组织型谷氨酰胺转移酶反义寡核苷酸对体外培养牛眼小梁细胞纤维连接蛋白、层粘连蛋白及Ⅳ胶原表达的影响

Effects of tissue transglutaminase antisense oligonucleotides on the expressions of fibronectin,laminin and collagen-Ⅳ in bovine trabecular meshwork cells

目的观察组织型谷氨酰胺转移酶(tissuetransglutaminase,tTG)反义寡核苷酸(antisenseoligonucleotides,ASODN)对牛眼小梁细胞(bovinetrabecularmeshworkcells,BTMC)纤维连接蛋白(fibronectin,FN)、层粘连蛋白(laminin,LN)、Ⅳ胶原(collagen-Ⅳ,CA-Ⅳ)表达的影响。方法在脂质体介导下,将0.3μmol·L-1的tTG-ASODN转染BTMC,无血清DMEM培养基为空白对照组。24h后,制作细胞爬片,利用免疫组化SP染色法检测tTG-ASODN对BTMC表达FN、LN、CA-Ⅳ的影响。结果空白对照组和A-SODN转染组FN、LN和CA-Ⅳ免疫组化染色均为阳性。通过对细胞爬片进行图像统计学分析,与空白对照组相比,ASODN1和ASODN2组中FN、LN、CA-Ⅳ的表达均明显下降(P<0.01),而ASODN1和ASODN22组之间没有显著性差异(P>0.05)。结论进一步证明了tTG可以促进BTMC表达细胞外基质。通过tTG-ASODN抑制BTMC表达FN、LN、CA-Ⅳ,从而有望从基因水平上防治原发性开角型青光眼。

Objective To observe the effects of antisense oligonucleotides （ASODN） against tissue transglutaminase （tTG） on the expression of fibronectin（ FN ）, laminin （ LN ） and collagen-iv （ CA-IV ） in bovine trabecular meshwork cells （BTMC）. Methods Using lipofectemtne 2000 reagent as vector, 0.3μmol·L^-1 antisense tTG was transfected into BTMC, and serum-free DMEM was set as control. Twenty-four hours after the transfection, immunohistochemistry staining SP method was used to detect the effects of antisense tTG on the expression of FN, LN and CA-Ⅳ. Results Immunohtstochemical stainings of FN,LN and CA-Ⅳ were positive both in control group and ASODN transfection group. Compared with control group, the expressions of FN, LN and CA-Ⅳwere significantly decreased in ASODN1 and ASODN2 group （P〈0.01）. However, there was no significantly difference between the group of ASODN1 and ASODN2 （P 〉0.05）. Conclusion It is further confirmed that tTG can promote the expression of extracellular matrix in BTMC. Antisense tTG may be expected to treat primary open angle glaucoma at gene level through inhibiting the expressions of FN, LN and CA-Ⅳ