G蛋白偶联受体107的缺失对HaCaT细胞生物学行为的影响  

Effect of deletion of G protein-coupled receptor 107 on the biological behaviour of HaCaT cells

作　　者：汪璟 赵韦 徐德苹 廖开楠 臧丹丹 周海胜[1,2] Wang Jing;Zhao Wei;Xu Deping;Liao Kainan;Zang Dandan;Zhou Haisheng(Dept of Biochemistry,Anhui Medical University,Hefei 230032;Center Scientific Research,Anhui Medical University,Hefei 230032)

机构地区：[1]安徽医科大学生物化学教研室,合肥230032 [2]安徽医科大学科研实验中心,合肥230032

出　　处：《安徽医科大学学报》2025年第3期385-391,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:82071832);安徽省高校学科(专业)拔尖人才学术资助项目(编号:gxbjZD2021046)。

摘　　要：目的构建稳定敲除G蛋白偶联受体107(GPR107)基因的人类角质形成细胞系(HaCaT),并初步探究GPR107的缺失对HaCaT细胞生物学行为的影响。方法利用CRISPR/Cas9基因编辑技术,构建敲除GPR107基因的HaCaT细胞,并通过有限稀释法获得GPR107缺失的单克隆细胞;利用Western blot和PCR扩增基因组DNA并测序验证验证敲除GPR107的单细胞克隆。运用流式细胞分析术检测细胞周期变化;利用CCK-8检测细胞增殖;运用流式细胞术检测细胞凋亡水平;运用Western blot检测细胞分化标记分子的变化;运用细胞划痕实验等方法分析细胞迁移能力。结果成功将LentiCas9-Blast与plenti-guide-RNA-GPR107质粒转染进HaCaT细胞,并通过有限稀释法得到了21株单克隆细胞,Western blot显示其中8株细胞GPR107表达显著降低;进一步采用细胞基因组PCR测序,显示获得了C4与2D82株GPR107-/-HaCaT单克隆细胞株。CCK-8检测、流式细胞术检测显示GPR107基因缺失导致HaCaT细胞出现G0G1期阻滞,增殖能力显著减弱,凋亡水平增加;Western blot显示敲除GPR107后HaCaT细胞分化加快;细胞划痕实验结果表明敲除GPR107后HaCaT细胞迁移能力增强。结论成功构建GPR107基因缺失的HaCaT细胞株;GPR107缺失阻滞HaCaT细胞G0G1期从而抑制了HaCaT细胞增殖并促进细胞凋亡,敲除GPR107后HaCaT细胞分化能力与迁移能力均增强。Objective To construct a human keratinocyte-forming cell line(HaCaT)with stable knockout of the G protein-coupled receptor 107(GPR107)gene,and to preliminarily investigate the effect of GPR107 deletion on the biological behaviour of HaCaT cells.Methods Using CRISPR/Cas9 gene editing technology,HaCaT cells with knockout of GPR107 gene were constructed and monoclonal cells with GPR107 deletion were obtained by limited dilution method.Genomic DNA was amplified using Western blot and PCR and sequenced to validate the single-cell clones with knockdown of GPR107.The cell cycle changes were detected by flow cytometry;cell proliferation was detected by CCK-8;apoptosis was detected by flow cytometry;changes in cell differentiation markers were detected by Western blot;cell migration ability was analyzed by cell scratch assay and other methods.Results LentiCas9-Blast and plenti-guide-RNA-GPR107 plasmids were successfully transfected into HaCaT cells,21 monoclonal cell lines were obtained by limited dilution,and Western blot showed that the GPR107 expression was significantly reduced in 8 of them;PCR sequencing of the cellular genome was used,which resulted in the obtainment of C4 and 2D8 GPR107-/-HaCaT monoclonal cell lines.CCK-8 assay and flow cytometry assay showed that GPR107 gene deletion resulted in G 0G 1 phase block,significantly weakened proliferation ability and increased apoptosis level of HaCaT cells.Western blot found that the differentiation of HaCaT cells accelerated after knockdown of GPR107.Additionally the results of the cell scratch assay indicated that the migration ability of HaCaT cells was enhanced after knockdown of GPR107.The results showed that the migration ability of HaCaT cells was enhanced after knockdown of GPR107.Conclusion HaCaT cell line with GPR107 gene deletion is successfully constructed,GPR107 deletion blocks the G 0G 1 phase of HaCaT cells,which inhibiting the proliferation of HaCaT cells and promoted apoptosis,and it was found that the differentiation and migration of HaCaT cells were

关 键 词：G蛋白偶联受体107 人类角质形成细胞 CRISPR/Cas9 细胞增殖 细胞周期 

分 类 号：R34[医药卫生—基础医学]