【摘要】：卵巢癌是最常見的婦科惡性腫瘤之一,早期發(fā)現難,治療效果差,預后不良,死亡率高。分子伴侶(molecular chaperone)是一類與其他蛋白的不穩(wěn)定構象相結合并使之穩(wěn)定的蛋白質,通過控制結合和釋放來幫助被結合多肽在細胞內進行折疊、組裝、轉運、降解等,維持細胞的內穩(wěn)態(tài)。Mortalin,又名Grp75/PBP74/mthsp70,是HSP70分子伴侶家族成員之一,其廣泛分布于多個細胞器和胞漿,參與細胞增殖和胞內運輸、調控應激反應、穩(wěn)定細胞骨架、抑制細胞凋亡等。研究發(fā)現在許多腫瘤中,如慢性粒細胞白血病、結腸、顱腦腫瘤、乳腺癌和肝癌中mortalin表達上調。過表達mortalin可以增加乳腺癌細胞的惡性程度,參與肝轉移。課題組先前通過對183例卵巢癌組織以及癌旁組織中的組織芯片檢測結果顯示,mortalin在卵巢癌組織中表達明顯高于癌旁組織；且mortalin的表達與卵巢癌的分期有關,分期越高的卵巢癌組織中,mortalin表達量越高。提示,mortalin高表達可能與卵巢癌的發(fā)生、發(fā)展有關。本課題選取8株卵巢癌細胞系A2780/cis、COC1、HO-8910、PM-8910、 OV-90、Caov-3、SKOV-3和ES-2,分別在蛋白質水平和mRNA水平檢測其mortalin的表達量。研究表明,在8株細胞系中,OV-90細胞中]mortalin的mRNA和蛋白的表達量最低,而順鉑耐藥型卵巢癌細胞系A2780/cis和腹水中提取的卵巢癌細胞COC1的mortalin的mRNA和蛋白的表達量最高。課題組之前的實驗結果顯示,人卵巢癌順鉑敏感型細胞系A2780中mortalin表達量明顯低于人卵巢癌順鉑耐藥型細胞系A2780/cis。以上結果提示,motalin的表達與卵巢癌細胞的惡性程度可能有關。隨后,選取人卵巢癌順鉑敏感型細胞系A2780和人卵巢癌順鉑耐藥型細胞系A2780/cis,通過構建mortalin上調、下調的慢病毒載體,分別感染兩株細胞,經流式篩選分別獲得mortalin上調表達、下調表達穩(wěn)定株。通過CCK-8(Cell Counting Kit-8)實驗、克隆形成實驗,細胞劃痕實驗和transwell細胞侵襲實驗分別檢測細胞活力、克隆形成率和細胞侵襲轉移能力。結果顯示,mortalin表達量增高可以促進卵巢癌細胞的細胞增殖、增加單個細胞克隆形成的大小和數量,并能促進細胞遷移,增加其轉移侵襲能力；而降低mortalin的表達,可以有效抑制卵巢癌細胞的細胞增殖、單個細胞克隆形成的大小和數量,并能抑制細胞遷移,降低其轉移侵襲能力。以上結果提示,mortalin的表達與卵巢癌細胞的發(fā)生、發(fā)展及侵襲有關,而下調mortalin表達能有效抑制卵巢癌細胞的侵襲,延緩卵巢癌的發(fā)生、發(fā)展。流式細胞儀檢測mortalin表達對細胞周期的影響,Western Blot檢測細胞周期蛋白的表達變化。結果顯示,mortalin高表達可以促進細胞快速通過G1/S期,進入G2/M期,同時會增加細胞內Cyclin-D1和C-myc的表達量,降低細胞內Cyclin-B1的表達量；而mortalin下調表達會延遲細胞通過G1/S期,同時會增加細胞內Cyclin-D1和C-myc的表達量,降低細胞內Cyclin-B1的表達量。為了研究mortalin在卵巢癌發(fā)生發(fā)展過程中的作用機制以其可能的信號通路,通過Western Blot實驗檢測一系列信號通路相關蛋白表達情況,結果顯示,mortalin可以特異性的促進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信號通路其促進卵巢癌細胞的增殖和侵襲遷移。綜上所述,mortalin上調表達參與了卵巢癌的發(fā)生、發(fā)展,下調mortalin可以通過減慢細胞從G1期向G2/M期過渡,有效降低卵巢癌細胞的生長速度和侵襲轉移率,并且這一過程與其對MAPK-ERK信號通路的激活有關。因此,mortalin可以作為治療卵巢癌的一個潛在靶點。
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.31

【共引文獻】

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