本文關(guān)鍵詞：關(guān)于CD147通過IGFBP2調(diào)控惡性黑色素瘤凋亡的體內(nèi)外相關(guān)研究　出處：《中南大學(xué)》2013年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CD147 惡性黑色素瘤 Akt/mTOR IGFBP2 人凋亡抗體芯片 凋亡

【摘要】：目的 惡性黑素瘤(malignant melanoma, MM)是來源于黑色素細(xì)胞的一種侵襲力強(qiáng)、轉(zhuǎn)移性高、進(jìn)展迅速、預(yù)后極差的高度惡性腫瘤,其發(fā)病率隨著近年來環(huán)境惡化呈上升趨勢(shì)。目前臨床上放化療的主要目的是誘導(dǎo)腫瘤細(xì)胞凋亡,凋亡的機(jī)制研究也就成為了腫瘤細(xì)胞生物學(xué)的研究熱點(diǎn)。CD147分子是一種在多種腫瘤中高表達(dá)的跨膜糖蛋白,屬于免疫球蛋白超家族。本研究組前期研究證實(shí)CD147在皮膚MM細(xì)胞中表達(dá)上調(diào),并參與了腫瘤組織侵襲、遠(yuǎn)處轉(zhuǎn)移和腫瘤血管新生。近年來的研究表明CD147參與了MM細(xì)胞的凋亡,而其機(jī)制尚不清楚。我們通過蛋白芯片技術(shù),探討CD147參與MM凋亡的作用及內(nèi)在機(jī)制,初步探討了CD147作為臨床腫瘤治療靶點(diǎn)的可能性。 本研究中,我們首先建立了CD147敲降的皮膚MM(A375-sh-CD147)細(xì)胞株,明確了CD147干擾對(duì)A375細(xì)胞凋亡的影響；通過人凋亡抗體芯片,找出了與CD147相互作用的凋亡相關(guān)蛋白譜,在這一系列蛋白中IGFBP2(Insulin-like Growth Factor Binding Protein2)受CD147調(diào)控最顯著；我們隨后證實(shí)了CD147可以通過Akt/mTOR途徑調(diào)控IGFBP2的表達(dá),進(jìn)而介導(dǎo)MM細(xì)胞的凋亡；同時(shí)通過裸鼠皮下成瘤模型進(jìn)一步在體內(nèi)水平明確CD147-IGFBP2與凋亡的相互關(guān)系。最后,我們利用MM組織芯片,在大樣本量臨床組織中證實(shí)了CD147和IGFBP2表達(dá)的相關(guān)性。本研究旨在明確CD147分子在皮膚MM凋亡中的作用及調(diào)控機(jī)制,為臨床MM抗凋亡治療提供創(chuàng)新的靶分子。 方法 1.運(yùn)用shRNA干擾技術(shù)抑制CD147的表達(dá),Western Blot鑒定轉(zhuǎn)染有效性,建立穩(wěn)定轉(zhuǎn)染細(xì)胞株(A375-sh-CD147-Cl/C2)。 2.運(yùn)用凋亡的相關(guān)檢測(cè)方法,包括Western Blot檢測(cè)cle-PARP和PTEN.電鏡檢測(cè)A375-sh-ctrl和A375-sh-CD147兩組細(xì)胞凋亡的形態(tài)學(xué)改變、Annexin V FITC/PI雙染流式細(xì)胞術(shù)檢測(cè)兩組細(xì)胞組的凋亡情況。 3.應(yīng)用人細(xì)胞凋亡的抗體芯片對(duì)A375-sh-ctrl和A375-sh-CD147兩組細(xì)胞的總蛋白進(jìn)行檢測(cè),運(yùn)用LuxScan10K-A掃描、并采用Cluster軟件比較分析找出受CD147調(diào)控的凋亡相關(guān)蛋白譜。 4.CD147和IGFBP2相互關(guān)系的研究：Western Blot和Real Time-PCR技術(shù)驗(yàn)證差異蛋白質(zhì)IGFBP2在各組細(xì)胞的表達(dá)情況；免疫共沉淀法檢測(cè)CD147和IGFBP2二者是否直接相互作用；WesternBlot檢測(cè)信號(hào)通路蛋白Akt/mTOR在A375-sh-ctrl和A375-sh-CD147細(xì)胞組中的表達(dá)；在A375-sh-ctrl細(xì)胞中加入Akt信號(hào)通路抑制劑后,檢測(cè)IGFBP2的表達(dá)是否有變化。 5.構(gòu)建裸鼠MM皮下異種移植模型,分為A375-sh-ctrl組和A375-sh-CD147組；運(yùn)用免疫組化檢測(cè)CD147和IGFBP2的表達(dá)；并運(yùn)用TUNEL技術(shù)檢測(cè)兩組的凋亡情況；免疫組化檢測(cè)cle-PARP的表達(dá)。 6.運(yùn)用皮膚MM組織芯片研究CD147和IGFBP2的表達(dá)。 結(jié)果 1.成功建立CD147shRNA穩(wěn)定轉(zhuǎn)染的細(xì)胞克隆(A375-sh-CD147-Cl, A375-sh-CD147-C2), Western Blot表明其抑制率分別為86.21%土8.30%,和28.9%土5.72%(P均0.01),選擇抑制效果較好的細(xì)胞克隆A375-sh-CD147-Cl (簡(jiǎn)寫為A375-sh-CD147)用于開展后續(xù)的研究工作。 2.用5氟尿嘧啶(150μg/mL)處理細(xì)胞,誘導(dǎo)凋亡 2.1)相對(duì)A375-sh-ctrl細(xì)胞,在A375-sh-CD147細(xì)胞中,cle-PARP的表達(dá)明顯增高3.12±0.22倍(P0.01),PTEN的表達(dá)也顯著增高3.42±0.28倍(P0.01)； 2.2)相對(duì)A375-sh-ctrl細(xì)胞,A375-sh-CD147細(xì)胞形態(tài)出現(xiàn)環(huán)狀、凝塊狀的核染色質(zhì)或星月狀小體(凋亡小體)等一系列凋亡細(xì)胞特征； 2.3)相對(duì)A375-sh-ctrl細(xì)胞,A375-sh-CD147細(xì)胞的凋亡率明顯增高4.38±0.25倍(P0.01)。 3.人細(xì)胞凋亡抗體芯片檢(?)A375-sh-ctrl和A375-sh-CD147細(xì)胞組,發(fā)現(xiàn)包括IGFBP2等9個(gè)凋亡相關(guān)蛋白的表達(dá)有明顯差異(P均0.01)。 4. CD147可以調(diào)控IGFBP2: 4.1)IGFBP2蛋白在A375-sh-CD147細(xì)胞中的表達(dá),相對(duì)A375-sh-ctrl細(xì)胞明顯降低2.91±0.23倍(P0.01),同時(shí)IGFBP2mRNA的表達(dá)也顯著降低3.42±0.28倍(P0.01)。 4.2)CD147和IGFBP2沒有直接發(fā)生相互作用； 4.3) p-Akt在A375-shRNA-CD147細(xì)胞中的表達(dá)降低了3.84±0.22倍,p-mTOR的表達(dá)也顯著降低2.42±0.20倍(P均0.01),證明CD147可能通過Akt/mTOP信號(hào)通路調(diào)控IGFBP2的表達(dá); 4.4)加入Akt信號(hào)通路抑制劑后,IGFBP2的表達(dá)明顯降低3.69±±0.74倍(P0.01),證實(shí)了Akt信號(hào)通路可以上游調(diào)控IGFBP2蛋白的產(chǎn)生。 5.5.1)成功構(gòu)建了裸鼠MM皮下異種移植模型； 5.2)相對(duì)于A375-sh-ctrl組的瘤體,CD147和IGFBP2的表達(dá)在A375-sh-CD147組的瘤體中都顯著降低(P0.01)； 5.3)A375-sh-CD147組的瘤體凋亡顯著增加(P0.01); 5.4)cle-PARP在A375-sh-CD147組的瘤體中的表達(dá)也明顯增加(P0.01)。 6.運(yùn)用皮膚MM組織芯片檢測(cè)了192例臨床組織中CD147和IGFBP2的表達(dá)情況,發(fā)現(xiàn)二者的表達(dá)正相關(guān),并且二者在轉(zhuǎn)移性MM中的表達(dá)都顯著高于原位MM(P0.05)。 結(jié)論 CD147可能通過Akt/mTOR途徑上調(diào)IGFBP2的表達(dá),從而抑制了皮膚MM的凋亡。
[Abstract]:Purpose Malignant melanoma ( MM ) is a highly invasive malignant melanoma derived from melanoma cells with high metastatic potential , rapid progress and poor prognosis . The main aim of this study is to induce apoptosis and apoptosis in tumor cells . In this study , we first established the CD147 - knock - down skin MM ( A375 - sh - CD147 ) cell line , and identified the effect of CD147 interference on A375 cell apoptosis . method 1 . The expression of CD147 was inhibited by shRNA interference technique , and the transfection efficiency was identified by Western Blot . A stable transfection cell line was established ( A375 - sh - CD147 - Cl / C2 ) . 2 . Apoptosis was detected by Western Blot . The apoptosis of A375 - sh - ctrl and A375 - sh - CD147 were detected by means of Western Blot . 3 . The total protein of the two groups of A375 - sh - ctrl and A375 - sh - CD147 cells was detected by using the antibody chip of human cell apoptosis . LuxScan10K - A was used to scan the total protein , and the apoptosis - related protein spectrum regulated by CD147 was found by cluster software . 4 . The relationship between CD147 and IGFBP2 was studied by Western Blot and Real Time - PCR . Western Blot and Real Time - PCR were used to detect the expression of IGFBP2 in each group . Western Blot was used to detect the direct interaction between CD147 and IGFBP2 . Western Blot was used to detect the expression of the signal pathway protein , the signal pathway protein , the signal pathway protein , the signal pathway protein , the signal pathway protein , the expression of the signal pathway protein , the signal pathway protein , A375 - sh - ctrl and A375 - sh - CD147 cells , and the expression of IGFBP2 was detected in A375 - sh - ctrl cells . 5.The expression of CD147 and IGFBP2 was detected by immunohistochemistry and TUNEL technique was used to detect the apoptosis of the two groups . 6 . The expression of CD147 and IGFBP2 was studied using skin MM tissue microarray . Results 1 . After successful establishment of CD147 shRNA stably transfected cell clones ( A375 - sh - CD147 - Cl , A375 - sh - CD147 - C2 ) , Western Blot showed that the inhibition rate was 86.21 % soil 8.30 % , and 28.9 % soil 5.72 % ( P < 0.01 ) , and the best cell clone A375 - sh - CD147 - Cl ( abbreviated as A375 - sh - CD147 ) was used to carry out the subsequent research . 2 . Cells were treated with 5 - fluorouracil ( 150 渭g / mL ) to induce apoptosis 2.1 ) Compared with A375 - sh - ctrl cells , in A375 - sh - CD147 cells , the expression of PTEN was significantly increased 3.12 鹵 0.22 times ( P0.01 ) , and the expression of PTEN was significantly increased by 3.42 鹵 0.28 times ( P0.01 ) . 2.2 ) A375 - sh - ctrl cells , A375 - sh - CD147 cells were characterized by a series of apoptotic cells , such as ring - shaped , massive nuclear chromatin or star - like small bodies ( apoptotic bodies ) ; 2.3 ) Compared with A375 - sh - ctrl cells , the apoptosis rate of A375 - sh - CD147 cells was significantly increased 4.38 鹵 0.25 times ( P0.01 ) . 3 . The expression of apoptosis - related proteins , including IGFBP2 , was found to be significantly different ( P < 0.01 ) . 4 . CD147 can regulate IGFBP2 :

4.1)IGFBP2铔嬬櫧鍦ˋ375-sh-CD147緇嗚優(yōu)涓殑琛ㄨ揪,鐩稿A375-sh-ctrl緇嗚優(yōu)鏄庢樉闄嶄綆2.91鹵0.23鍊，

本文編號(hào)：1399414