本文選題：重組質(zhì)粒 + IL-24�。� 參考：《延邊大學》2012年碩士論文

【摘要】：目的：研究重組質(zhì)粒pEGFP-N1-IL-24基因在小鼠黑色素瘤移植瘤體內(nèi)的表達及其對黑色素瘤的抑制作用。 方法： 1.B16細胞培養(yǎng)從液氮罐里取出并復蘇B16細胞,接種于含10%小牛血清的RPMI-1640培養(yǎng)液的培養(yǎng)瓶中進行培養(yǎng),傳代。 2.黑色素瘤B16細胞小鼠皮下移植瘤模型的建立待觀察C57BL/6小鼠一周后,一般狀況良好的情況下,收集對數(shù)生長期的B16細胞,在無菌條件下每只以5×10.cells/ml接種于小鼠背部皮下,觀察小鼠移植瘤的生長情況。 3.IL-24基因治療黑色素瘤B16細胞小鼠移植瘤的影響將荷瘤鼠隨機分為3組,每組6只,在瘤體內(nèi)注射脂質(zhì)體包裹的重組質(zhì)粒pEGFP-N1-IL-24(10μg)為實驗組,注射脂質(zhì)體包裹的空載質(zhì)粒pEGFP-N1(10μg)、注射PBS(0.2m1)為陰性對照組。每3天注射一次,共注射4次。每次注射藥物前先用游標卡尺測量腫瘤的長徑(L)和短徑(W),計算腫瘤體積,并繪制腫瘤的生長曲線。 4.在末次注射藥物后7天斷頸處死小鼠,剝離腫瘤組織。小部分組織冰凍用于RT-PCR鑒定,剩余部分組織冰凍用于Western蛋白印跡法檢測Bax蛋白的表達。 5. RT-PCR方法檢測移植瘤組織中IL-24mRNA的表達從各組移植瘤組織中提取總RNA,逆轉錄合成cDNA,與IL-24上下游引物,進行PCR反應,擴增產(chǎn)物進行瓊脂糖凝膠電泳,并查看目的條帶 6. Western蛋白印跡法檢測移植瘤組織中的Bax蛋白的表達提取各組移植瘤組織中的蛋白,進行聚丙烯酰胺凝膠電泳,轉膜,封閉,加入抗體與目的蛋白產(chǎn)生反應,顯影、定影后,觀察目的蛋白的表達水平。 結果： 1.成功建立了黑色素瘤移植瘤小鼠模型,接種細胞后第12天小鼠皮下腫瘤直徑均達5mm,腫瘤移植率達100%； 2.腫瘤生長曲線圖表明重組質(zhì)粒pEGFP-N1-IL-24組移植瘤的體積明顯小于空載質(zhì)粒pEGFP-N1組和PBS組,其差異有統(tǒng)計學意義(P0.05),而空載質(zhì)粒組和PBS組移植瘤體積則無明顯差異(P0.05)。與空載質(zhì)粒pEGFP-N1組和PBS組相比,重組質(zhì)粒pEGFP-Nl-IL-24組移植瘤生長緩慢,而空載質(zhì)粒pEGFP-N1組和PBS組移植瘤生長曲線幾乎平行； 3.各組的腫瘤組織中提取了RNA,進行RT-PCR檢測,擴增產(chǎn)物經(jīng)瓊脂糖凝膠電泳顯示重組質(zhì)粒pEGFP-N1-IL-24組在611bp處有一陽性條帶而空載質(zhì)粒pEGFP-N1組和PBS組未出現(xiàn)此預期的條帶； 4.各組移植瘤組織中提取蛋白進行Western蛋白印記法,結果在重組質(zhì)粒pEGFP-N1-IL-24組上出現(xiàn)了Bax蛋白的表達,而空載質(zhì)粒pEGFP-N1組和PBS組未出現(xiàn)Bax蛋白的表達。 結論： 1. IL-24基因在黑色素瘤組織中穩(wěn)定表達了其蛋白。 2. IL-24具有抑制黑色素瘤B16細胞小鼠移植瘤生長的作用。 3. IL-24對黑色素瘤組織有促進Bax蛋白分泌的作用。
[Abstract]:Aim: to investigate the expression of recombinant plasmid pEGFP-N1-IL-24 gene in murine melanoma xenografts and its inhibitory effect on melanoma. Methods: 1.B16 cells were extracted from liquid nitrogen tank and resuscitated. The B16 cells were cultured in RPMI-1640 culture flask containing 10% calf serum. 2. Establishment of subcutaneous tumor transplantation model of melanoma B16 cells in mice to be observed after one week after C57BL/6 mice were in good condition, B16 cells in logarithmic growth phase were collected and inoculated subcutaneously with 5 脳 10.cells/ml on the back of mice under aseptic condition. The growth of transplanted tumor in mice was observed. The effect of 3.IL-24 gene therapy on transplanted melanoma B16 cells mice were randomly divided into 3 groups, 6 mice in each group, and the recombinant plasmid pEGFP-N1-IL-24(10 渭 g encapsulated with liposome was injected into the tumor as experimental group. The blank plasmid pEGFP-N1(10 渭 g was injected with liposome, and the control group was injected with PBSX 0.2 m ~ (1). Once every 3 days, a total of 4 times. The tumor volume was calculated and the tumor growth curve was drawn by using Vernier caliper to measure the long diameter L) and the short diameter of the tumor before each injection. 4. The mice were killed 7 days after the last injection and the tumor tissue was removed. A small number of tissues were frozen for RT-PCR identification, while the rest were used for Western Western blot assay to detect the expression of Bax protein. 5. RT-PCR method was used to detect the expression of IL-24mRNA in transplanted tumor tissues. Total RNAs were extracted from each group of transplanted tumor tissues, then synthesized by reverse transcription, then reacted with IL-24 primers, reacted with PCR, amplified products were analyzed by agarose gel electrophoresis, and the target bands were examined. 6. Western Western blotting was used to detect the expression of Bax protein in the transplanted tumor tissue. The protein was extracted from the transplanted tumor tissue by polyacrylamide gel electrophoresis, transmembrane, blocking, reaction with the antibody and the target protein. The expression level of target protein was observed. Results: 1. The mouse model of melanoma transplanted tumor was established successfully. The diameter of subcutaneous tumor was 5 mm and the tumor transplant rate reached 100 mm on the 12th day after inoculation. 2. The tumor growth curve showed that the volume of transplanted tumor in the recombinant plasmid pEGFP-N1-IL-24 group was significantly smaller than that in the blank plasmid pEGFP-N1 group and the PBS group, and the difference was statistically significant (P 0.05), but there was no significant difference in the volume of the transplanted tumor between the no-load plasmid group and the PBS group. Compared with the no-load plasmid pEGFP-N1 group and PBS group, the growth of transplanted tumor was slower in the recombinant plasmid pEGFP-Nl-IL-24 group, but the growth curve was almost parallel between the no-load plasmid pEGFP-N1 group and the PBS group. 3. RNAs were extracted from tumor tissues of each group and detected by RT-PCR. Agarose gel electrophoresis showed that there was a positive band at 611bp in the recombinant plasmid pEGFP-N1-IL-24 group, but there was no such expected band in the pEGFP-N1 group and PBS group. 4. The expression of Bax protein was found in the recombinant plasmid pEGFP-N1-IL-24 group, but no Bax protein expression was found in the blank plasmid pEGFP-N1 group and PBS group. Conclusion: 1. IL-24 gene expressed its protein stably in melanoma tissue. 2. IL-24 can inhibit the growth of transplanted melanoma B 16 cells in mice. 3. IL-24 can promote the secretion of Bax protein in melanoma tissues.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.5

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