摘要
目的观察内皮素-1(ET-1)对人小梁网细胞(TMCs)细胞外基质纤维连接蛋白(FN)、Ⅰ型胶原(COLI)的影响。方法取死亡24h内尸眼小梁网组织,行TMCs原代和传代培养,并用FN、层黏连蛋白(LN)、神经元特异性烯醇化酶(NSE)和Ⅷ因子相关抗原(FⅧRAg)鉴定为人TMCs后,取传3代的人TMCs,给予不同浓度ET-1(10-9、10-8、10-7mol/L)孵育72h后,免疫荧光和Westernblot观察ET-1对TMCs中FN、COLI表达的影响;然后取传3代的人TMCs,分别给予10-7mol/LET-1、10-7mol/LET-1+10-7mol/LETA受体拮抗剂、10-7mol/LET-1+10-7mol/LETB受体拮抗剂后孵育72h,Westernblot观察ET受体拮抗剂对FN、COLI表达的影响。结果研究受到伦理委员会的批准,取材前征得供体家属的知情同意。培养的细胞形态多样,含少许色素颗粒,FN、LN、NSE染色均呈阳性反应,FⅧRAg呈阴性反应。人TMCs与10-9～10-7mol/LET-1共孵育后FN表达上调(F=18.41,P<0.05);与10-8mol/L、10-7mol/LET-1共孵育后COLI表达上调(F=39.81,P<0.05),并呈浓度依赖性,10-7mol/L时作用最明显;给予ETA受体拮抗剂后,与ET-1组相比,FN、COLI的表达均降低(qFN=7.213,P<0.05;qCOLI=6.187,P<0.05),但给予ETB受体拮抗剂后,FN、COLI的表达均无明显变化。结论 ET-1能够促进体外培养的人TMCs中FN、COLI的表达,其机制可能是通过激动ETA受体而非ETB受体发挥作用的。
Background Endothelin-1 （ET-1） has been proved to be a regulation factor for intraocular pressure （IOP） by playing role on the extracellular matrix of human trabecular meshwork cells （TMCs）. It has been reported that ET-1 plays an important role in extracellular matrix synthesis in many cells. Collagen type Ⅰ （ COL Ⅰ） and fibronectin （FN） are the main extracellular matrix in TMCs. Whether the increasing-IOP effect of ET-Ⅰ is related to COL Ⅰ and FN is unclear. Objective The present study was to observe the effect of ET-1 on the expressions of COL Ⅰ and FN in human TMCs. Methods Human trebecular meshwork tissue was obtained from the donor globes less than 24 hours post mortem. TMCs were primarily cultured and subcuhured in vitro and identified by FN, laminin （LM） , neuron-specific enolase （NSE） and factor VIH related antigen （FⅧIRag） staining. Cultured cells were co-ineubated with different concentrations of ET-Ⅰ （ 10^-9, 10^-8, 10^- 7 mol/L） for 72 hours respectively,and the effects of ET-1 on the expressions of the COL Ⅰ and FN in TMCs were evaluated by immunoinfluorescence and Western blot assay. The endothelin receptor antagonist （1×10^-7 mol/L ETA,1 × 10^-7 mol/L ETB） were added in DMEM/ F12 with 10^-7 mol/L ET-1 for 72 hours,and the influenees of ETA,ETB on the expressions of COL Ⅰ and FN in human TMCs were observed by Western blot analysis. This study was approved by Ethics Committee of this hospital. The oral informed consent was obtained from the family member of donor. Results Cultured cells presented the multiple shapes with less pigment and showed the positive responses for FN ,LN ,NSE staining and absent response for FⅧRag. The expression levels of FN and COL Ⅰ were significantly enhanced after treatment of ET-1 and appeared a concentration-dependent trend with the largest promoting- proliferation effeetiveness at the concentration of 10^-7 mol/L, showing statistically significant differences in different groups （FFN = 18.41 , P 〈 0.01 ;FCOLⅠ = 39.81 , P 〈 0.01 ）. After treatment of ETA antagonist, the expressions of COL I and FN in human TMCs were less than the ET-1 group （qFN = 6. 187, P 〈 0.05 ;qCOLⅠ = 6. 187 ,P 〈 0.05 ） , however, no a similar outcome was seen after treatment of ETB antagonist in compared with ET-1 group in expressions of COL Ⅰ and FN. Conclusion ET-1 can promote the expression of COL Ⅰ and FN of human TMCs in a dose-dependent manner in vitro. ETA antagonist instead of ETB antagonist can inhibit the effect of ET-1.
出处
《眼科研究》
CAS
CSCD
北大核心
2010年第5期421-425,共5页
Chinese Ophthalmic Research
