摘要
目的探讨转化生长因子β2反义寡核苷酸（TGF-β2艮ASON）在兔角膜创伤愈合过程中对瘢痕组织形成的作用。方法实验研究。新西兰大白兔192只，以随机数字表法，随机分为4组（A组、B组、C组、D组），每组实验动物48只。4组动物右眼均制备角膜穿孔伤模型。A组术后右眼滴TGF—β2ASON溶液（TGF—β2ASON组）；B组术后右眼滴地塞米松滴眼液（地塞米松组）；C组术后右眼滴去离子水（去离子水组），D组术后右眼不做任何处理（单纯手术组）。4组动物分别于术后4、7、14、28d取角膜组织，应用HE染色、免疫组织化学和荧光定量PCR等检测方法进行观察。多组间比较采用单因素方差分析，两两比较采用Bonferroni检验。结果HE染色：术后同一时间点A组与B组阳性成纤维细胞数均比C组和D组明显减少，差异具有统计学意义（F=510．79，178．12，79．14，P〈0．05），但A组与B组、C组与D组间对比差异无统计学意义（P〉0．05）。免疫组织化学观察：（1）α-平滑肌肌动蛋白（α-SMA）：A组的01．SMA染色阳性的成纤维细胞于术后4d开始增生（9．44±0．47个／高倍镜视野），7d达高峰（12．50±0．81个／高倍镜视野），14d回到基线水平（0．85±0．43个／高倍镜视野），同一时间点与B组（9．49±0．95，12．42±0．70，0．86±0．79个／高倍镜视野）相比差异无统计学意义（P〉0．05），与C组（20．14±0．78，18．19±1．28，4．87±0．58个／高倍镜视野）和D组（20．21±0．92，18．25±1．39，5．00±2．217个／高倍镜视野）相比均明显减少，差异具有统计学意义（P〈0．05）；（2）纤维连接蛋白：同一时间点，A、B组纤维连接蛋白染色强度较C、D组明显减弱。逆转录实时荧光PCR：A、B组各时间点TGF-β2的mRNA相对表达量均较C、D组明显降低，差异均有统计学意义（F=554．25，610．68，636．80，89．51，P〈0．05），但A组与B组、C与D组间比较差异均无统计学意义（P〉0．05）。结论TGF-β2 ASON可有效抑制TGF-β2的活性，减轻角膜损伤后结缔组织的增生而发挥抗瘢痕化的作用。在角膜损伤修复的早期，即术后7d内用TGF—β2 ASON进行干预调控是抑制术后瘢痕增生的关键。
Objective To investigate the influence of TGF-β2 antisense oligonucletide （ASON） on preventing corneal scar hyperplasia in rabbits and to provide experimental evidence for its clinical application. Methods It was an experimental study. One hundred and ninety two New Zealand white rabbits were randomly divided into 4 groups （groups A, B, C and D）. Corneal injury models were established in all groups. There were 48 experimental animals in each group. TGF-β2 ASON was dropped into right eyes in group A, dexamethasone was dropped into right eyes in group B, deionized water was dropped into right eyes in group C and nothing was dropped into right eyes in group D after the operation. The corneas were surgically removed and assessed by hematoxylin eosin （HE） staining, immunohistochemical study and real time PCR at four different time points （4 d, 7 d, 14 d and 28 d） after surgery. Results HE staining：at the same time point, fibroblasts in the groups A and B were significantlyfewer than that in the groups C and D, the difference was statistically significant （ P 〈 0.05 ） , but there was no significant difference between groups A and B or groups C and D. Immunohistochemical observation found that the expression of α-smooth muscle actin （α-SMA） positive fibroblasts could be observed by the 4th day （9. 44 ±0. 47/HP）, reached a climax by the 7th day（ 12. 50 ± 0. 81/HP）, and returned to the baseline levels by the 14th day（0. 85 ±0.43/HP） in the group A, which was similar to that in the group B（9.49 ±0. 95,12. 42 ± 0. 70,0. 86±0. 79/HP）at the same time point （P 〉 0. 05）, but it was significantly fewer than that in the group C （20. 14 ± 0.78,18.19 ± 1.28,4. 87 ± 0. 58/HP）and group D （20. 21 ± 0. 92,18.25 ± 1.39,5.00 ± 2. 217/HP）, which was statistical significant （P 〈 0. 05 ）. The staining intensity of fibronectin （FN） in groups A and B was significantly weaker than that in groups C and D. Real time PCR analysis showed that at each time point, the expression of TGF-β2 mRNAs in groups A and B was significantly lower than that in groups C and D （ P 〈 0. 05 ） . Conclusions TGF-β2 ASON can effectively prevent the proliferation of corneal tissue by inhibiting the activity of TGF-β2after injury. The early stage of corneal repair is 7 days after injury, so it is important to use TGF-β2 ASON at this stage to inhibit the scar hyperplasia. In addition, it is safe to apply TGF-β2ASON topically to protect the cornea from obvious side effects.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2013年第2期163-169,共7页
Chinese Journal of Ophthalmology
基金
河南省自然科学基金资助项目（102101310400）
关键词
角膜穿孔
瘢痕
增生
转化生长因子Β2
寡核苷酸类
反义
兔
Corneal pertoration
Cicatrix
Hyperplasia
Transforming growth factor beta 2
Oligonucletide,antisense
Rabbits
