本文選題：卵巢癌 + 孕激素��； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:探討醋酸甲羥孕酮(medroxyprogesterone acetate,MPA)、蛋白酶體抑制劑(Z-LLL-CHO,MG132)單獨(dú)及聯(lián)合應(yīng)用對(duì)不同PR表達(dá)卵巢癌細(xì)胞增殖、凋亡的影響及相關(guān)機(jī)制,為卵巢癌的臨床治療提供新的實(shí)驗(yàn)依據(jù)。方法:選取人卵巢癌細(xì)胞系HO-8910(PR高表達(dá))、SKOV3(PR低表達(dá))為研究對(duì)象。(1)不同濃度的MPA(0、1、20、50、100μmol/L)、MG132(0、1、2.5、5、10μmol/L)單獨(dú)干預(yù)24-72 h,分別于24 h、48 h、72 h采用CCK8法檢測(cè)兩種細(xì)胞的增殖情況。(2)取呈對(duì)數(shù)生長(zhǎng)的兩種細(xì)胞分為:空白對(duì)照組(0μmol/L)、MG132組(5μmol/L)、MPA組(100μmol/L)、MG132+MPA組(5+100μmol/L),均作用24 h:a、采用CCK8法檢測(cè)兩種細(xì)胞的增殖情況;b、倒置顯微鏡觀察細(xì)胞的形態(tài)學(xué)變化并攝片;c、采用Annexin V-FITC/PI雙染法通過(guò)流式細(xì)胞儀檢測(cè)藥物作用前后細(xì)胞的凋亡情況;d、利用免疫組化法(Immunohistochemistry,IHC)檢測(cè)藥物作用前后PR的表達(dá)情況;e、利用免疫印跡法(Westeron blot,WB)檢測(cè)藥物作用前后孕激素受體亞型PRA、PRB的表達(dá)情況。結(jié)果:(1)CCK8結(jié)果顯示:MPA組(不同濃度)的HO-8910細(xì)胞存活率(%)分別為:(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組(不同濃度)的SKOV3細(xì)胞存活率(%)分別為:(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對(duì)HO-8910細(xì)胞的增殖抑制作用明顯強(qiáng)于SKOV3細(xì)胞且細(xì)胞存活率均呈劑量-效應(yīng)和處理時(shí)間-效應(yīng)依賴性下降。MG132組(不同濃度)的HO-8910細(xì)胞的存活率(%)分別為:(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組(不同濃度)的SKOV3細(xì)胞的存活率(%)分別為:(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對(duì)HO-8910細(xì)胞和SKOV3細(xì)胞的增殖抑制作用相似且細(xì)胞存活率均呈劑量-效應(yīng)和處理時(shí)間-效應(yīng)依賴性下降。HO-8910細(xì)胞中,MG132和MPA聯(lián)合用藥組的細(xì)胞存活率(62.43%±3.56%)與單用MPA組的細(xì)胞存活率(63.05%±3.60%)相比差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),但在SKOV3細(xì)胞中MG132和MPA聯(lián)合用藥組的細(xì)胞存活率(65.52%±1.52%)明顯低于單用MPA組的細(xì)胞存活率(84.49%±2.29%),差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)細(xì)胞形態(tài)學(xué)觀察:control組細(xì)胞呈典型的上皮樣細(xì)胞特征,以類橢圓形及長(zhǎng)梭形為主,鋪路石樣生長(zhǎng),細(xì)胞排列規(guī)則緊湊;MG132組細(xì)胞數(shù)目減少,細(xì)胞間隙增大,視野內(nèi)見較多漂浮細(xì)胞,部分細(xì)胞體積變大,偽足細(xì)長(zhǎng),胞漿內(nèi)出現(xiàn)空泡;MG132+MPA組細(xì)胞數(shù)目減少更明顯,視野內(nèi)見大量漂浮細(xì)胞,胞漿內(nèi)的空泡更多;兩者M(jìn)PA組細(xì)胞形態(tài)存在明顯差異,HO-8910細(xì)胞MPA組細(xì)胞改變與MG132組細(xì)胞改變相似,而SKOV3細(xì)胞MPA組細(xì)胞數(shù)量稍減少,細(xì)胞排列較緊湊,部分細(xì)胞伸出偽足,呈多角形,未見明顯漂浮細(xì)胞及胞漿內(nèi)空泡。(3)流式細(xì)胞術(shù)結(jié)果顯示:HO-8910細(xì)胞中各組細(xì)胞凋亡率(%):control組0.42±0.04、MG132組7.49±0.83、MPA組11.95±0.52、MG132+MPA組12.54±0.42;SKOV3細(xì)胞中各組細(xì)胞凋亡率(%):control組0.64±0.06、MG132組7.29±0.41、MPA組6.37±0.54、MG132+MPA組14.67±0.65;聯(lián)合用藥時(shí),SKOV3細(xì)胞中,MG132+MPA組細(xì)胞凋亡率明顯大于單用MPA組(p0.05),但在HO-8910細(xì)胞中,MG132+MPA組凋亡率與單用MPA組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。(4)免疫組化法結(jié)果顯示:PR于胞核表達(dá)豐富,胞漿也可見表達(dá),HO-8910細(xì)胞中PR高表達(dá),SKOV3細(xì)胞中PR低表達(dá)。兩種細(xì)胞中MG132組、MG132+MPA組PR表達(dá)均高于control組(p0.05),而MPA組PR表達(dá)與control組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。(5)免疫印跡法結(jié)果顯示:HO-8910、SKOV3細(xì)胞中PRB的表達(dá)明顯高于PRA。MG132組、MG132+MPA組PRB、PRA的表達(dá)均高于control組(p0.05),而MPA組PRA、PRB的表達(dá)與control組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:(1)MPA對(duì)PR高表達(dá)細(xì)胞株HO-8910的增殖抑制及誘導(dǎo)凋亡作用明顯強(qiáng)于PR低表達(dá)細(xì)胞株SKOV3。(2)MG132對(duì)HO-8910、SKOV3均具有增殖抑制及誘導(dǎo)凋亡的作用,并可增強(qiáng)MPA對(duì)SKOV3細(xì)胞增殖抑制及誘導(dǎo)凋亡作用的敏感性。(3)MG132增強(qiáng)MPA對(duì)SKOV3細(xì)胞增殖抑制及誘導(dǎo)凋亡的作用可能與其提升PR的表達(dá)有關(guān)。
[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.

【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.31

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