）ESC＋P物质组伤后14 d创面愈合率达100.0％,明显早于ESC组、P物质组、对照组完全愈合时间（伤后17、17、23 d）.HE染色显示ESC＋P物质组创面愈合质量明显优于其余3组.（2）伤后10 d,ESC＋P物质组与P物质组创面组织中胶原着色深、面积广；其余2组胶原染色较浅、面积较小.随着伤后时间推移,各组创面Ⅰ型胶原沉积量逐渐升高,Ⅲ型胶原沉积量逐渐下降.伤后4、7、10 d,ESC＋P物质组Ⅰ型胶原沉积量明显高于ESC组（t值分别为32.72、118.21、26.71,P值均小于0.01）和对照组（t值分别为44.37、22 76、30.32,P值均小于0.01）；ESC＋P物质组与P物质组水平相对接近.伤后4、7、10d,ESC＋P物质组创面Ⅲ型胶原沉积量明显高于ESC组（t值分别为32.27、28 68、14.51,P值均小于0.01）和对照组（t值分别为35 68、22.52、22 24,P值均小于0.01）.（3）ESC ＋P物质组与P物质组创面组织中有大量PGP 9.5和P物质阳性神经纤维再生,创面深层部分神经纤维末梢向表皮延伸.ESC组、对照组仅见创面深层有少量PGP 9.5和P物质阳性神经纤维,且未向表皮延伸.伤后14、23 d,ESC＋P物质组创面PGP 9.5阳性神经纤维面积占（3.86±0.25）％、（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值均小于0.01]和对照组[（1 46±0 23）％、（2.84±0.29）％,t值分别为27.35、40.32,P值均小于0.01].伤后14、23 d,ESC＋P物质组创面P物质阳性神经纤维面积占（2.01±0 14）％、（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]. 结论 感觉神经肽P物质和ESC联合应用,可以有效促进糖尿病大鼠创面愈合与神经再生.

Abstract： Objective To observe the effect of sensory neuropeptide substance P combined with epidermal stem cells（ESC）on wound healing and nerve regeneration in diabetic rats. Methods 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. Results （ 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 0.0l ）,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,Pvalues all below0.01] 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 ofESC＋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 orP 〈0.01 ] and control group[（0.74 ±0.15）%,（1.30 ±0.17）%,with t value respectively 23.98,2.41,P 〈0.05 orP 〈0.01]. Conclusions Joint application of substance P and ESC can effectively promote healing of wound and nerve regeneration in diabetic rats.