Yuan Jishan, Li Tianzeng, Qi Shaohai
Department of Orthopedics, Affiliated People's Hospital of Jiangsu University, Zhenjiang Jiangsu 212002, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011 Jun;25(6):718-23.
Objective Col I A1 antisense oligodeoxynucleotide (ASODN) has inhibitory effect on collagen synthesis in cultured human hypertrophic scar fibroblasts. To investigate the effects of intralesional injection of Col I A1 ASODN on collagen synthesis in human hypertrophic scar transplanted nude mouse model.
The animal model of human hypertrophic scar transplantation was established in the 60 BALB/c-nunu nude mice (specific pathogen free grade, weighing about 20 g, and aged 6-8 weeks) by transplanting hypertrophic scar without epidermis donated by the patients into the interscapular subcutaneous region on the back, with 1 piece each mouse. Fifty-eight succeed models mice were randomly divided into 3 groups in accordance with the contents of injection. In group A (n = 20): 5 microL Col I A1 ASODN (3 mmol/L), 3 microL liposome, and 92 microL Opti-MEM I; in group B (n = 20): 3 microL liposome and 97 microL Opti-MEM I; in group C (n = 18): only 100 microL Opti-MEM I. The injection was every day in the first 2 weeks and once every other day thereafter. The scar specimens were harvested at 2, 4, and 6 weeks after injection, respectively and the hardness of the scar tissue was measured. The collagens type I and III in the scar were observed under polarized light microscope after sirius red staining. The ultrastructures of the scar tissues were also observed under transmission electronic microscope (TEM). Additionally, the Col I A1 mRNAs expression was determined by RT-PCR and the concentrations of Col I A1 protein were measured with ELISA method.
Seventeen mice died after intralesional injection. Totally 40 specimens out of 41 mice were suitable for nucleic acid and protein study, including 14 in group A, 13 in group B, and 14 in group C. The hardness of scars showed no significant difference (P > 0.05) among 3 groups at 2 weeks after injection, whereas the hardness of scars in group A was significantly lower than those in groups B and C at 4 and 6 weeks (P < 0.05), and there was no significant difference between groups B and C (P > 0.05). The collagen staining showed the increase of collagen type III in all groups, especially in group A with a regular arrangement of collagen type I fibers. TEM observation indicated that there was degeneration of fibroblasts and better organization of collagen fibers in group A, and the structures of collagen fibers in all groups became orderly with time. The relative expressions of Col I A1 mRNA and the concentrations of Col I A1 protein at 2 and 4 weeks after injection were significant difference among 3 groups (P < 0.05), and they were significantly lower in group A than in groups B and C (P < 0.05) at 6 weeks after injection, but no significant difference was found between groups B and C (P > 0.05).
Intralesional injection of Col I A1 ASODN in the nude mice model with human hypertrophic scars can inhibit the expression of Col I A1 mRNA and collagen type I, which enhances the mature and softening of the scar tissue. In this process, liposome shows some assistant effect.
目的 Ⅰ型胶原α1链反义寡脱氧核苷酸（ASODN）对培养的人增生性瘢痕成纤维细胞的胶原合成有抑制作用。探讨瘤内注射Ⅰ型胶原α1链ASODN对人增生性瘢痕移植裸鼠模型胶原合成的影响。
将患者捐献的无表皮增生性瘢痕移植到60只BALB/c-nu/nu裸鼠（无特定病原体级，体重约20 g，6-8周龄）背部肩胛间皮下区域，每只小鼠移植1块，建立人增生性瘢痕移植动物模型。58只成功建模的小鼠根据注射内容物随机分为3组。A组（n = 20）：5 μL Ⅰ型胶原α1链ASODN（浓度3 mmol/L）、3 μL脂质体和92 μL Opti-MEM I；B组（n = 20）：3 μL脂质体和97 μL Opti-MEM I；C组（n = 18）：仅100 μL Opti-MEM I。前2周每天注射，此后隔天注射1次。分别在注射后2、4和6周采集瘢痕标本，测量瘢痕组织硬度。天狼星红染色后在偏振光显微镜下观察瘢痕中的Ⅰ型和Ⅲ型胶原。同时在透射电子显微镜（TEM）下观察瘢痕组织的超微结构。另外，采用RT-PCR法检测Ⅰ型胶原α1链mRNA表达，用ELISA法测定Ⅰ型胶原α1链蛋白浓度。
瘤内注射后17只小鼠死亡。41只小鼠中共有40个标本适合进行核酸和蛋白质研究，其中A组14个，B组13个，C组14个。注射后2周时，3组瘢痕硬度差异无统计学意义（P > 0.05）；4周和6周时，A组瘢痕硬度显著低于B组和C组（P < 0.05），B组和C组之间差异无统计学意义（P > 0.05）。胶原染色显示所有组Ⅲ型胶原均增加，尤其是A组，Ⅰ型胶原纤维排列规则。TEM观察表明，A组成纤维细胞有退变，胶原纤维排列更有序，且所有组胶原纤维结构随时间推移变得更有序。注射后2周和4周时，3组Ⅰ型胶原α1链mRNA相对表达量和Ⅰ型胶原α1链蛋白浓度差异有统计学意义（P < 0.05）；注射后6周时，A组显著低于B组和C组（P < 0.05），B组和C组之间差异无统计学意义（P > 0.05）。
在人增生性瘢痕裸鼠模型中瘤内注射Ⅰ型胶原α1链ASODN可抑制Ⅰ型胶原α1链mRNA及Ⅰ型胶原表达，促进瘢痕组织成熟软化，在此过程中脂质体有一定辅助作用。
