Influence of human amniotic mesenchymal stem cells on macrophage phenotypes and inflammatory factors in full-thickness skin wounds of mice
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摘要: 目的 探讨人羊膜间充质干细胞(hAMSC)在体内外对小鼠全层皮肤缺损创面巨噬细胞表型的调控及对创面愈合相关炎症因子的影响。 方法 (1)取遵义医科大学附属医院妇产科5名足月分娩健康孕妇产后弃用的新鲜羊膜,酶消化法分离纯化培养hAMSC,取第3代细胞,经成骨、成脂诱导分化鉴定;取第4代细胞,经hAMSC表面标志物鉴定。取10只C57BL/6小鼠(均为雄性,6~8周龄,性别、鼠龄下同),腹腔灌洗法提取小鼠腹腔巨噬细胞,用含γ干扰素的DMEM培养基培养诱导制备M1型巨噬细胞。将M1型巨噬细胞分为hAMSC+巨噬细胞组及单纯巨噬细胞组。hAMSC+巨噬细胞组每孔巨噬细胞加入1×104个第4代hAMSC后加入DMEM培养基2 mL,常规培养;单纯巨噬细胞组每孔巨噬细胞仅加入2 mL DMEM培养基常规培养。于培养1、7 d,酶联免疫吸附测定法检测2组细胞培养上清液中白细胞介素12(IL-12)、精氨酸酶1及IL-10含量(样本数为6)。(2)取56只C57BL/6小鼠,建立背部全层皮肤缺损创面模型,按随机数字表法分为hAMSC组和磷酸盐缓冲液(PBS)组,每组28只。hAMSC组小鼠于创周皮下注射100 μL采用PBS悬浮的含1×107个/mL hAMSC的细胞悬液,PBS组小鼠创周仅注射100 μL PBS。于注射后1、3、7、14 d,2组分别取7只小鼠,处死后行苏木精-伊红染色组织病理学观察,免疫荧光染色检测创面巨噬细胞表面标志物[CD68与诱导型一氧化氮合酶(iNOS)双阳性、CD68与精氨酸酶1双阳性]表达,实时荧光定量反转录PCR检测创面IL-10、巨噬细胞炎症蛋白1α(MIP-1α)、MIP-2的mRNA表达。对数据行析因设计方差分析、
t 检验、Bonferroni校正。 结果 (1)培养1 d,2组细胞培养上清液中IL-12、精氨酸酶1含量相近(t =0.448、0.536,P >0.05),hAMSC+巨噬细胞组细胞培养上清液中IL-10含量明显低于单纯巨噬细胞组(t =14.722,P <0.01)。培养7 d,hAMSC+巨噬细胞组细胞培养上清液中IL-12含量明显低于单纯巨噬细胞组(t =13.226,P <0.01),精氨酸酶1和IL-10含量明显高于单纯巨噬细胞组(t =30.172、31.406,P <0.01)。(2)注射后1 d,2组小鼠创面均有大量炎性细胞浸润;注射后3 d,2组小鼠创面炎性细胞浸润均增多,hAMSC组炎性细胞浸润较PBS组少;注射后7 d,2组小鼠创面炎性细胞浸润均减少,hAMSC组炎性细胞浸润较PBS组少;注射后14 d,2组小鼠创面均未见明显炎性细胞浸润。(3)注射后1、14 d,2组小鼠创面CD68与iNOS双阳性细胞百分比、CD68与精氨酸酶1双阳性细胞百分比相近(t 1 d=0.134、0.693,t 14 d=1.146、2.585,P >0.05);注射后3、7 d,hAMSC组小鼠创面CD68与iNOS双阳性细胞百分比明显低于PBS组(t =6.396、4.787,P <0.01),CD68与精氨酸酶1双阳性细胞百分比明显高于PBS组(t =3.928、4.473,P <0.01)。(4)注射后1 d,2组小鼠创面IL-10的mRNA表达相近(t =2.005,P >0.05);注射后3、7、14 d,hAMSC组小鼠创面IL-10的mRNA表达明显高于PBS组(t =7.758、124.355、80.823,P <0.01)。注射后1、3、7、14 d,hAMSC组小鼠创面MIP-1α和MIP-2的mRNA表达(0.341±0.212、0.648±0.004、0.611±0.106、0.763±0.049,1.377±0.099、1.841±0.042、1.181±0.035、0.553±0.028)均明显低于PBS组(3.853±0.035、6.914±0.163、3.648±0.113、2.250±0.046,11.119±0.495、8.634±0.092、5.722±0.021、4.862±0.036,t =43.198、101.904、51.845、58.231,51.074、177.501、291.752、251.614,P <0.01)。 结论 hAMSC具有促进小鼠全层皮肤缺损创面M1型巨噬细胞向M2型巨噬细胞转化的生物学效应;可以上调抗炎及抗纤维化因子IL-10的表达,下调重要的炎症反应介导因子MIP-1α和MIP-2的表达。Abstract: Objective To explore the influence of human amniotic mesenchymal stem cells (hAMSCs) on the in vivo and in vitro regulation of macrophage phenotypes and inflammatory factors associated with wound healing of full-thickness skin wounds in mice. Methods Fresh amniotic membrane discarded from full-term delivery by 5 healthy pregnant women in the Department of Obstetrics and Gynecology of the Affiliated Hospital of Zunyi Medical University was used for the isolation and culture of hAMSCs by enzyme digestion method. The third passage of cells was used for identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of hAMSCs surface markers. Ten C57BL/6 mice (all male, aged 6 to 8 weeks, the same gender and age below) were selected for extracting mouse peritoneal macrophages by intraperitoneal lavage, and M1-type macrophages were induced by Dulbecco′s modified eagle medium (DMEM) medium containing interferon-γ. The M1-type macrophages were divided into hAMSCs+ macrophage group and macrophage alone group. Then 1×104 hAMSCs/per well of fourth passage were added to macrophage in hAMSCs+ macrophage group and cultured in 2 mL DMEM medium for routine culture. In macrophage alone group, each well was only added with 2 mL DMEM medium for routine culture. On day 1 and 7 in culture, the content of interleukin-12 (IL-12), arginase 1, and IL-10 in the cell culture supernatant of the 2 groups were detected by enzyme-linked immunosorbent assay with sample number of 6/per group. (2) Full-thickness skin wound model was reproduced in the back of 56 C57BL/6 mice, which were divided into hAMSCs group and phosphate buffer solution (PBS) group using the random number table, with 28 mice in each group. Mice in hAMSCs group were subcutaneously injected with 100 μL of cell suspension containing 1×107 hAMSCs per mL in PBS suspension along the wound edge. While mice in PBS group were only subcutaneously injected with 100 μL PBS along the wound edge. On post injection day (PID) 1, 3, 7, and 14, 7 mice in the two groups were sacrificed respectively. Histopathological observation was performed with hematoxylin-eosin staining. The expressions of macrophage surface markers [CD68 and inducible nitric oxide synthase (iNOS) double positive cells and CD68 and arginase 1 double positive] in the wounds were detected by immunofluorescent staining. The mRNA expressions of IL-10, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 in the wounds were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with analysis of variance for factorial design,t test, and Bonferroni correction. Results (1) On day 1 in culture, the content of IL-12 and arginase 1 in the cell culture supernatant of the two groups were similar (t =0.448, 0.536,P >0.05), and the content of IL-10 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group (t =14.722,P <0.01). On day 7 in culture, the content of IL-12 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group (t =13.226,P <0.01), and the content of arginase 1 and IL-10 was significantly higher than that in macrophage alone group (t =30.172, 31.406,P <0.01). (2) On PID 1, a large number of inflammatory cells infiltration were observed in the skin wounds of both groups. On PID 3, the inflammatory cells infiltration in the skin wounds increased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 7, the inflammatory cells infiltration in the wounds decreased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 14, no obvious inflammatory cells infiltration was observed in the wounds in the two groups. (3) On PID 1 and 14, the percentages of CD68 and iNOS double positive cells and CD68 and arginase 1 double positive cells in the wounds were similar in the two groups (t 1 d=0.134, 0.693,t 14 d=1.146, 2.585,P >0.05). On PID 3 and 7, the percentages of CD68 and iNOS double positive cells in the wounds in hAMSCs group were significantly lower than those of PBS group (t =6.396, 4.787,P <0.01), while the percentages of CD68 and arginase 1 double positive cells were significantly higher than those of PBS group (t =3.928, 4.473,P <0.01). (4) On PID 1, the mRNA expressions of IL-10 in the wounds of mice in the two groups were similar (t =2.005,P >0.05). On PID 3, 7, and 14, the mRNA expressions of IL-10 in the wounds of mice in hAMSCs group were significantly higher than those of PBS group (t =7.758, 124.355, 80.823,P <0.01). On PID 1, 3, 7, and 14, the mRNA expressions of MIP-1α and MIP-2 in the wounds of mice in hAMSCs group (0.341±0.212, 0.648±0.004, 0.611±0.106, 0.763±0.049, 1.377±0.099, 1.841±0.042, 1.181±0.035, 0.553±0.028) were significantly lower than those of PBS group (3.853±0.035, 6.914±0.163, 3.648±0.113, 2.250±0.046, 11.119±0.495, 8.634±0.092, 5.722±0.021, 4.862±0.036,t =43.198, 101.904, 51.845, 58.231, 51.074, 177.501, 291.752, 251.614,P <0.01). Conclusions hAMSCs demonstrates biological effects of promoting the transformation of M1-type macrophages into M2-type macrophages in full-thickness skin wounds of mice. They can up-regulate the expression of anti-inflammatory and anti-fibrotic factor IL-10, and down-regulate the expression of important inflammation mediated factors MIP-1α and MIP-2.-
Key words:
- Mesenchymal stem cells /
- Macrophages /
- Chemokines /
- Wound healing
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(1)腹股沟皮瓣供区位置隐蔽,于该处切取皮瓣造成的损伤小,该皮瓣有旋髂浅动脉和腹壁浅动脉双重供血,血供丰富,将其分叶移植、嵌合移植、与其他皮瓣联体移植等,有效修复了四肢电烧伤创面,此前鲜见报道。
(2)术前借助多普勒超声血流探测仪定位血管蒂位置与走行,降低皮瓣解剖风险;术中行吲哚菁绿血管造影评估皮瓣血运,指导血管吻合,改善皮瓣成活质量。
Highlights:
(1)The donor area of the inguinal flap is concealed, and the damage resulted from flap resection in this area is small. The flap is rich in blood supply with dual blood sourcing from the superficial circumflex iliac artery and the superficial abdominal wall artery. Its lobed transplantation, chimeric transplantation, cojoined transplantation with other flaps, etc., effectively repaired the electrical burn wounds of mice in the limbs, which has rarely been reported before.
(2)The position and course of vessel pedicle were determined by Doppler ultrasonic blood flow detector before operation to reduce the risk of flap anatomy; indocyanine green angiography was used to evaluate the blood supply of the flap during operation, to guide vascular anastomosis and improve the survival quality of the flap.
电烧伤创面损伤重,修复难度大 [ 1] ,愈合后常对受累肢体功能产生明显影响 [ 2] 。游离皮瓣移植是修复电烧伤创面的常用手术方式。腹股沟区因具有供区位置隐蔽、于该处切取皮瓣后供瓣区易闭合等优势,逐渐成为游离皮瓣较为理想的供区 [ 3] 。武汉大学同仁医院暨武汉市第三医院(以下简称本单位)2012年以来对收治的电烧伤患者四肢创面行腹股沟皮瓣游离移植修复,取得了较佳的效果。
1. 对象与方法
本回顾性观察性研究符合《赫尔辛基宣言》的基本原则。按照本单位伦理委员会临床研究暨伦理政策要求,患者对此项研究知情,并同意在不泄露其隐私的情况下对其病历资料进行分析、使用。
1.1 入选标准
纳入标准:四肢电烧伤患者,采用游离腹股沟皮瓣移植修复,术后随访6个月及以上。排除标准:临床资料不全者。
1.2 临床资料
2012年1月—2023年5月,本单位收治24例符合入选标准的四肢电烧伤患者,均为男性,年龄20~69岁,致伤电压为220 V~30 kV,烧伤深度为Ⅲ~Ⅳ度,伤后2 h~3周入院。共27个肢体受累,其中上肢18个、下肢9个。肢体创面基底均有不同程度肌腱、神经等重要结构的损伤和/或坏死,7个创面合并深部骨坏死。
1.3 治疗方法
1.3.1 术前准备
患者入院后对其创面行换药包扎,予以必要的抗感染、补液、内脏保护等支持治疗,对早期肢体张力偏大者行减张处理。完善术前相关检查,排除相关手术禁忌证,尽早行手术治疗。
1.3.2 受区处理
于全身麻醉下切除四肢电烧伤创面坏死组织,彻底止血,保留损伤后结构相对完整的肌腱和神经等重要结构,切除坏死骨,依次使用5 g/L碘伏和生理盐水反复冲洗创面。于显微镜下探查并解剖分离合适的受区动静脉,检查血管内膜损伤情况,如受区血管有附壁血栓或管内有絮状物,内膜有剥脱等,则向血管近端游离直至血管内膜结构基本正常。根据创面所处位置不同可选择的受区动脉包括上肢的肱动脉肌支、尺动脉、桡动脉的肌支、掌总动脉、指固有动脉等和下肢的胫前动脉的肌支、胫后动脉的肌支、足底内侧动脉、足背动脉、跖背动脉等,选择上述动脉的伴行静脉和/或肢体浅静脉作为受区静脉。
1.3.3 组织瓣切取与联合应用及供区处理
对受区清创后,肢体拟用腹股沟皮瓣修复的创面面积为5.0 cm×1.5 cm~20.0 cm×9.0 cm。以多普勒超声血流探测仪探测腹股沟区动脉血管位置及走行,以腹股沟韧带下方2 cm股动脉搏动点至髂前上棘连线为轴线,根据受区创面大小及形状设计皮瓣切取范围。按设计线切开皮瓣下外侧边缘,找到皮瓣血管蒂后,顺血管蒂分支走行分离进入皮瓣内的皮肤穿支。再逆行分离至血管蒂根部,明确进入皮瓣内的血管蒂来源,以及旋髂浅动脉与腹壁浅动脉是否共干及其位置关系,分离完毕,检查皮瓣血运。本组患者共行26次手术,切取27个腹股沟皮瓣,皮瓣面积为5.0 cm×3.5 cm~22.0 cm×12.0 cm、厚度为0.3~1.2 cm(平均0.76 cm),其中对2例足拇趾趾骨坏死患者同期嵌合移植同侧髂骨瓣延长足趾长度(共2处);对6例患者以血管蒂的不同皮穿支或分别以旋髂浅动脉、腹壁浅动脉为蒂进行腹股沟皮瓣分叶,以适应创面形状,或将腹股沟皮瓣分叶切取后拼接移植以增加移植皮瓣宽度(共6个腹股沟皮瓣);对4例患者肢体上使用腹股沟皮瓣不足以修复的大面积创面,联合其他皮瓣移植修复,其中3个腹股沟皮瓣与带蒂背阔肌肌皮瓣联体移植,1个腹股沟皮瓣与游离脐旁穿支皮瓣联体移植。将供区创面直接拉拢分层缝合,皮下放置引流管,用弹力腹带包扎固定。
1.3.4 组织瓣移植及血管吻合
将组织瓣移植于受区创面,将髂骨瓣用钢针固定于趾骨残端,端端吻合腹股沟皮瓣动静脉至受区动静脉,联合脐旁穿支皮瓣修复时,需另外吻合腹壁下动脉以保障皮瓣血运。当腹股沟皮瓣携带的旋髂浅动脉与腹壁浅动脉共干时,将共干血管与受区血管进行吻合(对本组5个腹股沟皮瓣行此操作);旋髂浅动脉与腹壁浅动脉不共干时,吻合两者中血管内径较粗者,必要时行血管增压吻合。术中行吲哚菁绿血管造影检查皮瓣血运,根据吻合后皮瓣远端动脉血流灌注情况决定是否需吻合另一条皮瓣动脉行增压。本组22个腹股沟皮瓣血管蒂的旋髂浅动脉与腹壁浅动脉不共干,其中13个皮瓣以单纯旋髂浅动脉为血管蒂,6个皮瓣以单纯腹壁浅动脉为血管蒂,3个皮瓣同时吻合旋髂浅动脉和腹壁浅动脉行动脉增压。另对1例示指远端血流中断者,使用旋髂浅动脉的深支作为桥接血管再通示指远节固有动脉,桥接血管长度为3.5 cm。检查皮瓣血运后,缝合皮瓣边缘,皮下放置引流条,包扎并预留皮瓣观察孔。
1.3.5 术后处理
术后常规予以心电监护、吸氧、镇痛、抗感染、抗凝、抗血管痉挛、补液等支持治疗。嘱制动并抬高患肢,皮瓣区予以烤灯照射保暖。术后72 h内每1~2小时、72 h~7 d每4~6小时观察1次皮瓣血运情况,若观察到血管危象等情况及时处理。供受区创面隔天换药,术后48 h左右拔除皮下引流条/管;术后10~14 d拆线,指导行渐进性康复锻炼。
1.4 疗效评价
记录腹股沟皮瓣切取完毕切断血管前与移植并吻合血管后即刻的血运状态,术后观察组织瓣成活情况、移植髂骨固定情况、供区继发缺损愈合情况,随访观察腹股沟皮瓣外观、受区功能恢复情况。末次随访时,使用臂、肩、手残障(DASH)评分表对受累上肢功能进行评分,评分表共涉及30项上肢活动功能(评分1~5分),每项活动功能无困难得1分,不能活动得5分,经公式计算最终得分,0分表示功能完全正常,100分表示功能极度受限 [ 4] ;参考利克特量表5级评分法 [ 5] ,调查患者对每次手术治疗效果的满意度,5分表示非常满意,4分表示比较满意,3分表示一般满意,2分表示有点不满意,1分表示很不满意。
2. 结果
2.1 总体情况
27个腹股沟皮瓣切取完毕切断血管前与移植并吻合血管后即刻动脉血流灌注与静脉回流均良好。1例患者术后12 d因前臂创面感染导致腹股沟皮瓣血管蒂栓塞后坏死,经创面换药培育肉芽组织后移植头部刃厚皮修复,其余26个腹股沟皮瓣及联体移植组织瓣术后完全成活,未发生血管危象,腹股沟皮瓣成活率为96.3%(26/27);移植髂骨术后固定牢靠;供区继发缺损术后均愈合良好。随访6~36个月,腹股沟皮瓣质地柔软,无明显臃肿,受区功能均较术前明显改善,腹股沟皮瓣均恢复保护性感觉。末次随访时,18个受累上肢功能的DASH评分表评分为0~100分(平均27分),患者对17次手术治疗效果表示非常满意、对9次手术治疗效果表示比较满意。
2.2 典型病例
例1
男,30岁,双手被220 V电烧伤,伤后3 d入院。入院时,可见双手多指Ⅳ度创面,其中左手示指中远节干枯坏死,中指部分骨外露,环指、小指近节指背肌腱损伤;右手示指中远节指腹皮肤干性坏死,远节皮肤冰凉,甲床逐渐发暗、无充盈。术前吲哚菁绿血管造影显示左手示指中远节无血流灌注,右手示指末节无血流灌注。伤后4 d,同期切取右、左侧腹股沟皮瓣分别修复左、右手创面。右手清创后拟用游离腹股沟皮瓣移植修复创面大小为5.0 cm×3.0 cm。切取6.0 cm×3.0 cm左侧腹股沟皮瓣移植修复右手示指创面,将旋髂浅动静脉分别与示指尺侧固有动脉及其伴行静脉吻合,并以旋髂浅动脉的深支桥接末节指固有动脉。切取右侧腹股沟皮瓣,将皮瓣以旋髂浅动脉的2条皮穿支为蒂,分成大小为8.0 cm×3.0 cm与6.0 cm×2.0 cm的2叶,分别移植修复创面大小为9.0 cm×2.0 cm与5.0 cm×1.5 cm的左手中指创面与环指、小指指背清创后创面,将旋髂浅动静脉分别与掌总动脉及手背静脉吻合,左手示指中远节干枯坏死部分予以截除。术后皮瓣各分叶存活良好,吲哚菁绿血管造影显示,各皮瓣血流灌注和静脉回流均良好,血管桥接移植后右手示指末节血运恢复。术后皮瓣供受区均愈合良好,右手示指保指成功。术后3个月行左手皮瓣修整术。皮瓣移植术后随访2年,皮瓣质地柔软,外形可,手功能均恢复良好。末次随访时,左手功能的DASH评分表评分为7分,右手功能的DASH评分表评分为0分;患者对手术治疗效果表示非常满意。见 图1。
例2
男,30岁,右足被380 V电烧伤,伤后3周入院。入院时,右足跟坏死干痂,右第1、2趾背侧皮肤皮革样坏死,第1趾远节干枯坏死,创面深度为Ⅲ~Ⅳ度。伤后26 d,清创见右足第1跖骨内侧、第1趾近节及第2趾背侧部分骨质外露,给予切除坏死骨质和第1趾骨末节,清创完毕,创面大小14.0 cm×5.0 cm。切取3.0 cm×2.0 cm髂前上棘外侧髂骨瓣和15.0 cm×6.0 cm右侧腹股沟皮瓣,以旋髂浅动脉的2条皮穿支为蒂对皮瓣远侧进行分叶,并分别移植修复第1、2趾创面,用钢针固定髂骨瓣于第1趾近节,吻合皮瓣的旋髂浅动静脉至跖背动脉和足背静脉。术后组织瓣供受区均愈合良好。术后随访8个月时,皮瓣质地柔软,外形可,患肢功能恢复良好,患者可正常行走;患者对手术治疗效果表示非常满意。见 图2。
3. 讨论
电烧伤患者在所有烧伤患者中占4%~6% [ 6] ,在烧伤住院患者中约占10% [ 7] 。电烧伤以四肢损伤多见,常导致肢体组织严重毁损,其截肢率高、预后差,明显影响患者的生存质量,给社会造成沉重的经济负担 [ 8, 9] 。电烧伤创面常累及深层骨骼、肌腱、神经及血管等重要结构,需行皮瓣移植修复。受血管蒂长度以及受区血管损伤的限制,带蒂皮瓣移植仅适合修复少部分电烧伤创面。随着显微外科技术的日益成熟,游离皮瓣移植因其血运佳、手术次数少、有利于创面的早期愈合和患肢的康复 [ 10] ,逐渐成为修复电烧伤创面的首选方式 [ 11] 。有学者报道使用游离上臂外侧穿支皮瓣修复手足部电烧伤创面 [ 12] 以及应用股前外侧皮瓣修复严重高压电烧伤创面 [ 13] ,均取得了较为满意的结果,但部分供区创面无法直接缝合关闭,需行皮片移植修复。为尽可能减少供区损伤,足部皮瓣也仅适用于修复较小创面。腹股沟皮瓣带蒂移植也是修复手腕和上肢远端深度烧伤创面的经典术式 [ 14] 。刘安等 [ 15] 报道总结了采用腹股沟分叶皮瓣带蒂移植修复6例虎口电烧伤患者的经验,该皮瓣具有切取操作容易、创面修复满意、手功能恢复良好等优点。既往有学者研究报道了采用游离腹股沟皮瓣修复四肢软组织缺损、口腔鳞状细胞癌根治术后缺损、拇指环形电烧伤后小型创面的经验,结果均取得了较为满意的临床疗效 [ 16, 17, 18] 。然而,由于腹股沟皮瓣解剖结构存在血管蒂变异较大、口径细小等特点,限制了该类皮瓣作为游离皮瓣的使用。随着显微外科技术的进步和穿支解剖技术的发展,口径<0.5 mm血管的吻合质量得以保障,腹股沟皮瓣的穿支解剖内涵也被逐渐理清,使得在临床应用中克服其弊端的同时,充分利用其优势实现各类缺血创面的良好修复成为可能。
本研究观察了使用游离腹股沟皮瓣修复四肢电烧伤创面的临床效果,结果表明成功切取的27个皮瓣移植修复电烧伤创面后成活率达96.3%,提示修复效果稳定。切取并移植腹股沟皮瓣后对供区造成的损伤小、瘢痕位置隐蔽、可达到皮瓣移植良好的得失比 [ 4, 19, 20] ,而游离移植该皮瓣修复电烧伤创面,还具有如下特点和优势:(1)与股前外侧皮瓣相比较,在供区能直接闭合的前提下,腹股沟皮瓣最大切取宽度常更大,如本组病例游离皮瓣宽度最大值为12.0 cm。(2)与股前外侧皮瓣相比较,腹股沟皮瓣更薄,还可顺血管蒂的皮肤穿支薄层切取,从而获取超薄皮瓣 [ 21, 22] ,避免术后皮瓣过于臃肿,便于在修复手等处创面时的皮瓣塑形。本组病例皮瓣最薄仅约3 mm。(3)电烧伤创面往往中央损伤更为严重,腹股沟皮瓣血管蒂位于皮瓣一侧,移植后血管蒂常远离损伤区域,相较于股前外侧皮瓣,降低了因坏死损伤组织残留和继发感染导致血管蒂栓塞的风险 [ 23] 。(4)腹股沟皮瓣可由旋髂浅动脉和腹壁浅动脉双重供血,直接皮动脉走行也具有明显方向性,适合切取长条形皮瓣而远端血运丰富,必要时可同时吻合旋髂浅动脉和腹壁浅动脉对皮瓣动脉进行增压,甚至可以将肋下动脉外侧支作为皮瓣的外增压血管 [ 4, 24] ,从而切取更大面积皮瓣。(5)腹股沟皮瓣血管的皮肤穿支多、分布广,便于进行分叶设计 [ 25, 26] ,可结合局部穿支解剖特点,根据创面形状来设计皮瓣,适用于电烧伤所致不规则创面的修复。本组有6例患者进行皮瓣分叶设计及切取,分别以各血管穿支为蒂,各尖端血运丰富,修复异形创面优势明显。(6)联合或联体移植。腹股沟区域的旋髂浅动脉与腹壁浅动脉存在交叉供血区,若创面面积过大,单使用腹股沟皮瓣不足以修复时,可联合切取下腹部皮瓣扩大修复面积,而不必跨区选择其他皮瓣供区,实现皮瓣移植“利益”最大化。皮瓣面积仍不足时,还可与脐旁穿支皮瓣、背阔肌肌皮瓣等联合切取、联体移植修复大面积电烧伤创面 [ 27] 。(7)嵌合移植 [ 28, 29] 。电烧伤常导致肌肉、骨等深层组织受损,此时可一并切取血运丰富的腹外斜肌填塞腔隙或切取带血运的髂骨延长受累肢体远端长度。本组病例中2例联合切取髂骨瓣,用于延长残端长度,治疗效果较佳。(8)其他。针对本组例1患者,切取旋髂浅动脉的深支作为指动脉的桥接血管挽救了远节手指,该方法可作为血管桥接的手段之一。
电烧伤肢体血管损伤常较明显,吻合血管蒂时,应选择内膜结构良好、射血明显的动脉进行吻合,以保障皮瓣蒂部远端跨穿支动脉间吻合网灌注存活 [ 30] 。腹股沟皮瓣同样存在其局限性,主要是以下2个方面:(1)腹股沟皮瓣的血管蒂动脉口径相对细小,并不适合与受区口径差异较大的动脉血管进行吻合,对于肢体毁损严重、受区血管损伤明显的电烧伤创面,仍应采用血管蒂口径与受区相匹配的其他皮瓣移植修复。本组1例患者腹股沟皮瓣术后坏死可能与肢体电烧伤创面毁损严重,吻合口口径细小增加了术后动脉血管危象发生概率有关,在临床选择皮瓣供区时应将该因素考虑在内。(2)腹股沟皮瓣无确切的皮神经支配,术后仅能恢复保护性感觉,薄型腹股沟皮瓣或经皮瓣修整去薄后感觉恢复相应增强,但仍与吻合神经的足部皮瓣存在差距,在修复承重部位或手功能区域等对皮肤感觉重建要求高的区域时,应考虑该因素。
综上所述,腹股沟皮瓣供区位置隐蔽,于该处切取皮瓣造成的损伤小,该皮瓣有旋髂浅动脉和腹壁浅动脉双重供血,血运丰富,利用其解剖学特点,可分叶移植、嵌合移植、与其他皮瓣联体移植等,适合修复形状不规则、面积大小不等、多组织缺损的肢体电烧伤创面。术前可用多普勒超声血流探测仪初探腹股沟皮瓣血管蒂位置及走行、术中借助吲哚菁绿血管造影来评估皮瓣血流灌注和静脉回流,指导血管吻合,降低术后血管危象发生率,保障手术效果。总之,本研究表明,游离移植腹股沟皮瓣修复四肢电烧伤创面后,受区外形与功能恢复较佳,患者对手术治疗效果的满意度较高,该术式值得临床推广。
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