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目的:通过新兴的纳米粒子基因载体观察p21cip1基因对视网膜色素上皮(retinal pigment epithelium,RPE)细胞增殖的抑制情况。方法:制备p21cip1基因纳米粒子,将其转染到培养的人RPE细胞,通过免疫组织化学检测p21cip1蛋白的表达,流式细胞仪检测细胞周期的相关细胞量的变化。结果:p21cip1纳米粒子的DNA含量为3%,包封效率为78%,p21cip1基因在体内由于PLGA-PVA载体的保护作用,可以维持比质粒更长时间的有效期,减少质粒易被生物体内核酸酶降解的问题。流式细胞仪检测显示转染了目的基因的RPE细胞发生G1期阻滞,细胞增殖明显受到抑制。免疫组织化学检测结果显示转染了目的基因的RPE细胞p21cip1蛋白表达明显增强。结论:p21cip1基因有可能作为一个目的基因,借助新兴的基因载体纳米粒子用于抑制细胞增殖的基因治疗。
OBJECTIVE: To observe the inhibitory effect of p21cip1 gene on the proliferation of retinal pigment epithelium (RPE) cells through the emerging nanoparticle gene carrier. Methods: The p21cip1 gene nanoparticles were prepared and transfected into cultured human RPE cells. The expression of p21cip1 protein was detected by immunohistochemistry. The cell cycle related cell number was detected by flow cytometry. Results: The DNA content of p21cip1 nanoparticles was 3%, and the encapsulation efficiency was 78%. The p21cip1 gene could maintain the expiration date longer than the plasmid due to the protective effect of PLGA-PVA carrier in vivo, and reduce the plasmids susceptibility to in vivo nuclease The problem of degradation. The results of flow cytometry showed that G1 phase arrest occurred in RPE cells transfected with the target gene and cell proliferation was significantly inhibited. The results of immunohistochemistry showed that the expression of p21cip1 protein in RPE cells transfected with the target gene was significantly increased. CONCLUSION: The p21cip1 gene may serve as a gene of interest for the gene therapy of inhibiting cell proliferation with the help of novel gene-carrier nanoparticles.