人脂肪间充质干细胞多层膜片对促进裸鼠皮肤缺损愈合的效果观察

doi:10.3877/cma.j.issn.1673-9450.2024.04.012

目的

探讨人脂肪间充质干细胞(ADSCs)膜片对BALB/c-nu裸鼠全层皮肤缺损的治疗效果。

方法

体外分离培养人ADSCs，制备成3层的人ADSCs膜片。选择健康无特殊病原体级雄性BALB/c-nu裸鼠36只，建立背部直径为12 mm的圆形全层皮肤缺损创面。将裸鼠随机分为2组，对照组单纯使用羊脱细胞真皮基质(ADM)覆盖创面；ADSCs膜片组在3层ADSCs膜片移植后使用羊ADM覆盖创面。术后7 d进行组织学评价，测量创面肉芽组织厚度。术后7、12、17 d，比较两组创面愈合率和创面组织表皮生长因子(EGF)的表达。各组各时间点样本数均为6。对数据行t检验。

结果

从人脂肪组织分离培养的细胞呈梭形，漩涡状排列，流式细胞术检测和分化实验证实为ADSCs。经过成膜培养，ADSCs形成3层膜片。术后7 d，ADSCs膜片组创面肉芽组织厚度为(456.35±57.05)μm，明显大于对照组的(295.08±38.86)μm(t＝5.721，P<0.001)。术后7、12、17 d，ADSCs膜片组创面愈合率[(45.07±1.58)%、(83.29±1.54)%、(98.49±0.85)%]均高于对照组[(29.99±0.90)%、(78.18±1.02)%、(92.38±0.54)%]，差异均有统计学意义(t＝20.294、6.777、14.733，P<0.001)。术后7、12、17 d，ADSCs膜片组创面组织EGF表达[(90.32±2.36)pg/ml、(107.84±1.82)pg/ml、(111.93±2.25)pg/ml]均高于对照组[(81.14±2.15)pg/ml、(102.46±2.18)pg/ml、(106.14±2.10)pg/ml]，差异均有统计学意义(t＝7.012、4.621、4.630，P<0.001)。

结论

人ADSCs膜片能显著加速裸鼠创面愈合，其机制可能与EGF表达增加从而促进肉芽组织生长有关。

关键词: 脂肪间充质干细胞, 细胞膜片技术, 创面愈合

Objective

To investigate the therapeutic effect of human adipose-derived stem cells (ADSCs) sheet on full-thickness skin defect in BALB/c-nu nude mice. The sample number was 6 in each group at each time point. Data were statistically analyed with t test.

Methods

Human ADSCs were isolated and cultured in vitro. Three layers of human ADSCs sheet were prepared. A 12 mm-diameter, round, full-thickness cutaneous wound was made on the back of 36 nude mice. The nude mice were divided into two groups, sheep acelluar dermal matrix (ADM) was used alone to cover the wound in control group, and ADSCs sheet combined with sheep ADM were used in ADSCs sheet group. On 7 days after surgery, the thickness of granulation tissue was measured. On 7, 12 and 17 days after surgery, the wound healing rate and epidermal growth factor (EGF) expression in wound tissue were compared between the two groups.

Results

The cells isolated and cultured from adipose tissue were spindle shaped, arranged in whirlpool. Flow cytometry and differentiation experiments confirmed that the cells were ADSCs. After sheet formation culture, ADSCs formed membrane. On 7 days after surgery, the thickness of wound granulation tissue in ADSCs sheet group was (456.35±57.05)μm, which was significantly larger than (295.08±38.86)μm in control group(t=5.721, P<0.001). On 7, 12 and 17 days after surgery, the wound healing rates in ADSCs sheet group [(45.07±1.58)%, (83.29±1.54)%, (98.49±0.85)%] were significantly higher than those in control group [(29.99±0.90)%, (78.18±1.02)%, (92.38±0.54)%], the differences were statistically significant (t=20.294, 6.777, 14.733; P<0.001). On 7, 12 and 17 days after surgery, EGF expressions in wound tissue in ADSCs sheet group [(90.32±2.36)pg/ml, (107.84±1.82)pg/ml, (111.93±2.25)pg/ml] were significantly higher than those in control group [(81.14±2.15)pg/ml, (102.46±2.18)pg/ml, (106.14±2.10)pg/ml], with statistically significant difference(t=7.012, 4.621, 4.630; P<0.001).

Conclusion

Human ADSCs sheet could accelerate wound healing in nude mice, its mechanism may be related to the increase of EGF expression, which promotes the growth of granulation tissue.

Key words: Adipose-derived stem cells, Cell sheet technology, Wound healing

图1 ADSCs的形态。A示ADSCs原代细胞(×50)；B示ADSCs P₃代细胞(×50)

图2 ADSCs生长曲线

图3 ADSCs表面标记物表达

图4 ADSCs成脂、成骨诱导实验。A示成脂诱导实验细胞，B示成脂诱导对照细胞，油红O染色(×200)；C示成骨诱导实验细胞，D示成骨诱导对照细胞，茜素红染色(×100)

图5 ADSCs膜片。A示培养第8天大体外观；B示培养第8天显微镜下所见(×50)；C示培养第21天大体外观；D示培养第21天显微镜下所见(×50)；E示培养第21天苏木精-伊红染色(×400)

图6 2组术后7 d创面肉芽组织生长情况(苏木精-伊红染色，×40)。A示对照组创面肉芽组织生长情况；B示ADSCs膜片组创面肉芽组织生长情况

图7 2组术后各时间点创面情况。A-C示对照组术后7、12、17 d创面情况；D-F示ADSCs膜片组术后7、12、17 d创面情况

表1 两组术后7、12、17 d创面愈合率比较(%,

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	45.07±1.58	83.29±1.54	98.49±0.85
对照组	6	29.99±0.90	78.18±1.02	92.38±0.54
t值		20.294	6.777	14.733
P值		<0.001	<0.001	<0.001

表1 两组术后7、12、17 d创面愈合率比较(%,

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	45.07±1.58	83.29±1.54	98.49±0.85
对照组	6	29.99±0.90	78.18±1.02	92.38±0.54
t值		20.294	6.777	14.733
P值		<0.001	<0.001	<0.001

表2 两组术后7、12、17 d创面组织EGF表达量(pg/ml，

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	90.32±2.36	107.84±1.82	111.93±2.25
对照组	6	81.14±2.15	102.46±2.18	106.14±2.10
t值		7.012	4.621	4.630
P值		<0.001	<0.001	<0.001

表2 两组术后7、12、17 d创面组织EGF表达量(pg/ml，

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	90.32±2.36	107.84±1.82	111.93±2.25
对照组	6	81.14±2.15	102.46±2.18	106.14±2.10
t值		7.012	4.621	4.630
P值		<0.001	<0.001	<0.001

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Observation on the efficacy of human adipose-derived stem cells multilayer sheet promoting wound healing in nude mice

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Observation on the efficacy of human adipose-derived stem cells multilayer sheet promoting wound healing in nude mice