人源pEGFP-C1-p38γ真核表达质粒的构建及其功能研究

Construction of eukaryotic expression of human derived pEGFP-C1-p38γ plasmids and its function research

目的构建人源pEGFP-C1-p38γ表达质粒,并观察其对乙醇刺激的L0-2肝细胞的增殖和凋亡及炎症因子分泌的影响。方法在人源L0-2肝细胞中提取RNA并且逆转录为cDNA,同时将引物稀释到10μmol/L,其余作为储液备用。采用PCR技术扩增p38γ并鉴定,使用AxyPrep DNA凝胶回收试剂盒进行PCR产物的纯化回收,再对PCR产物及载体进行酶切回收,酶切片段连接后,进行连接产物的转化、挑菌、摇菌、质粒小抽。酶切鉴定后,进行测序鉴定。将构建好的质粒转染至乙醇刺激的人源L0-2细胞中,通过MTT实验和流式细胞术检测对其增殖和凋亡的影响,并用Western blot技术检测炎症因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)在L0-2肝细胞中的表达。结果测序显示pEGFP-C1-p38γ真核表达质粒构建成功。同时,MTT实验显示,乙醇刺激L0-2细胞24 h后p38γ过表达组细胞的增殖率为(0.42±0.08)%,低于转染空载体的对照组(0.60±0.03)%;流式细胞术检测结果显示,p38γ过表达组细胞的凋亡率为(17.46±1.52)%,高于转染空载体质粒的对照组(13.18±1.34)%;Western blot检测显示p38γ过表达组中的IL-6和TNF-α表达较对照组升高。结论 p38γ能够抑制乙醇刺激的L0-2肝细胞的增殖,并促进其凋亡。同时,p38γ能够促进乙醇刺激后的L0-2肝细胞中IL-6和TNF-α的表达。

Objective To construct a human derived pEGFP-C1-p38γ expression plasmid and observe its expression of inflammatory factors in alcohol-induced L0-2 cells and its effect on cell proliferation and apoptosis. Methods RNA was extracted from L0-2 cells and reversely transcribed into cDNA. At the same time, the primers were diluted to 10 μmol/L and the rest were used as storage fluid. P38γ was amplified by PCR technology and identified. The DNA gel recovery kit AxyPrep was used for the purification and recovery of PCR products. Then the PCRproducts and carriers were recovered by enzyme digestion. After enzyme digestion and identification, they were sent to sequencing for identification and sequencing. The constructed plasmids were transfected into alcohol-induced L0-2 cells of human mononuclear macrophage cell line. The effects on cell proliferation and apoptosis were detected by MTT assay and flow cytometry, and the expressions of inflammatory cytokines IL-6 and TNF-α in alcohol-induced L0-2 cells were detected by Western blot. Results Sequencing results showed that pEGFP-C1-p38γ eukaryotic expression plasmid was successfully constructed. The results of MTT assay showed that 24 h later, the cell proliferation rate of the p38γ overexpressed group(0.42±0.08) % was significantly lower than that of the control group(0.60±0.03)%. The results of flow cytometry showed that the apoptosis rate of p38γ overexpressed cells group was(17.46±1.52) %, significantly higher than that in the control group(13.18±1.34) %(P<0.05). Western blot result showed that the expression of IL-6 and TNF-α in L0-2 cells transfected with pEGFP-C1-p38γ plasmid was higher than that in the control group. Conclusion P38γ can significantly inhibit the proliferation and promote the apoptosis of alcohol-induced L0-2 liver cells. P38γ promotes the expression of inflammatory cytokines IL-6 and TNF-α in alcohol-induced L0-2 cells.