负压创面治疗技术对糖尿病大鼠创面血管生成的影响

Influence of negative pressure wound therapy on the angiogenesis of wounds in diabetic rats

目的观察负压创面治疗技术对糖尿病大鼠创面血管生成的影响。方法取40只sD大鼠，按65mg／kg腹腔注射20g／L链脲佐菌素诱导糖尿病模型。2周后按随机数字表法将大鼠分为对照组和负压组，每组20只，在大鼠背部正中切除2cm×2cm全层皮肤制备创面。伤后即刻，对照组创面常规换药；负压组创面予以每天4h持续负压（-16．0kPa）治疗，连续7d。（1）治疗前及治疗后1、2周，分别采用血糖仪及电子秤检测2组大鼠血糖及体质量。（2）治疗前及治疗后1、3、7d，每组取5只大鼠，采用激光多普勒血流成像仪检测创面血流量。（3）治疗后3、7d，每组取5只大鼠处死后切取创面组织并分成两部分，取左侧组织行免疫组织化学染色观察血管形成情况，计算微血管密度。（4）取治疗前制备创面时切取的全层皮肤及治疗后3、7d冻存的右侧组织，治疗后1、14d同前切取创面组织，采用实时荧光定量PCR法检测组织中血管内皮生长因子（VEGF）、血管内皮生长因子受体1（Fit—1）、血管生成素1（Ang—1）、Ang-2以及酪氨酸激酶受体2（Tie-2）mRNA的表达。对数据行双因素方差分析或LSD—t检验。结果（1）2组大鼠血糖及体质量水平总体或各时相点比较无明显差异（F值分别为0．667、0．176，t值为0．311～0．707，P值均大于0．05）。（2）2组大鼠创面血流量总体比较有明显差异（F=24．66，P〈0．05）。治疗后1、3、7d，负压组创面血流量分别为（179±24）、（219±12）、（192±30）灌注单位，显著高于对照组的（127±16）、（179±8）、（144±17）灌注单位（t值分别为3．71、5．57、2．77，P〈0．05或P〈0．01）。（3）2组大鼠创面微血管密度总体比较有明最差异（F=33．25，P〈0．05）。治疗后3d，负压组创面每100倍视野下微血管密度为（80±12）个，明显高于对照组的（38±4）个（t=9．257，P〈0．05）。治疗后7d，2组大鼠创面微血管密度相近（t=1．159，P〉0．05），此时负压组血管排列规律、管腔宽畅，而对照组血管排列紊乱、管腔狭窄。（4）治疗后1、3d，负压组VEGF、Fit-1及Ang-1mRNA表达水平均明显高于对照组（t值为1．28-11．60，P值均小于0．01）；治疗后7d，负压组Ang-1mRNA表达水平（27．59±3．55）明显高于对照组（19．87±1．86，t=7．23，P〈0．001），其拮抗剂Ang-2mRNA表达水平（5．79±0．61）明显低于对照组（17．62±0．85，t=19．88，P〈0．001）。治疗后3～14d，负压组Tie-2mRNA表达水平均低于对照组（t值为8．92～15．60，P值均小于0．01）。结论负压创面治疗技术可能通过增强创面愈合后期Ang-1表达以及降低Ang-2表达，促进糖尿病大鼠创面血管形成。

Objective To observe the influence of negative pressure wound therapy on the angio- genesis of wounds in diabetic rats. Methods Diabetes model was reproduced by intraperitoneal injection of 20 g/L streptozotocin in the dosage of 65 mg/kg in 40 SD rats. Two weeks later, rats were divided into control group （C） and negative pressure group （NP） according to the random number table, with 20 rats in each group. A piece of full-thickness skin in the center of the back of each rat in the size of 2 cm ×2 cm was excised to produce a wound. Immediately after injury, wounds in group C were given conventional dressing change; wounds in group NP were treated with continuous negative pressure （ -16.0 kPa） therapy for four hours a day, which lasted for seven days. （ 1 ） Blood glucose and body weight of rats in two groups were re-spectively measured by glucose meter and electronic scale before treatment, and 1 and 2 week （s） after. （2） Wound blood flow was detected by laser Doppler perfusion imager before treatment and on post treatment day （PTD） 1, 3, 7, with 5 rats at each time point. （3） On PTD 3 and 7, respectively, five rats from each group were sacrificed. The wound tissue was excised and divided into two parts. The angiogenesis in the left part tissue was observed with immunohistochemical staining. The microvessel density was calculated. （4） The full-thickness skin excised before treatment and the right part tissue freezed on PTD 3 and 7 were col- lected. On PTD 1 and 14, wound tissue was excised in the above-mentioned method. The mRNA levels of the vascular endothelial growth factor （VEGF） , vascular endothelial growth factor receptor 1 （Fit-1） , angio- poietin 1 （ Ang-1 ） , Ang-2, and tyrosine kinase receptor 2 （Tie-2） were determined with real-time fluores- cence quantification PCR. Data were processed with two-way analysis of variance or LSD- t test. Results （ 1 ） No significant difference was observed between two groups in blood glucose level and body weight as a whole or at each time point （ with F values respectively 0. 667, 0. 176, t values from 0.311 to 0. 707, P val- ues all above 0.05 ）. （2） The difference in the overall wound blood flow of rats between two groups was sig- nificant （ F =24.66, P 〈0.05）. On PTD 1, 3, 7, values of wound blood flow of rats in group NP were （179±24）, （219 ± 12）, （192 ± 30） perfusion unit, significantly higher than those of rats in group C [（127±16）, （179±8）, （144±17） perfusion unit, with t values respectively 3.71, 5.57, 2.77, P 〈 0.05 or P 〈 0.01 ]. （3） The difference in the overall microvessel density in the wound of rats between two groups was significant （ F = 33.25, P 〈 0.05）. On PTD 3, the microvessel density in the wound of rats in group NP was （80 ± 12） per 100-time visual field, which was significantly higher than that of group C [ （38 ±4） per 100-time visual field, t =9. 257, P 〈0.05]. On PTD 7, the microvessel density in the wound of rats in two groups were close （ t = 1. 159, P 〉0.05） , but the vessels in group NP were regularly arranged with spacious lumen, while the vessels in group C were disorderly arranged with narrow lumen. （4） On PTD 1 , 3, mRNA expression levels of VEGF, Fit-1, and Ang-1 in group NP were obviously higher than those in group C （withtvalues from 1.28 to 11.60, Pvalues all below0.01）. On PTD 7, the mRNA ex- pression level of Ang-1 （27.59 ± 3.55） in group NP was obviously higher than that in group C （19.87 ± 1.86, t = 7.23, P 〈 0. 001 ） , while the mRNA level of its antagonist Ang-2 （5.79 ± 0.61 ） in group NP was obviously lower than that in group C （17.62±0.85, t =19.88, P 〈0.001）. On PTD 3, 7, 14, mRNA levels of Tie-2 in group NP were obviously lower than those in group C （ with t values from 8.92 to 15.60, P values all below 0.01 ）. Conclusions Negative pressure wound therapy may promote wound angiogenesis by enhancing the expression of Ang-1 and lowering the expression of Ang-2 in diabetic rats.