本文選題：L-arginine + CAT-2　； 參考：《南京大學(xué)》2011年碩士論文

【摘要】：目的利用RNA干擾(RNA interference, RNAi)技術(shù),設(shè)計并構(gòu)建可以表達CAT-2 siRNA的重組質(zhì)粒載體,將其轉(zhuǎn)染RAW264.7細胞后,觀察RNAi技術(shù)抑制CAT-2表達后iNOS下游調(diào)控途徑的變化。 方法1.根據(jù)基因庫收錄的小鼠CAT-2基因全序列及RNAi原理,設(shè)計并構(gòu)建可以表達CAT-2 siRNA的四組重組質(zhì)粒載體及陰性對照質(zhì)粒,pSilencer-CAT-2-1、pSilencer-CAT-2-2、pSilencer-CAT-2-3、pSilencer-CAT-2-4、pSilencer-control。2.將5種質(zhì)粒分別轉(zhuǎn)染RAW264.7細胞后,篩選出穩(wěn)定轉(zhuǎn)染且CAT-2抑制效率高的細胞系。3.檢測CAT-2B抑制率高的細胞系經(jīng)LPS活化后NO合成、L-arginine的跨膜轉(zhuǎn)運、iNOS及CAT-2表達的變化。 結(jié)果1.成功構(gòu)建表達CAT-2 siRNA質(zhì)粒載體,pSilencer-CAT-2-1、pSilencer-CAT-2、pSilencer-CAT-2-3、pSilencer-CAT-2-4、pSilencer-control; 2篩選出CAT-2抑制率高的穩(wěn)定轉(zhuǎn)染細胞系pSilencer-CAT2-4。3.與對照組比較,pSilencer-CAT2-4細胞系經(jīng)LPS活化后,NO合成、L-arginine跨膜轉(zhuǎn)運、CAT-2表達明顯下降,而iNOS表達無明顯變化。 結(jié)論pSilencer-CAT2-4重組質(zhì)粒表達的siRNA可以沉默CAT-2基因表達,進而阻斷L-arginine的跨膜轉(zhuǎn)運,從而在底物水平上抑制NO合成。為進一步實驗研究提供理論及技術(shù)基礎(chǔ)。
[Abstract]:Objective to design and construct a recombinant plasmid vector expressing CAT-2 siRNA by RNA interference RNA interference (RNAi) technique, and to observe the change of downstream regulation pathway of iNOS after RNAi technique inhibited CAT-2 expression in RAW264.7 cells. Method 1. According to the whole sequence of mouse CAT-2 gene and the principle of RNAi, four groups of recombinant plasmids expressing CAT-2 siRNA were designed and constructed, and the negative control plasmids, pSilencer-CAT-2-2, pSilencer-CAT-2-3, pSilencer-CAT-2-4, pSilencer-control.2were designed and constructed. After transfection of five plasmids into RAW264.7 cells, a stable transfection cell line. 3. 3 was selected with high inhibition efficiency of CAT-2. The transmembrane transport of L-arginine and the expression of CAT-2 in the cell lines with high inhibition rate of CAT-2B were detected after the activation of LPS. Result 1. The expression of CAT-2 siRNA plasmid pSilencer-CAT-2-1 pSilencer-CAT-2 + pSilencer-CAT-2-3 pSilencer-CAT-2-4 pSilencer-control. 2 stable transfection cell line pSilencer-CAT2-4.3 with high inhibition rate of CAT-2 was successfully constructed. Compared with the control group, the expression of L-arginine transporter CAT-2 was significantly decreased in pSilencer-CAT2-4 cell line activated by LPS, but the expression of iNOS was not changed. Conclusion siRNA expressed by pSilencer-CAT2-4 recombinant plasmid can silence the expression of CAT-2 gene and block the transmembrane transport of L-arginine, thus inhibiting the synthesis of no at substrate level. To provide theoretical and technical basis for further experimental research.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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