摘要
目的探讨microRNAs的差异性表达在原发性及复发性翼状胬肉发病机制中的作用。方法收集2012年12月至2013年12月在广州医科大学附属第六医院（清远市人民医院）眼科行住院手术治疗的翼状胬肉与眼外伤患者共30例（30只眼）的临床资料进行研究。其中,男性16例（16只眼）,女性14例（14只眼）。根据病种分为原发组、复发组及对照组等共3组,各10例（10只眼）患者。原发组和复发组患者各10例（10只眼）均行单纯翼状胬肉切除术;对照组患者10例（10只眼）术中取少量正常结膜组织。所有手术由同一术者操作。采用MicroRNA芯片和荧光定量PCR（qRT-PCR）技术检测microRNAs的表达水平。计量资料以均数±标准差（x珋±s）的形式表示,计数资料以例数和百分比形式表示。三组间年龄和microRNAs表达水平的比较采用方差分析,当差异有统计学意义时,进一步两两比较。三组间性别、职业的比较采用χ2检验。使用聚类分析的方法,探索不同组MicroRNA表达量的关系。绘制不同组MicroRNA表达量的散点图,计算pearson相关系数,看各组之间的相关性。结果原发组、复发组及对照组患者的平均年龄分别为（41.3±6.7）岁、（47.3±8.7）岁及（39.3±11.7）岁,三组之间的年龄差别无统计学意义（F=3.08,P〉0.05）。在上述三组中,性别为男性的患者例数与百分比分别为5例（5只眼）,占50.0%;6例（6只眼）,占60.0%及4例（4只眼）占40.0%。职业为从事户外职业的患者例数与百分比分别为6例（6只眼）,占60.0%;7例（7只眼）,占70.0%及3例（3只眼）,占30.0%。三组差别无统计学意义（χ~2=0.88,1.09;P〉0.05）。通过对MicroRNA芯片结果进行聚类分析,发现复发组的MicroRNA表达谱和对照组分为一组,不同于原发组。复发组与原发组患者的MicroRNA表达谱无相关性（r=0.57,P〉0.05）,复发组与对照组有相关性（r=0.67,P〈0.05）。三组间的组织的microRNAs表达谱差别被MicroRNA芯片检测,随后用qRT-PCR技术验证芯片结果。与原发组及对照组相比,复发组患者的MicroRNA谱与前两者显著不同,有24个microRNAs显著差异性表达。其中14个microRNAs（miR-126、-15b、-135b、-199b、-214、-301a、-20a、-4 496、-93、-21、-29a、-181a、-146b及-99b）在组织中被上调,而11个microRNAs（miR-4 687、-4 279、-483、-296、-652、-622、-1、-3 127、-328、-361及-4 533）被下调。通过蛋白印迹试验（Western blot）发现,在复发性翼状胬肉组织中血管内皮生长因子通路被明显激活,可能是microRNAs参与了调控。结论 microRNAs的差异性表达与翼状胬肉复发密切相关,而某些高表达的microRNAs与血管新生、上皮-间质转化以及基质成纤维细胞增加关系密切。因此,我们认为抑制某些特定MicroRNA表达可能是今后防治翼状胬肉复发的有效方法。
Objective In this study, we evaluate the ditterence expression levels and possible ettects of microRNAs on primary or recurrent pterygium. Methods We continuously collected surgically excised paired human pterygium and conjunctival tissues and information regarding patient age, gander and occupation from records in December 2012 to December 2013. This study enrolled 20 pterygium and 10 normal eonjunctival samples,including 16 male patients （ 16 eyes） and 14 females （ 14 eyes）. 10 patients （ 10 eyes） of primary or recurrent pterygium group underwent pterygium excision alone, and in the ocular trauma group （10 cases, 10 eyes） ,only a small amount of conjunctival tissue was taken during the operation. All surgical procedures were done by the same surgeon. Expression levels of microRNAs were obtained through examination of excised pterygium and conjunctival tissues by MicroRNA Microarray and quantitative real time polymerase chain reaction （qRT-PCR）. Differences between age, gender, occupation and microRNAs expression levels were evaluated by Kruskal-Wallis or chi-square test. Results This study evaluated 20 patients （44. 3 ±6. 7 ） years and 10 normal contrast （39. 3±11.7 ） years. There was no statistical difference between the three groups in age, sex and occupation （ F = 3.08, P〉 0.05 ）. In the above three groups,the gender of the number of patients with the percentage of patients were 5 cases 5 （50%） eyes, 6 cases 6 （60%） eyes,and 4 cases 4 （40%） eyes. Number and percentage of patients who were engaged in outdoor occupations were 6 cases 6 （60%） eyes,7 cases 7 （70%） eyes,and 3 cases 3 （30%） eyes. The three groups had no significant difference （X2 =0.88,1.09 ;P〉0.05 ）. Through cluster analysis on MicroRNA chip results, found the expression profile and control group was divided into a group of recurrence group MicroRNA, different from the primary group. The recurrent group and primary group were no correlation between the expression profile of MicroRNA （ r = 0. 57, P 〉 0. 05 ）, there is a correlation between the recurrence group and the control group （r = 0.67,P〈0.05）. Between the three groups of tissue microRNAs expression profile difference is MicroRNA chip detection, then using qRT-PCR technology to verify the results of microarray. With the primary group and control group, the recurrence group were significantly different from the MicroRNA spectra of the first two,there are significant differences of the expression of 24 microRNAs. There are 24 microRNAs significantly differentially expressed, and 14 microRNAs were up- regulated （ miR-126, -15b, -135b, -199b, -214, -301 a, -20a, -4 496, -93, -21, -29a, -181a, 146b, and -99b）, whereas 10 down-regulated （miR-4 687,4 279, -483,-296,-652,-622,-1,-3 127,-328,-361, and -4 533 ）. By Western blot analysis,it was found that the vascular endothelial growth factor（VEGF） pathway was significantly activated in the recurrent pterygium, and microRNAs may be involved in this process. Conclusion We demonstrated an association between microRNAs and pterygium recurrence,consistent with its known angiogenesis, epithelial-mesenchymal transition and increased fibroblastie production effects. Because the management of pterygium recurrence is often difficult, we suggest that microRNAs should be further studied as a potential treatment.
出处
《中华眼科医学杂志（电子版）》
2016年第1期24-30,共7页
Chinese Journal of Ophthalmologic Medicine(Electronic Edition)
基金
广东省科技计划项目(No.2013B021800185)
清远市科技计划项目(No.2012B011204013)
