视网膜内质网应激免疫组织化学抗体浓度选择的实验研究
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中国人民解放军联勤保障部队第九○○医院院内课题杰出青年培育专项(No.2018Q02); 福建省科技计划引导项目(No.2020Y0076); 福建省自然科学基金项目(No.2020J05282); 中国人民解放军南京军区福州总医院博士后课题(No.48678)


Experimental study on concentration selection of immunohistochemical antibodies for retinal endoplasmic reticulum stress
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the Outstanding Youth Training Project of the 900th Hospital of Joint Logistic of Support Force Chinese PLA(No.2018Q02); Science and Technology Project of Fujian Province(No.2020Y0076); Youth Project of Natural Science Foundation of Fujian Province(No.2020J05282); Post-doctoral Project of Fuzhou General Hospital of Nanjing Military Region of Chinese People's Liberation Army(No.48678)

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    摘要:

    目的:探索小鼠视网膜色素变性(RP)模型视网膜内质网应激免疫组织化学(IHC)染色抗体的最适浓度,为研究RP发病机制及干预措施提供相应的指标检测方法。

    方法:给予清洁级健康雄性C57BL/6J小鼠腹腔注射N-甲基-N-亚硝基脲(MNU,60mg/kg)制备RP小鼠模型,造模后第7d行视网膜电图(ERG)检测和苏木精-伊红(HE)染色验证造模成功,摘取眼球并采用IHC染色法检测视网膜内质网应激相关蛋白(IRE1、ATF6、PERK、GRP78、Caspase-12)表达情况。

    结果:IRE1、ATF6、PERK、GRP78、Caspase-12蛋白均在RP小鼠视网膜呈阳性表达,IRE1抗体浓度为1:1000、ATF6抗体浓度为1:500和1:1000(此抗体两种浓度阳性表达无差异,P>0.05)、PERK抗体浓度为1:1500、GRP78抗体浓度为1:200、Caspase-12抗体浓度为1:100时,阳性表达强度适宜,且与各自其余抗体浓度相应蛋白阳性表达均有差异(P<0.05)。

    结论:内质网应激相关蛋白抗体浓度IRE1为1:1000、ATF6为1:500和1:1000、PERK为1:1500、GRP78为1:200、Caspase-12为1:100时,其在小鼠RP模型视网膜IHC染色阳性表达程度较适宜。

    Abstract:

    AIM: To explore the optimal concentration of endoplasmic reticulum stress immunohistochemical(IHC)staining antibody in mouse retinitis pigmentosa(RP)model, which provides the corresponding index detection method for studying the pathogenesis and intervention measures of RP.

    METHODS: Clean male C57BL/6J mice were intraperitoneally injected with N-methyl-N-nitrosourea(MNU, 60mg/kg)to prepare RP mouse model. Electroretinogram(ERG)and hematoxylin-eosin(HE)staining were performed on 7d after modeling to verify the successful modeling. The expression of endoplasmic reticulum stress-related proteins(IRE1, ATF6, PERK, GRP78, Caspase-12)was detected by IHC staining.

    RESULTS: The following proteins, including IRE1, ATF6, PERK, GRP78 and Caspase-12, were positively expressed in retina of RP mouse. The optimal concentrations of the above proteins were as follows: IRE1 antibody concentration was 1:1000, ATF6 antibody concentration was 1:500 and 1:1000(with no difference in positive expression, P>0.05), PERK antibody concentration was 1:1500, GRP78 antibody concentration was 1:200 and Caspase-12 antibody concentration was 1:100, the proteins were well expressed at the above concentrations, and the positive expressions of corresponding proteins were different from those of other antibody concentrations(P<0.05).

    CONCLUSION: The optimal concentrations for IHC staining in different proteins of mouse RP models were as follows: the concentrations of endoplasmic reticulum stress-related protein antibodies were 1:1000 in IRE1, 1:500 and 1:1000 in ATF6, 1:1500 in PERK, 1:200 in GRP78, and 1:100 in Caspase-12.

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陈小红,梁婉娇,黄诗舒,等.视网膜内质网应激免疫组织化学抗体浓度选择的实验研究.国际眼科杂志, 2023,23(1):32-38.

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  • 收稿日期:2022-01-23
  • 最后修改日期:2022-12-08
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  • 在线发布日期: 2023-01-04
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