發(fā)布時(shí)間：2018-02-27 12:32

  本文關(guān)鍵詞： 蛋白酶體抑制劑 卵巢癌 乳胞素 SKOV3細(xì)胞 免疫組化　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：實(shí)驗(yàn)?zāi)康?因?yàn)槁殉舶┰缙跓o明顯癥狀,加之其缺乏早期的診斷方法和依據(jù)、病情進(jìn)展快等特點(diǎn),大部分卵巢癌預(yù)后很差,高居死亡率首位。而在所有原發(fā)性卵巢癌中,70%-90%的病理類型屬于上皮性卵巢癌,是臨床上最為常見的一種病理類型。本文通過探討蛋白酶體抑制劑乳胞素對(duì)卵巢癌SKOV3細(xì)胞體外存活率及增殖率的影響,并檢測(cè)細(xì)胞內(nèi)相關(guān)物質(zhì)信號(hào)水平的變化,初步探討其可能的作用機(jī)制,為卵巢癌的治療研究提供新的理論思路與研究方向,希望能夠改變臨床上卵巢癌死亡率居高不下的狀況,造福于患者。實(shí)驗(yàn)方法:MTT染色、流式細(xì)胞儀檢測(cè)從細(xì)胞層面檢測(cè)蛋白酶體抑制乳胞素作用于體外生長(zhǎng)的卵巢癌SKOV3細(xì)胞后,對(duì)其在細(xì)胞增殖及凋亡方面產(chǎn)生的影響Western Blot,RT-PCR則分別檢測(cè)細(xì)胞內(nèi)蛋白質(zhì)及基因水平的變化,從微觀的層面檢測(cè)乳胞素引起細(xì)胞損傷后,細(xì)胞內(nèi)相關(guān)物質(zhì)信號(hào)表達(dá)水平發(fā)生的變化。實(shí)驗(yàn)結(jié)果:(1)顯微鏡觀察細(xì)胞形態(tài)變化:對(duì)照組SKOV3細(xì)胞生長(zhǎng)狀態(tài)良好,細(xì)胞形態(tài)飽滿,呈多邊形樣式,以貼壁的方式生長(zhǎng),各細(xì)胞之間邊界清晰、連接緊密。而經(jīng)不同濃度的乳胞素作用后的各組細(xì)胞,生長(zhǎng)狀態(tài)較差,細(xì)胞萎縮,形狀多變?yōu)閳A形或橢圓形,細(xì)胞與細(xì)胞之間的界限也不十分清楚,細(xì)胞體內(nèi)因充滿大量顆粒狀物質(zhì)而使其透光變差,培養(yǎng)基中漂浮的無活性細(xì)胞明顯增多。隨著乳胞素濃度的增高,上述差別表現(xiàn)的更加明顯。(2)MTT染色檢測(cè)結(jié)果顯示:對(duì)比于對(duì)照組,經(jīng)不同濃度的乳胞素作用后,體外生長(zhǎng)的SKOV3細(xì)胞的生長(zhǎng)受到明顯抑制,增殖率降低[0.5umol/L組(65.5±0.10%)、1.0umol/L組(54.4±0.11%),2.5umol/L組(42.5±0.09%),5.0umol/L組(29.2±0.12%)及10.0umol/L組(18.9±0.13%)](差異有統(tǒng)計(jì)學(xué)意義:P0.05)。(3)流式細(xì)胞儀檢測(cè)結(jié)果表明,對(duì)比于對(duì)照組,乳胞素可以促使SKOV3細(xì)胞凋亡的發(fā)生,并且在單位時(shí)間內(nèi),其凋亡率隨著藥物作用濃度的增高而增加,與藥物濃度呈正相關(guān),濃度依賴性明顯(差異有統(tǒng)計(jì)學(xué)意義:P0.05)。(4)Western Blot檢測(cè)結(jié)果表明:對(duì)比于對(duì)照組,隨著乳胞素濃度的增高,在相同作用時(shí)間條件下,卵巢癌細(xì)胞內(nèi)Bax蛋白的表達(dá)水平逐漸增強(qiáng),而Bcl-2蛋白的表達(dá)水平逐漸減弱(差異有統(tǒng)計(jì)學(xué)意義:P0.05)。(5)RT-PCR電泳結(jié)果表明:對(duì)比于對(duì)照組,隨著乳胞素濃度的增加,在相同作用時(shí)間條件下,卵巢癌細(xì)胞內(nèi)Bax m RNA的表達(dá)水平明顯增加,而Bcl-2 m RNA的表達(dá)水平逐漸減少(差異有統(tǒng)計(jì)學(xué)意義:P0.05)。實(shí)驗(yàn)結(jié)論:蛋白酶體抑制劑乳胞素對(duì)體外生長(zhǎng)的卵巢癌SKOV3細(xì)胞,具有抑制其增殖和誘導(dǎo)其凋亡的作用,推斷其主要內(nèi)部機(jī)制可能是通過抑制泛素-蛋白酶體通路的活性,從而導(dǎo)致細(xì)胞內(nèi)Bax、bcl-2等與細(xì)胞凋亡密切相關(guān)的基因及蛋白的表達(dá)水平的失衡,進(jìn)而影響線粒體途徑參與的細(xì)胞凋亡,誘導(dǎo)卵巢癌SKOV3細(xì)胞凋亡的發(fā)生。
[Abstract]:Objective: because ovarian cancer has no obvious symptoms at the early stage and lacks of early diagnostic methods and evidence, the prognosis of most ovarian cancer is poor. The highest mortality rate. And in all primary ovarian cancer, 70 to 90 percent of the pathological types belong to epithelial ovarian cancer, In this paper, we studied the effects of proteasome inhibitor lactin on the survival rate and proliferation rate of ovarian cancer SKOV3 cells in vitro, and detected the changes of intracellular signal level of related substances. In order to provide a new theoretical approach and research direction for the treatment of ovarian cancer, we hope to change the high mortality rate of ovarian cancer in clinic and benefit the patients. Flow cytometry was used to detect the inhibitory effect of proteasome on ovarian cancer SKOV3 cells in vitro. The effects of Western blottir RT-PCR on cell proliferation and apoptosis were detected by reverse transcription-polymerase chain reaction (RT-PCR). The changes of protein and gene levels in cells were detected by RT-PCR, and the changes of cell damage induced by lactin were detected at the microcosmic level. Changes in signal expression level of related substances in cells. Results: microscopic observation of cell morphology: SKOV3 cells in the control group were in good growth state, full shape, polygonal pattern, and grew in the form of adherent to the wall. The boundary between the cells is clear and the connection is tight. However, the cells treated with different concentrations of lactin have a poor growth state, the cells atrophy, the shape is varied into a round or oval shape, and the boundary between the cells is not very clear. When the cells were filled with a large number of granular substances, the light transmittance of the cells became worse, and the number of inactive cells floating in the culture medium increased obviously with the increase of the concentration of lactin. The results of MTT staining showed that the growth of SKOV3 cells in vitro was significantly inhibited after the treatment of different concentrations of lactin, as compared with the control group. The proliferation rate was decreased [0.5umolr / L group 65.5 鹵0.10umol.L group 54.4 鹵0.11mg / L + 2.5umoll / L group 42.5 鹵0.09umoll / L group 29.2 鹵0.12cm / L, and 10.0umolol / L group 18.9 鹵0.135mm / L respectively] (the difference was statistically significant (P 0.05 / L).) flow cytometry analysis showed that compared with the control group, lactin could induce apoptosis of SKOV3 cells, and in a unit time, lactin could induce apoptosis of SKOV3 cells. The apoptosis rate increased with the increase of drug concentration, and was positively correlated with drug concentration. The results of Western Blot analysis showed that compared with the control group, the apoptosis rate increased with the increase of the concentration of lactin. At the same time, the expression of Bax protein in ovarian cancer cells was gradually increased, while the expression level of Bcl-2 protein was gradually decreased (the difference was statistically significant). The results of RT-PCR electrophoresis showed that: compared with the control group, the expression of Bax protein in ovarian cancer cells was significantly lower than that in control group. At the same time, the expression level of Bax m RNA in ovarian cancer cells increased significantly with the increase of the concentration of lactin. However, the expression of Bcl-2 m RNA was gradually decreased (P 0.05). Conclusion: the proteasome inhibitor lactin can inhibit the proliferation and induce apoptosis of ovarian cancer SKOV3 cells in vitro. It is inferred that its main internal mechanism may be to inhibit the activity of the ubiquitin proteasome pathway, resulting in the imbalance of the expression level of genes and proteins closely related to cell apoptosis, such as Baxanbcl-2. The apoptosis of ovarian cancer SKOV3 cells was induced by mitochondrial pathway.
【學(xué)位授予單位】：吉林大學(xué)
【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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