亨廷顿蛋白相关蛋白1基因对小鼠成纤维细胞增殖的影响

曹媛媛; 杨桂枝; 龚艳菊; 赵佳; 冯颖; 郑翔; 周雪

亨廷顿蛋白相关蛋白1基因对小鼠成纤维细胞增殖的影响

Effect of Huntingtin-associated Protein 1 Gene on Proliferation of Mouse Fibroblasts

摘要

摘要:
目的研究亨廷顿蛋白相关蛋白1（Huntingtin-associated protein 1，Hap1）基因对成纤维细胞增殖的影响。
方法体外分离培养获得Hap1基因敲除（Hap1^-/-）的原代成纤维细胞，鉴定后采用EdU增殖实验、细胞周期（流式细胞术）检测验证Hap1成纤维细胞增殖的改变；将野生型和Hap1^-/-成纤维细胞送转录组测序筛选增殖相关基因，实时荧光定量PCR（qPCR）验证相关基因表达水平的改变。对小鼠进行皮肤损伤造模检测Hap1敲减（Hap1^+/-）小鼠皮肤损伤修复情况；PCNA免疫组化染色检测损伤修复过程中成纤维细胞的增殖水平。
结果成功培养原代Hap1^-/-成纤维细胞；与野生型成纤维细胞相比，Hap1^-/-成纤维细胞EdU阳性比例减少，进入S期的细胞比例减少；原代成纤维细胞转录组测序筛选出Cdc25C、E2f7、E2f8和Ccl5四个差异表达的增殖相关基因，qPCR验证发现E2f7在Hap1敲除后表达增多。小鼠皮肤损伤结果显示，Hap1^+/-小鼠伤口面积较相同时间点野生型小鼠伤口面积大且愈合速度减慢，成纤维细胞增殖阳性密度低于野生型小鼠。
结论 Hap1可能通过抑制细胞周期负性调节因子E2f7的表达对成纤维细胞增殖起正向调节作用，其缺失将抑制成纤维细胞进入S期，从而减少细胞增殖，影响伤口修复。

Abstract:
Objective To determine the effect of Huntingtin-associated protein 1 (Hap1) on fibroblast proliferation.
Methods Hap1 knockout (Hap1^-/-) primary fibroblasts were isolated and cultured in vitro. The proliferation of Hap1^-/- fibroblasts was detected by EdU proliferation assay and cell flow assay. Transcriptome sequencing of the wild-type and Hap1^-/- fibroblasts was screened for proliferation-related genes. Real-time quantitative PCR (qPCR) was performed to verify changes in expressions of related genes. Skin repair was examined in Hap1 knockdown mice with skin wounds. The proliferation of fibroblasts during wound repair was detected by PCNA immunohistochemical staining.
Results Hap1^-/- fibroblasts were successfully cultured. Compared with WT, EdU-positive fibroblasts decreased in Hap1^-/-,with less cells entering the S phase. Transcriptome sequencing of primary fibroblasts identified genes of Cdc25C, E2f7, E2f8 and Ccl5. qPCR confirmed that Hap1 knockout increased E2f7 expression. Hap1^+/- mice had larger skin lesions, slower healing and lower positive density of fibroblast proliferation than those of wild type mice.
Conclusion Hap1 may positively regulate fibroblast proliferation by inhibiting the expression of cell cycle negative regulator E2f7.Its deletion inhibits fibroblasts entering the S phase, thereby reducing cell proliferation and affecting wound repair.

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