超顺磁性氧化铁-短发夹RNA双功能分子探针体外转染卵巢癌细胞的最佳浓度

Optimal concentration of superparamagnetic iron oxide-short hairpin RNA dual functional molecular probe transfected into ovarian cancer cells in vitro

目的：采用不同质量浓度的超顺磁性氧化铁-短发夹RNA（superparamagnetic iron oxide-short hairpin RNA,SPIO-ShRNA）分子探针转染卵巢癌SKOV3细胞,观察细胞形态,检测细胞的生物学行为,寻找最佳的转染浓度。方法：将铁浓度分别为5、15、30、45、75、100 mg/L的探针转染SKOV3细胞后,采用荧光倒置显微镜观察探针进入细胞情况,普鲁士蓝染色及透射电镜观察细胞形态及细胞内铁颗粒,原子吸收光谱仪法测定细胞内铁含量,cell counting kit-8（CCK-8）检测细胞活性,流式细胞术观察细胞凋亡,Western blot检测细胞内表皮生长因子受体（epidermal growth factor receptor,EGFR）蛋白的表达,磁共振成像（magnetic resonance imaging,MRI）检测细胞内信号强度变化。结果：在5∽30 mg/L铁浓度范围内,细胞摄取量随探针铁质量浓度增加而增大,当探针铁浓度为45 mg/L时,进入细胞达到饱和,标记率接近100%;细胞活性随探针质量浓度的增加而降低,各实验组细胞活性分别为94.626%±1.050%、93.373%±1.180%、91.700%±3.122%、75.100%±4.362%、72.983%±3.233%、71.010%±2.910%,5、15、30 mg/L组细胞活性未见明显下降,差异无统计学意义（P=0.475,P=0.226,P=0.068）;≥45 mg/L细胞活性明显下降（P〈0.001）;探针浓度≥45 mg/L时,探针对SKOV3细胞EGFR蛋白表达抑制率明显增加,45 mg/L组蛋白表达率为68.905%±3.510%,与5、15、30 mg/L组相比差异具有统计学意义（P〈0.001,P=0.001,P=0.003,P均〈0.01）;MRI显示随着探针质量浓度增加,细胞信号强度逐渐降低,45 mg/L组细胞信号强度为165.55±4.92,信号强度明显降低,45 mg/L组与空白组（相同体积的磷酸盐缓冲液）、正常细胞组（未标记的卵巢癌SKOV3细胞）、5、15、30 mg/L各组信号强度相比,差异均有统计学意义（P均〈0.001）。结论：当SPIO-ShRNA分子探针铁浓度为45 mg/L时,可有效转染并特异性抑制卵巢癌SKOV3细胞的表达,能被MRI所检测,初步证实具有诊断与治疗的双重作用。

Objective： To explore the effects of superparamagnetic iron oxide-short hairpin RNA（ SPIOShRNA） dual functional molecular probes of different concentrations on morphology and biological behavior of ovarian cancer SKOV3 cells in vitro. Methods： The dual functional molecular probes at an iron concentration of 5,15,30,45,75,and 100 mg / L were transfected into SKOV3 cells. The transfection rate of the probe was observed by fluorescence microscope. The distribution and content of iron particles in SKOV3 cells were determined by Prussian blue staining,atomic adsorption spectrometer and electron microscopy. Cell viability was observed by cell counting kit-8（ CCK-8）. The apoptosis was detected by flow cytometry. The expression of protein within the cells was detected by Western blot. The changes of the signal intensity were measured by magnetic resonance imaging（ MRI）. Results： The SPIO-ShRNA dual functional molecular probe was uptaken in aconcentration-dependence manner within a certain range（ 5- 30 mg /L）. When the concentration of the probe was 45 mg /L,the labeling rate of the cell was close to 100%; With the increase of the concentration of probe,the cell survival rate decreased gradually. The cell survival rate of each experimental group were 94. 626% ± 1. 050%,93. 373% ± 1. 180%,91. 700% ± 3. 122%,75. 100% ± 4. 362%,72. 983% ± 3. 233%,71. 010% ± 2. 910%,5,15,30 mg / L cell survival rate was not significantly decreased,the difference was not statistically significant（ P =0. 226,P = 0. 068,P = 0. 475）; When the concentration of the probe was greater than or equal to 45 mg / L,the survival rate decreased obviously（ P〈0. 001）; Group of 45 mg / L protein expression rate was 68. 905% ±3. 510%,When the concentration of the probe was greater than or equal to 45 mg / L,the inhibition rate of the protein expression level of epidermal growth factor receptor was obviously higher than those of 5,15,and30 mg / L groups,the difference was statistically significant（ P〈0. 001,P = 0. 001,P = 0. 003,all P〈0. 01）; the MRI displayed that the signal intensity was decreased with increasing concentrations of the probe. The signal intensity of 45 mg / L group was 165. 55 ± 4. 92,compared with the blank control group（ same volume of phosphate buffer saline）,normal group（ unlabeled ovarian cancer SKOV3 cells）,5,15,and 30 mg / L groups,the signal intensity of 45 mg / L group decreased significantly（ all P〈0. 001）. Conclusion： The dual functional molecular probe can effectively transfect and specifically inhibit the expression of SKOV3 cell lines at the iron concentration of 45 mg / L,and can also be detected by MRI. The role of diagnosis and treatment of the dual functional molecular probe has been initially confirmed.