微孔化羊ADM复合hUCMSC促进创面修复的机制研究

doi:10.3877/cma.j.issn.1673-9450.2021.02.002

目的

探讨微孔化羊脱细胞真皮基质(ADM)复合人脐带间充质干细胞(hUCMSC)促进全层损伤皮肤创面愈合的治疗机制。

方法

将hUCMSC种植于微孔化羊ADM上共培养，形成复合生物敷料。选用72只裸鼠制作成皮肤全层损伤模型，按随机数字表法分为3组，每组24只，hUCMSC种植于微孔化羊ADM上治疗全层损伤创面组(hUCMSC+微孔化羊ADM组)；羊ADM治疗全层损伤组(羊ADM组)；碘伏纱布治疗全层损伤创面组(碘伏纱布组)。术后14、21、28 d检测各组裸鼠创面愈合率，运用qRT-PCR技术检测创面组织中Bax和Bcl-2的mRNA的表达，免疫组织化学染色检测创面愈合相关蛋白细胞胶原蛋白Ⅰ和血管内皮生长因子(VEGF)的表达。数据比较采用单因素方差分析和t检验。

结果

术后14 d, hUCMSC+微孔化羊ADM组的愈合率为(65.34±14.72)%，明显高于碘伏纱布组[(37.54±10.21)%]，也高于羊ADM组[(49.08±11.16)%]，差异均有统计学意义(t＝19.52、14.72，P<0.05)。随着损伤创面的逐步愈合，术后28 d, hUCMSC+微孔化羊ADM组的愈合率为(98.63±15.41)%，明显高于羊ADM组[(81.74±16.27)%]和碘伏纱布组[(63.47±14.80)%]，差异均有统计意义(t＝-16.42、20.35，P<0.05)。Bax在hUCMSC+微孔化羊ADM组的创面组织中的表达显著降低，尤其在术后21 d的表达量为0.25±0.06，明显低于碘伏纱布组(0.53±0.16)和羊ADM组(0.41±0.12)，差异均有统计学意义(t＝3.52、-2.83，P<0.05)。Bcl-2在hUCMSC+微孔化羊ADM组的表达显著高于其他2组，尤其是在术后21 d的表达量为0.63±0.19，明显高于碘伏纱布组(0.34±0.09)和羊ADM组(0.46±0.13)，差异均有统计学意义(t＝5.31、-6.07，P<0.05)。免疫组织化学技术检测发现hUCMSC+微孔化羊ADM组胶原蛋白Ⅰ和VEGF的表达深棕色细胞虽然比羊ADM组和碘伏纱布组稍微有所增多，但是效果并不显著。

结论

hUCMSC+微孔化羊ADM复合敷料，通过携带hUCMSC更能促进皮肤全层损伤的愈合并减少凋亡细胞的产生。

关键词: 小鼠, 创伤和损伤, 间质干细胞, 微孔化, 羊脱细胞真皮基质, 伤口愈合

Objective

To explore the therapeutic mechanism of microporous sheep acellular dermal matrix (ADM) combined with human umbilical cord mesenchymal stem cell(hUCMSC) to promote the healing of full-thickness wounds.

Methods

hUCMSC was co-cultured on microporous sheep ADM to form composite biological dressings. Seventy-two nude mice were selected to make a full-thickness skin injury model and randomly divide them into 3 groups, each group of 24. hUCMSC+ microporous sheep ADM group, sheep ADM group and iodophor gauze group. The wound healing rate of each group was detected at 14, 21 and 28 days after operation, qRT-PCR technique was used to detect the expression of Bax and Bcl-2 in wound tissue, and immunohistochemical technique was used to detect the expression of wound healing related proteins. Data were analyzed with one-way analysis of variance and t test.

Results

On the 14 days after operation, the healing rate of the hUCMSC+ microporous sheep ADM group was (65.34±14.72)%, which was significantly higher than that of the Iodophor gauze group (37.54±10.21)%, and also higher than that of the sheep ADM group [(49.08±11.16)%], the differences were statistically significant (t=19.52, 14.72; P<0.05). With the gradual healing of the wound, the healing rate of the hUCMSC+ microporous sheep ADM group was (98.63±15.41)% at 28 days after operation, which was significantly higher than that of the sheep ADM group [(81.74±16.27)%] and the Lodophor gauze group [(63.47±14.80)%], the differences were statistically significant (t=-16.42, 20.35; P<0.05). The expression of Bax in the wound tissue of the hUCMSC+ microporous sheep ADM group was significantly reduced, especially on the 21 days after operation, the expression level was 0.25±0.06, which was significantly lower than the Lodophor gauze group (0.53±0.16) and sheep ADM group (0.41±0.12), the differences were statistically significant(t=3.52, -2.83; P<0.05). The expression of Bcl-2 in the hUCMSC+ microporous sheep ADM group was significantly higher than that of the other two groups, especially at the 21 days after operation postoperative day, the expression level was 0.63±0.19, which was significantly higher than the Lodophor gauze group (0.34±0.09) and sheep ADM group (0.46±0.13), the differences were statistically significant (t=5.31, -6.07; P<0.05). Immunohistochemical detection showed that the expression of CollagenⅠand VEGF in hUCMSC+ microporous sheep ADM group was slightly more than that of sheep ADM group and iodophor gauze group, but the effect was not significant.

Conclusion

hUCMSC+ microporous sheep ADM composite dressing can promote the healing of full-layer skin injury and reduce the production of apoptotic cells by carrying hUCMSC.

Key words: Mice, Wounds and injuries, Mesenchymal stem cells, Microporation, Sheepskin acellular dermal matrix, Wound healing

表1 各组全层损伤裸鼠术后不同时间创面愈合率比较(%, ±s)

组别	鼠数(只)	术后14 d	术后21 d	术后28 d
碘伏纱布组	12	37.54±10.21	52.36±13.54	63.47±14.80
羊ADM组	12	49.08±11.16^a	71.53±15.40^a	81.74±16.27^a
hUCMSC+微孔化羊ADM组	12	65.34±14.72^ab	84.53±19.24^ab	98.63±15.41^ab
F值		167.25	172.43	186.48
P值		<0.05	<0.05	<0.05

表1 各组全层损伤裸鼠术后不同时间创面愈合率比较(%, ±s)

组别	鼠数(只)	术后14 d	术后21 d	术后28 d
碘伏纱布组	12	37.54±10.21	52.36±13.54	63.47±14.80
羊ADM组	12	49.08±11.16^a	71.53±15.40^a	81.74±16.27^a
hUCMSC+微孔化羊ADM组	12	65.34±14.72^ab	84.53±19.24^ab	98.63±15.41^ab
F值		167.25	172.43	186.48
P值		<0.05	<0.05	<0.05

表2 各组全层损伤裸鼠创面组织中Bax的mRNA表达量比较(±s)

组别	鼠数(只)	术后14 d	术后21 d	术后28 d
碘伏纱布组	8	0.83±0.19	0.53±0.16	0.74±0.21
羊ADM组	8	0.56±0.13^a	0.41±0.12^a	0.52±0.18^a
hUCMSC+微孔化羊ADM组	8	0.38±0.09^ab	0.25±0.06^ab	0.41±0.15^a
F值		9.21	17.58	2.45
P值		<0.05	<0.05	<0.05

表2 各组全层损伤裸鼠创面组织中Bax的mRNA表达量比较(±s)

组别	鼠数(只)	术后14 d	术后21 d	术后28 d
碘伏纱布组	8	0.83±0.19	0.53±0.16	0.74±0.21
羊ADM组	8	0.56±0.13^a	0.41±0.12^a	0.52±0.18^a
hUCMSC+微孔化羊ADM组	8	0.38±0.09^ab	0.25±0.06^ab	0.41±0.15^a
F值		9.21	17.58	2.45
P值		<0.05	<0.05	<0.05

表3 各组全层损伤裸鼠创面组织中Bcl-2的mRNA表达量比较(±s)

组别	鼠数(只)	术后14 d	术后21 d	术后28 d
碘伏纱布组	8	0.25±0.07	0.34±0.09	0.26±0.05
羊ADM组	8	0.39±0.11^a	0.46±0.13^a	0.37±0.10^a
hUCMSC+微孔化羊ADM组	8	0.51±0.16^ab	0.63±0.19^ab	0.54±0.15^ab
F值		4.57	3.17	6.24
P值		<0.05	<0.05	<0.05

表3 各组全层损伤裸鼠创面组织中Bcl-2的mRNA表达量比较(±s)

组别	鼠数(只)	术后14 d	术后21 d	术后28 d
碘伏纱布组	8	0.25±0.07	0.34±0.09	0.26±0.05
羊ADM组	8	0.39±0.11^a	0.46±0.13^a	0.37±0.10^a
hUCMSC+微孔化羊ADM组	8	0.51±0.16^ab	0.63±0.19^ab	0.54±0.15^ab
F值		4.57	3.17	6.24
P值		<0.05	<0.05	<0.05

图1 各组创面组织胶原蛋白Ⅰ免疫组织化学染色 (×400)；A、B、C分别为术后14 d碘伏纱布组，羊ADM组和hUCMSC+微孔化羊ADM组；D、E、F分别为术后21 d碘伏纱布组，羊ADM组和hUCMSC+微孔化羊ADM组；G、H、I分别为术后28 d碘伏纱布组，羊ADM组和hUCMSC+微孔化羊ADM组；各组创面间质内均可看到胶原蛋白Ⅰ表达，hUCMSC+微孔化羊ADM组表达略强，碘伏纱布组表达略弱，但3个时间点各组内表达强度均相对一致，没有明显变化。ADM为脱细胞真皮基质；hUCMSC为人脐带间充质干细胞

图2 各组创面组织VEGF免疫组织化学染色 (×400)。A、B、C分别为术后14 d碘伏纱布组，羊ADM组和hUCMSC+微孔化羊ADM组，可见创面组织内成纤维细胞及血管内皮均表达VEGF，A(碘伏纱布组)表达最弱，C(hUCMSC+微孔化羊ADM组)表达增强；D、E、F分别为术后21 d碘伏纱布组，羊ADM组和hUCMSC+微孔化羊ADM组；术后21 d, 3组VEGF表达较术后14 d 3组表达略有增强；G、H、I分别为术后28 d碘伏纱布组，羊ADM组和hUCMSC+微孔化羊ADM组，术后28 d, 3组可见血管形成明显，VEGF表达较术后21 d与术后14 d有所升高，I示VEGF表达数量和强度均最高；VEGF为血管内皮生长因子；ADM为脱细胞真皮基质；hUCMSC为人脐带间充质干细胞

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Study on the mechanism of microporous sheep ADM combined with hUCMSC in promoting wound healing

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Study on the mechanism of microporous sheep ADM combined with hUCMSC in promoting wound healing