MPA、MG132对不同PR表达卵巢癌细胞增殖、凋亡影响的实验研究

发布时间：2018-04-27 22:12

本文选题：卵巢癌 + 孕激素　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:探讨醋酸甲羟孕酮(medroxyprogesterone acetate,MPA)、蛋白酶体抑制剂(Z-LLL-CHO,MG132)单独及联合应用对不同PR表达卵巢癌细胞增殖、凋亡的影响及相关机制,为卵巢癌的临床治疗提供新的实验依据。方法:选取人卵巢癌细胞系HO-8910(PR高表达)、SKOV3(PR低表达)为研究对象。(1)不同浓度的MPA(0、1、20、50、100μmol/L)、MG132(0、1、2.5、5、10μmol/L)单独干预24-72 h,分别于24 h、48 h、72 h采用CCK8法检测两种细胞的增殖情况。(2)取呈对数生长的两种细胞分为:空白对照组(0μmol/L)、MG132组(5μmol/L)、MPA组(100μmol/L)、MG132+MPA组(5+100μmol/L),均作用24 h:a、采用CCK8法检测两种细胞的增殖情况;b、倒置显微镜观察细胞的形态学变化并摄片;c、采用Annexin V-FITC/PI双染法通过流式细胞仪检测药物作用前后细胞的凋亡情况;d、利用免疫组化法(Immunohistochemistry,IHC)检测药物作用前后PR的表达情况;e、利用免疫印迹法(Westeron blot,WB)检测药物作用前后孕激素受体亚型PRA、PRB的表达情况。结果:(1)CCK8结果显示:MPA组(不同浓度)的HO-8910细胞存活率(%)分别为:(作用24 h)100.00±5.47、101.65±5.24、86.52±0.89、77.38±0.94、63.05±3.60;(作用48 h)100.00±0.94、104.45±0.37、71.06±3.22、55.70±0.74、48.08±0.08;(作用72 h)100.00±0.38、105.52±2.15、58.63±0.21、43.30±0.57、29.43±0.39。MPA组(不同浓度)的SKOV3细胞存活率(%)分别为:(作用24 h)100.00±3.46、101.53±3.3、95.39±2.02、89.15±2.51、84.49±2.29;(作用48 h)100.00±2.70、99.58±0.29、89.22±1.67、86.37±0.22、78.44±5.08;(作用72 h)100.00±3.59、103.22±6.12、87.21±3.32、78.68±3.30、65.56±0.79。20-100μmol/L的MPA对HO-8910细胞的增殖抑制作用明显强于SKOV3细胞且细胞存活率均呈剂量-效应和处理时间-效应依赖性下降。MG132组(不同浓度)的HO-8910细胞的存活率(%)分别为:(作用24 h)100.00±1.30、98.73±1.09、83.29±0.43、71.36±3.10、66.54±8.33;(作用48 h)100±0.06、89.35±2.05、73.39±1.86、59.46±0.69、47.40±0.98;(作用72 h)100±2.70、79.67±2.80、54.72±3.56、48.42±0.20、34.90±0.25。MG132组(不同浓度)的SKOV3细胞的存活率(%)分别为:(作用24 h)100.00±4.58、94.39±4.33、86.12±3.18、75.73±2.57、61.41±1.11;(作用48 h)100.00±3.40、80.18±2.73、62.11±3.89、51.76±3.99、46.97±1.75;(作用72 h)100.00±0.35、62.86±8.79、62.86±8.79、43.39±2.10、37.36±0.36、29.18±1.62。1-10μmol/L的MG132对HO-8910细胞和SKOV3细胞的增殖抑制作用相似且细胞存活率均呈剂量-效应和处理时间-效应依赖性下降。HO-8910细胞中,MG132和MPA联合用药组的细胞存活率(62.43%±3.56%)与单用MPA组的细胞存活率(63.05%±3.60%)相比差异无统计学意义(P0.05),但在SKOV3细胞中MG132和MPA联合用药组的细胞存活率(65.52%±1.52%)明显低于单用MPA组的细胞存活率(84.49%±2.29%),差异有统计学意义(p0.05)。(2)细胞形态学观察:control组细胞呈典型的上皮样细胞特征,以类椭圆形及长梭形为主,铺路石样生长,细胞排列规则紧凑;MG132组细胞数目减少,细胞间隙增大,视野内见较多漂浮细胞,部分细胞体积变大,伪足细长,胞浆内出现空泡;MG132+MPA组细胞数目减少更明显,视野内见大量漂浮细胞,胞浆内的空泡更多;两者MPA组细胞形态存在明显差异,HO-8910细胞MPA组细胞改变与MG132组细胞改变相似,而SKOV3细胞MPA组细胞数量稍减少,细胞排列较紧凑,部分细胞伸出伪足,呈多角形,未见明显漂浮细胞及胞浆内空泡。(3)流式细胞术结果显示:HO-8910细胞中各组细胞凋亡率(%):control组0.42±0.04、MG132组7.49±0.83、MPA组11.95±0.52、MG132+MPA组12.54±0.42;SKOV3细胞中各组细胞凋亡率(%):control组0.64±0.06、MG132组7.29±0.41、MPA组6.37±0.54、MG132+MPA组14.67±0.65;联合用药时,SKOV3细胞中,MG132+MPA组细胞凋亡率明显大于单用MPA组(p0.05),但在HO-8910细胞中,MG132+MPA组凋亡率与单用MPA组相比,差异无统计学意义(p0.05)。(4)免疫组化法结果显示:PR于胞核表达丰富,胞浆也可见表达,HO-8910细胞中PR高表达,SKOV3细胞中PR低表达。两种细胞中MG132组、MG132+MPA组PR表达均高于control组(p0.05),而MPA组PR表达与control组相比,差异无统计学意义(p0.05)。(5)免疫印迹法结果显示:HO-8910、SKOV3细胞中PRB的表达明显高于PRA。MG132组、MG132+MPA组PRB、PRA的表达均高于control组(p0.05),而MPA组PRA、PRB的表达与control组相比,差异无统计学意义(p0.05)。结论:(1)MPA对PR高表达细胞株HO-8910的增殖抑制及诱导凋亡作用明显强于PR低表达细胞株SKOV3。(2)MG132对HO-8910、SKOV3均具有增殖抑制及诱导凋亡的作用,并可增强MPA对SKOV3细胞增殖抑制及诱导凋亡作用的敏感性。(3)MG132增强MPA对SKOV3细胞增殖抑制及诱导凋亡的作用可能与其提升PR的表达有关。
[Abstract]:Objective: To investigate the effect of medroxyprogesterone acetate (MPA) and proteasome inhibitor (Z-LLL-CHO, MG132) on the proliferation and apoptosis of ovarian cancer cells with different PR expressions, and to provide a new experimental basis for the clinical treatment of ovarian cancer. Method: HO-8910 (high expression of PR) in human ovarian cancer cell line (PR high expression) (1) SKOV3 (PR low expression) as the research object. (1) different concentrations of MPA (0,1,20,50100 mu mol/L), MG132 (0,1,2.5,5,10 mu mol/L) alone intervention 24-72 h, respectively, 24 h, 48 h, 72 h to detect the proliferation of two cells. (2) the logarithmic growth of two cells are divided into the blank control group (5 mu), 100 group (5 mu), 100 micron group (5 mu) /L), group MG132+MPA (5+100 mu mol/L), both acting 24 h:a, using CCK8 to detect the proliferation of two kinds of cells; B, inverted microscope to observe the morphological changes of the cells and the film; C, using the Annexin V-FITC/PI double staining method to detect the apoptosis of the cells before and after the action of the drug by flow cytometry; D, using immunohistochemistry (Immunohistochemistry. C) detection of the expression of PR before and after the action of drugs; E, using Westeron blot (WB) to detect the expression of PRA and PRB of progesterone receptor subtype before and after the action of drugs. Results: (1) CCK8 results showed that the survival rate of HO-8910 cells (%) in MPA group (24 h) was 100 +. 63.05 + 3.60; (action 48 h) 100 + 0.94104.45 + 0.37,71.06 + 3.22,55.70 + 0.74,48.08 + 0.08; (effect 72 h) 100 + 0.38105.52 + 2.15,58.63 + 0.21,43.30 + 0.57,29.43 + 0.39.MPA group (%) of SKOV3 cell survival rate (%) was 100 + + + + + + + 2.29; (action 48) H) 100 + 2.70,99.58 + 0.29,89.22 + 1.67,86.37 + 0.22,78.44 + 5.08; (action 72 h) 100 + 3.59103.22 + 6.12,87.21 + 3.32,78.68 + 3.30,65.56 + 3.30,65.56 + 0.79.20-100 micron The survival rate of HO-8910 cells (24 h) was 100 + 1.30,98.73 + 1.09,83.29 + 0.43,71.36 + 3.10,66.54 + 8.33, (action 48 h) 100 + 0.06,89.35 + 2.05,73.39 + 1.86,59.46 + 0.69,47.40 + 0.98, (72 h) 100 + The survival rate of V3 cells (24 h) was 100 + 4.58,94.39 + 4.33,86.12 + 3.18,75.73 + 2.57,61.41 + 1.11, (action 48 h) 100 + 3.40,80.18 + 2.73,62.11 + 3.89,51.76 + 3.99,46.97 + 1.75, and 72 h) 100 The proliferation inhibition of SKOV3 cells was similar and the cell survival rate was dose-dependent and time effect dependence decreased in.HO-8910 cells. The cell survival rate (62.43% + 3.56%) in the combination group of MG132 and MPA was not significantly different from that of the MPA group (63.05% + 3.60%) (P0.05), but M in SKOV3 cells. The cell survival rate of G132 and MPA group (65.52% + 1.52%) was significantly lower than that of the single MPA group (84.49% + 2.29%), and the difference was statistically significant (P0.05). (2) morphological observation of cell morphology: the cells in the control group showed typical epithelioid cell characteristics, mainly like ellipsoidal and long spindle shape, paving stone like growth, and the cell arrangement rules tight The number of cells in the MG132 group was reduced, the cell space was enlarged, more floating cells were seen in the field of vision, the volume of some cells became larger, the pseudo foot was slender, the vacuoles appeared in the cytoplasm, the number of cells in the group MG132+MPA decreased more clearly, there were a large number of floating cells in the field of vision and more vacuoles within the cytoplasm; the cell morphology of the two group MPA group was significantly different, and the MPA group of HO-8910 cells was in MPA group. Cell change was similar to that in MG132 group, while the number of cells in group MPA of SKOV3 cells decreased slightly, the cell arrangement was more compact, some cells protruded in the polygon, and there was no obvious floating cells and intracellular vacuoles. (3) flow cytometry results showed that the apoptosis rate of each group in HO-8910 cells (%): control group 0.42 + 0.04, MG132 group 7.49 + 0.83, Group MPA was 11.95 + 0.52, group MG132+MPA was 12.54 + 0.42, and the apoptosis rate of each group in SKOV3 cells (%) was 0.64 + 0.06, MG132 group 7.29 + 0.41, MPA group 6.37 + 0.54, MG132+MPA group 14.67 + 0.65. In SKOV3 cells, the apoptosis rate of MG132+MPA group was obviously greater than that of MPA group (P0.05), but in HO-8910 cells, apoptosis rate and The difference was not statistically significant (4) in the MPA group. (4) the results of immunohistochemical staining showed that the expression of PR was abundant in the nucleus and the expression of cytoplasm, the expression of PR in the HO-8910 cells and the low expression of PR in the SKOV3 cells. The expression of PR in the two cells was higher than that in the control group (P0.05). Statistical significance (P0.05). (5) the results of immunoblotting showed that the expression of PRB in HO-8910, SKOV3 cells was significantly higher than that in group PRA.MG132, and the expression of PRB and PRA in group MG132+MPA was higher than that in group control (P0.05), while MPA group PRA, the difference was not statistically significant. Inhibition and induction of apoptosis is significantly stronger than PR low expression cell line SKOV3. (2) MG132 on HO-8910, SKOV3, which can inhibit proliferation and induce apoptosis, and enhance the sensitivity of MPA to the proliferation inhibition and apoptosis induction of SKOV3 cells. (3) the effect of MG132 enhanced MPA on proliferation inhibition and apoptosis of SKOV3 cells may be promoted by PR The expression is related.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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