Expression of microRNA-296 in rabbit hypertrophic scars and its role to human fibroblasts
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摘要:
目的 探讨微小RNA-296（miR-296）在兔增生性瘢痕中的表达及对人成纤维细胞（HFb）的作用。 方法 采用实验研究方法。将12只雌雄不限成年新西兰长耳兔按完全随机法分为正常对照组和瘢痕组，每组6只。瘢痕组根据文献制作兔耳增生性瘢痕模型，正常对照组兔不行任何处理。于瘢痕组建模后60 d，采用苏木精-伊红染色法观察2组兔耳瘢痕/皮肤组织中成纤维细胞（Fb）生长与排列，采用实时荧光定量反转录PCR法检测2组兔耳瘢痕/皮肤组织中miR-296和转化生长因子β1（TGF-β1）的mRNA表达量，另对miR-296与TGF-β1mRNA表达量的相关性进行Pearson回归分析。取2批HFb，第1批分为TGF-β1野生型+miR-296阴性对照组和TGF-β1野生型+miR-296模拟物组，第2批分为TGF-β1突变型+miR-296阴性对照组和TGF-β1突变型+miR-296模拟物组，分别转染对应的序列。转染后48 h，采用荧光素酶报告基因检测试剂盒检测每组细胞TGF-β1的荧光素酶和肾荧光素酶的表达，以其比值反映基因表达水平。取2批HFb，每批细胞均分为miR-296阴性对照组和miR-296模拟物组，分别转染对应序列。第1批细胞转染后0（即刻）、12、24、36、48 h，采用噻唑蓝法检测细胞增殖情况。第2批细胞转染后24 h，采用蛋白质印迹法检测细胞中TGF-β1和Ⅰ型胶原蛋白表达情况。细胞实验中样本数均为3。对数据行析因设计方差分析、独立样本t检验。 结果 瘢痕组建模后60 d，瘢痕组兔耳瘢痕组织中Fb过度增生且排列紊乱，正常对照组兔耳皮肤组织中Fb生长与排列无异常；瘢痕组兔耳瘢痕组织中miR-296的mRNA表达量（0.65±0.11）显著低于正常对照组兔耳皮肤组织（1.19±0.12，t=5.175，P<0.01），瘢痕组兔耳瘢痕组织中TGF-β1的mRNA表达量（1.47±0.06）显著高于正常对照组兔耳皮肤组织（1.10±0.03，t=12.410，P<0.01）。Pearson回归分析显示，12只兔耳瘢痕/皮肤组织中miR-296和TGF-β1的mRNA表达量呈明显负相关（F=7.278，P<0.05）。转染后48 h，TGF-β1野生型+miR-296模拟物组细胞TGF-β1的基因表达明显低于TGF-β1野生型+miR-296阴性对照组（t=35.190，P<0.01），2个TGF-β1突变型组TGF-β1基因表达相近（P>0.05）。miR-296模拟物组细胞增殖能力在转染后12、24、36、48 h明显低于miR-296阴性对照组（t=3.275、11.980、10.460、17.260，P<0.05或P<0.01）。转染后24 h，miR-296阴性对照组细胞TGF-β1和Ⅰ型胶原的蛋白表达量均明显高于miR-296模拟物组（t=3.758、29.390，P<0.05或P<0.01）。 结论 兔增生性瘢痕中miR-296表达下调，miR-296能够通过下调TGF-β1的表达抑制HFb的增殖和Ⅰ型胶原蛋白的表达。 Abstract:Objective To investigate the expression of microRNA-296 (miR-296) in rabbit hypertrophic scars and its role in human fibroblasts (HFbs). Methods The experimental method was used. Twelve healthy adult New Zealand long-eared rabbits regardless gender were randomly divided into normal control group and scar group, with 6 rabbits in each group. The rabbit ear hypertrophic scar model was created in scar group according to the literature, and the rabbits in normal control group did not receive any treatment. On 60 days after setting up the models in scar group, hematoxylin-eosin staining was performed to observe the growth and arrangement of fibroblasts (Fbs) in the ear scars and skin tissue of rabbits in the two groups. The mRNA expressions of miR-296 and transforming growth factor-β1 (TGF-β1) in ear scars and skin tissue of rabbits in the two groups were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction, and the correlation of mRNA between miR-296 and TGF-β1 was performed with Pearson regression analysis. Two batches of HFbs were used and transfected respectively with corresponding sequences, with the 1st batch being divided into TGF-β1 wild type+miR-296 negative control group and TGF-β1 wild type+miR-296 mimic group and the 2nd batch being divided into TGF-β1 mutant type+miR-296 negative control group and TGF-β1 mutant type+miR-296 mimic group. At 48 h after transfection, luciferase reporter gene detection kit was used to detect the luciferase and renal luciferase expression of TGF-β1 in the cells of each group, with their ratio being used to reflect the gene expression level. Two batches of HFbs were used, and each batch of cells were divided into miR-296 negative control group and miR-296 mimic group, being transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfecting the first batch of cells, the cell proliferation was detected by thiazolyl blue method. At 24 h after transfecting the second batch of cells, the expression of TGF-β1 and collagen type Ⅰ was detected by Western blotting. The number of samples in cell experiments was 3. Data were statistically analyzed with analysis of variance for factorial design, independent sample t test. Results On 60 days after setting up the models in scar group, the Fbs of rabbit ear scar tissue in scar group proliferated and arranged disorderly, while the growth and arrangement of Fbs in rabbit ear skin tissue in normal control group were normal. The mRNA expression of miR-296 of rabbit scar tissue in scar group (0.65±0.11) was significantly lower than 1.19±0.12 of rabbit ear skin tissue in normal control group (t=5.175, P<0.01). The mRNA expression of TGF-β1 of rabbit ear scar tissue in scar group (1.47±0.06) was significantly higher than 1.10±0.03 of rabbit ear skin tissue in normal control group (t=12.410, P<0.01). Pearson regression analysis showed that there was a negative correlation between the mRNA expression of miR-296 and TGF-β1 in the ear scars and skin tissue of 12 rabbits (F=7.278, P<0.05). At 48 h after transfection, the gene expression of TGF-β1 of cells in TGF-β1 wild type+miR-296 mimic group was significantly lower than that in TGF-β1 wild type+miR-296 negative control group (t=35.190, P<0.01), while the gene expression of TGF-β1 of cells in the two TGF-β1 mutant type groups were close (P>0.05). The HFbs proliferation ability in miR-296 mimic group was significantly lower than that in miR-296 negative control group at 12, 24, 36, and 48 h after transfection(t=3.275, 11.980, 10.460, 17.260, P<0.05 or P<0.01). At 24 h after transfection, the protein expressions of TGF-β1 and type Ⅰ collagen of cells in miR-296 negative control group were significantly higher than those in miR-296 mimic group (t=3.758, 29.390, P<0.05 or P<0.01). Conclusions The miR-296 expression in rabbit hypertrophic scars is down-regulated; miR-296 can inhibit the proliferation of HFbs and the expression of type Ⅰ collagen by down regulating the expression of TGF-β1. -
Key words:
- Cicatrix /
- Fibroblasts /
- Transforming growth factor beta 1 /
- microRNA
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证明超声清创法可显著减轻烧伤残余创面的细菌负荷，减少术后血肿形成，促进移植皮片成活，从而缩短患者病程。
烧伤，尤其是深度烧伤会遗留严重瘢痕，对患者身心造成损害［1, 2, 3］。深度烧伤创面难以愈合或一次封闭困难，均会导致残余创面形成。临床上修复残余创面常采用清创+植皮术，而传统清创手术易误伤创面周围健康组织［4］，违背现代精准医学的治疗理念。近年来，超声清创技术在糖尿病足、下肢静脉溃疡、压疮、骨外露等创面修复［5, 6, 7, 8］中取得了良好效果，但目前国内鲜见将超声清创技术应用于烧伤残余创面的相关报告。解放军联勤保障部队第980医院（以下简称本单位）近年来将超声清创技术应用于烧伤残余创面，取得了良好的临床效果。
1. 对象与方法
本回顾性队列研究符合《赫尔辛基宣言》的基本原则，根据本单位伦理委员会政策，临床资料可以在不泄露患者身份信息的前提下分析、使用。
1.1 入选标准
纳入标准：（1）原发深Ⅱ度或Ⅲ度烧伤经清创植皮后仍存在残余创面；（2）创面分布相对集中，创面总面积≥1%TBSA，且至少存在1处残余创面直径≥5 cm。（3）对残余创面行传统清创法或超声清创法+植皮治疗者。排除标准：资料不全者。
1.2 患者一般资料及烧伤创面情况
2017年8月—2021年8月，本单位收治64例符合入选标准的患者，其中男41例、女23例，年龄为9个月~67岁，平均年龄31岁，烧伤总面积25%~90%TBSA，残余创面总面积1%~17%TBSA，致伤原因：火焰烧伤者36例，热液烫伤者23例，电烧伤者5例。根据创面清创方法，将患者分为超声清创组（34例）和传统清创组（30例）。2组患者性别、年龄、致伤原因、烧伤部位等一般情况比较，差异均无统计学意义（P>0.05）；2组患者烧伤总面积、残余创面面积、伤后清创时间等创面情况均相似（P>0.05）。见表1。清创前，2组患者的所有创面中均有细菌感染：超声清创组创面中检出金黄色葡萄球菌者12例、铜绿假单胞菌者15例、鲍曼不动杆菌者5例、粪肠球菌者3例、阴沟肠杆菌者2例，其中复合感染者3例；传统清创组创面检出金黄色葡萄球菌者11例、铜绿假单胞菌者15例、鲍曼不动杆菌者3例、肺炎克雷伯菌者2例、阴沟肠杆菌者2例，其中复合感染者3例。
表1 2组烧伤残余创面患者一般资料及其烧伤创面情况比较组别 例数 性别（例） 年龄（岁， 致伤原因（例） 烧伤部位（例） 烧伤总面积（%TBSA， 残余创面面积（%TBSA， 伤后清创时间（d， 男 女 火焰烧伤 热液烫伤 电烧伤 头面颈部 前后躯干 四肢 超声清创组 34 22 12 31 19 12 3 4 20 10 51 10.9 32 传统清创组 30 19 11 32 17 11 2 3 19 8 50 10.1 33 统计量值 χ2=0.01 t=0.30 χ2=0.10 χ2=0.14 t=0.29 t=0.89 t=0.71 P值 0.909 0.972 0.949 0.932 0.707 0.907 0.169 注：TBSA为体表总面积 1.3 治疗方法
2组患者入院后均行常规补液、换药等治疗，术前纠正电解质紊乱、低蛋白血症、贫血、血糖和血压异常等情况，选择敏感抗生素防治感染，改善创面微循环。术中全身麻醉下对创面进行清创：（1）超声清创组创面，采用超声清创机根据创面大小使用500~2 000 mL生理盐水清除坏死组织或陈旧性肉芽至新鲜点状出血，清创后以1∶250 000肾上腺素盐水浸润的纱布覆盖创面以减少出血。超声机功率设置（50%~80%额定功率）及清创时长根据肉芽生长、坏死组织残存及感染情况确定。（2）传统清创组创面，采用传统锐性清创的方式，清创同时以1∶250 000肾上腺素盐水浸润的纱布湿敷，必要时行电凝止血。清创结束后，2组创面均使用过氧化氢、氯已定、生理盐水等冲洗3~5遍后植皮。功能部位或对容貌影响较大的暴露区创面首选大张皮片植皮；非功能部位创面，尤其是供皮困难者，选择移植邮票皮。植皮后四肢等部位采用无菌纱布适当加压包扎；面部等不易包扎的部位予缝线打包加压固定。术后行抗生素治疗，每2~3天换药1次。对于Ⅰ期手术后未愈合创面行二次清创+植皮术，2组创面的清创方式均分别同其Ⅰ期。
1.4 观察指标
1.4.1 创面分泌物细菌阳性率
术后3 d，于创面分泌物较多处选取标本行微生物培养及药物敏感试验检测细菌情况并计算细菌阳性率。创面细菌阳性率=细菌阳性患者例数÷患者总例数×100%。
1.4.2 皮片成活率及皮下血肿发生率
术后7 d，对于采用邮票皮治疗者，若面积较大，则统计5处约5 cm×5 cm植皮区内成活皮片数量；若面积较小，则统计所有植皮区域内成活皮片数量［4］。对于采用大张皮植皮者，行大体观察直接评估皮片成活情况。皮片成活标准为贴附良好、颜色红润、按压后充血明显、无明显滑动。皮片成活率=成活皮片数÷移植皮片总数×100%。术后7 d，大体观察创面皮下血肿发生率，皮下血肿发生率=皮下血肿形成患者例数÷患者总例数×100%。
1.4.3 创面愈合时间及二次清创率
出院时，统计患者创面愈合时长（二次清创者以Ⅱ期创面愈合时间为准）及清创次数并计算二次清创率，二次清创率=二次清创患者例数÷患者总例数×100%。
1.5 统计学处理
采用SPSS 17.0统计软件对数据进行分析。符合正态分布的计量资料数据以
2. 结果
2.1 创面分泌物细菌阳性率
术后3 d，超声清创组创面感染金黄色葡萄球菌者2例、铜绿假单胞菌者2例，传统清创组创面感染金黄色葡萄球菌者5例、铜绿假单胞菌者3例、鲍曼不动杆菌者1例、肺炎克雷伯菌者1例、阴沟肠杆菌者1例；超声清创组患者创面细菌阳性率为11.8%（4/34）明显低于传统清创组的36.7%（11/30），χ2=5.51，P=0.019。
2.2 皮片成活率及皮下血肿发生率
术后7 d，超声清创组创面的移植皮片成活率为（92
2.3 创面愈合时间及二次清创率
出院时，超声清创组创面愈合时长为（11.0
2.4 典型病例
患者男，38岁，致伤原因为电烧伤，全身多处均为Ⅲ度烧伤，总面积为26%TBSA，主要位于左下肢（14%TBSA）及头面部（3%TBSA）、左上肢（3%TBSA）、背部（4%TBSA）、左臀部（2%TBSA），伤后被送至当地医院治疗，其间行全身多处创面削痂+Meek植皮术。患者于伤后25 d转入本单位。入院时，患者部分创面已经基本封闭，已愈合区域与肉芽创面相互交错；残余创面主要位于背部，面积为2%TBSA，其表面可见肉芽组织增生及稀薄分泌物。入院后，从患者创面分泌物中检出金黄色葡萄球菌，后续采用静脉输注抗生素抗感染、纠正电解质紊乱等措施改善患者全身状况，创面每2~3天换药1次。入院后第7天，在全身麻醉下采用超声清创机对患者背部残余创面进行清创，清创机功率设置为75%额定功率，使用约500 mL生理盐水冲洗创面。然后选择右侧背部皮肤为供皮区，切取相应大小刃厚皮片行邮票皮移植术并采用无菌纱布适当加压包扎。术后行抗生素治疗，每2~3天换药1次。术后3 d，取创面中少量分泌物行微生物培养，未检出细菌。术后7 d，创面皮片成活率约为98%，皮片下无明显血肿。术后12 d创面完全愈合。见图1。
1 超声清创法+邮票皮移植治疗背部烧伤患者残余创面的疗效。1A.伤后25 d入院时，创面中较少肉芽组织增生；1B.伤后32 d背部创面清创术前，见肉芽组织增生明显，突出皮面；1C.清创术中；1D.术后12 d，邮票皮片存活良好3. 讨论
浅Ⅱ度烧伤经适当处理后可愈合，但深Ⅱ度及Ⅲ度等深度烧伤创面愈合困难，勉强愈合后也会遗留瘢痕或反复破溃，严重影响机体外观和功能［9, 10, 11］。因此，对深度烧伤常在早期行外科手术修复，常形成残余创面需在Ⅱ期行清创植皮术。理想的清创方式应具备高效且尽量保留健康组织、减少出血、降低住院费用、减轻创面中细菌负荷及提高愈合率等特点［12］。烧伤残余创面存在已愈合区域与坏死组织或肉芽组织相互交叉、镶嵌分布、边缘不规则等特点，清创难度大，传统锐性清创法的应变效能差，会造成周围正常组织不必要的损伤，出血较多。这导致经验较少的年轻医师在治疗残余创面的过程中存在较多突出问题［4］。
超声清创法是近年来兴起的清创方式，为残余创面清创提供了新的选择［13］。高频超声以热效应为主，低频超声以机械效应为主，即分别具有空化效应与声流作用［14, 15, 16］。超声清创机采用低频、高能超声波加载喷射流技术，在冲洗创面过程中会产生空化效应，即通过超声波形成的空化泡崩塌产生的微射流及高压可清除坏死组织［17, 18, 19］。超声清创机操作灵活，可用于平面或腔隙等不同类型创面；还可根据坏死组织或肉芽组织情况选择不同的功率，达到最佳清创效果。同时，超声清创机可将液体以雾化的形式喷出，使液体全面接触创面以更精确地清除非活性组织，又避免了对周围正常组织的附带损伤［5，17，20］。
创面细菌负荷的减轻程度是反映清创效果的一个重要指标。创面细菌负荷与细菌生物膜形成密切相关，生物膜可保护细菌免受抗生素、防腐剂和宿主免疫的影响，还可逐步释放浮游细菌导致创面持续感染，严重影响创面皮片成活率［21, 22, 23, 24, 25, 26］。有研究显示超声不仅对革兰阳性、革兰阴性、需氧和厌氧细菌具有明显抗菌作用［27, 28］，还可以有效清除细菌生物膜［21］，显著减少细菌负荷，改善创面条件［29, 30, 31］。本研究结果显示，超声清创组术后3 d创面细菌阳性率显著低于传统清创组，同时前者的二次清创率也显著低于后者，提示超声清创法在清除创面坏死组织，减轻细菌负荷等方面具有显著作用，其结果与前述既往国内外研究结果一致。
此外，超声清创法可有效减轻术后炎症反应和疼痛，还可促进伤口组织中碱性FGF和EGF的高表达，改善创面微循环，促进血管生成和胶原沉积，提升组织再生和修复能力，这可能与超声产生的空化效应和声流作用等机械能刺激信号转导，继发广泛的细胞效应有关［18, 19，32, 33, 34］。本研究中超声清创组的皮片成活率显著高于传统清创组，且创面愈合时间显著短于传统清创组。移植皮片的成活率与皮下积血、积液等密切相关。相关研究指出，超声清创机可通过热效应达到减少出血的效果［5，35, 36］，但由于术中出血与组织液、膨胀液或冲洗液等混合在一起，难以精确估计出血量。但是本研究显示，超声清创组皮下血肿发生率显著低于传统清创组，提示减少术中出血和皮下血肿形成可能是超声清创法提高皮片成活率，促进创面愈合的机制之一。
综上，与传统清创法相比，超声清创法可在烧伤残余创面清创中彻底清除坏死组织和陈旧性肉芽组织、减轻细菌负荷、降低皮下血肿发生率，促进移植皮片成活，从而缩短创面愈合时间，为烧伤残余创面的修复提供了新的方法和选择。超声清创法对于提高烧伤残余创面的修复效果具有重要意义，值得临床推广。
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参考文献
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1 瘢痕组建模后60 d，2组兔耳瘢痕/皮肤组织中成纤维细胞（Fb）排列 苏木精-伊红×200。1A.正常对照组兔耳皮肤组织中Fb生长正常且排列均匀；1B. 瘢痕组兔耳瘢痕组织中Fb增生且排列异常
2 蛋白质印迹法检测2组人成纤维细胞转染后24 h转化生长因子β1和Ⅰ型胶原蛋白表达
注：1、2分别为微小RNA-296（miR-296）阴性对照组和miR-296模拟物组
表1 2组人成纤维细胞转染后各时间点细胞增殖情况比较（
组别 样本数 0 h 12 h 24 h 36 h 48 h miR-296阴性对照组 15 0.140±0.034 0.350±0.041 0.548±0.030 0.764±0.044 0.815±0.021 miR-296模拟物组 15 0.142±0.022 0.258±0.025 0.330±0.010 0.488±0.006 0.570±0.013 t值 0.065 3.275 11.980 10.460 17.260 P值 0.952 0.031 0.001 0.001 0.001 注：miR为微小RNA；各组各时间点样本数均为3；处理因素主效应，F=26.760，P<0.001；时间因素主效应，F=269.400，P<0.001；两者交互作用，F=404.200，P<0.001 
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