本文選題：CRISPR/CAS9 + p53�。� 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：第一部分P53敲除A375細(xì)胞系的建立目的：利用CRISPR/CAS9系統(tǒng)建立p53敲除的細(xì)胞系方法：設(shè)計兩條針對人的p53基因的gRNA,分別將兩條gRNA插入pBT-U6-Cas9-2A-GFP表達質(zhì)粒,然后將含有不同gRNA的質(zhì)粒分別轉(zhuǎn)染進入K562細(xì)胞,用SUVEYOR突變檢測試劑盒,對兩條設(shè)計的gRNA的剪切效率進行比較,選取效率較高gRNA進行后續(xù)p53敲除。在A375細(xì)胞中轉(zhuǎn)染GFP標(biāo)記含有g(shù)RNA和CAS9核酸酶的質(zhì)粒,驗證突變,同時流式分選出GFP陽性的細(xì)胞。將分選出的細(xì)胞,分別單個接種于96孔板,待其克隆形成。部分克隆形成之后,將其擴增傳代,形成單克隆細(xì)胞系。為從中篩選出p53敲除細(xì)胞,首先我們使用x-ray處理所有獲得克隆,然后免疫電泳檢測所有克隆中p53的表達情況,選出p53表達陰性的克隆進行測序,以驗證p53敲除的可靠性。同時也對X射線刺激細(xì)胞,然后對p53下游基因p21進行檢測,以在功能上確認(rèn)p53的敲除。結(jié)果：SUVEYOR結(jié)果顯示設(shè)計的兩條gRNA均可以造成突變,編輯效率第一條gRNA較好。使用gRNA 1轉(zhuǎn)染A375細(xì)胞,SUVEYOR檢測到突變發(fā)生。Western檢測p53在各個克隆中的表達發(fā)現(xiàn),在獲得的23個克隆中,有3個克隆在p53位置出現(xiàn)了缺失,敲除效率為3/23.同時使用PCR對敲除位點進行了擴增,相比正常WT p53條帶,所獲得疑似敲除克隆的p53條帶位置均發(fā)生了變化。測序結(jié)果顯示,所獲得三個陽性克隆p53基因段均發(fā)生了明顯的基因插入或者缺失,導(dǎo)致了p53表達的失調(diào)。最后對p53下游基因p21的表達進行檢測,發(fā)現(xiàn)所獲克隆中,在p53已被激活的情況下,p21的表達全部缺失。結(jié)論：利用CRISPR/CAS9系統(tǒng),成功敲除p53基因,獲得了穩(wěn)定遺傳的p53完全敲除和p53變異細(xì)胞株。第二部分p53通過microRNAs抑制B7-H1的表達目的：研究p53敲除后是否對細(xì)胞B7-H1的表達造成影響以及通過何種機制影響其表達方法：分別用流式檢測A375.HCT116和其p53敲除前后的B7-H1表達；使用RT-PCR檢測B7-H1的信使RNA在A375, HCT116和其p53敲除細(xì)胞系中的表達：使用microRNA芯片檢測A375和A375Δp53細(xì)胞之間的microRNA表達差異,并結(jié)合B7-H1靶點預(yù)測,從而篩選出可能具有對B7-H1具有調(diào)控功能的microRNAs;將篩選出來的microRNAs mimics轉(zhuǎn)染A375Δp53細(xì)胞,觀察B7-H1在各個不同mimics轉(zhuǎn)染后的表達情況,選擇出調(diào)控B7-H1表達的microRNA;使用特異性inhibitors同mimics一同轉(zhuǎn)染A375Δp53細(xì)胞,以確認(rèn)microRNA作用的特異性;使用qRT-PCR檢測A375,HCT116和其p53敲除前后以及使用siRNA敲除p53之后的microRNA-34a以及microRNA-200b表達,以驗證p53對microRNA-34a以及microRNA-200b表達的調(diào)控作用,并使用免疫電泳對p53以及B7-H1的表達進行檢測。構(gòu)建雙熒光報告載體驗證microRNA-34a以及microRNA-200b靶向作用于B7-Hl的3UTR。結(jié)果：當(dāng)p53缺失時,通過流式檢測發(fā)現(xiàn)B7-H1的表達在A375, HCT116細(xì)胞中均明顯上升；之后,對B7-H1的信使RNA表達水平進行檢測,發(fā)現(xiàn)p53的敲除,并不影響B(tài)7-H1 mRNA的表達,結(jié)果無顯著性差異；通過篩選,并結(jié)合靶向預(yù)測,我們發(fā)現(xiàn)在p53敲除前后,有8個候選microRNAs可能調(diào)控B7-H1表達。轉(zhuǎn)染mimics后發(fā)現(xiàn),microRNA-34a和microRNA-200b可下調(diào)B7-H1在A375Δp53細(xì)胞中的表達,差異明顯。在加入microRNA-34a和microRNA-200b inhibitors共轉(zhuǎn)染時,這種下調(diào)效果可被消除。實時定量PCR檢測發(fā)現(xiàn),在p53敲除或敲低的情況下,microRNA-34a和microRNA-200b表達明顯下降,同時B7-H1的表達上調(diào)。雙熒光素報告載體檢測發(fā)現(xiàn),microRNA-34a和microRNA-200b可特異性結(jié)合域B7-H1的3'UTR端調(diào)控轉(zhuǎn)錄。結(jié)論：p53的敲除下調(diào)microRNA-34a和microRNA-200b的表達,從而抑制microRNA-34a和microRNA-200b對B7-H1信使RNA的作用,導(dǎo)致B7-H1蛋白表達上升。第三部分microRNA-34a和microRNA-200b調(diào)控Jurkat增殖和細(xì)胞因子分泌目的：研究microRNA-34a和microRNA-200b通過調(diào)控B7-H1分子從而影響Jurkat細(xì)胞的功能,介導(dǎo)免疫抑制。方法：培養(yǎng)Jurkat細(xì)胞,用TPA刺激,誘導(dǎo)其表達PD-1受體,免疫電泳檢測誘導(dǎo)表達效果;將誘導(dǎo)表達PD-1的Jurkat細(xì)胞同A375Δp53細(xì)胞共培養(yǎng),利用B7-H1阻斷性抗體抑制B7-H1/PD-1通路的激活,觀察Jurkat細(xì)胞的增殖情況。在使用microRNA-34a和microRNA-200b的mimics和inhibitors分別轉(zhuǎn)染A375Ap53細(xì)胞后同Jurkat細(xì)胞共培養(yǎng),以觀察microRNA-34a和microRNA-200b對細(xì)胞造成的增殖改變。同時收集共培養(yǎng)細(xì)胞的上清培養(yǎng)液,檢測microRNA-34a和microRNA-200b對細(xì)胞分泌IFN-gamma, IL-2細(xì)胞因子的影響。結(jié)果：jurkat細(xì)胞在TPA刺激后,其PD-1的表達明顯增強。將刺激后的細(xì)胞同A375Δp53細(xì)胞共培養(yǎng),其增殖受到明顯抑制,在用B7-H1抗體阻斷后,此種增值抑制效應(yīng)可以得到解除；同時,結(jié)果顯示,A375Ap53細(xì)胞相對于A375細(xì)胞更能明顯抑制Jurkat細(xì)胞增殖。在A375Δp53細(xì)胞中轉(zhuǎn)染進microRNA-34a和microRNA-200b的mimics后可以部分消除對Jurkat細(xì)胞的增殖抑制效應(yīng),加入對應(yīng)inhibitors,可以恢復(fù)對Jurkat細(xì)胞的抑制。同時對細(xì)胞因子的檢測發(fā)現(xiàn),A375Δp53細(xì)胞表達B7-H1抑制Jurkat細(xì)胞的細(xì)胞因子分泌,microRNA-34a和microRNA-200b的mimics轉(zhuǎn)染可以有效解除抑制效果,共轉(zhuǎn)染對應(yīng)inhibitors可以使得分泌再次被抑制。結(jié)論：PD-1/B7-H1通路的激活可以抑制Jurkat細(xì)胞的增殖。A375Ap53細(xì)胞相比A375細(xì)胞具有更強的免疫抑制能力,此種抑制效應(yīng)同microRNA-34a和microRNA-200b對B7-H1表達的調(diào)控具有聯(lián)系。microRNA-34a和microRNA-200b的表達可以明顯抑制對Jurkat細(xì)胞功能的抑制。
[Abstract]:The first part of the P53 knockout A375 cell line: using the CRISPR/CAS9 system to establish the p53 knockout cell line method: design two gRNA for human p53 gene, insert two gRNA into pBT-U6-Cas9-2A-GFP expression plasmid, and then transfer the plasmid containing different gRNA into K562 cells, and use SUVEYOR mutation to detect the reagent. In the box, the shear efficiency of the two designed gRNA was compared, and the efficient gRNA was selected for subsequent p53 knockout. The plasmid containing gRNA and CAS9 nuclease was transfected in the A375 cell to verify the mutation, and the flow formula was used to separate the GFP positive cells. After the formation of the lung, the PCR was amplified to form a monoclonal cell line. In order to screen out the p53 knockout cells, we first used X-ray to treat all the clones, and then the expression of p53 in all the clones was detected by immunoelectrophoresis, and the clones with negative p53 expression were sequenced to verify the reliability of the p53 knockout. At the same time, the X ray stimulation was also used. Cells, then detection of the downstream gene p21 of p53 to confirm the knockout of p53 in function. Results: SUVEYOR results showed that the two gRNA of the designed gRNA could cause the mutation, the efficiency of the first gRNA was better. GRNA 1 was used to transfect A375 cells, and SUVEYOR detected the expression of.Western detection p53 in the individual clones. Of the 23 clones, 3 clones were missing in the p53 position, the knockout efficiency was 3/23. and the PCR knockout loci were amplified. Compared to the normal WT p53 strip, the position of the suspected knockout clone p53 bands had changed. The results showed that the three positive clones p53 gene segments had obvious genes. The insertion or deletion resulted in the maladjustment of p53 expression. Finally, the expression of the p53 downstream gene p21 was detected. In the clones, the expression of p21 was completely missing in the case of p53 being activated. Conclusion: the p53 gene was successfully knocked out by the CRISPR/CAS9 system, and the stable hereditary p53 knockout and p53 variant cell lines were obtained. Second parts were obtained. P53 suppression of the expression of B7-H1 through microRNAs: the study of whether p53 knockout has an impact on the expression of B7-H1, and the mechanism to affect its expression: A375.HCT116 and B7-H1 expression before and after the p53 knockout, respectively, and the messenger and its knockout cells using RT-PCR to detect B7-H1. Expression in the system: microRNA chip was used to detect the difference of microRNA expression between A375 and A375 delta p53 cells, and the B7-H1 target prediction was used to screen the microRNAs that may have regulatory function to B7-H1, and the selected microRNAs mimics transfected to A375 delta p53 cells. In addition, microRNA was selected to regulate the expression of B7-H1, and A375 delta p53 cells were transfected with mimics with specific inhibitors to confirm the specificity of microRNA action, and qRT-PCR was used to detect A375, HCT116 and its p53 knockout, as well as the expression after the use of siRNA knockout. The expression of croRNA-200b and the expression of p53 and B7-H1 were detected by immunoelectrophoresis. A double fluorescent report vector was constructed to verify the 3UTR. results of microRNA-34a and microRNA-200b targeting B7-Hl. When p53 was missing, the expression of B7-H1 was obviously increased in A375, HCT116 cells by flow detection; after that, the expression of B7-H1 was obviously increased. To detect the expression level of B7-H1's messenger RNA, it is found that p53 knockout does not affect the expression of B7-H1 mRNA, and there is no significant difference in the results. Through screening and targeting prediction, we found that 8 candidate microRNAs may regulate the expression of B7-H1 before and after p53 knockout. After transfection of mimics, microRNA-34a and microRNA-200b can downregulate B7-H1 The expression in A375 delta p53 cells was distinctly different. The down regulation effect could be eliminated when CO transfected with microRNA-34a and microRNA-200b inhibitors. Real-time quantitative PCR detection found that the expression of microRNA-34a and microRNA-200b decreased significantly in the case of p53 knockout or knock down, while the expression of B7-H1 was up. It is found that the 3'UTR terminal of the microRNA-34a and microRNA-200b specific binding domain B7-H1 controls the transcription. Conclusion: p53 knocks down the expression of microRNA-34a and microRNA-200b, thus inhibiting the effect of microRNA-34a and microRNA-200b on B7-H1 messenger RNA, leading to the increase of the expression of the B7-H1 protein. The purpose of Kat proliferation and cytokine secretion is to study the effect of microRNA-34a and microRNA-200b on the function of Jurkat cells by regulating B7-H1 molecules and mediated immunosuppression. Methods: Jurkat cells were cultured and stimulated by TPA to induce the expression of PD-1 receptors, and immunoelectrophoresis was used to detect the induction of expression; Jurkat cells expressing PD-1 were induced with A375 delta p53. Cell co culture was used to observe the proliferation of Jurkat cells by using B7-H1 blocking antibodies to inhibit the activation of the B7-H1/PD-1 pathway. The proliferation of A375Ap53 cells was co cultured with A375Ap53 cells using mimics and inhibitors in microRNA-34a and microRNA-200b, respectively, to observe the proliferation of microRNA-34a and microRNA-200b on the cells. The effect of microRNA-34a and microRNA-200b on the secretion of IFN-gamma and IL-2 cytokine was detected by the supernatant culture of co cultured cells. Results: the expression of PD-1 in Jurkat cells increased obviously after TPA stimulation. The stimulated cells were co cultured with A375 delta p53 cells, and the proliferation of Jurkat cells was significantly inhibited. This type of cells was blocked by B7-H1 antibodies. At the same time, the results showed that A375Ap53 cells could significantly inhibit the proliferation of Jurkat cells compared with A375 cells. The proliferation inhibition response to Jurkat cells could be partially eliminated after A375 delta p53 cells were transferred to microRNA-34a and microRNA-200b mimics, and the corresponding inhibitors could be restored to Jurkat. At the same time, the detection of cytokines showed that A375 delta p53 cells expressed B7-H1 to inhibit the secretion of cytokines in Jurkat cells, and mimics transfection of microRNA-34a and microRNA-200b could effectively relieve the inhibitory effect. Co transfection of corresponding inhibitors could cause the secretion of secretions to be suppressed again. Conclusion: the activation of PD-1/B7-H1 pathway can inhibit J. The proliferating.A375Ap53 cells of urkat cells have stronger immunosuppressive ability than A375 cells. This inhibitory effect is associated with the regulation of microRNA-34a and microRNA-200b on the expression of B7-H1 and the expression of.MicroRNA-34a and microRNA-200b can obviously inhibit the inhibition of Jurkat cell function.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.2

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7 查若鵬;肝癌轉(zhuǎn)移相關(guān)microRNA的鑒定及其分子機制研究[D];復(fù)旦大學(xué);2013年

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9 辛成齊;椰棗microRNA鑒定及其在果實發(fā)育過程中的表達譜研究[D];中國科學(xué)院北京基因組研究所;2015年

10 李棟;先天性心臟病血漿microRNA表達譜及與GATA4靶序列單核苷酸多態(tài)性的關(guān)聯(lián)研究[D];山東大學(xué);2015年

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1 高智紅;應(yīng)用多樣性增量方法識別人類基因組microRNA前體序列[D];內(nèi)蒙古大學(xué);2010年

2 王娜娜;microRNA進化關(guān)系及編碼特性研究[D];內(nèi)蒙古大學(xué);2007年

3 王玫;小麥microRNA的鑒定與分析[D];南昌大學(xué);2007年

4 秦保東;原發(fā)性膽汁性肝硬化microRNA表達譜的檢測及其功能研究[D];第二軍醫(yī)大學(xué);2013年

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