【摘要】：目的：通過細(xì)胞實(shí)驗(yàn)研究超聲微泡促進(jìn)雙基因聯(lián)合轉(zhuǎn)染（Rb94、P53基因）HXO-RB44細(xì)胞的研究，以探討兩種抑癌基因聯(lián)合對(duì)視網(wǎng)膜母細(xì)胞瘤細(xì)胞的效果。初步探討單獨(dú)轉(zhuǎn)染RB94基因后HXO-RB44細(xì)胞株上MICA、MICB配體的表達(dá)情況。 方法： 1.構(gòu)建重組質(zhì)粒載體pVIVO1-p53， pVIVO1-Rb94，pVIVO1-p53-Rb94表達(dá)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(xì)胞后，對(duì)視網(wǎng)膜母細(xì)胞瘤細(xì)胞生長活性的影響。 3.單獨(dú)轉(zhuǎn)染Rb94基因?qū)XO-Rb44細(xì)胞上NKG2D配體表達(dá)的影響。 結(jié)果： 1.重新構(gòu)建的載體內(nèi)的目標(biāo)基因序列與Rb94基因和P53基因序列完全一致。RT-PCR檢測(cè)結(jié)果表明Rb94基因及P53基因分別得到了有效表達(dá)。 2.以連續(xù)波超聲（參數(shù)：0.5W/cm2聲強(qiáng)，輻照時(shí)間30s），，對(duì)HXO-Rb44細(xì)胞進(jìn)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(xì)胞418bp的wtp53基因強(qiáng)表達(dá)，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(xì)胞內(nèi)，且目標(biāo)基因mRNA得到有效表達(dá)。RT-PCR檢測(cè)到pVIVO1-Rb94和pVIVO1-p53-Rb94轉(zhuǎn)染組細(xì)胞581bp的RB94基因表達(dá)，其余3組則無該基因表達(dá)，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(xì)胞，且目標(biāo)基因在mRNA水平穩(wěn)定表達(dá)。檢測(cè)HXO-Rb44細(xì)胞經(jīng)外源基因轉(zhuǎn)染后對(duì)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(xì)胞儀檢測(cè)細(xì)胞凋亡率結(jié)果顯示pVIVO1-p53-Rb94組經(jīng)超聲+微泡處理后細(xì)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(dá)量聯(lián)合轉(zhuǎn)染組（0.322±0.028）較單獨(dú)轉(zhuǎn)染組顯著較高。 3.單獨(dú)轉(zhuǎn)染組（只轉(zhuǎn)RB94基因），WESTERNBLOT顯示NKG2D配體：MICA、MICB蛋白表達(dá)均高于空白對(duì)照組及空質(zhì)粒組，差異具有統(tǒng)計(jì)學(xué)意義（P值均＜0.01）。MICA、MICB蛋白在空白對(duì)照組和pEGFP-C1質(zhì)粒組表達(dá)差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 結(jié)論:本實(shí)驗(yàn)采用超聲爆破微泡作為促進(jìn)基因轉(zhuǎn)染的手段，以視網(wǎng)膜母細(xì)胞瘤細(xì)胞為對(duì)象分別進(jìn)行抑癌基因P53、RB94以及P53聯(lián)合RB94進(jìn)行轉(zhuǎn)染，檢測(cè)了各組基因的表達(dá)，以及對(duì)視網(wǎng)膜母細(xì)胞瘤細(xì)胞生長活性的影響，并檢測(cè)了單獨(dú)轉(zhuǎn)染Rb94基因?qū)σ暰W(wǎng)膜母細(xì)胞瘤細(xì)胞上兩種NKG2D配體：MICA、MICB影響。實(shí)驗(yàn)結(jié)果提示P53聯(lián)合RB94基因轉(zhuǎn)染組對(duì)視網(wǎng)膜母細(xì)胞瘤細(xì)胞的抑制高于分別轉(zhuǎn)染P53及分別轉(zhuǎn)染RB94組。HXO-RB44細(xì)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(xì)胞瘤的基因治療尚處于起步階段，且視網(wǎng)膜母細(xì)胞瘤細(xì)胞株上NKG2D配體上調(diào)的因素尚處需進(jìn)一步探索，本研究僅在體外實(shí)驗(yàn)為視網(wǎng)膜母細(xì)胞瘤基因治療的初步研究做了一定的探索，為進(jìn)一步進(jìn)行動(dòng)物實(shí)驗(yàn)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.72

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