本文選題：TGF-β1 + 上皮-間質(zhì)轉(zhuǎn)化　； 參考：《中南大學(xué)》2014年博士論文

【摘要】：第一部分構(gòu)建經(jīng)TGF-β1誘導(dǎo)的EMT化的膀胱尿路上皮細(xì)胞癌T24細(xì)胞并驗(yàn)證 目的探索利用TGF-β1因子提高人膀胱尿路上皮癌T24細(xì)胞系EMT水平的最佳誘導(dǎo)濃度和誘導(dǎo)時(shí)間,并進(jìn)行基因表達(dá)、生物學(xué)行為等的驗(yàn)證。 方法培養(yǎng)人膀胱尿路上皮癌T24細(xì)胞系并傳代,使用不同濃度的TGF-β1因子進(jìn)行誘導(dǎo),然后在不同時(shí)間點(diǎn)對(duì)細(xì)胞形態(tài)變化進(jìn)行觀察。利用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)上皮細(xì)胞-間質(zhì)轉(zhuǎn)化的標(biāo)志物(如：Vimentin、MMP2、E-cadherin、slug、ZEB1等等)的表達(dá)水平進(jìn)行檢測。利用細(xì)胞劃痕試驗(yàn)、Transwell小室遷移試驗(yàn)等對(duì)誘導(dǎo)前后細(xì)胞的侵襲、穿透、遷移等能力進(jìn)行檢測和評(píng)估,進(jìn)一步驗(yàn)證TGF-β1因子在T24細(xì)胞EMT進(jìn)程中的最佳誘導(dǎo)濃度和誘導(dǎo)時(shí)間。 結(jié)果濃度為lOng/ml的TGF-β1誘導(dǎo)T24細(xì)胞36h后,T24細(xì)胞的細(xì)胞形態(tài)變化提示有上皮細(xì)胞間質(zhì)化發(fā)生；同時(shí),EMT標(biāo)志基因的表達(dá)水平亦出現(xiàn)相應(yīng)改變,如：間質(zhì)細(xì)胞標(biāo)志物Vimentin和MMP2表達(dá)水平上調(diào),E-cadherin等上皮細(xì)胞標(biāo)志物表達(dá)下調(diào),slug、 ZEB1等促EMT因子表達(dá)上調(diào)；另外,細(xì)胞劃痕試驗(yàn)顯示,lOng/ml的TGF-β1因子誘導(dǎo)前后兩組的平均愈合面積(百分比)差異有顯著性(0.81vs1,P0.05)；同樣,Transwell小室遷移試驗(yàn)檢測T24-N.C.組和T24-TGF-β1組細(xì)胞的侵襲情況發(fā)現(xiàn)10ng/mlT24-TGF-β1組細(xì)胞侵襲能力明顯增強(qiáng),(每視野平均細(xì)胞數(shù)54.6vs97.65,P0.05)。 結(jié)論膀胱尿路上皮癌T24細(xì)胞經(jīng)濃度為10ng/ml的TGF-β1因子誘導(dǎo)36小時(shí)后其細(xì)胞形態(tài)學(xué)呈現(xiàn)較為明顯的上皮細(xì)胞間質(zhì)化改變。并且其誘導(dǎo)后細(xì)胞的基因呈現(xiàn)EMT化的相關(guān)表達(dá),其細(xì)胞的侵襲、穿透、遠(yuǎn)處遷移等能力均有明顯增強(qiáng)。 第二部分構(gòu)建TGF-β1誘導(dǎo)的EMT化的膀胱尿路上皮癌T24細(xì)胞的mRNA和microRNA的表達(dá)譜 目的構(gòu)建TGF-β1因子誘導(dǎo)的膀胱尿路上皮癌T24細(xì)胞的microRNA表達(dá)譜和mRNA的表達(dá)譜。 方法對(duì)TGF-β1誘導(dǎo)的EMT化的膀胱尿路上皮癌T24細(xì)胞的總RNA進(jìn)行提取,并予以質(zhì)檢。質(zhì)檢合格后進(jìn)行RNA純化、孵育加尾、標(biāo)記生物素?zé)晒�、芯片雜交、清洗染色、掃描芯片、數(shù)據(jù)提取以及數(shù)據(jù)標(biāo)準(zhǔn)化等,所用芯片為目前較為先進(jìn)GeneChip(?)PrimeView M Human Gene Expression Array芯片以及Multispecies miRNA2.0array芯片。然后注冊(cè)并利用分子功能注釋3.0系統(tǒng)(Capital Bio(?) Molecule Annotation System V3.0, MAS3.0)對(duì)芯片結(jié)果進(jìn)行相關(guān)分析(如GO, KEGG pathway,靶基因預(yù)測,列聯(lián)分析等等)。 結(jié)果經(jīng)10ng/ml TGF-β1因子誘導(dǎo)36小時(shí)后的膀胱尿路上皮癌T24細(xì)胞共有包括SPC25等在內(nèi)的1062個(gè)基因的表達(dá)下調(diào),以及包括ASNS基因在內(nèi)的572個(gè)基因的表達(dá)上調(diào)。經(jīng)KEGG pathway平臺(tái)分析發(fā)現(xiàn),共有包括Cell cycle等在內(nèi)的40條信號(hào)傳導(dǎo)通路與之密切相關(guān)。利用GO數(shù)據(jù)庫對(duì)該基因集進(jìn)行闡釋,共運(yùn)算出1250條相關(guān)的GO信息,其中多與細(xì)胞周期、細(xì)胞分裂、黏附、代謝調(diào)控、免疫反應(yīng)、運(yùn)動(dòng)等密切相關(guān)。microRNA芯片掃描結(jié)果則顯示,microRNA-1184等在內(nèi)的6個(gè)轉(zhuǎn)錄本表達(dá)下調(diào),而以及包括microRNA-21等在內(nèi)的8個(gè)microRNA的表達(dá)上調(diào)。MicroRNA芯片與mRNA芯片列聯(lián)分析發(fā)現(xiàn)包括microRNA-21、microRNA-146a以及microRNA-638在內(nèi)的3個(gè)microRNAs與部分差異表達(dá)的基因的表達(dá)趨勢(shì)存在相關(guān),其中包括ACVR1C、NOG、THBS1、TUBB2A等基因與EMT過程相關(guān)性較高。 結(jié)論獲得了TGF-β1誘導(dǎo)的膀胱尿路上皮癌T24細(xì)胞系EMT過程中的mRNA以及microRNA的差異表達(dá)譜；T24細(xì)胞系EMT進(jìn)程中涉及到包括Cell cycle等在內(nèi)的多個(gè)分子信號(hào)通路,這些分子信號(hào)通路多與細(xì)胞的生長、連接、黏附、運(yùn)動(dòng)、侵襲等生物學(xué)行為相關(guān)；microRNA-21、microRNA-146a以及microRNA-638很可能通過靶向調(diào)控ACVR1C等基因的方式在T24細(xì)胞的EMT進(jìn)程中發(fā)揮作用。 第三部分對(duì)TGF-β1因子誘導(dǎo)的膀胱尿路上皮癌T24細(xì)胞的生物芯片分析結(jié)果的驗(yàn)證 目的對(duì)生物信息學(xué)分析的基因芯片及microRNA芯片的結(jié)果進(jìn)行初步的實(shí)驗(yàn)室驗(yàn)證,以期提高該分析結(jié)果的可信性。 方法利用microRNA mimics以及microRNA inhibitor對(duì)microRNA進(jìn)行過表達(dá)以及抑制表達(dá)。Real-time PCR試驗(yàn)驗(yàn)證三個(gè)microRNA的表達(dá)趨勢(shì)是否與芯片結(jié)果相同、mimics和inhibitor的表達(dá)效果、以及對(duì)靶基因表達(dá)的調(diào)節(jié)。劃痕試驗(yàn)、Transwell小室遷移實(shí)驗(yàn)驗(yàn)證細(xì)胞是否發(fā)生相應(yīng)的侵襲、轉(zhuǎn)移等生物學(xué)行為的改變。 結(jié)果PCR結(jié)果顯示,microRNA-21、microRNA-146a以及microRNA-638等microRNAs的表達(dá)趨勢(shì)與芯片結(jié)果相同,microRNA的mimics和inhibitor達(dá)到預(yù)期效果,并且這些microRNAs的靶基因表達(dá)與篩選出的生物信息學(xué)分析結(jié)果相同；劃痕試驗(yàn)顯示除陰性對(duì)照組(N.C.)外的mimics組侵襲能力明顯增強(qiáng),并且各inhibitor組(除inhibitor N.C.組外)的侵襲能力有所減弱。Transwell小室遷移實(shí)驗(yàn)提示除陰性對(duì)照組(N.C.)外的mimics組遷移轉(zhuǎn)移能力明顯增強(qiáng),inhibitor組的遷移轉(zhuǎn)移能力有所減弱。 結(jié)論microRNA-21、microRNA-146a以及microRNA-638等可通過靶向調(diào)控ACVR1C等基因的方式來在膀胱尿路上皮癌T24細(xì)胞的EMT進(jìn)程中發(fā)揮作用,抑制這些microRNAs的表達(dá)則可抑制膀胱尿路上皮癌細(xì)胞的上皮間質(zhì)化進(jìn)程,進(jìn)而影響其細(xì)胞的侵襲、遷移的能力。
[Abstract]:The first part was constructed by transforming growth factor - 尾1 - induced EMT - induced bladder urological epithelial cell carcinoma

Objective To explore the optimal induction concentration and induction time of EMT in human bladder urinary tract epithelial carcinoma ( BTCC ) by transforming growth factor - 尾1 ( TGF - 尾1 ) , and to verify the gene expression , biological behavior and so on .

Methods The expression level of TGF - 尾1 was detected by means of real - time fluorescence quantitative PCR , and the optimal concentration and induction time of TGF - 尾1 in EMT process were further verified by means of cell scratch test , Transwell chamber migration test and so on .

Results The changes of cellular morphological changes of 24 cells showed that the epithelial cells were interstitial after 36 h of TGF - 尾1 induced by lOng / ml .
At the same time , the expression level of EMT marker genes also changed accordingly , such as up - regulation of the expression levels of vimentin and MMP2 in the interstitial cell markers , downregulation of epithelial markers such as E - cadherin , and up - regulation of EMT factors such as downregulation of epithelial markers such as E - cadherin and the like ;
In addition , the cell scratch test showed that the difference of average healing area ( percentage ) between the two groups was significant ( 0.81 vs1 , P0.05 ) after lOng / ml TGF - 尾1 .
In the same way , the invasion of the cells in the group 24 - N . C . and 24 - TGF - 尾1 was detected by the Transwell chamber migration test , and the invasion ability of the cells was significantly increased in 10 ng / ml 24 - TGF - 尾1 group ( 54.6 vs 97.65 , P < 0.05 ) .

Conclusion The expression of EMT in the cells after induction of TGF - 尾1 at 10 ng / ml for 24 hours after induction of TGF - 尾1 with 10 ng / ml has been shown to be related to the expression of EMT , the invasion , penetration and far - distance migration of the cells .

In the second part , TGF - 尾1 - induced EMT - induced expression profiles of mRNA and microRNA in human EMT 24 cells were detected .

Objective To construct the expression profiles of microRNA expression profiles and mRNA expression in bladder urinary tract epithelial cancer cell line 24 induced by TGF - 尾1 factor .

鏂規(guī)硶瀵筎GF-尾1璇卞鐨凟MT鍖栫殑鑶，

本文編號(hào)：1782135