摘要
目的人Ⅰ型前胶原基因(ColⅠA1)反义寡核苷酸(antisense oligodeoxyneucleotide,ASODN)对体外培养的人增生性瘢痕成纤维细胞有抑制胶原合成作用,拟进一步探讨ColⅠA1 ASODN对裸鼠移植模型体内的人增生性瘢痕的胶原合成作用。方法 SPF级BALB/c-nunu品系6～8周龄雌性裸鼠60只,体重约20 g;取行瘢痕切除手术患者自愿捐赠的瘢痕组织块去表皮后,移植至裸鼠背部肩胛内侧皮下,每只裸鼠移植1块,制备人增生性瘢痕裸鼠移植模型。将58只成功制备模型的裸鼠根据处理方法不同,随机分成3组,于瘢痕移植2周根据分组行经皮穿刺瘢痕内注射。A组(n=20):5μL浓度为3 mmol/L ColⅠA1 ASODN、3μL脂质体、92μL Opti-MEMⅠ减血清培养基;B组(n=20):3μL脂质体、97μL Opti-MEMⅠ减血清培养基;C组(n=18):100μL Opti-MEMⅠ减血清培养基。实验最初2周每天注射1次,之后隔天1次,至实验取材。注射后2、4、6周,对存活裸鼠进行瘢痕硬度测量后,处死裸鼠取瘢痕组织行胶原染色组织学观察以及透射电镜观察;提取细胞总RNA后行RT-PCR测定ColⅠA1 mRNA相对含量;ELISA法行ColⅠA1蛋白定量分析。结果注射后6周内17只裸鼠死亡;共41只裸鼠40块瘢痕组织符合实验标准,纳入实验;A、B、C组各14、13、14只。注射后2周,3组间瘢痕硬度比较,差异均无统计学意义(P>0.05);4、6周时A组与B、C组比较,差异有统计学意义(P<0.05),B、C组间比较差异无统计学意义(P>0.05)。随时间延长,组织学观察示3组Ⅲ型胶原表达上升,A组最明显,Ⅰ型胶原排列规则。透射电镜观察示A组成纤维细胞退化,胶原纤维排列更趋于一致;B、C组胶原纤维排列也逐渐规则。注射后2、4周3组间ColⅠA1 mRNA和蛋白表达量比较,差异均有统计学意义(P<0.05);6周时A组与B、C组比较,差异有统计学意义(P<0.05),B、C组间比较差异无统计学意义(P>0.05)。结论 在人增生性瘢痕裸鼠移植模型的瘢痕内注射ColⅠA1 ASODN可抑制ColⅠA1 mRNA及Ⅰ型胶原表达,促进瘢痕组织成熟、软化,脂质体可促进该抑制效果。
Objective Col I A1 antisense oligodeoxyneucleotide（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.Methods 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 μL Col I A1 ASODN（3 mmol/L）,3 μL liposome,and 92 μL Opti-MEM I;in group B（n=20）： 3 μL liposome and 97 μL Opti-MEM I;in group C（n=18）： only 100 μL 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.Results 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）.Conclusion 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.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2011年第6期718-723,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
广东省重点科研项目(199827817)
广东省医学科研联合攻关项目(98002)~~
