核酶阻断IGF－Ⅰ及IGF－ⅠR表达抑制病理性瘢痕形成的研究

doi:10.3877/cma.j.issn.1673-9450.2017.01.006

目的

研究胰岛素样生长因子-Ⅰ(IGF-Ⅰ)及胰岛素样生长因子Ⅰ受体(IGF-ⅠR)核酶抑制病理性瘢痕形成的作用。

方法

选取100只SPF级4～6周龄裸鼠，另选取2015年5月至2016年7月暨南大学医学院附属广州市红十字会医院18例烧伤整形科住院患者手术切除的组织病理性瘢痕标本。将瘢痕组织体外培养成纤维细胞并分为a～g 7组，分别转染IGF-Ⅰ及IGF-ⅠR核酶后，检测培养基上清中羟脯氨酸含量。利用real-time PCR检测IGF-Ⅰ及IGF-ⅠR mRNA水平。将小鼠随机分成A～H 8组，每组10只，分别散点注射不同浓度IGF-Ⅰ及IGF-ⅠR核酶后，检测瘢痕增生组织体积大小，应用内参物竞争性PCR定量分析裸鼠瘢痕组织中Ⅰ、Ⅲ型前胶原mRNA水平的改变。利用Western Immunoblotting法分析经处理后Ⅰ、Ⅲ型胶原蛋白水平的变化。

结果

在成纤维细胞中转染不同浓度IGF-Ⅰ及IGF-ⅠR核酶后，上清中羟脯氨酸含量和IGF-Ⅰ、IGF-ⅠR mRNA水平均有所降低。单独转染IGF-ⅠR核酶及单独转染IGF-Ⅰ核酶对IGF-ⅠR mRNA水平无显著影响。在人增生性瘢痕裸鼠动物模型中注射IGF-Ⅰ及IGF-ⅠR核酶后，裸鼠瘢痕组织体积减小，瘢痕增生组织中Ⅰ型前胶原mRNA水平和蛋白水平降低，Ⅲ型前胶原mRNA水平和蛋白水平均有所增高。

结论

IGF-Ⅰ及IGF-ⅠR核酶可用于治疗病理性瘢痕形成。

关键词: 胰岛素样生长因子Ⅰ, 受体，IGF 1型, 瘢痕, 核酶

Objective

To investigate the function of insulin-like growth factor-Ⅰ(IGF-Ⅰ) and insulin-like growth factor-Ⅰ receptor(IGF-ⅠR) ribozyme in inhibiting pathological scar formation.

Methods

To select 100 SPF grade 4 to 6 week-old nude mice, and to select another 18 cases of orthopedics in the department of burns and plastics, Guangzhou Red Cross hospital affiliated to Jinan university from May, 2015 to July, 2016 for histopathological scar specimens. The fibroblasts were cultured in vitro and divided into 7 groups. After transfection of IGF-Ⅰ and IGF-ⅠR ribozymes, the hydroxyproline content in the culture supernatant was detected. The mRNA levels of IGF-Ⅰ and IGF-ⅠR were detected by real-time PCR. The mice were divided into A to H groups (n=10 each) randomly. The IGF-Ⅰ and IGF-ⅠR ribozymes were injected at different concentrations. The size of hypertrophic scar was detected by competitive PCR and the changes of type Ⅰ and Ⅲ procollagen mRNA levels in scar tissue. The levels of type Ⅰ and Ⅲ collagen were analysed by Western immunoblotting.

Results

The expression of IGF-Ⅰ and IGF-ⅠR mRNA in supernatant were decreased after transfection of IGF-Ⅰ and IGF-ⅠR ribozymes in fibroblasts. The expression of IGF-ⅠR mRNA was not significantly affected by transfection of IGF-ⅠR ribozyme alone or IGF-Ⅰ ribozyme alone. The expression of procollagen mRNA and type Ⅰ procollagen mRNA in the hypertrophic scars of nude mice was significantly lower than that of the control group (P<0.05). The expression of procollagen mRNA Levels and protein levels were increased.

Conclusion

IGF-Ⅰ and IGF-ⅠR ribozyme can be used for the treatment of pathological scar formation.

Key words: Insulin-like growth factor Ⅰ, Receptor, IGF type 1, Cicatrix, Ribozyme

图1 核酶转染后成纤维细胞中IGF-Ⅰ相对表达量

图2 核酶转染后成纤维细胞中IGF-ⅠR相对表达量

图3 核酶转染后成纤维细胞后羟脯氨酸含量

表1 人增生性瘢痕裸鼠动物模型中裸鼠瘢痕增生组织体积大小(mm³)

组别	实验前	实验后
A组	28.74±1.85	25.93±2.15^ab
B组	30.78±2.51	27.58±2.66^ab
C组	28.74±1.06	26.77±1.97^ab
D组	30.25±3.61	29.28±2.87
E组	29.33±2.77	25.04±2.41^ab
F组	29.17±2.68	23.79±1.32^ab
G组	31.45±2.82	30.57±2.65
H组	32.58±1.37	32.98±1.99

表1 人增生性瘢痕裸鼠动物模型中裸鼠瘢痕增生组织体积大小(mm³)

组别	实验前	实验后
A组	28.74±1.85	25.93±2.15^ab
B组	30.78±2.51	27.58±2.66^ab
C组	28.74±1.06	26.77±1.97^ab
D组	30.25±3.61	29.28±2.87
E组	29.33±2.77	25.04±2.41^ab
F组	29.17±2.68	23.79±1.32^ab
G组	31.45±2.82	30.57±2.65
H组	32.58±1.37	32.98±1.99

表2 裸鼠瘢痕增生组织中胶原纤维蛋白水平和mRNA水平变化

组别	蛋白水平		mRNA水平
组别	Ⅰ型胶原	Ⅲ型胶原	Ⅰ型胶原	Ⅲ型胶原
A组	57.23±0.37^a	24.81±0.55^a	0.995±0.041^a	1.571±0.047^a
B组	59.56±0.24^a	25.42±0.49^a	0.942±0.032^a	1.485±0.059^a
C组	61.01±0.27^a	28.57±0.33^a	0.971±0.038^a	1.442±0.073^a
D组	63.34±0.61^a	29.53±0.25^a	0.947±0.021^a	1.647±0.094^a
E组	49.74±0.34^a	41.55±0.26^a	0.752±0.053^a	2.501±0.023^a
F组	45.76±0.17^a	41.39±0.31^a	0.772±0.048^a	2.615±0.095^a
G组	72.87±0.39	11.25±0.15	1.175±0.027	1.187±0.068
H组	82.75±0.36	19.31±0.22	1.259±0.064	1.377±0.081

表2 裸鼠瘢痕增生组织中胶原纤维蛋白水平和mRNA水平变化

组别	蛋白水平		mRNA水平
组别	Ⅰ型胶原	Ⅲ型胶原	Ⅰ型胶原	Ⅲ型胶原
A组	57.23±0.37^a	24.81±0.55^a	0.995±0.041^a	1.571±0.047^a
B组	59.56±0.24^a	25.42±0.49^a	0.942±0.032^a	1.485±0.059^a
C组	61.01±0.27^a	28.57±0.33^a	0.971±0.038^a	1.442±0.073^a
D组	63.34±0.61^a	29.53±0.25^a	0.947±0.021^a	1.647±0.094^a
E组	49.74±0.34^a	41.55±0.26^a	0.752±0.053^a	2.501±0.023^a
F组	45.76±0.17^a	41.39±0.31^a	0.772±0.048^a	2.615±0.095^a
G组	72.87±0.39	11.25±0.15	1.175±0.027	1.187±0.068
H组	82.75±0.36	19.31±0.22	1.259±0.064	1.377±0.081

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Inhibition of IGF-Ⅰ and IGF-ⅠR expression by ribozyme in inhibiting pathological scar formation

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Inhibition of IGF-Ⅰ and IGF-ⅠR expression by ribozyme in inhibiting pathological scar formation