【摘要】：研究背景與目的： 卵巢癌是婦科腫瘤中死亡率非常高的惡性腫瘤，在近三十年中死亡率并沒(méi)有呈下降趨勢(shì)。由于其發(fā)病隱匿，早期診斷相當(dāng)困難，一旦發(fā)現(xiàn)已有遠(yuǎn)處轉(zhuǎn)移。Hedgehog(Hh)信號(hào)通路參與胚胎的發(fā)育及成人體內(nèi)穩(wěn)態(tài)的維持，在機(jī)體發(fā)育中扮演重要的角色。近年來(lái)研究顯示多種惡性腫瘤的發(fā)生、發(fā)展與Hh信號(hào)通路的異常激活有關(guān)，包括卵巢癌。但Hh信號(hào)通路怎么參與調(diào)控卵巢癌的侵襲與轉(zhuǎn)移的分子機(jī)制仍不清楚。本實(shí)驗(yàn)從細(xì)胞分子水平及動(dòng)物模型水平來(lái)初步探討Hh信號(hào)通路參與卵巢癌侵襲與轉(zhuǎn)移的分子機(jī)制及尋找卵巢癌的早期診斷治療靶點(diǎn)，為卵巢癌新藥研發(fā)提供理論根據(jù)。 研究方法： (1)根據(jù)NCBI查詢到的MMP7cDNA序列在Invitrogen網(wǎng)站上設(shè)計(jì)3個(gè)靶向MMP7的干擾序列，連接到pcDNA6.2-GW/EmGFP-miR載體中構(gòu)建重組質(zhì)粒，分別命名為miRMMP7-158、314、688。用脂質(zhì)體2000將MMP7干擾質(zhì)粒轉(zhuǎn)染人卵巢癌SKO-V3細(xì)胞，并用倒置熒光顯微鏡觀察熒光強(qiáng)度以確定轉(zhuǎn)染效率。蛋白免疫印跡（Western blot）篩選有效的干擾片段。 (2)以優(yōu)化了的轉(zhuǎn)染條件【plasmid（ug）：Lipofectamine2000(ul)=1:3】轉(zhuǎn)染人卵巢癌細(xì)胞SKO-V3，并通過(guò)劃痕實(shí)驗(yàn)和Transwell細(xì)胞侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞遷移與侵襲能力。 (3)分別用GANT61（Hh信號(hào)通路的抑制劑）與SHH信號(hào)通路配體條件培養(yǎng)液（Hh信號(hào)通路激活劑）處理人卵巢癌細(xì)胞SKO-V3，然后通過(guò)real-TimePCR和Western blot實(shí)驗(yàn)檢測(cè)MMP7mRNA水平和蛋白水平的表達(dá)。 (4)用GANT61裸鼠皮下注射后檢測(cè)人卵巢癌裸鼠皮下移植瘤組織中MMP7蛋白表達(dá)水平的改變。 結(jié)果： (1)成功構(gòu)建干擾質(zhì)粒（miRMMP7-158、314、688），該質(zhì)粒轉(zhuǎn)染SKO-V3細(xì)胞的熒光率大于80%。經(jīng)Western blot檢測(cè)MMP7蛋白表達(dá)，，其中MMP7-158為最佳干擾質(zhì)粒（p＜0.05）。 (2)轉(zhuǎn)染陰性對(duì)照質(zhì)粒組其侵襲與轉(zhuǎn)移能力均明顯強(qiáng)于轉(zhuǎn)染干擾質(zhì)粒組（p＜0.05）；轉(zhuǎn)染陰性對(duì)照質(zhì)粒的SKO-V3細(xì)胞，加入SHH配體條件培養(yǎng)液組其侵襲與遷移能力明顯強(qiáng)于加入對(duì)照培養(yǎng)液組（p＜0.05）；轉(zhuǎn)染miRNAMMP7-158質(zhì)粒的SKO-V3細(xì)胞，加入SHH配體條件培養(yǎng)液組其侵襲與遷移能力與加入對(duì)照培養(yǎng)液組無(wú)差異（p＞0.05）。 (3)經(jīng)SHH配體條件培養(yǎng)液刺激后，SKO-V3細(xì)胞中MMP7的mRNA及蛋白水平的表達(dá)隨處理時(shí)間延長(zhǎng)逐漸增高（p＜0.05）；而經(jīng)GANT61（20μM）處理后的SKO-V3細(xì)胞中MMP7的mRNA及蛋白表達(dá)水平隨處理時(shí)間延長(zhǎng)逐漸降低（p＜0.05）。 (4)免疫組化結(jié)果顯示，經(jīng)GANT61皮下裸鼠注射的人卵巢癌裸鼠皮下移植瘤組織中，MMP7蛋白表達(dá)明顯低于對(duì)照組（p＜0.05）。 結(jié)論： 1. SHH配體條件培養(yǎng)液激活Hh信號(hào)通路，卵巢癌細(xì)胞的侵襲與遷移能力明顯增強(qiáng)，干擾MMP7表達(dá)后，能有效抑制卵巢癌細(xì)胞的侵襲與遷移能力，在干擾掉MMP7的SKO-V3細(xì)胞中無(wú)論是否激活Hh信號(hào)通路，卵巢癌細(xì)胞的侵襲與遷移能力皆減弱，以上結(jié)果提示Hh信號(hào)通路很可能是通過(guò)MMP7的表達(dá)來(lái)調(diào)控卵巢癌的侵襲與轉(zhuǎn)移。 2.采用Gli特異性小分子抑制劑GANT61處理SKO-V3細(xì)胞后， MMP7的mRNA及蛋白表達(dá)水平下降；用SHH配體條件培養(yǎng)液激活Hh信號(hào)通路后，卵巢癌細(xì)胞SKO-V3中MMP7的mRNA及蛋白表達(dá)水平增強(qiáng)；GANT61可能有效抑制人卵巢癌裸鼠皮下移植瘤模型中腫瘤的體積及重量，且實(shí)驗(yàn)組中MMP7的蛋白表達(dá)水平下降；提示MMP7可能是Hh信號(hào)通路的下游靶基因，Hh信號(hào)通路有望成為卵巢癌診療策略的新靶點(diǎn)，為抗癌新藥研發(fā)提供理論依據(jù)。
[Abstract]:Background and purpose:
Ovarian cancer is a very high mortality malignancy in gynecological tumors. The mortality rate has not shown a downward trend in the past 30 years. Owing to its latent onset, early diagnosis is very difficult. Once distant metastasis is found, the Hedgehog (Hh) signaling pathway plays an important role in embryonic development and maintenance of homeostasis in adults. Recent studies have shown that the occurrence and development of many malignant tumors are related to the abnormal activation of Hh signaling pathways, including ovarian cancer. However, the molecular mechanism of how Hh signaling pathways participate in the regulation of ovarian cancer invasion and metastasis remains unclear. The molecular mechanism of invasion and metastasis of nest cancer and the target of early diagnosis and treatment of ovarian cancer will provide theoretical basis for the development of new drugs for ovarian cancer.
Research methods:
(1) According to the sequence of MMP7 cDNA inquired by NCBI, three interference sequences targeting MMP7 were designed on Invitrogen website and linked to pcDNA6.2-GW/EmGFP-miR vector to construct recombinant plasmids, which were named microMMP7-158,314,688 respectively. MMP7 interference plasmids were transfected into human ovarian cancer SKO-V3 cells by liposome 2000, and the fluorescence intensity was observed by inverted fluorescence microscope. To determine the transfection efficiency, Western blot was used to screen effective interference fragments.
(2) Human ovarian cancer cell SKO-V3 was transfected with optimized transfection conditions [plasmid (ug): Lipofectamine 2000 (ul) = 1:3], and the ability of migration and invasion was tested by scratch test and Transwell cell invasion test.
(3) Human ovarian cancer cells SKO-V3 were treated with GANT61 (inhibitor of Hh signaling pathway) and SHH signaling ligand conditioned medium (Hh signaling pathway activator) respectively. The expression of MMP7 mRNA and protein was detected by real-time PCR and Western blot.
(4) After subcutaneous injection of GANT61 into nude mice, the expression of MMP7 protein in human ovarian cancer xenografts was detected.
Result:
(1) The interfering plasmid (MiMMP7-158,314,688) was successfully constructed, and the fluorescence rate of the plasmid transfected SKO-V3 cells was more than 80%. The expression of MMP7 protein was detected by Western blot, and MMP7-158 was the best interfering plasmid (p < 0.05).
(2) The invasive and metastatic abilities of SKO-V3 cells transfected with negative control plasmids were significantly stronger than those of SKO-V3 cells transfected with interfering plasmids (p < 0.05); the invasive and metastatic abilities of SKO-V3 cells transfected with negative control plasmids and cultured with SHH ligand conditioned medium were significantly stronger than those of SKO-V3 cells transfected with MiNAMMP7-158 plasmids (p < 0.05). The invasion and migration ability of the ligand conditioned medium group was not different from that of the control medium (P > 0.05).
(3) After SHH ligand conditioned medium stimulation, the expression of MMP7 mRNA and protein in SKO-V3 cells increased gradually with the prolongation of treatment time (p < 0.05), while the expression of MMP7 mRNA and protein in SKO-V3 cells treated with GANT61 (20 mu M) decreased gradually with the prolongation of treatment time (p < 0.05).
(4) Immunohistochemical staining showed that the expression of MMP7 protein in subcutaneous transplanted human ovarian cancer tissues of nude mice injected with GANT61 was significantly lower than that of control group (p < 0.05).
Conclusion:
1. SHH ligand conditioned medium activates the Hh signaling pathway, and the invasion and migration of ovarian cancer cells are significantly enhanced. Interference with MMP7 expression can effectively inhibit the invasion and migration of ovarian cancer cells. In SKO-V3 cells interfering with MMP7, the invasion and migration of ovarian cancer cells are weakened whether or not the Hh signaling pathway is activated. The results suggest that Hh signaling pathway may regulate the invasion and metastasis of ovarian cancer through the expression of MMP7.
2. After the SKO-V3 cells were treated with Gli-specific small molecule inhibitor GANT61, the expression of MMP7 mRNA and protein decreased; after the Hh signaling pathway was activated by SHH ligand conditioned medium, the expression of MMP7 mRNA and protein in ovarian cancer SKO-V3 cells increased; GANT61 may effectively inhibit the tumor in human ovarian cancer xenograft model in nude mice. The results showed that MMP7 may be the downstream target gene of Hh signaling pathway, and Hh signaling pathway may become a new target of ovarian cancer diagnosis and treatment strategy, providing theoretical basis for the development of new anticancer drugs.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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