IL-24对小鼠黑色素瘤B16细胞抑制作用的实验研究

发布时间：2019-03-13 07:35

【摘要】：目的：构建含有目的基因IL-24的真核表达质粒pEGFP-N1-IL-24,将其转染小鼠黑色素瘤B16细胞,研究IL-24在细胞内表达对肿瘤细胞的凋亡和体外增殖的影响。方法：将pUC57-IL-24用限制性核酸内切酶双酶切后,经琼脂糖凝胶电泳回收、纯化,与pEGFP-N1相连接,将连接产物转化至感受态细胞E.coli DH5a中,提取质粒,再次酶切电泳,检查IL-24插入pEGFP-N1的准确性,并进行测序验证。用脂质体法将pEGFP-N1-IL-24转染B16细胞(B16/pEGFP-N1-IL-24组),同时设未处理的B16细胞(B16组)作对照组,另外将只加入脂质体的B16细胞(B16/Lip组)作脂质体组,pEGFP-N1转染的B16细胞(B16/pEGFP-N1组)作空载体组,加入长春新碱的B16细胞(B16/VCR组)作化疗药组,24h后在荧光显微镜下观察各组细胞的表达,于转染48h后在倒置显微镜高倍镜下观察细胞的形态学变化,用吖啶橙/溴化乙锭(AO/EB)法检测细胞凋亡,用MTT法检测B16细胞的体外增殖能力,实验结果用x±s表示,用统计软件做t检验,P0.05时有显著性差异。结果： 1.将构建的含有IL-24的真核表达质粒经酶切、电泳,发现了4.7kb和660bp的条带,与预期值相符,经测序验证为pEGFP-N1-IL-24基因序列。 2.将B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五组细胞分别于转染后24h在荧光显微镜下观察,可见B16/pEGFP-N1组细胞和B16/pEGFP-N1-IL-24组细胞发出绿色荧光,说明EGFP基因在B16细胞中有表达,而其他组细胞未见绿色荧光。 3.观察B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五组细胞在转染后48h的细胞形态,可见,B16组、B16/Lip组和B16/pEGFP-N1组细胞,因细胞分裂增殖而满视野,细胞大小类似,呈圆形、椭圆形或短梭型,细胞外周清晰,立体感强。B16/pEGFP-N1-IL-24组和B16/VCR组细胞,大多细胞的周边较模糊,成团紧密聚集,细胞数量少,有碎片,大小不规则。 4. B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五组细胞分别进行AO/EB双荧光染色,结果表明B16组、B16/Lip组和B16/pEGFP-N1组细胞大多为核染色质着绿色,并呈正常结构,有少量死亡细胞,即核染色质着红色并呈正常结构；B16/pEGFP-N1-IL-24组和B16/VCR组细胞大多为凋亡细胞,核染色质为橘黄色或橙红色,并呈固缩状或圆珠状。 5.将B16、B16/Lip、B16/pEGFP-N1、B16/pEGFP-N1-IL-24、B16/VCR五组细胞用MTT进行比色法测定,B16/pEGFP-N1-IL-24组与B16/Lip组、B16/pEGFP-N1-IL-24与B16/pEGFP-N1的抑制率有显著性差异(P0.05,P0.05),而B16/pEGFP-N1-IL-24组与B16/VCR组的抑制率无明显差异(P=0.991)。由转染后48h,B16细胞由抑制率而做的直方图可以看出,B16/pEGFP-N1-IL-24组和B16/VCR组细胞的增殖与B16/Lip组和B16/pEGFP-N1组相比明显受到抑制。结论： 1.成功构建了重组表达质粒pEGFP-N1-IL-24,并通过酶切、电泳和基因测序验证了其基因序列的正确性。 2.外源性IL-24基因可以体外转染小鼠黑色素瘤B16细胞,并在B16细胞中表达。 3.外源性IL-24基因可诱导小鼠黑色素瘤B16细胞发生凋亡,抑制其在体外增殖。 4.IL-24基因作为黑色素瘤的治疗基因,具有广阔的应用前景。
[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
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

【相似文献】