【摘要】：目的：構(gòu)建含有目的基因IL-24的真核表達(dá)質(zhì)粒pEGFP-N1-IL-24,將其轉(zhuǎn)染小鼠黑色素瘤B16細(xì)胞,研究IL-24在細(xì)胞內(nèi)表達(dá)對腫瘤細(xì)胞的凋亡和體外增殖的影響。 方法：將pUC57-IL-24用限制性核酸內(nèi)切酶雙酶切后,經(jīng)瓊脂糖凝膠電泳回收、純化,與pEGFP-N1相連接,將連接產(chǎn)物轉(zhuǎn)化至感受態(tài)細(xì)胞E.coli DH5a中,提取質(zhì)粒,再次酶切電泳,檢查IL-24插入pEGFP-N1的準(zhǔn)確性,并進(jìn)行測序驗證。用脂質(zhì)體法將pEGFP-N1-IL-24轉(zhuǎn)染B16細(xì)胞(B16/pEGFP-N1-IL-24組),同時設(shè)未處理的B16細(xì)胞(B16組)作對照組,另外將只加入脂質(zhì)體的B16細(xì)胞(B16/Lip組)作脂質(zhì)體組,pEGFP-N1轉(zhuǎn)染的B16細(xì)胞(B16/pEGFP-N1組)作空載體組,加入長春新堿的B16細(xì)胞(B16/VCR組)作化療藥組,24h后在熒光顯微鏡下觀察各組細(xì)胞的表達(dá),于轉(zhuǎn)染48h后在倒置顯微鏡高倍鏡下觀察細(xì)胞的形態(tài)學(xué)變化,用吖啶橙/溴化乙錠(AO/EB)法檢測細(xì)胞凋亡,用MTT法檢測B16細(xì)胞的體外增殖能力,實驗結(jié)果用x±s表示,用統(tǒng)計軟件做t檢驗,P0.05時有顯著性差異。 結(jié)果： 1.將構(gòu)建的含有IL-24的真核表達(dá)質(zhì)粒經(jīng)酶切、電泳,發(fā)現(xiàn)了4.7kb和660bp的條帶,與預(yù)期值相符,經(jīng)測序驗證為pEGFP-N1-IL-24基因序列。 2.將B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五組細(xì)胞分別于轉(zhuǎn)染后24h在熒光顯微鏡下觀察,可見B16/pEGFP-N1組細(xì)胞和B16/pEGFP-N1-IL-24組細(xì)胞發(fā)出綠色熒光,說明EGFP基因在B16細(xì)胞中有表達(dá),而其他組細(xì)胞未見綠色熒光。 3.觀察B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五組細(xì)胞在轉(zhuǎn)染后48h的細(xì)胞形態(tài),可見,B16組、B16/Lip組和B16/pEGFP-N1組細(xì)胞,因細(xì)胞分裂增殖而滿視野,細(xì)胞大小類似,呈圓形、橢圓形或短梭型,細(xì)胞外周清晰,立體感強。B16/pEGFP-N1-IL-24組和B16/VCR組細(xì)胞,大多細(xì)胞的周邊較模糊,成團緊密聚集,細(xì)胞數(shù)量少,有碎片,大小不規(guī)則。 4. B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五組細(xì)胞分別進(jìn)行AO/EB雙熒光染色,結(jié)果表明B16組、B16/Lip組和B16/pEGFP-N1組細(xì)胞大多為核染色質(zhì)著綠色,并呈正常結(jié)構(gòu),有少量死亡細(xì)胞,即核染色質(zhì)著紅色并呈正常結(jié)構(gòu)；B16/pEGFP-N1-IL-24組和B16/VCR組細(xì)胞大多為凋亡細(xì)胞,核染色質(zhì)為橘黃色或橙紅色,并呈固縮狀或圓珠狀。 5.將B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五組細(xì)胞用MTT進(jìn)行比色法測定,B16/pEGFP-N1-IL-24組與B16/Lip組、B16/pEGFP-N1-IL-24與B16/pEGFP-N1的抑制率有顯著性差異(P0.05,P0.05),而B16/pEGFP-N1-IL-24組與B16/VCR組的抑制率無明顯差異(P=0.991)。由轉(zhuǎn)染后48h,B16細(xì)胞由抑制率而做的直方圖可以看出,B16/pEGFP-N1-IL-24組和B16/VCR組細(xì)胞的增殖與B16/Lip組和B16/pEGFP-N1組相比明顯受到抑制。 結(jié)論： 1.成功構(gòu)建了重組表達(dá)質(zhì)粒pEGFP-N1-IL-24,并通過酶切、電泳和基因測序驗證了其基因序列的正確性。 2.外源性IL-24基因可以體外轉(zhuǎn)染小鼠黑色素瘤B16細(xì)胞,并在B16細(xì)胞中表達(dá)。 3.外源性IL-24基因可誘導(dǎo)小鼠黑色素瘤B16細(xì)胞發(fā)生凋亡,抑制其在體外增殖。 4.IL-24基因作為黑色素瘤的治療基因,具有廣闊的應(yīng)用前景。
[Abstract]:Objective: To construct the eukaryotic expression plasmid pEGFP-N1-IL-24 containing the target gene IL-24 and to transfect the mouse melanoma B16 cells, and to study the effect of IL-24 on the apoptosis and in vitro proliferation of the tumor cells. Methods: pUC57-IL-24 was digested with restriction endonuclease and purified by agarose gel electrophoresis and then connected with pEGFP-N1, and the ligation product was transformed into competent cell E. coli DH5a. The plasmid was extracted, and the plasmid was extracted again, and the expression of IL-24 into pEGFP-N1 was examined. The pEGFP-N1-IL-24 was transfected into B16 cells (B16/ pEGFP-N1-IL-24 group) by the liposome method, and the untreated B16 cells (B16 groups) were also provided as the control group, and the B16 cells (B16/ Lip group), which were only added to the liposomes, were used as the liposome group, and the B16 cells transfected with pEGFP-N1 (B16/ pEGFP-N1 group) were used as the empty vectors. In the group, B16 cells (B16/ VCR group) with vincristine were added as the group of chemotherapeutic drugs. After 24 h, the expression of each group of cells was observed under a fluorescence microscope. After 48 h of transfection, the morphological changes of the cells were observed at high magnification in an inverted microscope, and the cells were detected by the method of orange/ ethidium bromide (AO/ EB). The in vitro proliferation ability of B16 cells was detected by MTT method, and the results of the experiment were expressed by x-% s. The statistical software was used for t-test, and there was a significant difference in P0.05. I'm different. Results:1. The constructed eukaryotic expression plasmid containing IL-24 was digested and electrophoresed, and the bands of 4.7 kb and 660bp were found to be consistent with the expected value, and the pEGFP-N1-IL-2 was verified by sequencing. 4 gene sequence.2. B16, B16/ Lip, B16/ pEGFP-N1, B16/ pEGFP-N1-IL-24 and B16/ VCR five-group cells were observed under a fluorescence microscope at 24 h after transfection, and the cells of B16/ pEGFP-N1 group and B16/ pEGFP-N1-IL-24 group were observed to emit green fluorescence, indicating that the EGFP gene was expressed in B16 cells, while the other groups were thin The cells were not seen with green fluorescence.3. Observe the cell morphology of B16, B16/ Lip, B16/ pEGFP-N1, B16/ pEGFP-N1-IL-24, B16/ VCR 5-group cells for 48 h after transfection, and the cells of the B16/ pEGFP-N1-IL-24 and B16/ VCR 5-group cells are full of field of view due to cell division and proliferation, and the cell size is similar, circular, elliptical or short-shuttle type, cell, the periphery of the B16/ pEGFP-N1-IL-24 and B16/ VCR group cells is clear, the periphery of the majority of the cells is relatively vague, the aggregation is closely gathered, the number of cells is small, The cells of B16, B16/ Lip, B16/ pEGFP-N1, B16/ pEGFP-N1-IL-24 and B16/ VCR were stained with AO/ EB double fluorescence respectively. The results showed that the cells in B16, B16/ Lip and B16/ pEGFP-N1 group were mostly nuclear chromatin with green and normal structure, with a small number of dead cells, i.e., nuclear staining. The color is red and the normal structure; B16/ pEGFP-N1-IL-24 and B16/ VCR group cells are mostly apoptotic cells, and the nuclear chromatin is orange or orange The ratio of B16, B16/ Lip, B16/ pEGFP-N1, B16/ pEGFP-N1-IL-24, B16/ VCR 5-group cells was determined by MTT, and the B16/ pEGFP-N1-IL-24 and B16/ Lip groups, B16/ pEGFP-N1-IL-24 and B16/ pEGFP-N1 had a significant difference (P0.05, P0.05), and the B16/ pEGFP-N1-IL-24 group and B16/ VCR group had no inhibition rate. Significant differences (P = 0.991). The histograms of B16/ pEGFP-N1-IL-24 and B16/ VCR group cells were seen to be different from B16/ Lip and B16/ pEGF in the B16/ pEGFP-N1-IL-24 group and B16/ VCR group cells, as can be seen from the histograms of the inhibition rate at 48 h after transfection. P-N Conclusion:1. The recombinant expression plasmid pEGFP-N1-IL-24 was successfully constructed and the recombinant expression plasmid pEGFP-N1-IL-24 was successfully constructed. And the exogenous IL-24 gene can be transfected into the mouse in vitro. Cytochrome B16 cells and expressed in B16 cells.3. Exogenous IL-24 gene can induce mouse black Cytochrome B16 cells apoptosis and inhibit their proliferation in vitro.4. IL-24
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.5

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