肝素化羊脱细胞真皮基质对深Ⅱ度烧伤创面组织中血管内皮生长因子和层粘连蛋白的影响

doi:10.3877/cma.j.issn.1673-9450.2018.05.002

中华损伤与修复杂志(电子版) ›› 2018, Vol. 13 ›› Issue (05) : 325 -330. doi: 10.3877/cma.j.issn.1673-9450.2018.05.002

所属专题：文献；

论著

肝素化羊脱细胞真皮基质对深Ⅱ度烧伤创面组织中血管内皮生长因子和层粘连蛋白的影响

郝春光¹, 李俊亮², 段霞光³, 符雪², 王凌峰²^,^†(

)

^1. 014010　包头，内蒙古医科大学第三附属医院麻醉科；014010　包头，内蒙古医科大学第三附属医院内蒙古烧伤研究所
^2. 014010　包头，内蒙古医科大学第三附属医院内蒙古烧伤研究所；014010　包头，内蒙古医科大学第三附属医院烧伤科
^3. 014010　包头，内蒙古医科大学第三附属医院麻醉科

收稿日期:2018-07-24 出版日期:2018-10-01
通信作者: 王凌峰
基金资助:
国家自然科学基金项目(81060154); 内蒙古自治区自然科学基金项目(2010Zd27)

Effect of heparinized sheep acellular dermal matrix on vascular endothelial growth factor and laminin in deep partial-thickness burn wound tissue

Chunguang Hao¹, Junliang Li², Xiaguang Duan³, Xue Fu², Lingfeng Wang²^,^†()

^1. Department of Anesthesiology, Third Affiliated Hospital of Inner Mongolia Medical University, Baotou 014010, China; Inner Mongolia Institute of Burn Research, Third Affiliated Hospital of Inner Mongolia Medical University, Baotou 014010, China
^2. Inner Mongolia Institute of Burn Research, Third Affiliated Hospital of Inner Mongolia Medical University, Baotou 014010, China; Department of Burns, Third Affiliated Hospital of Inner Mongolia Medical University, Baotou 014010, China
^3. Department of Anesthesiology, Third Affiliated Hospital of Inner Mongolia Medical University, Baotou 014010, China

Received:2018-07-24 Published:2018-10-01
Corresponding author: Lingfeng Wang
About author:
Corresponding author: Wang Lingfeng, Email: wlf7413@vip.sina.com

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郝春光, 李俊亮, 段霞光, 符雪, 王凌峰. 肝素化羊脱细胞真皮基质对深Ⅱ度烧伤创面组织中血管内皮生长因子和层粘连蛋白的影响[J/OL]. 中华损伤与修复杂志(电子版), 2018, 13(05): 325-330.

Chunguang Hao, Junliang Li, Xiaguang Duan, Xue Fu, Lingfeng Wang. Effect of heparinized sheep acellular dermal matrix on vascular endothelial growth factor and laminin in deep partial-thickness burn wound tissue[J/OL]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2018, 13(05): 325-330.

目的

研究肝素化羊脱细胞真皮基质(ADM)促进深Ⅱ度烧伤创面愈合的作用机制。

方法

制作大鼠深Ⅱ度烧伤模型，随机分为碘伏纱布组、猪ADM组、羊ADM组和肝素化羊ADM组，每组40只。在伤后不同时间段，观察各组创面愈合率，同时在各个时间段处死大鼠8只，取创面组织标本做RT-qPCR和蛋白质印迹法检测各组创面组织中血管内皮生长因子(VEGF)和层粘连蛋白(LN)的mRNA与蛋白表达情况。对数据行单因素方差分析和t检验。

结果

肝素化羊ADM组深Ⅱ度创面的愈合率优于碘伏纱布组。RT-qPCR检测发现，VEGF的mRNA在各组伤后3 d的创面组织中表达较对照组高，各组间的比较发现，伤后7 d，肝素化羊ADM组的VEGF的mRNA表达量明显高于碘伏纱布组(t＝80.83，P<0.05)，羊ADM组和猪ADM组在伤后7 d也较碘伏纱布组的表达量高(t＝60.80、53.42，P值均小于0.05)；蛋白质印迹法检测肝素化羊ADM组伤后3 d时VEGF蛋白表达量较多，随后便逐渐减少，伤后21 d时蛋白表达较少。伤后3 d,碘伏纱布组LN在创面的表达明显下降，在之后的治疗过程中，LN的表达逐渐恢复，伤后14 d的表达量明显高于伤后3 d(t＝19.5，P<0.001)，伤后21、28 d的表达量与伤后14 d相差不明显。蛋白质印迹法检测发现肝素化羊ADM组LN在伤后3 d便开始出现表达量逐渐增多，伤后21 d时出现蛋白表达较多。

结论

肝素化羊ADM能够保护创面及促进创面组织中VEGF和LN的表达，进而促进创面愈合。

关键词: 肝素, 烧伤, 血管内皮生长因子, 层粘连蛋白, 羊脱细胞真皮基质

Objective

To investigate the mechanism of heparin-selective sheep acellular dermal matrix in healing wound healing of deep partial-thickness burn.

Methods

Rat deep partial-thickness models were made and randomly divided into iodophor gauze group, pig acellular dermal matrix (ADM) group, sheep ADM group and heparinized sheep ADM group, each group of 40 rats. The wound healing rate in the group was also sacrificed at various time points. Eight rats were killed at different times. The wound tissue samples were taken for RT-qPCR and Western-blotting to detect vascular endothelial growth factor (VEGF) and laminin(LN) in the wound tissues of each group. Data were processed with one-way analysis of variance and t test.

Results

The wound healing rate of the heparinized sheep ADM group in the treatment was better than that of the iodophor gauze group. RT-qPCR analysis showed that the expression of VEGF in the wound tissue at 3 days post-operation after scald in each group was higher than in the control group. The comparison between the groups showed that the expression of VEGF in heparinized sheep ADM group was significantly higher than that in iodophor group (t=80.83, P<0.05). The sheep ADM group and the pig ADM group were also higher than the iodophor group at 7 days post-operation (t=60.80, 53.42; with P values below 0.05). Western-blotting was used to detect the VEGF wound in the heparinized sheep ADM group. At 3 days post-operation, the expression was more, and then gradually decreased. At 21 days post-operation, the protein expression was less. At 3 days post-operation, the expression of LN in the iodophor group decreased significantly. During the subsequent treatment, the expression of LN gradually recovered, and the expression level at 14 days post-operation was significantly higher than that at 3 days post-operation group (t=19.5, P<0.001). The expression levels of 21 days post-operation and 28 days post-operation were not significantly different from those of 14 days post-operation. The Western-blotting showed that the LN of the heparinized sheep ADM group began to increase gradually at 3 days post-operation after the wound surface, and the protein expression appeared more on the 21 days post-operation.

Conclusion

Heparinized sheep ADM can protect the wound surface and promote the expression of VEGF and LN in the wound tissue, which in turn promotes wound healing.

Key words: Heparin, Burns, Vascular endothelial growth factor, Laminin, Sheepskin acellular dermal matrix

表1 VEGF、LN及β-肌动蛋白的引物序列

基因	引物序列
VEGF	上游5′- CGACAGAAGGGGAGCAGAAAG- 3
?	下游5′- GCAAGTACGTTCGTTTAACTC- 3′
LN	上游5′- CCTGTGGCTGATGACTGC- 3′
?	下游5′- ACTCTGTTGTGCTCCCGA- 3′
β-肌动蛋白	上游5′- GTAAAGACCTCTATGCCAACA- 3′
?	下游5′-GGACTCATCGTACTCCTG CT- 3′

表1 VEGF、LN及β-肌动蛋白的引物序列

基因	引物序列
VEGF	上游5′- CGACAGAAGGGGAGCAGAAAG- 3
?	下游5′- GCAAGTACGTTCGTTTAACTC- 3′
LN	上游5′- CCTGTGGCTGATGACTGC- 3′
?	下游5′- ACTCTGTTGTGCTCCCGA- 3′
β-肌动蛋白	上游5′- GTAAAGACCTCTATGCCAACA- 3′
?	下游5′-GGACTCATCGTACTCCTG CT- 3′

表2 各组深Ⅱ度烫伤大鼠伤后不同时相点创面愈合率比较(%, ±s)

组别	鼠数(只)	创面愈合率
组别	鼠数(只)	伤后7 d	伤后14 d	伤后21 d	伤后28 d
碘伏纱布组	8	16.75±4.61	35.58±7.89	74.33±16.24	84.42±4.80
猪ADM组	8	18.47±3.25	46.73±7.96^a	87.58±11.83^a	92.04±6.00^a
羊ADM组	8	17.92±3.60	46.58±7.74^a	87.42±12.63^a	91.28±5.90^a
肝素化羊ADM组	8	18.83±4.51	49.08±6.72^a	93.83±10.19^abc	96.83±14.70^ac

表2 各组深Ⅱ度烫伤大鼠伤后不同时相点创面愈合率比较(%, ±s)

组别	鼠数(只)	创面愈合率
组别	鼠数(只)	伤后7 d	伤后14 d	伤后21 d	伤后28 d
碘伏纱布组	8	16.75±4.61	35.58±7.89	74.33±16.24	84.42±4.80
猪ADM组	8	18.47±3.25	46.73±7.96^a	87.58±11.83^a	92.04±6.00^a
羊ADM组	8	17.92±3.60	46.58±7.74^a	87.42±12.63^a	91.28±5.90^a
肝素化羊ADM组	8	18.83±4.51	49.08±6.72^a	93.83±10.19^abc	96.83±14.70^ac

图1 RT-qPCR检测深Ⅱ度烧伤大鼠各治疗组VEGF mRNA的表达。VEGF为血管内皮生长因子；ADM为脱细胞真皮基质

图2 RT-qPCR检测深Ⅱ度烧伤大鼠各治疗组LN mRNA的表达。LN为层粘连蛋白；ADM为脱细胞真皮基质

图3 蛋白质印迹法检测各组中VEGF和LN蛋白的表达。A、B、C、D、E分别为伤后3、7、14、21、28 d碘伏纱布组、猪ADM组、羊ADM组、肝素化羊ADM组蛋白表达

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Effect of heparinized sheep acellular dermal matrix on vascular endothelial growth factor and laminin in deep partial-thickness burn wound tissue

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Effect of heparinized sheep acellular dermal matrix on vascular endothelial growth factor and laminin in deep partial-thickness burn wound tissue