血红素氧合酶-CO-cGMP通路在体外培养人眼小梁细胞中的表达

Existence of heme oxygenase-carbon monoxide-cyclic guanosine monophosphate pathway in human trabecular meshwork cells in vitro

目的探讨体外培养人眼小梁细胞是否存在血红素氧合酶(HO)-一氧化碳(CO)-环磷酸鸟苷(cGMP)通路以及氯化血红素(hemin)对其的诱导作用。方法应用RTPCR和免疫组织化学方法检测体外培养人眼小梁细胞中HO1和HO2mRNA及蛋白的表达。向细胞培养液中加入氯化血红素,RTPCR检测小梁细胞HO1mRNA,分光光度法检测培养上清液中碳氧血红蛋白(HbCO)浓度,并用放射免疫法检测细胞中cGMP浓度。结果体外培养人眼小梁细胞存在HO1和HO2mRNA及蛋白。氯化血红素对小梁细胞HO1mRNA、HbCO和cGMP浓度的诱导呈浓度依赖性。结论人眼小梁细胞存在HOCOcGMP通路,并受氯化血红素诱导。药理诱导这一通路,可能为青光眼的治疗提供一个有效的新方法。

Objective To identify the expression of heme oxygenase （HO）-carbon monoxide （CO）-cyclic guanosine monophosphate（cGMP） pathway in the cultured human trabecular meshwork cells （HTMC） in vitro and evaluate the cGMP activation by hemin. Methods HTMC of the third to fourth generation were used in the study. 0. 1,1,10 and 100 μmol/L of hemin was added in B,E, D, C group separately and equal amount of saline was served as control in the A group for 6 hours. Reverse transcripase - polymerase chain reaction （RT-PCR） was employed for the detection of HO-1 or HO-2 mRNA. Immunohistochemical stain was used to detect HO-lor HO-2 protein. The relative amounts of carbon monoxide releasing into the media were measured with the quantifying carbon monoxide hemoglobin （HbCO） by spectrophotometry. Radioimmunoassay was used to determine the changes of cGMP in HTMC. Results Cultured cells had the specific characteristic of HTMC. Genes and proteins of HO-1 and HO-2 were stained as brown immunohistochemically in the cytoplasm of cultured HTMC. The expression level of HO-1 mRNA was 0. 941 ±0. 072,1. 383 ±0. 107,1. 875 ±0. 093 and 2. 150 ±0. 138 in B,E,D,C group respectively with a statistically significant difference in intergroup and remarkable difference in comparison with the A group（0. 702 ± 0. 065）. The level of HbCO in B,E,D,C group was 0.32 ± 0.02,0.37 ± 0.02,0.45 ± 0.03 and 0.54 ± 0.03 respectively, showing a significantly intergroup difference and a statistical difference compared to the A group （ 0. 27 ± 0.02 ）. The level of cGMP in HTMC was 0. 582 ±0. 116,0. 817 ± 0. 124,1. 364 ±0. 152 and 2. 193 ±0. 145 in B,E,D,C group and that of A group was 0. 331 ± 0. 082 pmol/mg, pro with a significant difference among them. Conclusion Hemin induces the expression of HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. HO-CO-cGMP pathway is existed in the cultured HTMC. Pharmacological stimulation of HO-CO-cGMP pathway may provide a novel therapeutic approach to rescuing glaucoma.