發(fā)布時間：2018-04-21 06:14

  本文選題：表皮生長因子受體 + 短發(fā)卡狀RNA　； 參考：《天津醫(yī)科大學(xué)》2011年博士論文

【摘要】：目的:設(shè)計有效抑制表皮生長因子受體(epidermal growth factor receptor,EGFR)基因表達(dá)的小干擾RNA(short interfering RNA,siRNA)序列,構(gòu)建相應(yīng)的真核表達(dá)載體,轉(zhuǎn)染到人晶狀體上皮細(xì)胞(Human lens epithelial cells,HLEs)中,研究其對HLEs細(xì)胞生物學(xué)特性的影響;同時篩選出促進(jìn)HLEs細(xì)胞增長的重組表皮生長因子(epidermal growth factor,EGF)的最適宜濃度,進(jìn)一步研究在最適宜濃度的EGF刺激下,EGFR的siRNA對HLEs細(xì)胞生長的影響,從而明確EGFR的siRNA的抑制效果,并推斷出si-EGFR抑制HLEs細(xì)胞增長的分子機(jī)制。方法:1、根據(jù)siRNA設(shè)計原則,設(shè)計并體外化學(xué)合成靶向EGFR基因的特異性短發(fā)卡狀 RNA(short hairpin RNA,shRNA),克隆到 pSilencer2.1-U6neo 載體中,構(gòu)建si-EGFR的表達(dá)載體,同時以pSilencer2.1-U6neo空轉(zhuǎn)載體為陰性對照,在脂質(zhì)體介導(dǎo)下分別轉(zhuǎn)染HLEs細(xì)胞,以免疫熒光技術(shù)定位EGFR;實時熒光定量PCR(Real time FQ-PCR)檢測HLEs中EGFR的mRNA水平上表達(dá)的變化;Western-blot蛋白印跡法檢測EGFR在蛋白表達(dá)上的變化;MTS法及細(xì)胞生長曲線分別檢測細(xì)胞增殖活性;流式細(xì)胞術(shù)檢測細(xì)胞生長周期變化。2、體外傳代HLEs細(xì)胞,以不同濃度(0.1、1、10ng/mL)的外源性重組EGF分別刺激HLEs,觀察HLEs細(xì)胞的形態(tài)變化和生長情況,自加入重組EGF即刻起,通過MTS法連續(xù)4d測量各組細(xì)胞光密度(Optical Density,OD)490及570的值;同時連續(xù)7d計數(shù)細(xì)胞個數(shù),繪制細(xì)胞生長曲線以檢測細(xì)胞增殖活性,從而尋找能促進(jìn)HLEs細(xì)胞生長的重組EGF的最適宜濃度。3、分別轉(zhuǎn)染pSilencer2.1-shRNA-EGFR及pSilencer2.1-U6neo空轉(zhuǎn)載體兩組質(zhì)粒到HLEs細(xì)胞,并在兩組轉(zhuǎn)染后的細(xì)胞中加入最適宜濃度的等量重組EGF,通過MTS法連續(xù)4d檢測細(xì)胞OD490和OD570數(shù)值,并連續(xù)7d計數(shù)細(xì)胞個數(shù),繪制細(xì)胞生長曲線,觀察在EGF干擾下,EGFR的siRNA對HLEs細(xì)胞增殖的影響。結(jié)果:1、Western-blot蛋白印跡法與Real time PCR檢測法的結(jié)果一致,轉(zhuǎn)染細(xì)胞48h后,si-EGFR實驗組中EGFR的mRNA表達(dá)水平是陰性對照組的0.450625倍;蛋白水平si-EGFR實驗組是陰性對照組的0.1194倍,是空白對照組的0.1149倍,組間比較差異具有統(tǒng)計學(xué)意義(P＜0.01)。這些結(jié)果表明構(gòu)建的si-EGFR表達(dá)載體pSilencer2.1-shRNA-EGFR無論在分子水平還是蛋白水平均明顯抑制EGFR的表達(dá)。2、流式細(xì)胞檢測技術(shù)結(jié)果表明,72h之后,轉(zhuǎn)染了 si-EGFR質(zhì)粒的HLEs,G1期細(xì)胞占到41.6%,而未進(jìn)行基因沉默的陰性對照組G1期的細(xì)胞占到29.5%;同時轉(zhuǎn)染了 si-EGFR質(zhì)粒的HLEs細(xì)胞,G2期占到27.5%,而陰性對照組G2期的細(xì)胞占到43.7%;轉(zhuǎn)染了 si-EGFR質(zhì)粒的HLEs細(xì)胞,其增殖指數(shù)為140.4%,而陰性對照組的增殖指數(shù)為239%。以上結(jié)果顯示:si-EGFR影響了 HLEs細(xì)胞從G1期進(jìn)入S期,從而作用于細(xì)胞周期進(jìn)程。MTS法及細(xì)胞生長曲線的結(jié)果一致,證實了轉(zhuǎn)染了 si-EGFR質(zhì)粒的HLEs細(xì)胞自轉(zhuǎn)染后24小時開始,生長活性明顯降低,且隨著時間推移,生長活性降低的更為明顯(P＜0.01)。3、以不同濃度的重組EGF刺激HLEs細(xì)胞發(fā)現(xiàn):在一定范圍內(nèi),重組EGF濃度增高,細(xì)胞的增殖活性也隨之增高,當(dāng)重組EGF濃度為1ng/mL時,HLEs細(xì)胞的增殖活性最強(qiáng),但是超過了這個范圍,高濃度的EGF反而會抑制細(xì)胞生長;細(xì)胞生長曲線檢測結(jié)果與MTS結(jié)果一致。4、轉(zhuǎn)染了 si-EGFR質(zhì)粒的HLEs細(xì)胞盡管受到了最適宜濃度重組EGF的刺激,但是細(xì)胞增殖不明顯,而陰性對照組在接受了 1ng/mL重組EGF的刺激后,細(xì)胞增殖活性顯著提高,MTS檢測結(jié)果與細(xì)胞生長曲線一致,兩組組間比較差異具有統(tǒng)計學(xué)意義(P0.01)。結(jié)論:1、體外化學(xué)合成的pSilencer2.1-shRNA-EGFR表達(dá)載體在分子水平及蛋白水平上均能明顯降低HLEs細(xì)胞中EGFR的表達(dá),并因而有效抑制了 HLEs細(xì)胞的增殖活性。靶向si-EGFR為預(yù)防后發(fā)性白內(nèi)障提供了新的思路。2、適宜濃度的重組EGF可以刺激HLEs細(xì)胞的生長,過高濃度的重組EGF反而會抑制細(xì)胞的生長,1ng/ml的重組EGF最有利于促進(jìn)HLEs細(xì)胞的生長。3.、適宜濃度的重組EGF也不能促進(jìn)si-EGFR作用過的HLEs細(xì)胞的增殖,因此我們推斷轉(zhuǎn)染了 pSilencer2.1-shRNA-EGFR的HLEs細(xì)胞,其增殖活性的降低是通過EGF-EGFR通路的阻斷得以實現(xiàn)的。
[Abstract]:Objective: to design a small interfering RNA (short interfering RNA, siRNA) sequence that effectively inhibits the expression of epidermal growth factor receptor (EGFR) gene, and to construct a corresponding eukaryotic expression vector and transfect into the human lens epithelial cells (Human lens). At the same time, the optimum concentration of the recombinant epidermal growth factor (epidermal growth factor, EGF) to promote the growth of HLEs cells was screened, and the effect of siRNA on the growth of HLEs cells was further studied under the optimum concentration of EGF, and the inhibitory effect of EGFR siRNA was determined, and the molecular machine for inhibiting the growth of si-EGFR to inhibit the cell growth was deduced. Methods: 1, according to the design principle of siRNA, the specific short hairpin RNA (short hairpin RNA, shRNA) targeting EGFR gene was designed and synthesized in vitro, and was cloned into pSilencer2.1-U6neo vector to construct the expression vector of si-EGFR, while pSilencer2.1-U6neo empty reloading body was negative control, and transfected into HLEs thin under liposome. Cell, immunofluorescence technique was used to locate EGFR; real-time quantitative PCR (Real time FQ-PCR) was used to detect the changes in the mRNA level of EGFR in HLEs; Western-blot protein blotting was used to detect the changes of EGFR in protein expression; MTS method and cell growth curve were used to detect cell proliferation activity; flow cytometry was used to detect cell growth cycle changes.2, body The external HLEs cells were stimulated by exogenous recombinant EGF with different concentrations (0.1,1,10ng/mL). The morphological changes and growth of HLEs cells were observed. The values of the light density (Optical Density, OD) 490 and 570 of each group were measured by MTS method from the instant 4D, and the number of cells was counted by continuous 7d, and cell growth was plotted. The curve was used to detect cell proliferation activity, so as to find the most suitable concentration.3 of recombinant EGF that could promote HLEs cell growth, transfect two groups of plasmids of pSilencer2.1-shRNA-EGFR and pSilencer2.1-U6neo empty reload bodies to HLEs cells, and add the most suitable concentration of recombinant EGF in the two groups of transfected cells, and continuous 4D detection by MTS method. The number of cells OD490 and OD570, counting the number of cells in continuous 7d, plotting the cell growth curve, and observing the effect of EGFR siRNA on the proliferation of HLEs cells under the interference of EGF. Results: 1, the result of Western-blot blot and Real time PCR detection method is consistent. After transfection of cell 48h, the level of expression is negative control The protein level si-EGFR experimental group was 0.1194 times more than the negative control group and 0.1149 times the blank control group, and the difference between the groups was statistically significant (P < 0.01). These results showed that the constructed si-EGFR expression vector, pSilencer2.1-shRNA-EGFR, significantly inhibited the expression of EGFR in the level of molecular water and protein, and the expression of.2 in EGFR was obviously inhibited. Flow cytometry showed that after 72h, the transfection of si-EGFR plasmid HLEs, G1 phase cells accounted for 41.6%, while the negative control group without gene silencing accounted for 29.5% in G1 stage, HLEs cells of si-EGFR plasmids, G2 period 27.5%, and negative group G2 phase 43.7%; si-EGFR plasmid transfected. The proliferation index of HLEs cells was 140.4%, while the proliferation index of the negative control group was above 239%. showed that si-EGFR affected HLEs cells to enter S phase from G1 phase, thus the result of.MTS method and cell growth curve in cell cycle process was consistent, which confirmed that the HLEs cells transfected with si-EGFR plasmids began to grow at 24 hours after the transfection of si-EGFR plasmids. The activity decreased obviously, and the growth activity decreased more obviously over time (P < 0.01).3. The recombinant EGF stimulated HLEs cells with different concentrations. In a certain range, the concentration of the recombinant EGF increased and the proliferation activity of the cells increased. When the concentration of the recombinant EGF was 1ng/mL, the proliferation activity of HLEs cells was the strongest, but it exceeded that. In the range, high concentration of EGF could inhibit cell growth, and the results of cell growth curve were consistent with the results of MTS.4. The HLEs cells transfected with si-EGFR plasmids were stimulated by the most suitable concentration of recombinant EGF, but the cell proliferation was not obvious, while the negative control group had a significant proliferation activity after the stimulation of the 1ng/mL recombinant EGF. The results of MTS detection were in accordance with the cell growth curve, and the difference between the two groups was statistically significant (P0.01). Conclusion: 1, the pSilencer2.1-shRNA-EGFR expression vector synthesized in vitro could significantly reduce the expression of EGFR in the HLEs cells, and thus effectively inhibit the proliferation activity of HLEs cells. Targeted si-EGFR provides a new way of thinking.2 for the prevention of post cataract. The suitable concentration of recombinant EGF can stimulate the growth of HLEs cells. The high concentration of recombinant EGF will inhibit the growth of cells. The recombinant EGF of 1ng/ml is most conducive to promoting the growth of HLEs cells, and the suitable concentration of recombinant EGF can not promote HLEs fined by si-EGFR action. Cell proliferation, we concluded that the transfection of pSilencer2.1-shRNA-EGFR HLEs cells reduced the proliferation activity through EGF-EGFR pathway blocking.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R776.1

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