中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2013年
2期
163-169
,共7页
郑广瑛%郝莉莉%王瑞娜%连元君%谭楠
鄭廣瑛%郝莉莉%王瑞娜%連元君%譚楠
정엄영%학리리%왕서나%련원군%담남
角膜穿孔%瘢痕%增生%转化生长因子β2%寡核苷酸类,反义%兔
角膜穿孔%瘢痕%增生%轉化生長因子β2%寡覈苷痠類,反義%兔
각막천공%반흔%증생%전화생장인자β2%과핵감산류,반의%토
Corneal pertoration%Cicatrix%Hyperplasia%Transforming growth factor beta 2%Oligonucletide,antisense%Rabbits
目的 探讨转化生长因子β2反义寡核苷酸(TGF-β2 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-β2 ASON溶液(TGF-β2 ASON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、28 d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α-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),14 d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组与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.
目的 探討轉化生長因子β2反義寡覈苷痠(TGF-β2 ASON)在兔角膜創傷愈閤過程中對瘢痕組織形成的作用.方法 實驗研究.新西蘭大白兔192隻,以隨機數字錶法,隨機分為4組(A組、B組、C組、D組),每組實驗動物48隻.4組動物右眼均製備角膜穿孔傷模型.A組術後右眼滴TGF-β2 ASON溶液(TGF-β2 ASON組)；B組術後右眼滴地塞米鬆滴眼液(地塞米鬆組)；C組術後右眼滴去離子水(去離子水組),D組術後右眼不做任何處理(單純手術組).4組動物分彆于術後4、7、14、28 d取角膜組織,應用HE染色、免疫組織化學和熒光定量PCR等檢測方法進行觀察.多組間比較採用單因素方差分析,兩兩比較採用Bonferroni檢驗.結果 HE染色:術後同一時間點A組與B組暘性成纖維細胞數均比C組和D組明顯減少,差異具有統計學意義(F =510.79,178.12,79.14,P＜0.05),但A組與B組、C組與D組間對比差異無統計學意義(P＞0.05).免疫組織化學觀察:(1)α-平滑肌肌動蛋白(α-SMA):A組的α-SMA染色暘性的成纖維細胞于術後4d開始增生(9.44±0.47箇/高倍鏡視野),7d達高峰(12.50±0.81箇/高倍鏡視野),14 d迴到基線水平(0.85±0.43箇/高倍鏡視野),同一時間點與B組(9.49 ±0.95,12.42±0.70,0.86±0.79箇/高倍鏡視野)相比差異無統計學意義(P＞0.05),與C組(20.14 ±0.78,18.19±1.28,4.87 ±0.58箇/高倍鏡視野)和D組(20.21±0.92,18.25±1.39,5.00±2.217箇/高倍鏡視野)相比均明顯減少,差異具有統計學意義(P＜0.05)；(2)纖維連接蛋白:同一時間點,A、B組纖維連接蛋白染色彊度較C、D組明顯減弱.逆轉錄實時熒光PCR:A、B組各時間點TGF-β2的mRNA相對錶達量均較C、D組明顯降低,差異均有統計學意義(F=554.25,610.68,636.80,89.51,P＜0.05),但A組與B組、C組與D組間比較差異均無統計學意義(P＞0.05).結論 TGF-β2 ASON可有效抑製TGF-β2的活性,減輕角膜損傷後結締組織的增生而髮揮抗瘢痕化的作用.在角膜損傷脩複的早期,即術後7d內用TGF-β2 ASON進行榦預調控是抑製術後瘢痕增生的關鍵.
목적 탐토전화생장인자β2반의과핵감산(TGF-β2 ASON)재토각막창상유합과정중대반흔조직형성적작용.방법 실험연구.신서란대백토192지,이수궤수자표법,수궤분위4조(A조、B조、C조、D조),매조실험동물48지.4조동물우안균제비각막천공상모형.A조술후우안적TGF-β2 ASON용액(TGF-β2 ASON조)；B조술후우안적지새미송적안액(지새미송조)；C조술후우안적거리자수(거리자수조),D조술후우안불주임하처리(단순수술조).4조동물분별우술후4、7、14、28 d취각막조직,응용HE염색、면역조직화학화형광정량PCR등검측방법진행관찰.다조간비교채용단인소방차분석,량량비교채용Bonferroni검험.결과 HE염색:술후동일시간점A조여B조양성성섬유세포수균비C조화D조명현감소,차이구유통계학의의(F =510.79,178.12,79.14,P＜0.05),단A조여B조、C조여D조간대비차이무통계학의의(P＞0.05).면역조직화학관찰:(1)α-평활기기동단백(α-SMA):A조적α-SMA염색양성적성섬유세포우술후4d개시증생(9.44±0.47개/고배경시야),7d체고봉(12.50±0.81개/고배경시야),14 d회도기선수평(0.85±0.43개/고배경시야),동일시간점여B조(9.49 ±0.95,12.42±0.70,0.86±0.79개/고배경시야)상비차이무통계학의의(P＞0.05),여C조(20.14 ±0.78,18.19±1.28,4.87 ±0.58개/고배경시야)화D조(20.21±0.92,18.25±1.39,5.00±2.217개/고배경시야)상비균명현감소,차이구유통계학의의(P＜0.05)；(2)섬유련접단백:동일시간점,A、B조섬유련접단백염색강도교C、D조명현감약.역전록실시형광PCR:A、B조각시간점TGF-β2적mRNA상대표체량균교C、D조명현강저,차이균유통계학의의(F=554.25,610.68,636.80,89.51,P＜0.05),단A조여B조、C조여D조간비교차이균무통계학의의(P＞0.05).결론 TGF-β2 ASON가유효억제TGF-β2적활성,감경각막손상후결체조직적증생이발휘항반흔화적작용.재각막손상수복적조기,즉술후7d내용TGF-β2 ASON진행간예조공시억제술후반흔증생적관건.
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 significantly fewer 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 ±O.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 significanrly 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-β2 after 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-β2 ASON topically to protect the cornea from obvious side effects.
