CAJ | 학술논문

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。目的：採用不同質量濃度的超順磁性氧化鐵-短髮夾RNA（ superparamagnetic iron oxide-short hairpin RNA ， SPIO-ShRNA）分子探針轉染卵巢癌SKOV3細胞，觀察細胞形態，檢測細胞的生物學行為，尋找最佳的轉染濃度。方法：將鐵濃度分彆為5、15、30、45、75、100 mg／L的探針轉染SKOV3細胞後，採用熒光倒置顯微鏡觀察探針進入細胞情況，普魯士藍染色及透射電鏡觀察細胞形態及細胞內鐵顆粒，原子吸收光譜儀法測定細胞內鐵含量，cell counting kit-8（CCK-8）檢測細胞活性，流式細胞術觀察細胞凋亡，Western blot 檢測細胞內錶皮生長因子受體（epidermal growth factor receptor，EGFR）蛋白的錶達，磁共振成像（magnetic resonance imaging，MRI）檢測細胞內信號彊度變化。結果：在5～30 mg／L鐵濃度範圍內，細胞攝取量隨探針鐵質量濃度增加而增大，噹探針鐵濃度為45 mg／L時，進入細胞達到飽和，標記率接近100％；細胞活性隨探針質量濃度的增加而降低，各實驗組細胞活性分彆為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組細胞活性未見明顯下降，差異無統計學意義（P＝0．475，P＝0．226，P＝0．068）；≥45 mg／L細胞活性明顯下降（P＜0．001）；探針濃度≥45 mg／L時，探針對SKOV3細胞EGFR蛋白錶達抑製率明顯增加，45 mg／L組蛋白錶達率為68．905％±3．510％，與5、15、30 mg／L組相比差異具有統計學意義（ P＜0．001，P＝0．001，P＝0．003， P均＜0．01）；MRI顯示隨著探針質量濃度增加，細胞信號彊度逐漸降低，45 mg／L組細胞信號彊度為165．55±4．92，信號彊度明顯降低，45 mg／L組與空白組（相同體積的燐痠鹽緩遲液）、正常細胞組（未標記的卵巢癌SKOV3細胞）、5、15、30 mg／L各組信號彊度相比，差異均有統計學意義（P均＜0．001）。結論：噹SPIO-ShRNA分子探針鐵濃度為45 mg／L時，可有效轉染併特異性抑製卵巢癌SKOV3細胞的錶達，能被MRI所檢測，初步證實具有診斷與治療的雙重作用。
목적：채용불동질량농도적초순자성양화철-단발협RNA（ superparamagnetic iron oxide-short hairpin RNA ， SPIO-ShRNA）분자탐침전염란소암SKOV3세포，관찰세포형태，검측세포적생물학행위，심조최가적전염농도。방법：장철농도분별위5、15、30、45、75、100 mg／L적탐침전염SKOV3세포후，채용형광도치현미경관찰탐침진입세포정황，보로사람염색급투사전경관찰세포형태급세포내철과립，원자흡수광보의법측정세포내철함량，cell counting kit-8（CCK-8）검측세포활성，류식세포술관찰세포조망，Western blot 검측세포내표피생장인자수체（epidermal growth factor receptor，EGFR）단백적표체，자공진성상（magnetic resonance imaging，MRI）검측세포내신호강도변화。결과：재5～30 mg／L철농도범위내，세포섭취량수탐침철질량농도증가이증대，당탐침철농도위45 mg／L시，진입세포체도포화，표기솔접근100％；세포활성수탐침질량농도적증가이강저，각실험조세포활성분별위94．626％±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／L조세포활성미견명현하강，차이무통계학의의（P＝0．475，P＝0．226，P＝0．068）；≥45 mg／L세포활성명현하강（P＜0．001）；탐침농도≥45 mg／L시，탐침대SKOV3세포EGFR단백표체억제솔명현증가，45 mg／L조단백표체솔위68．905％±3．510％，여5、15、30 mg／L조상비차이구유통계학의의（ P＜0．001，P＝0．001，P＝0．003， P균＜0．01）；MRI현시수착탐침질량농도증가，세포신호강도축점강저，45 mg／L조세포신호강도위165．55±4．92，신호강도명현강저，45 mg／L조여공백조（상동체적적린산염완충액）、정상세포조（미표기적란소암SKOV3세포）、5、15、30 mg／L각조신호강도상비，차이균유통계학의의（P균＜0．001）。결론：당SPIO-ShRNA분자탐침철농도위45 mg／L시，가유효전염병특이성억제란소암SKOV3세포적표체，능피MRI소검측，초보증실구유진단여치료적쌍중작용。
Objective:To explore the effects of superparamagnetic iron oxide-short hairpin RNA ( SPIO-ShRNA) dual functional molecular probes of different concentrations on morphology and biological beha -vior 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 gradual-ly.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, 30mg/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, and 30 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).Con-clusion: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 .