Effects of local transplantation of autologous adipose-derived mesenchymal stem cells on the formation of hyperplastic scar on rabbit ears
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摘要: 目的 探讨兔自体脂肪源性间充质干细胞(ADSC)局部移植对兔耳增生性瘢痕形成的影响。 方法 取6只新西兰大耳白兔腹股沟处脂肪组织,分离ADSC并进行传代培养。取每只兔的第3代ADSC进行以下实验。在每只兔每侧耳腹造成6个直径为6 mm的全层皮肤缺损创面,观察创面上皮化情况及局部组织增生情况,记录创面完全上皮化时间和增生性瘢痕形成时间。选择兔左耳创面为ADSC组,右耳创面为对照组,每组36个创面。于创面完全上皮化后(伤后25 d),ADSC组创面注射0.2 mL溴脱氧尿苷(BrdU)标记的自体ADSC悬液(浓度为5×106个/mL),对照组创面注射等量PBS。每5天注射1次,共注射3次(后2次注射于创面愈合后形成的瘢痕内)。于第3次注射后5 d分别切取2组增生性瘢痕组织,HE染色观察组织形态,VG染色观察增生性瘢痕中胶原排列情况,荧光显微镜下观察增生性瘢痕中BrdU标记的ADSC分布,ELISA法检测增生性瘢痕中Ⅰ、Ⅲ型胶原及TGF-β1、核心蛋白多糖的蛋白含量,实时荧光定量RT-PCR法检测增生性瘢痕中TGF-β1、核心蛋白多糖的mRNA表达。对数据行配对
t 检验。 结果 (1)兔耳创面完全上皮化时间为伤后(20.0±2.0)d,伤后(35.0±2.2)d增生性瘢痕形成。伤后40 d,对照组增生性瘢痕仍维持较明显的增生状态,ADSC组增生性瘢痕体积缩小、变平、质地变软、色泽稍变浅。(2)与对照组比较,ADSC组增生性瘢痕中上皮细胞层数增多,可见上皮脚样及真皮乳突样结构形成,真皮层有核细胞数量明显增多。对照组增生性瘢痕中胶原紧密,排列较紊乱;ADSC组增生性瘢痕中胶原密度较对照组下降,排列较规整。(3)伤后40 d,ADSC组增生性瘢痕中仍可见BrdU标记的ADSC。(4)ADSC组增生性瘢痕中Ⅰ、Ⅲ型胶原及TGF-β1、核心蛋白多糖蛋白含量分别为(1.40±0.04)、(8.18±0.23)μg/L及(25.1±0.7)ng/L、(4.872±0.101)ng/mL,对照组增生性瘢痕中Ⅰ、Ⅲ型胶原及TGF-β1、核心蛋白多糖蛋白含量分别为(2.29±0.05)、(12.20±0.38)μg/L及(37.2±1.1)ng/L、(4.143±0.024)ng/mL。与对照组比较,ADSC组增生性瘢痕中Ⅰ、Ⅲ型胶原及TGF-β1蛋白含量明显降低(t 值为-33.66~-22.84,P 值均小于0.001),核心蛋白多糖蛋白含量明显增高(t =10.41,P <0.001)。(5)与对照组比较,ADSC组增生性瘢痕中TGF-β1 mRNA表达量明显降低(t =4.45,P <0.01),核心蛋白多糖的mRNA表达量明显升高(t =5.61,P <0.01)。 结论 兔增生性瘢痕形成早期瘢痕内移植自体ADSC可抑制增生性瘢痕的形成,改善创面愈合质量,其机制可能与ADSC下调TGF-β1及Ⅰ、Ⅲ型胶原的表达,上调核心蛋白多糖的表达有关。-
关键词:
- 伤口愈合 /
- 瘢痕 /
- 间质干细胞移植 /
- 脂肪源性间充质干细胞
Abstract: Objective To investigate the effects of local transplantation of autologous adipose-derived mesenchymal stem cells (ADSCs) on the formation of hyperplastic scar on rabbit ears. Methods ADSCs were isolated from inguinal fat of six New Zealand rabbits and then sub-cultured. ADSCs of the third passage of each rabbit were used in the following experiments. Six full-thickness skin defect wounds with diameter of 6 mm on the ventral surface of every rabbit ear were made. Wound healing and local-tissue proliferation were observed, and complete epithelization time of wounds and formation time of hyperplastic scar were recorded. The wounds on left ears were selected as group ADSCs, and the wounds on right ears were selected as control group, with 36 wounds in each group. After the complete epithelization of wounds (post injury day 25), 0.2 mL bromodeoxyuridine (BrdU) labeled autologous ADSCs with the concentration of 5×106 per milliliter were injected into each wound of the rabbit of group ADSCs, while the same amount of phosphate buffer solution was injected into each wound of the rabbit of control group. The frequency of injection was once every 5 days, totally for 3 times, and the latter 2 times were injected into scars generated from healed wound. Hyperplastic scars of rabbits of two groups were harvested on the fifth day after the third injection, then the morphology was observed by HE staining, and the arrangement of collagen in hyperplastic scar was observed by VG staining. The distribution of BrdU-labeled ADSCs in the hyperplastic scar was observed with fluorescence microscope. The protein content of type Ⅰ collagen, type Ⅲ collagen, transforming growth factor β1 (TGF-β1), and decorin in hyperplastic scar were detected by enzyme-linked immunosorbent assay, and the mRNA expression of decorin and TGF-β1 in hyperplastic scar were tested by real-time fluorescent quantitative reverse transcription-polymerase chain reaction. Data were processed with pairedt test. Results (1) The complete epithelization time of wounds of rabbits' ears was (20.0±2.0) d post injury, and hyperplastic scars were formed on post injury day 35.0±2.2. On post injury day 40, hyperplastic scars of rabbits of control group were still obvious, while those of group ADSCs became smaller, flat, soft, and light colored. (2) Compared with those in control group, epithelial cell layers and the number of nucleated cells in corium layer of hyperplastic scars of rabbits of group ADSCs were increased, and epithelium foot like and dermal papilla like structures were observed. The collagen density of hyperplastic scars of rabbits of control group was tight and arranged disorderly, while that of group ADSCs were decreased significantly and arranged regularly as compared with that of control group. (3) On post injury day 40, BrdU-labeled ADSCs were still observed in the hyperplastic scars of rabbits of group ADSCs. (4) The protein content of type Ⅰ collagen, type Ⅲ collagen, TGF-β1, and decorin in hyperplastic scars of rabbits of group ADSCs were respectively (1.40±0.04) and (8.18±0.23) μg/L, (25.1±0.7) ng/L, and (4.872±0.101) ng/mL, and those in hyperplastic scars of rabbits of control group were respectively (2.29±0.05) and (12.20±0.38) μg/L, (37.2±1.1) ng/L, and (4.143±0.024) ng/mL. Compared with those in control group, the protein content of type Ⅰ collagen, type Ⅲ collagen, and TGF-β1 in hyperplastic scars of rabbit of group ADSCs were significantly decreased (witht values from -33.66 to -22.84,P values below 0.001), while the protein content of decorin were significantly increased (t =10.41,P <0.001). (5) Compared with those in control group, the mRNA expression of TGF-β1 in hyperplastic scars of rabbits of group ADSCs was significantly decreased (t =4.45,P <0.01), while the mRNA expression of decorin was significantly increased (t =5.61,P <0.01). Conclusions Autologous transplantation of ADSCs into scar of rabbit at the early stage can inhibit the formation of hyperplastic scar, promote the quality of wound healing, and the mechanism may relate to the down-regulation of TGF-β1, type Ⅰ collagen, and type Ⅲ collagen and the up-regulation of decorin induced by ADSCs.
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