超声微泡介导野生型P53联合RB94基因转染HXO-Rb44细胞的实验研究

发布时间：2018-10-24 12:55

【摘要】：目的：通过细胞实验研究超声微泡促进双基因联合转染（Rb94、P53基因）HXO-RB44细胞的研究，以探讨两种抑癌基因联合对视网膜母细胞瘤细胞的效果。初步探讨单独转染RB94基因后HXO-RB44细胞株上MICA、MICB配体的表达情况。方法： 1.构建重组质粒载体pVIVO1-p53， pVIVO1-Rb94，pVIVO1-p53-Rb94表达载体。 2.利用UTMD方法分别转染P53组、Rb94组、以及二者联合组处理HXO-Rb44细胞后，对视网膜母细胞瘤细胞生长活性的影响。 3.单独转染Rb94基因对HXO-Rb44细胞上NKG2D配体表达的影响。结果： 1.重新构建的载体内的目标基因序列与Rb94基因和P53基因序列完全一致。RT-PCR检测结果表明Rb94基因及P53基因分别得到了有效表达。 2.以连续波超声（参数：0.5W/cm2声强，辐照时间30s），，对HXO-Rb44细胞进行P53、Rb94、以及P53联合Rb94基因转染。pVIVO1-p53与pVIVO1-p53-Rb94转染组细胞418bp的wtp53基因强表达，显著高于空白组，pVIVO1组及pVIVO1-Rb94组（P0.05），说明携带有wtp53基因的pVIVO1-p53与pVIVO1-p53-Rb94组质粒已成功转染入细胞内，且目标基因mRNA得到有效表达。RT-PCR检测到pVIVO1-Rb94和pVIVO1-p53-Rb94转染组细胞581bp的RB94基因表达，其余3组则无该基因表达，说明携带有Rb94基因的pVIVO1-Rb94和pVIVO1-p53-Rb94质粒成功转染入细胞，且目标基因在mRNA水平稳定表达。检测HXO-Rb44细胞经外源基因转染后对其生长活性的影响。双质粒联合组经过超声爆破微泡处理后的MTT值明显小于其余各组。流式细胞仪检测细胞凋亡率结果显示pVIVO1-p53-Rb94组经超声+微泡处理后细胞早期凋亡率（35.3%±1.51%），明显高于其他各组。凋亡蛋白BAX的表达量联合转染组（0.322±0.028）较单独转染组显著较高。 3.单独转染组（只转RB94基因），WESTERNBLOT显示NKG2D配体：MICA、MICB蛋白表达均高于空白对照组及空质粒组，差异具有统计学意义（P值均＜0.01）。MICA、MICB蛋白在空白对照组和pEGFP-C1质粒组表达差异无统计学意义（P＞0.05）。结论:本实验采用超声爆破微泡作为促进基因转染的手段，以视网膜母细胞瘤细胞为对象分别进行抑癌基因P53、RB94以及P53联合RB94进行转染，检测了各组基因的表达，以及对视网膜母细胞瘤细胞生长活性的影响，并检测了单独转染Rb94基因对视网膜母细胞瘤细胞上两种NKG2D配体：MICA、MICB影响。实验结果提示P53联合RB94基因转染组对视网膜母细胞瘤细胞的抑制高于分别转染P53及分别转染RB94组。HXO-RB44细胞上MICA、MICB在单转RB94基因后有上调的作用。然而视网膜母细胞瘤的基因治疗尚处于起步阶段，且视网膜母细胞瘤细胞株上NKG2D配体上调的因素尚处需进一步探索，本研究仅在体外实验为视网膜母细胞瘤基因治疗的初步研究做了一定的探索，为进一步进行动物实验积累一定理论依据，最终用于临床尚需更加深入研究。
[Abstract]:Aim: to investigate the effects of two tumor suppressor genes on retinoblastoma cells by using ultrasound microbubbles to promote the transfection of Rb94,P53 gene into HXO-RB44 cells. To investigate the expression of MICA,MICB ligand on HXO-RB44 cell line after transfection of RB94 gene alone. Methods: 1. Construction of recombinant plasmid pVIVO1-p53, pVIVO1-Rb94,pVIVO1-p53-Rb94 expression vector. 2. The effects of UTMD transfection on the growth activity of retinoblastoma cells in P 53 group, Rb94 group and combined group were compared. Effect of Rb94 gene transfection on NKG2D ligand expression in HXO-Rb44 cells. Results: 1. The target gene sequence of the reconstructed vector was completely consistent with that of Rb94 gene and p53 gene. The results of RT-PCR analysis showed that Rb94 gene and p53 gene were effectively expressed. 2. By continuous wave ultrasound (parameters: 0.5W/cm2 intensity, irradiation time 30s), the HXO-Rb44 cells were transfected with P53 rb94 and p53 combined with Rb94 gene. The wtp53 gene of 418bp was strongly expressed in the pVIVO1-p53 and pVIVO1-p53-Rb94 transfection groups. Significantly higher than the blank group, pVIVO1 group and pVIVO1-Rb94 group (P0.05), indicating that the pVIVO1-p53 and pVIVO1-p53-Rb94 plasmid carrying wtp53 gene had been successfully transfected into the cells, and the target gene mRNA was effectively expressed. 581bp RB94 gene expression in pVIVO1-Rb94 and pVIVO1-p53-Rb94 transfected cells was detected by RT-PCR. The other three groups did not express the gene, indicating that the pVIVO1-Rb94 and pVIVO1-p53-Rb94 plasmids carrying Rb94 gene were successfully transfected into the cells, and the target gene was stably expressed at the mRNA level. The effect of exogenous gene transfection on the growth activity of HXO-Rb44 cells was detected. The MTT value of double plasmids combined group was significantly lower than that of other groups. The results of flow cytometry showed that the early apoptosis rate in pVIVO1-p53-Rb94 group was significantly higher than that in other groups (35.3% 卤1.51%). The expression of apoptotic protein BAX in co-transfection group (0.322 卤0.028) was significantly higher than that in single transfection group. The expression of NKG2D ligand: MICA,MICB protein was significantly higher in single transfection group than in blank control group and blank plasmid group (P < 0. 01). There was no significant difference in the expression of MICA,MICB protein between blank control group and pEGFP-C1 plasmid group (P > 0. 05). Conclusion: in this experiment, the tumor suppressor gene P53 / RB94 and p53 combined with RB94 were transfected into retinoblastoma cells by using ultrasound blasting microbubbles as a means to promote gene transfection, and the expression of each group of genes was detected. The effect of Rb94 gene transfection on the growth activity of retinoblastoma cells was also studied. Two NKG2D ligands on retinoblastoma cells: MICA,MICB were detected. The results suggested that the inhibition of retinoblastoma cells by p53 combined with RB94 gene transfection group was higher than that by p53 transfection and RB94 transfection group. MICA,MICB on HXO-RB44 cells was up-regulated after single transfer of RB94 gene. However, the gene therapy of retinoblastoma is still in its infancy, and the factors of up-regulation of NKG2D ligand in retinoblastoma cell line need to be further explored. In this study, only in vitro experiments for the preliminary study of retinoblastoma gene therapy have been made a certain exploration, for further animal experiments to accumulate a certain theoretical basis, the final clinical needs to be more in-depth research.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.72

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