敲除TLR2基因对同种异体小鼠角膜移植排斥反应的影响

目的　探讨敲除Ｔｏｌｌ样受体２（ｔｏｌｌ-ｌｉｋｅｒｅｃｅｐｔｏｒ２，ＴＬＲ２）基因后是否抑制同种异体小鼠角膜排斥反应的发生。方法　以ＢＡＬＢ／ｃ为供体，各取３０只Ｃ５７ＢＬ／６和同背景ＴＬＲ２基因敲除鼠为受体行右眼角膜移植术，设为野生组（ＷＴ）和基因敲除组（ＫＯ）；取１９只Ｃ５７ＢＬ／６行右眼自体角膜移植术，为自体组（ＩＳＯ）；各取９只Ｃ５７ＢＬ／６和ＴＬＲ２基因敲除鼠分别设为野生对照组（ＷＴｃｏｎｔｒｏｌ）和基因敲除对照组（ＫＯｃｏｎｔｒｏｌ）。术后每周两次观察植片并记录免疫排斥的发生时间；术后１４ｄ，收集术眼同侧颈部淋巴结，行流式细胞术分析ＣＤ４＋Ｔ细胞百分比；收集术眼角膜，行免疫组织化学染色和实时荧光定量ＰＣＲ检测角膜干扰素（ｉｎｔｅｒ-ｆｅｒｏｎ，ＩＦＮ）-γ、ＴＬＲ２和ＭｙＤ８８的表达。结果　ＷＴ组和ＫＯ组小鼠角膜移植术后中位生存时间ＫＯ组（３５．０±３．８）ｄ长于ＷＴ组（２１．０±１．５）ｄ（Ｐ＜０．０５）。流式细胞术分析显示，ＷＴ组同侧颈部淋巴结ＣＤ４＋Ｔ细胞百分比较ＷＴｃｏｎｔｒｏｌ组明显增高（Ｐ＜０．０５），而ＩＳＯ和ＫＯ组相对于其对应的ｃｏｎｔｒｏｌ组（ＷＴ／ＫＯｃｏｎｔｒｏｌ）差异均无统计学意义（均为Ｐ＞０．０５）；免疫组织化学结果显示，ＩＳＯ和ＫＯ组间角膜ＩＦＮ-γ和ＭｙＤ８８分子表达无差异，但两者均低于ＷＴ组。ＫＯ组角膜几乎不表达ＴＬＲ２分子，ＩＳＯ组有微量表达，ＷＴ组表达明显增高；ＰＣＲ检测显示，ＩＳＯ和ＫＯ组角膜ＩＦＮ-γ和ＴＬＲ２ｍＲＮＡ相对表达量差异均无统计学意义（均为Ｐ＞０．０５），但两者均低于ＷＴ组（均为Ｐ＜０．０５）；ＫＯ组角膜ＭｙＤ８８ｍＲＮＡ相对表达量比ＩＳＯ组高（Ｐ＜０．０５），但两者均低于ＷＴ组（均为Ｐ＜０．０５）。结论　敲除ＴＬＲ２基因可以一定程度抑制同种异体小鼠角膜排斥反应的发生。

Variation of immune response after allogeneic corneal transplantation in TLR2 knock-out mice

Objective To explore whether toll-like receptor 2 ( TLR2 ) knock-out can attenuate immune rejection after allogeneic corneal transplantation. Methods BALB/c mice were used as donors.30 C57BL/6 mice and 30 TLR2 knock-out mouse were chosen as receptors to establish allograft corneal transplantation models, which were grouped as the wide type ( WT) mouse group and knock-out ( KO) mouse group. Another 19 C57BL/6 mice were performed isograft corneal transplantation and were termed as isograft ( ISO) group. Nine normal mice of wild-type C57BL/6 ( WT control) or TLR2 knock-out ( KO control) were respectively involved as negative controls. The edema and transparency were observed twice per week under slit lamp nucroscope after surgery and the rejection times were recorded according to Sonoda’ s criteria. At 14 days after transplantation , the frequencies of CD4 + T cells in ipsilateral cervical drairung lymph nodes were analyzed by flow cytometry, and the expression of IFN-7 , TLR2 and MyD88 were detected by immunohistochemical staining and qPCR. Results The median survival time ( MST) in KO group was ( 35. 0 +3. 8) days , while in WT group was ( 21. 0 + 1. 5 ) days, there was significant difference between WT and KO group (P < 0. 05 ) . Lymphoid CD4 + T cells percentage of WT group was significantly higher than that of WT control group ( P < 0. 05 ) . There was no statistical difference between ISO and WT control group , as well as KO and KO control group ( all P > 0. 05 ) . Immunohistochemistry staining revealed that IFN-7 and MyD88 expressed almost alike in ISO and KO group, but there was a significant decreasing in ISO and KO group compared with WT group. No TLR2 could be detected in KO group, while expressed weakly in ISO group, and increased in WT group. The mRNA expression of IFN-7 and MyD88 in ISO and KO group were nearly the same ( all P > 0. 05 ) ,but there was a marked decrease in ISO and KO group while compared with WT group ( P < 0. 05 ) . The MyD88 mRNA expression level in KO group was much higher than that in ISO group (P < 0. 05 ) , and both of them were lower than that in WT group ( all P < 0. 05 ) . Conclusion TLR2 knock-out may attenuate immune rejection after allogeneic corneal transplantation.