摘要
目的观察青光眼滤过手术后滤过泡瘢痕内成纤维细胞中反义寡核苷酸(ASODN)对结缔组织生长因子(CTGF)的调节,为抗青光眼术后滤过泡区瘢痕形成提出新的解决思路。方法取兔眼小梁切除术后第5天的手术区结膜及板层巩膜行成纤维细胞的原代培养。培养的细胞根据干预方式的不同分为CTGFASODN-脂质体复合物组、CTGFASODN对照组、CTGF随机序列寡核苷酸(SCODN)-脂质体复合物组和空白对照组。利用脂质体介导,将经异硫氰酸荧光素(FITC)标记的CTGFASODN转染至兔抗青光眼滤过手术后滤过泡瘢痕组织成纤维细胞中,利用半定量逆转录-聚合酶链反应(RT-PCR)检测CTGFmRNA在成纤维细胞中的表达,采用Westernblot法检测CTGF蛋白的合成量,以相对灰度值为指标,观察CTGFASODN对CTGF在手术区结膜及板层巩膜行成纤维细胞中表达的抑制效果。结果组织块培养法培养的成纤维细胞在13～14d达到融合。CTGFASODN-脂质体复合物组转染6h后可见培养的细胞质中出现少量黄绿色荧光,12h后荧光依然存在;CTGFmRNA在成纤维细胞中表达的相对灰度值明显低于空白对照组(0.457±0.035vs0.790±0.037),差异有统计学意义(P<0.01);CTGF蛋白水平表达也呈相同趋势(0.177±0.022vs0.557±0.024)(P<0.01)。CTGFASODN对照组、CTGFSCODN-脂质体复合物组CTGF在成纤维细胞中的表达与空白对照组相比差异均无统计学意义(P>0.05)。结论脂质体能有效地将CTGFASODN转染进入体外培养的成纤维细胞;CTGF在青光眼滤过手术后的瘢痕形成过程中发挥重要作用;CTGFASODN能在脂质体介导下抑制CTGF在成纤维细胞中的表达。
Background The pathological proliferation of fibroblast is the primary cause of fail in filter after filtering surgery,and connective tissue growth factor （CTGF） plays important effect in scarring of conjunctival follicles.Objective Present study was to observe the influence of CTGF antisense oligonucleotide （ASODN） and discuss the effect of liposome-mediated CTGF ASODN in scaring after glaucoma filtering surgery.Methods The lamellar sclera tissue was harvested from filtering bleb area during the filtering surgery in 32 inbred line New Zealand white rabbits and primary culture was carried out using explants method in the fifth day after glaucoma filtering surgery.Cultured cells were assigned to CTGF ASODN＋liposome group,CTGF ASODN group,CTGF scrambled control oligodeoxynucleotide（SCODN）-liposome group and blank control group based on the different intervene ways.CTGF ASODN conjugated with isothiocyananate fluorescence was encapsulated by liposome and transfected into fibroblast.The distribution of CTGF ASODN labeled by fluorescein isothiocyanate （FITC） in cytoplasm was detected under the fluorescencemicroscope.Expression of CTGF protein and mRNA was assessed by reverse transcription PCR （RT-PCR） and Western-blot saperately.Results Cultured cells showed the shape of fibroblasts in 3-4 days and confluented in 13-14 days.A few fluorescence spots were found in cytoplasm at 6 hours but enhanced at 12 hours after transfection in CTGF ASODN＋liposome group.Little fluorescence spots were exhibited in cultured fibroblasts of CTGF ASODN group.Expression level of CTGF mRNA （mRNA index） was lower in CTGF ASODN＋liposome group compared with blank control group （0.457±0.035 vs 0.790±0.037）（P0.01）.Expression of CTGF protein was reduced in CTGF ASODN＋liposome group,shwoing a statistically significant difference in comparison with blank control group （0.177±0.022 vs 0.557±0.024）（P0.01）.No significant differences in expression levels of CTGF mRNA and protein were found between CTGF ASODN group and CTGF SCODN＋liposome group with blank control group （P0.05）.Conclusion Liposome can effectively transfect CTGF ASODN into fibroblast in vitro.CTGF plays an important role in scarring and wound contracture after filtering surgery.And CTGF ASODN can inhibit the expression of CTGF in fibroblast.
出处
《眼科研究》
CAS
CSCD
北大核心
2010年第10期937-940,共4页
Chinese Ophthalmic Research
基金
江西省卫生厅科技计划项目(20051022)
关键词
结缔组织生长因子
反义寡核苷酸
青光眼滤过手术
成纤维细胞
connective tissue growth factor
antisense oligonucleotides
glaucoma filtering surgery
fibroblast
