【摘要】：卵巢癌是最常見的婦科惡性腫瘤之一,早期發(fā)現(xiàn)難,治療效果差,預(yù)后不良,死亡率高。分子伴侶(molecular chaperone)是一類與其他蛋白的不穩(wěn)定構(gòu)象相結(jié)合并使之穩(wěn)定的蛋白質(zhì),通過控制結(jié)合和釋放來幫助被結(jié)合多肽在細(xì)胞內(nèi)進(jìn)行折疊、組裝、轉(zhuǎn)運(yùn)、降解等,維持細(xì)胞的內(nèi)穩(wěn)態(tài)。Mortalin,又名Grp75/PBP74/mthsp70,是HSP70分子伴侶家族成員之一,其廣泛分布于多個(gè)細(xì)胞器和胞漿,參與細(xì)胞增殖和胞內(nèi)運(yùn)輸、調(diào)控應(yīng)激反應(yīng)、穩(wěn)定細(xì)胞骨架、抑制細(xì)胞凋亡等。研究發(fā)現(xiàn)在許多腫瘤中,如慢性粒細(xì)胞白血病、結(jié)腸、顱腦腫瘤、乳腺癌和肝癌中mortalin表達(dá)上調(diào)。過表達(dá)mortalin可以增加乳腺癌細(xì)胞的惡性程度,參與肝轉(zhuǎn)移。課題組先前通過對(duì)183例卵巢癌組織以及癌旁組織中的組織芯片檢測結(jié)果顯示,mortalin在卵巢癌組織中表達(dá)明顯高于癌旁組織；且mortalin的表達(dá)與卵巢癌的分期有關(guān),分期越高的卵巢癌組織中,mortalin表達(dá)量越高。提示,mortalin高表達(dá)可能與卵巢癌的發(fā)生、發(fā)展有關(guān)。本課題選取8株卵巢癌細(xì)胞系A(chǔ)2780/cis、COC1、HO-8910、PM-8910、 OV-90、Caov-3、SKOV-3和ES-2,分別在蛋白質(zhì)水平和mRNA水平檢測其mortalin的表達(dá)量。研究表明,在8株細(xì)胞系中,OV-90細(xì)胞中]mortalin的mRNA和蛋白的表達(dá)量最低,而順鉑耐藥型卵巢癌細(xì)胞系A(chǔ)2780/cis和腹水中提取的卵巢癌細(xì)胞COC1的mortalin的mRNA和蛋白的表達(dá)量最高。課題組之前的實(shí)驗(yàn)結(jié)果顯示,人卵巢癌順鉑敏感型細(xì)胞系A(chǔ)2780中mortalin表達(dá)量明顯低于人卵巢癌順鉑耐藥型細(xì)胞系A(chǔ)2780/cis。以上結(jié)果提示,motalin的表達(dá)與卵巢癌細(xì)胞的惡性程度可能有關(guān)。隨后,選取人卵巢癌順鉑敏感型細(xì)胞系A(chǔ)2780和人卵巢癌順鉑耐藥型細(xì)胞系A(chǔ)2780/cis,通過構(gòu)建mortalin上調(diào)、下調(diào)的慢病毒載體,分別感染兩株細(xì)胞,經(jīng)流式篩選分別獲得mortalin上調(diào)表達(dá)、下調(diào)表達(dá)穩(wěn)定株。通過CCK-8(Cell Counting Kit-8)實(shí)驗(yàn)、克隆形成實(shí)驗(yàn),細(xì)胞劃痕實(shí)驗(yàn)和transwell細(xì)胞侵襲實(shí)驗(yàn)分別檢測細(xì)胞活力、克隆形成率和細(xì)胞侵襲轉(zhuǎn)移能力。結(jié)果顯示,mortalin表達(dá)量增高可以促進(jìn)卵巢癌細(xì)胞的細(xì)胞增殖、增加單個(gè)細(xì)胞克隆形成的大小和數(shù)量,并能促進(jìn)細(xì)胞遷移,增加其轉(zhuǎn)移侵襲能力；而降低mortalin的表達(dá),可以有效抑制卵巢癌細(xì)胞的細(xì)胞增殖、單個(gè)細(xì)胞克隆形成的大小和數(shù)量,并能抑制細(xì)胞遷移,降低其轉(zhuǎn)移侵襲能力。以上結(jié)果提示,mortalin的表達(dá)與卵巢癌細(xì)胞的發(fā)生、發(fā)展及侵襲有關(guān),而下調(diào)mortalin表達(dá)能有效抑制卵巢癌細(xì)胞的侵襲,延緩卵巢癌的發(fā)生、發(fā)展。流式細(xì)胞儀檢測mortalin表達(dá)對(duì)細(xì)胞周期的影響,Western Blot檢測細(xì)胞周期蛋白的表達(dá)變化。結(jié)果顯示,mortalin高表達(dá)可以促進(jìn)細(xì)胞快速通過G1/S期,進(jìn)入G2/M期,同時(shí)會(huì)增加細(xì)胞內(nèi)Cyclin-D1和C-myc的表達(dá)量,降低細(xì)胞內(nèi)Cyclin-B1的表達(dá)量；而mortalin下調(diào)表達(dá)會(huì)延遲細(xì)胞通過G1/S期,同時(shí)會(huì)增加細(xì)胞內(nèi)Cyclin-D1和C-myc的表達(dá)量,降低細(xì)胞內(nèi)Cyclin-B1的表達(dá)量。為了研究mortalin在卵巢癌發(fā)生發(fā)展過程中的作用機(jī)制以其可能的信號(hào)通路,通過Western Blot實(shí)驗(yàn)檢測一系列信號(hào)通路相關(guān)蛋白表達(dá)情況,結(jié)果顯示,mortalin可以特異性的促進(jìn)c-Raf和Erk1/2的磷酸化,但并沒有影響JNK的活化。因此推測,mortalin可以通過MAPK-ERK信號(hào)通路其促進(jìn)卵巢癌細(xì)胞的增殖和侵襲遷移。綜上所述,mortalin上調(diào)表達(dá)參與了卵巢癌的發(fā)生、發(fā)展,下調(diào)mortalin可以通過減慢細(xì)胞從G1期向G2/M期過渡,有效降低卵巢癌細(xì)胞的生長速度和侵襲轉(zhuǎn)移率,并且這一過程與其對(duì)MAPK-ERK信號(hào)通路的激活有關(guān)。因此,mortalin可以作為治療卵巢癌的一個(gè)潛在靶點(diǎn)。
[Abstract]:Ovarian cancer is one of the most common gynecological malignancies. It is difficult to detect early, poor therapeutic effect, poor prognosis and high mortality. Mortalin, also known as Grp75/PBP74/mthsp70, is a member of the HSP70 chaperone family. It is widely distributed in many organelles and cytoplasm, and participates in cell proliferation and intracellular transport, regulates stress response, stabilizes cytoskeleton and inhibits cell apoptosis. Overexpression of mortalin in granulocytic leukemia, colon, brain tumors, breast cancer and liver cancer can increase the malignant degree of breast cancer cells and participate in liver metastasis. The expression of mortalin was higher in ovarian cancer tissues than in adjacent tissues, and the higher the stage, the higher the expression of mortalin. It was suggested that the high expression of mortalin might be related to the occurrence and development of ovarian cancer. Eight ovarian cancer cell lines, A2780/cis, COC1, HO-8910, PM-8910, OV-90, Caov-3, SKOV-3 and ES-2, were selected in this study, respectively. The expression of]mortalin mRNA and protein was the lowest in OV-90 cells, while that in cisplatin-resistant ovarian cancer cell line A2780/cis and ascites was the highest. The results showed that the expression of mortalin in human ovarian cancer cell line A2780 was significantly lower than that in human ovarian cancer cell line A2780 / cis. These results suggest that the expression of motalin may be related to the malignancy of ovarian cancer cells. Subsequently, human ovarian cancer cell line A2780 and human ovarian cancer cell line A2780 were selected for cisplatin resistance. Drug-type cell line A2780/cis was infected with two mortalin-up-regulated and down-regulated lentiviral vectors respectively, and the stable mortalin-up-regulated and down-regulated strains were obtained by flow cytometry. Cell viability was detected by CCK-8 (Cell Counting Kit-8) assay, clone formation assay, cell scratch assay and Transwell cell invasion assay, respectively. The results showed that the increased expression of mortalin could promote the proliferation of ovarian cancer cells, increase the size and number of single cell clones, promote cell migration, increase the ability of metastasis and invasion, and decrease the expression of mortalin could effectively inhibit ovarian cancer cells. These results suggest that the expression of mortalin is related to the occurrence, development and invasion of ovarian cancer cells, and down-regulation of mortalin expression can effectively inhibit the invasion of ovarian cancer cells, delay the occurrence and development of ovarian cancer. Western Blot was used to detect the effect of mortalin expression on cell cycle, and Western Blot was used to detect the expression of cyclin. Delayed cell passage through G1/S phase increased the expression of Cyclin-D1 and C-myc, and decreased the expression of Cyclin-B1. In order to study the mechanism of mortalin in ovarian cancer development and progression, Western Blot assay was used to detect the expression of a series of signal pathway related proteins. These results suggest that mortalin can specifically promote the phosphorylation of c-Raf and Erk1/2, but does not affect the activation of JNK. Therefore, mortalin may promote the proliferation and invasion and migration of ovarian cancer cells through MAPK-ERK signaling pathway. The transition from G1 phase to G2/M phase can effectively reduce the growth rate and invasion and metastasis rate of ovarian cancer cells, and this process is related to the activation of MAPK-ERK signaling pathway.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

【共引文獻(xiàn)】

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