EGFR靶向單鏈抗體制備及介導siRNA內(nèi)化NSCLC細胞的生物學效應
發(fā)布時間：2019-06-18 09:08
【摘要】：目的:EGFR-TKIs耐藥引起腫瘤進展是當前NSCLC治療的主要重點和難點,siRNA可沉默耐藥相關(guān)基因表達但缺乏靶向性。為提高siRNA的靶向性,本研究設計并優(yōu)化EGFR靶向單鏈抗體核苷酸序列,在大腸桿菌中表達后,純化人源性抗EGFR單鏈抗體(scFv,s-9R)融合蛋白,并對純化產(chǎn)物的抗原結(jié)合活性、細胞內(nèi)化活性及核算攜帶能力進行鑒定;研究s-9R攜帶siRNA在體內(nèi)、外的抗腫瘤活性。方法:1.根據(jù)人源性抗EGFR抗體輕、重鏈可變區(qū)氨基酸序列,設計、合成其單鏈抗體核酸表達序列,將輕鏈與重鏈核酸序列通過G4S連接肽相連并在核酸序列末端加入九聚精氨酸及His.tag表達序列,以構(gòu)建scFv及s-9R融合基因。將測序正確的融合基因連接入原核表達載體pGEX-4T-1,轉(zhuǎn)化大腸桿菌BL21(DE3),誘導表達后行親和層析純化并酶切GST.tag。表達產(chǎn)物經(jīng)SDS-PAGE和Western blot鑒定后,ELISA分析融合蛋白的抗原結(jié)合活性,凝膠遷移阻滯實驗檢測s-9R與核酸的結(jié)合活性,間接免疫熒光和流式細胞術(shù)檢測檢測融合蛋白的內(nèi)化活性。2.scfv及s-9r與egfr-sirna,met-sirna,kras-sirna及her2-sirna分別混合后,加入h1975,h1993,a549,spc-a1,pc-9及h69細胞培養(yǎng)液中共孵育,同時加入/不加入gefitinib。通過rt-qpcr,westernblot實驗分析基因表達水平;通過mtt實驗、流式細胞學實驗、平板克隆實驗分析egfr-tkis細胞藥物敏感性。3.使用近紅外染料dylight800nhsester對單鏈抗體融合蛋白進行標記,經(jīng)鼠尾靜脈對h1975細胞荷瘤裸鼠注射標記后的融合蛋白,活體成像觀察融合蛋白在腫瘤部位富集及全身代謝過程。使用cy5熒光染料標記sirna,經(jīng)h1975細胞荷瘤裸鼠鼠尾靜脈注射融合蛋白/cy5-sirna,活體成像觀察融合蛋白/cy5-sirna在腫瘤部位富集及全身代謝過程。經(jīng)鼠尾靜脈注射融合蛋白/egfr-sirna混合物,并同時gefitinib灌胃,觀察裸鼠荷瘤變化;經(jīng)鼠尾靜脈注射融合蛋白/her2-sirna混合物,觀察裸鼠荷瘤變化。剝離荷瘤組織后對組織切片進行免疫組化實驗,觀察egfr、her2、ki67表達情況;對組織切片進行tunel標記,觀察細胞凋亡情況。結(jié)果:1.pgex-4t-1-scfv及pgex-4t-1-s-9r質(zhì)粒轉(zhuǎn)化大腸桿菌bl21(de3)后,iptg誘導表達融合蛋白scfv及s-9r,經(jīng)sds-page及westernblot證實表達成功;elisa分析證實兩條單鏈抗體融合蛋白具有良好的egfr結(jié)合活性;凝膠遷移阻滯實驗表明s-9r具有核酸結(jié)合活性;間接免疫熒光及流式細胞術(shù)實驗顯示兩條單鏈抗體融合蛋白能夠特異性結(jié)合并內(nèi)化入egfr陽性的腫瘤細胞中,而不能內(nèi)化入egfr表達陰性腫瘤細胞中;s-9r可以攜帶fam-sirna結(jié)合并內(nèi)化入egfr陽性的腫瘤細胞中,而scfv則無此功能。2.s-9r可以攜帶egfr-sirna,met-sirna,kras-sirna及her2-sirna內(nèi)化入腫瘤細胞中,并下調(diào)細胞中egfr、met、kras、her2基因的表達;聯(lián)合應用gefitinib后h1975、h1993及a549細胞的凋亡水平較對照組明顯提高;s-9r/her2-sirna混合物可以使spc-a1,pc-9增殖能力、克隆形成能力降低,細胞周期出現(xiàn)g1期停滯。3.小動物活體成像顯示兩條單鏈抗體融合蛋白本身能夠通過循環(huán)系統(tǒng)富集于裸鼠荷瘤部位,其中s-9r可以攜帶cy5/fam熒光素標記的sirna富集于裸鼠荷瘤部位,并內(nèi)化入瘤細胞中;經(jīng)鼠尾靜脈注射s-9r/egfr-sirna混合物聯(lián)合gefitinib灌胃,或單獨經(jīng)鼠尾靜脈注射s-9R/HER2-siRNA混合物可以使裸鼠荷瘤生長受到明顯抑制,病理切片顯示腫瘤細胞靶蛋白表達減少,增殖能力下降,凋亡細胞增多,荷瘤血管形成能力降低。實驗組裸鼠生存時間延長,移植瘤成瘤率下降,荷瘤體積變小、重量減輕。結(jié)論:所設計、構(gòu)建的EGFR靶向單鏈抗體scFv及s-9R可以在體內(nèi)、外特異性的識別結(jié)合EGFR表達陽性NSCLC細胞。s-9R可以攜帶特異性siRNA內(nèi)化入腫瘤細胞之中抑制靶蛋白表達,發(fā)揮siRNA的生物學作用,具有恢復Gefitinib敏感性,抑制腫瘤細胞增殖、促進細胞凋亡的作用。我們的研究為解決當前NSCLC臨床治療中所面對的酪氨酸激酶抑制劑耐藥問題,以及治療以HER2為驅(qū)動基因的NSCLC,提供了一條新的方法和思路。
[Abstract]:Objective: EGFR-TKIs resistance-induced tumor progression is the main focus and difficulty in the treatment of NSCLC, and siRNA can be used to silence resistance-related gene expression but lacks targeting. In order to improve the targeting of the siRNA, the present study designs and optimizes the EGFR-targeting single-chain antibody nucleosonic acid sequence, and after the expression in E. coli, the humanized anti-EGFR single-chain antibody (scFv, s-9R) fusion protein is purified, and the antigen binding activity of the purified product is carried out, The cell-internalization activity and the carrying capacity of the cell were identified; and the anti-tumor activity of the siRNA in vivo and in vitro was studied by the study of s-9R. Method:1. according to the human-derived anti-EGFR antibody light and heavy chain variable region amino acid sequence, the single-chain antibody nucleic acid expression sequence is synthesized, the light chain and the heavy-chain nucleic acid sequence are connected through a G4S connection peptide and a 9-polyarginine and a His.tag expression sequence are added at the end of the nucleic acid sequence, In ord to construct that fusion gene of scFv and s-9R. The correct fusion gene was ligated into the prokaryotic expression vector pGEX-4T-1, transformed into E. coli BL21 (DE3), the expression product was purified by affinity chromatography, and the enzyme-digested GST.tag. expression product was identified by SDS-PAGE and Western blot, and the antigen binding activity of the fusion protein was analyzed by ELISA. The binding activity of s-9R and the nucleic acid, the indirect immunofluorescence and flow cytometry were used to detect the internalized active .2.scfv of the fusion protein and s-9r and egfr-sirna, met-sirna, kras-sirna and her2-sirna, respectively, and then added to the culture medium of h1975, h1993, a549, spc-al, pc-9 and h69. At the same time, gefitinib was added/ not added. The expression level of gene was analyzed by rt-qpcr and western blot. The fusion protein of the single-chain antibody fusion protein was labeled by using the near-infrared dye dylight800nhester, and the fusion protein after the injection of the h1975 cell tumor-bearing nude mice was injected via the mouse tail vein, and the fusion protein was concentrated in the tumor site and the whole body metabolism process. Cy5 fluorescent dye was used to mark the sirna, and the fusion protein/ cy5-sirna was injected into the tumor-bearing nude mouse tail vein by the h1975 cell tumor-bearing nude mouse tail, and the fusion protein/ cy5-sirna was observed in the tumor site and the whole body metabolism process. The mice were injected with the fusion protein/ egfr-sirna mixture and gefitinib was given intragastric administration to observe the tumor-bearing changes of the nude mice, and the tumor-bearing changes of the nude mice were observed by the combination of the fusion protein/ her2-sirna mixture in the tail of the mice. The expression of egfr, her2 and ki67 was observed after the tumor-bearing tissue was peeled off and the expression of egfr, her2 and ki67 was observed. Results :1.pgex-4t-1-scfv and pgex-4t-1-s-9r plasmids were transformed into E. coli bl21 (de3), and iptg induced the expression of the fusion protein scfv and s-9r. The expression of the fusion protein scfv and s-9r was confirmed by sds-page and western blot. indirect immunofluorescence and flow cytometry show that two single-chain antibody fusion proteins can be specifically combined and internalized into egfr-positive tumor cells, and can not be internalized into egfr expression-negative tumor cells; s-9r can carry the fam-sirna binding and internalized into egfr-positive tumor cells, scfv does not have such a function. s-9r can carry egfr-sirna, met-sirna, kras-sirna and her2-sirna into the tumor cells and down-regulate the expression of egfr, met, kras, and her2 in the cells, and the level of apoptosis in h1975, h1993 and a549 cells after the combined application of gefitinib is significantly higher than that of the control group; s-9r/ her2-sirna mixtures may enable the proliferation of spc-a1, pc-9, The ability of the clone formation decreased and the cell cycle showed a phase-1 stagnation. in vivo imaging of small animals, two single-chain antibody fusion proteins can be enriched in a tumor-bearing part of a nude mouse through a circulating system, wherein s-9r can carry a cy5/ fam fluorescein-labeled sirna to be enriched in a tumor-bearing part of a nude mouse, And the tumor cell target protein expression is reduced, the proliferation ability is reduced, the apoptosis cell is increased, and the tumor-bearing blood vessel forming capacity is reduced. The survival time of the nude mice in the experimental group was prolonged, the tumor-forming rate of the transplanted tumor was decreased, the tumor-bearing volume was small, and the weight was reduced. Conclusion: The designed and constructed EGFR-targeting single-chain antibody scFv and s-9R can identify and bind EGFR-expressing positive NSCLC cells in vivo and in vitro. S-9R can carry specific siRNA into the tumor cells to inhibit the expression of the target protein, play the biological function of the siRNA, have the function of restoring the sensitivity of the Gefitinib, inhibiting the proliferation of the tumor cells and promoting the apoptosis of the cells. Our study provides a new approach and thought to address the problem of the resistance of tyrosine kinase inhibitors in the clinical treatment of NSCLC and to treat NSCLC with HER2 as a driving gene.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
本文編號：2501357
[Abstract]:Objective: EGFR-TKIs resistance-induced tumor progression is the main focus and difficulty in the treatment of NSCLC, and siRNA can be used to silence resistance-related gene expression but lacks targeting. In order to improve the targeting of the siRNA, the present study designs and optimizes the EGFR-targeting single-chain antibody nucleosonic acid sequence, and after the expression in E. coli, the humanized anti-EGFR single-chain antibody (scFv, s-9R) fusion protein is purified, and the antigen binding activity of the purified product is carried out, The cell-internalization activity and the carrying capacity of the cell were identified; and the anti-tumor activity of the siRNA in vivo and in vitro was studied by the study of s-9R. Method:1. according to the human-derived anti-EGFR antibody light and heavy chain variable region amino acid sequence, the single-chain antibody nucleic acid expression sequence is synthesized, the light chain and the heavy-chain nucleic acid sequence are connected through a G4S connection peptide and a 9-polyarginine and a His.tag expression sequence are added at the end of the nucleic acid sequence, In ord to construct that fusion gene of scFv and s-9R. The correct fusion gene was ligated into the prokaryotic expression vector pGEX-4T-1, transformed into E. coli BL21 (DE3), the expression product was purified by affinity chromatography, and the enzyme-digested GST.tag. expression product was identified by SDS-PAGE and Western blot, and the antigen binding activity of the fusion protein was analyzed by ELISA. The binding activity of s-9R and the nucleic acid, the indirect immunofluorescence and flow cytometry were used to detect the internalized active .2.scfv of the fusion protein and s-9r and egfr-sirna, met-sirna, kras-sirna and her2-sirna, respectively, and then added to the culture medium of h1975, h1993, a549, spc-al, pc-9 and h69. At the same time, gefitinib was added/ not added. The expression level of gene was analyzed by rt-qpcr and western blot. The fusion protein of the single-chain antibody fusion protein was labeled by using the near-infrared dye dylight800nhester, and the fusion protein after the injection of the h1975 cell tumor-bearing nude mice was injected via the mouse tail vein, and the fusion protein was concentrated in the tumor site and the whole body metabolism process. Cy5 fluorescent dye was used to mark the sirna, and the fusion protein/ cy5-sirna was injected into the tumor-bearing nude mouse tail vein by the h1975 cell tumor-bearing nude mouse tail, and the fusion protein/ cy5-sirna was observed in the tumor site and the whole body metabolism process. The mice were injected with the fusion protein/ egfr-sirna mixture and gefitinib was given intragastric administration to observe the tumor-bearing changes of the nude mice, and the tumor-bearing changes of the nude mice were observed by the combination of the fusion protein/ her2-sirna mixture in the tail of the mice. The expression of egfr, her2 and ki67 was observed after the tumor-bearing tissue was peeled off and the expression of egfr, her2 and ki67 was observed. Results :1.pgex-4t-1-scfv and pgex-4t-1-s-9r plasmids were transformed into E. coli bl21 (de3), and iptg induced the expression of the fusion protein scfv and s-9r. The expression of the fusion protein scfv and s-9r was confirmed by sds-page and western blot. indirect immunofluorescence and flow cytometry show that two single-chain antibody fusion proteins can be specifically combined and internalized into egfr-positive tumor cells, and can not be internalized into egfr expression-negative tumor cells; s-9r can carry the fam-sirna binding and internalized into egfr-positive tumor cells, scfv does not have such a function. s-9r can carry egfr-sirna, met-sirna, kras-sirna and her2-sirna into the tumor cells and down-regulate the expression of egfr, met, kras, and her2 in the cells, and the level of apoptosis in h1975, h1993 and a549 cells after the combined application of gefitinib is significantly higher than that of the control group; s-9r/ her2-sirna mixtures may enable the proliferation of spc-a1, pc-9, The ability of the clone formation decreased and the cell cycle showed a phase-1 stagnation. in vivo imaging of small animals, two single-chain antibody fusion proteins can be enriched in a tumor-bearing part of a nude mouse through a circulating system, wherein s-9r can carry a cy5/ fam fluorescein-labeled sirna to be enriched in a tumor-bearing part of a nude mouse, And the tumor cell target protein expression is reduced, the proliferation ability is reduced, the apoptosis cell is increased, and the tumor-bearing blood vessel forming capacity is reduced. The survival time of the nude mice in the experimental group was prolonged, the tumor-forming rate of the transplanted tumor was decreased, the tumor-bearing volume was small, and the weight was reduced. Conclusion: The designed and constructed EGFR-targeting single-chain antibody scFv and s-9R can identify and bind EGFR-expressing positive NSCLC cells in vivo and in vitro. S-9R can carry specific siRNA into the tumor cells to inhibit the expression of the target protein, play the biological function of the siRNA, have the function of restoring the sensitivity of the Gefitinib, inhibiting the proliferation of the tumor cells and promoting the apoptosis of the cells. Our study provides a new approach and thought to address the problem of the resistance of tyrosine kinase inhibitors in the clinical treatment of NSCLC and to treat NSCLC with HER2 as a driving gene.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
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相關(guān)期刊論文 前2條
1 付勇;蘇雪梅;劉彥仿;趙君;李鍇男;楊守京;鄒賽英;;抗肝癌重組免疫毒素對荷人肝癌裸鼠移植瘤生長的抑制作用[J];中華腫瘤防治雜志;2007年04期
2 任新玲,汪靜,金伯泉,鄧敬蘭,戚好文,喬宏慶,沈麗英;~(99)Tc~m標記EGF受體單抗肺癌放免顯像的實驗研究[J];中華核醫(yī)學雜志;2001年02期
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