首页|肾脏特异性ALR基因敲除小鼠模型的制备及ALR对肾纤维化的影响

肾脏特异性ALR基因敲除小鼠模型的制备及ALR对肾纤维化的影响

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目的 利用Cre/loxp基因敲除技术构建肾脏特异性肝再生增强因子(ALR)基因敲除小鼠,为研究ALR基因在肾脏中的生物学功能提供重要的动物模型,并初步探讨了肾脏特异性ALR敲除对小鼠肾脏纤维化的影响.方法 将ALRflox/flox小鼠与肾脏肾小管特异性表达Ggt1重组酶的小鼠进行交配和鉴定,筛选出子代中ALRflox/+/Ggt1-Cre小鼠,再与ALRflox/flox小鼠进行交配并鉴定,获得ALRflox/flox/Ggt1-Cre小鼠,将ALRflox/flox/Ggt1-Cre小鼠与ALRflox/flox/Ggt1-Cre小鼠进行交配,其子代基因型为ALRflox/flox/Ggt1-Cre的小鼠即为本实验所构建的肾脏特异性ALR敲除小鼠,ALRflox/flox小鼠即为对照小鼠.提取小鼠鼠尾基因组DNA,通过PCR鉴定子代小鼠的基因型;提取小鼠肾脏和肝脏中的总蛋白,利用Western blot技术和Real-Time PCR技术分别检验ALR在小鼠肾脏组织中总蛋白和mRNA的表达情况;使用免疫组化检测ALR在小鼠肾脏中的表达;对小鼠肾脏进行苏木精-伊红、PAS和Masson染色,分析组织病理变化;流式、免疫荧光、免疫组化、Real-Time PCR分别分析小鼠肾脏组织巨噬细胞浸润情况.结果 Real-Time PCR结果表明制备出的小鼠基因型符合ALRflox/flox/Ggt1-Cre;肾脏特异性ALR基因敲除小鼠肾脏组织中ALR蛋白的表达水平显著低于ALRflox/flox型小鼠;与对照组相比,肾脏特异性ALR基因敲除小鼠肾间质炎症细胞增加,肾小管基底膜断裂,刷状缘脱落更明显,小鼠间质纤维化明显;肾脏特异性ALR基因敲除后,小鼠肾脏组织巨噬细胞浸润增加,巨噬细胞M1向M2极化进程减缓.结论 本研究利用Cre/Loxp技术成功构建了肾脏特异性ALR基因敲除小鼠,为在动物水平上研究ALR基因在肾脏中的作用提供了重要的动物模型,且肾脏特异性敲除ALR后,促进了小鼠肾脏纤维化的发展.
Preparation of kidney-specific ALR knockout mice and the effect of ALR on renal tubulointerstitial fibrosis
Objective To generate a stable kidney-specific deletion of augmenter of liver regeneration(ALR)mice and provide an animal model for further studying the biological function of ALR in the kidney.In addition,we further explored the effect of ALR on renal tubulointerstitial fibrosis.Methods Mating and identification of ALRflox/+/Ggt1-Cre mice with ALRflox/flox mice was carried out,and the ALRflox/flox/Ggt1-Cre mice were screened.Mating and identification of ALRflox/flox/Ggt1-Cre mice with ALRflox/flox mice were carried out,and screening the ALRflox/flox/Ggt1-Cre mice was performed.The ALRflox/flox/Ggt1-Cre mouse was a kidney-specific ALR knockout mouse and the ALRflox/flox mouse was a control mouse.The mouse genotypes were identified by PCR;the expression of ALR protein and mRNA levels in mouse liver were detected by Western blotting and real-time PCR;ALR expression in mouse kidney was detected by immunofluorescence.The kidney tissue morphology of the mouse kidney was observed using hematoxylin-eosin staining,periodic acid Schiff's reaction and Masson stain.Flow cytometry,immunohistochemistry,immunofluorescence and real time PCR analyses were used to determine macrophage phenotype.Results PCR results indicate that mouse genotypes are consistent with ALRflox/flox/Ggt1-Cre.Compared with ALRflox/flox mice,ALRflox/flox/Ggt1-Cre mice showed lower levels of ALR mRNA and protein,more interstitial inflammatory cells in kidney tissue,broken basement membrane of the renal tubules,the brush-border damage and significant interstitial fibrosis.After the kidney-specific ALR gene was knocked out,macrophages infiltrated the kidney tissue o and the polarization process of macrophages from M1 to M2 was reduced.Conclusion In this study,the kidney-specific deletion of ALR mouse was successfully constructed using the Cre/Loxp technology,which providing an important animal model for further study of the role of ALR genes.And ALR deficiency promotes renal fibrosis in physiological conditions.

ALRKidney gene knockoutKidney fibrosisMacrophage polarization

黄丽利、马怡欣、谭方彦、杨鹏飞、刘杞、张玲、廖晓辉

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400010,重庆医科大学附属第二医院肾内科

400010,重庆医科大学附属第二医院病毒性肝炎研究所

肝再生增强因子 肾脏基因敲除 肾脏纤维化 巨噬细胞极化

2024

免疫学杂志
第三军医大学,中国免疫学会

免疫学杂志

CSTPCD
影响因子:0.704
ISSN:1000-8861
年,卷(期):2024.40(8)