关于CD147通过IGFBP2调控恶性黑色素瘤凋亡的体内外相关研究

发布时间：2018-01-09 01:14

  本文关键词：关于CD147通过IGFBP2调控恶性黑色素瘤凋亡的体内外相关研究　出处：《中南大学》2013年博士论文　论文类型：学位论文

  更多相关文章： CD147 恶性黑色素瘤 Akt/mTOR IGFBP2 人凋亡抗体芯片 凋亡

【摘要】：目的 恶性黑素瘤(malignant melanoma, MM)是来源于黑色素细胞的一种侵袭力强、转移性高、进展迅速、预后极差的高度恶性肿瘤,其发病率随着近年来环境恶化呈上升趋势。目前临床上放化疗的主要目的是诱导肿瘤细胞凋亡,凋亡的机制研究也就成为了肿瘤细胞生物学的研究热点。CD147分子是一种在多种肿瘤中高表达的跨膜糖蛋白,属于免疫球蛋白超家族。本研究组前期研究证实CD147在皮肤MM细胞中表达上调,并参与了肿瘤组织侵袭、远处转移和肿瘤血管新生。近年来的研究表明CD147参与了MM细胞的凋亡,而其机制尚不清楚。我们通过蛋白芯片技术,探讨CD147参与MM凋亡的作用及内在机制,初步探讨了CD147作为临床肿瘤治疗靶点的可能性。 本研究中,我们首先建立了CD147敲降的皮肤MM(A375-sh-CD147)细胞株,明确了CD147干扰对A375细胞凋亡的影响；通过人凋亡抗体芯片,找出了与CD147相互作用的凋亡相关蛋白谱,在这一系列蛋白中IGFBP2(Insulin-like Growth Factor Binding Protein2)受CD147调控最显著；我们随后证实了CD147可以通过Akt/mTOR途径调控IGFBP2的表达,进而介导MM细胞的凋亡；同时通过裸鼠皮下成瘤模型进一步在体内水平明确CD147-IGFBP2与凋亡的相互关系。最后,我们利用MM组织芯片,在大样本量临床组织中证实了CD147和IGFBP2表达的相关性。本研究旨在明确CD147分子在皮肤MM凋亡中的作用及调控机制,为临床MM抗凋亡治疗提供创新的靶分子。 方法 1.运用shRNA干扰技术抑制CD147的表达,Western Blot鉴定转染有效性,建立稳定转染细胞株(A375-sh-CD147-Cl/C2)。 2.运用凋亡的相关检测方法,包括Western Blot检测cle-PARP和PTEN.电镜检测A375-sh-ctrl和A375-sh-CD147两组细胞凋亡的形态学改变、Annexin V FITC/PI双染流式细胞术检测两组细胞组的凋亡情况。 3.应用人细胞凋亡的抗体芯片对A375-sh-ctrl和A375-sh-CD147两组细胞的总蛋白进行检测,运用LuxScan10K-A扫描、并采用Cluster软件比较分析找出受CD147调控的凋亡相关蛋白谱。 4.CD147和IGFBP2相互关系的研究：Western Blot和Real Time-PCR技术验证差异蛋白质IGFBP2在各组细胞的表达情况；免疫共沉淀法检测CD147和IGFBP2二者是否直接相互作用；WesternBlot检测信号通路蛋白Akt/mTOR在A375-sh-ctrl和A375-sh-CD147细胞组中的表达；在A375-sh-ctrl细胞中加入Akt信号通路抑制剂后,检测IGFBP2的表达是否有变化。 5.构建裸鼠MM皮下异种移植模型,分为A375-sh-ctrl组和A375-sh-CD147组；运用免疫组化检测CD147和IGFBP2的表达；并运用TUNEL技术检测两组的凋亡情况；免疫组化检测cle-PARP的表达。 6.运用皮肤MM组织芯片研究CD147和IGFBP2的表达。 结果 1.成功建立CD147shRNA稳定转染的细胞克隆(A375-sh-CD147-Cl, A375-sh-CD147-C2), Western Blot表明其抑制率分别为86.21%土8.30%,和28.9%土5.72%(P均0.01),选择抑制效果较好的细胞克隆A375-sh-CD147-Cl (简写为A375-sh-CD147)用于开展后续的研究工作。 2.用5氟尿嘧啶(150μg/mL)处理细胞,诱导凋亡 2.1)相对A375-sh-ctrl细胞,在A375-sh-CD147细胞中,cle-PARP的表达明显增高3.12±0.22倍(P0.01),PTEN的表达也显著增高3.42±0.28倍(P0.01)； 2.2)相对A375-sh-ctrl细胞,A375-sh-CD147细胞形态出现环状、凝块状的核染色质或星月状小体(凋亡小体)等一系列凋亡细胞特征； 2.3)相对A375-sh-ctrl细胞,A375-sh-CD147细胞的凋亡率明显增高4.38±0.25倍(P0.01)。 3.人细胞凋亡抗体芯片检(?)A375-sh-ctrl和A375-sh-CD147细胞组,发现包括IGFBP2等9个凋亡相关蛋白的表达有明显差异(P均0.01)。 4. CD147可以调控IGFBP2: 4.1)IGFBP2蛋白在A375-sh-CD147细胞中的表达,相对A375-sh-ctrl细胞明显降低2.91±0.23倍(P0.01),同时IGFBP2mRNA的表达也显著降低3.42±0.28倍(P0.01)。 4.2)CD147和IGFBP2没有直接发生相互作用； 4.3) p-Akt在A375-shRNA-CD147细胞中的表达降低了3.84±0.22倍,p-mTOR的表达也显著降低2.42±0.20倍(P均0.01),证明CD147可能通过Akt/mTOP信号通路调控IGFBP2的表达; 4.4)加入Akt信号通路抑制剂后,IGFBP2的表达明显降低3.69±±0.74倍(P0.01),证实了Akt信号通路可以上游调控IGFBP2蛋白的产生。 5.5.1)成功构建了裸鼠MM皮下异种移植模型； 5.2)相对于A375-sh-ctrl组的瘤体,CD147和IGFBP2的表达在A375-sh-CD147组的瘤体中都显著降低(P0.01)； 5.3)A375-sh-CD147组的瘤体凋亡显著增加(P0.01); 5.4)cle-PARP在A375-sh-CD147组的瘤体中的表达也明显增加(P0.01)。 6.运用皮肤MM组织芯片检测了192例临床组织中CD147和IGFBP2的表达情况,发现二者的表达正相关,并且二者在转移性MM中的表达都显著高于原位MM(P0.05)。 结论 CD147可能通过Akt/mTOR途径上调IGFBP2的表达,从而抑制了皮肤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 卤 0.22 times ( P0.01 ) , and the expression of PTEN was significantly increased by 3.42 卤 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 卤 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 :

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