發(fā)布時間：2018-10-24 12:55

【摘要】：目的：通過細胞實驗研究超聲微泡促進雙基因聯(lián)合轉(zhuǎn)染（Rb94、P53基因）HXO-RB44細胞的研究，以探討兩種抑癌基因聯(lián)合對視網(wǎng)膜母細胞瘤細胞的效果。初步探討單獨轉(zhuǎn)染RB94基因后HXO-RB44細胞株上MICA、MICB配體的表達情況。 方法： 1.構(gòu)建重組質(zhì)粒載體pVIVO1-p53， pVIVO1-Rb94，pVIVO1-p53-Rb94表達載體。 2.利用UTMD方法分別轉(zhuǎn)染P53組、Rb94組、以及二者聯(lián)合組處理HXO-Rb44細胞后，對視網(wǎng)膜母細胞瘤細胞生長活性的影響。 3.單獨轉(zhuǎn)染Rb94基因?qū)XO-Rb44細胞上NKG2D配體表達的影響。 結(jié)果： 1.重新構(gòu)建的載體內(nèi)的目標基因序列與Rb94基因和P53基因序列完全一致。RT-PCR檢測結(jié)果表明Rb94基因及P53基因分別得到了有效表達。 2.以連續(xù)波超聲（參數(shù)：0.5W/cm2聲強，輻照時間30s），，對HXO-Rb44細胞進行P53、Rb94、以及P53聯(lián)合Rb94基因轉(zhuǎn)染。pVIVO1-p53與pVIVO1-p53-Rb94轉(zhuǎn)染組細胞418bp的wtp53基因強表達，顯著高于空白組，pVIVO1組及pVIVO1-Rb94組（P0.05），說明攜帶有wtp53基因的pVIVO1-p53與pVIVO1-p53-Rb94組質(zhì)粒已成功轉(zhuǎn)染入細胞內(nèi)，且目標基因mRNA得到有效表達。RT-PCR檢測到pVIVO1-Rb94和pVIVO1-p53-Rb94轉(zhuǎn)染組細胞581bp的RB94基因表達，其余3組則無該基因表達，說明攜帶有Rb94基因的pVIVO1-Rb94和pVIVO1-p53-Rb94質(zhì)粒成功轉(zhuǎn)染入細胞，且目標基因在mRNA水平穩(wěn)定表達。檢測HXO-Rb44細胞經(jīng)外源基因轉(zhuǎn)染后對其生長活性的影響。雙質(zhì)粒聯(lián)合組經(jīng)過超聲爆破微泡處理后的MTT值明顯小于其余各組。流式細胞儀檢測細胞凋亡率結(jié)果顯示pVIVO1-p53-Rb94組經(jīng)超聲+微泡處理后細胞早期凋亡率（35.3%±1.51%），明顯高于其他各組。凋亡蛋白BAX的表達量聯(lián)合轉(zhuǎn)染組（0.322±0.028）較單獨轉(zhuǎn)染組顯著較高。 3.單獨轉(zhuǎn)染組（只轉(zhuǎn)RB94基因），WESTERNBLOT顯示NKG2D配體：MICA、MICB蛋白表達均高于空白對照組及空質(zhì)粒組，差異具有統(tǒng)計學意義（P值均＜0.01）。MICA、MICB蛋白在空白對照組和pEGFP-C1質(zhì)粒組表達差異無統(tǒng)計學意義（P＞0.05）。 結(jié)論:本實驗采用超聲爆破微泡作為促進基因轉(zhuǎn)染的手段，以視網(wǎng)膜母細胞瘤細胞為對象分別進行抑癌基因P53、RB94以及P53聯(lián)合RB94進行轉(zhuǎn)染，檢測了各組基因的表達，以及對視網(wǎng)膜母細胞瘤細胞生長活性的影響，并檢測了單獨轉(zhuǎn)染Rb94基因?qū)σ暰W(wǎng)膜母細胞瘤細胞上兩種NKG2D配體：MICA、MICB影響。實驗結(jié)果提示P53聯(lián)合RB94基因轉(zhuǎn)染組對視網(wǎng)膜母細胞瘤細胞的抑制高于分別轉(zhuǎn)染P53及分別轉(zhuǎn)染RB94組。HXO-RB44細胞上MICA、MICB在單轉(zhuǎn)RB94基因后有上調(diào)的作用。然而視網(wǎng)膜母細胞瘤的基因治療尚處于起步階段，且視網(wǎng)膜母細胞瘤細胞株上NKG2D配體上調(diào)的因素尚處需進一步探索，本研究僅在體外實驗為視網(wǎng)膜母細胞瘤基因治療的初步研究做了一定的探索，為進一步進行動物實驗積累一定理論依據(jù)，最終用于臨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% 鹵1.51%). The expression of apoptotic protein BAX in co-transfection group (0.322 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.72

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