靶向survivin的siRNA及其改性穿膜肽纳米给药系统的研究
发布时间：2018-07-29 18:35
【摘要】：肿瘤是严重威胁人类健康的一类疾病,据世界卫生组织报告,全球新确诊和死于肿瘤的人数都在逐年增加,肿瘤的治疗已引起全世界的广泛关注。小干扰核糖核酸(Small interfering RNA,si RNA)是通过诱导核糖核酸干扰(RNA interference,RNAi)效应,在细胞质激发与之互补的目标信使核糖核酸(Messenger RNA,m RNA)沉默,进而调节蛋白的表达,为肿瘤治疗提供了一种有前景的手段。但是,si RNA本身易被核酸酶降解,所以稳定性较差;同时si RNA的亲水性强且带负电,所以不易透过带负电荷的细胞膜进入细胞质,最终导致难以发挥高效的RNAi作用;因此缺乏高效的si RNA以及能够有效结合、保护并递送si RNA载体的现状严重限制了其应用。本论文合成了一条经2′-甲氧基修饰的si RNA序列,对其RNA干扰效果进行评价;同时在穿膜肽八聚精氨酸的基础上合成了多种脂肪酸修饰的八聚精氨酸,经过一系列的试验筛选出能够高效结合、保护并递送si RNA的改性穿膜肽纳米粒;为了克服改性穿膜肽特异性差、靶向配体穿膜效率低的缺点,应用筛选出的改性穿膜肽和靶向配体制备多功能脂质体用于递送si RNA,并考察其体内外递送si RNA的效果。论文具体内容概括如下:1.si RNA在肿瘤细胞中RNAi效果评价设计了一条靶向survivin基因的2′-甲氧基修饰的si RNA序列,通过实时荧光定量PCR、Western blot、流式细胞术等方法检测了si RNA的活性,结果显示20 n M的si RNA作用72 h对survivin m RN A的表达抑制率达到了90%,对蛋白的表达抑制率也达到了80%以上;40 n M的si RNA能够有效地抑制肿瘤细胞的增殖,将细胞周期阻滞在G2/M期,使细胞凋亡率达到40%以上,而且能够导致多种细胞凋亡蛋白表达量和线粒体膜电位的变化。2.改性穿膜肽的合成、鉴定及细胞毒性评价通过固相肽合成的方法,分别将4种脂肪酸(辛酸、硬脂酸、油酸和亚油酸)共价结合到八聚精氨酸分子链游离氨基末端,制备两亲性的穿膜肽,并利用高效液相色谱法(HPLC)和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对其进行分离纯化和鉴定。通过MTT试验对改性穿膜肽进行细胞毒性评价,结果表明各组改性穿膜肽的细胞毒性相对较低,可以作为递送药物的载体材料。3.改性穿膜肽纳米粒的制备及递送si RNA的研究改性穿膜肽自身具备两亲性,能够形成自组装体系,同时带有正电荷,可以通过静电相互作用结合si RNA形成载si RNA的纳米粒,通过对其性质表征、细胞摄取效果和沉默靶基因能力等的研究,筛选出对si RNA结合效率高、保护能力强、递送效果好的改性穿膜肽纳米粒。各试验结果显示,OA-R8(油酸修饰的八聚精氨酸)纳米粒与si RNA的电荷比为1:1时,就可以结合全部的si RNA,具有最高的结合效率;在电荷比为4:1,具有良好的血清和聚阴离子稳定性;OA-R8载si RNA纳米粒的平均粒径为191.9±17.2 nm,Zeta电位为13.2±5.4 m V,粒径及电位分布均匀、稳定;体外细胞试验结果显示,通过网格蛋白和肌动蛋白介导的内吞作用,OA-R8载si RNA纳米粒能够成功地将si RNA转染进入到肿瘤细胞内,将Hep G2和A549细胞中survivin m RN A的表达量降低至30.2%和38.9%,survivin蛋白的表达量降低至42.7%和54.6%,说明改性穿膜肽OA-R8能够有效地穿过细胞膜,递送si RNA进入到肿瘤细胞质中,发挥高效的RNAi作用,进而抑制肿瘤细胞的增殖、阻滞细胞周期以及促进细胞凋亡等。4.基于改性穿膜肽的多功能脂质体的制备及体内外抗肿瘤研究虽然改性穿膜肽细胞毒性小、穿膜效果好,能高效地递送si RNA,但是改性穿膜肽缺少细胞特异性,不能很好地靶向到肿瘤组织,且带有大量正电荷,易被血液快速清除。因此本论文将转铁蛋白(Transferrin,Tf)作为靶向配体,改性穿膜肽OA-R8作为阳离子配体,共同修饰到脂质体表面制成Tf和OA-R8双修饰的多功能脂质体(TOLP),装载survivin si RNA后制成载si RNA的多功能脂质体(s TOLP)。s TOLP利用Tf高度的肿瘤细胞特异性和OA-R8较强的穿膜能力,可以靶向到肿瘤细胞,穿透细胞膜,可以将其用于体内外输送si RNA进行抗肿瘤的研究。s TOLP带有正电荷约3.4±2.3 m V,平均粒径在150.5±14.6 nm,粒度分布均匀,形状多为规则的球形。体外细胞试验结果表明,多功能脂质体对人正常细胞和肿瘤细胞的活力无影响,说明脂质体中引入改性穿膜肽OA-R8不会产生任何细胞毒性;引入OA-R8的载si RNA脂质体(s TOLP和OA-R8修饰的脂质体s OLP)与未经修饰的脂质体(s LP)和单独用Tf修饰的脂质体(s TLP)相比,细胞摄取效果更好,激光共聚焦观察细胞内化也得到了相同的结果;因此OA-R8的引入能够在体外提高脂质体向细胞内递送si RNA的能力,并且无任何毒副作用。体内的试验中,人肝癌异种移植裸小鼠经尾静脉注射脂质体,通过监测肿瘤体积、小鼠体重等变化,发现s TOLP组小鼠肝肿瘤的生长受到显著抑制,与生理盐水组相比肿瘤抑制率达到了61.7%;小动物活体成像及激光共聚焦观察si RNA在荷瘤小鼠体内的组织分布,发现经s TOLP和s TLP递送的si RNA主要蓄积在肝脏和肿瘤组织,s TOLP递送的到达肿瘤部位的si RNA比s TLP更多;经病理切片分析各组织器官的病理变化,发现s TOLP组小鼠的主要器官与对照组小鼠相比,没有明显的组织差异和病变,说明s TOLP在小鼠体内没有全身毒性,可用于体内递送si RNA进而抑制肿瘤的生长。综上所述,本论文合成的靶向survivin的si RNA序列能够在肿瘤细胞中发挥高效的RNAi作用,显著地抑制survivin基因的表达;合成的改性穿膜肽OA-R8具有低毒性,自组装形成的纳米粒对si RNA的结合效率高、保护能力强、递送效果好,提高了si RNA的稳定性和入胞效率;制备的OA-R8和Tf共同修饰的载si RNA多功能脂质体,克服了改性穿膜肽特异性差、转铁蛋白的穿膜效率低的缺点,可以跨越体内重重屏障将si RNA成功地递送到肿瘤组织,发挥高效的RNA干扰作用,最终达到抑制肿瘤生长的效果。
[Abstract]:Cancer is a kind of disease that seriously threatens human health. According to the WHO, the number of new diagnosis and deaths in the world is increasing year by year. The treatment of cancer has attracted worldwide attention. The Small interfering RNA (Si RNA) is a RNA interference, RNAi effect. Messenger RNA (m RNA) is silent in cytoplasm excitation and complementation, and then regulates the expression of protein, which provides a promising means for cancer treatment. However, Si RNA itself is easily degraded by nuclease, so the stability is poor; meanwhile, Si RNA is highly hydrophilic and negative, so it is not easy to pass the negative charge. The cell membrane enters the cytoplasm and eventually leads to the difficult to play an efficient RNAi effect. Therefore, the lack of efficient Si RNA and the effective binding, and the protection and delivery of Si RNA carriers have severely restricted its application. This paper synthesizes a 2 '- methoxy Modified Si RNA sequence to evaluate the effect of RNA interference; at the same time, the membrane peptide is a membrane peptide. On the basis of eight polyarginine, a variety of fatty acid modified eight polyarginine was synthesized. After a series of experiments, a modified membrane peptide nanoparticle, which could be efficiently combined and protected and delivered Si RNA, was screened. In order to overcome the shortcomings of the modified membrane peptide specificity and the low membrane efficiency of the target ligand, the modified membrane peptide and target matching were used. The multifunctional liposomes were prepared to deliver Si RNA and investigate the effect of delivery of Si RNA in vivo and in vitro. The specific content of the paper is summarized as follows: 1.si RNA designed a Si RNA sequence of 2 '- methoxy modified by the target survivin gene in the tumor cells, and by real time fluorescent quantitative PCR, Western, and flow cytometry. The activity of Si RNA was detected by the method. The results showed that the inhibition rate of the expression of Si RNA of 20 N M to survivin m RN A was 90%, and the inhibition rate of protein expression reached 80%. 40 n can effectively inhibit the proliferation of tumor cells. The synthesis, identification and cytotoxicity evaluation of.2. modified transmembrane peptides that lead to the changes in the expression of a variety of apoptotic protein and mitochondrial membrane potential, combined with the method of solid phase peptide synthesis, combined 4 fatty acids (octanoic acid, stearic acid, oleic acid and linoleic acid) together to the free amino terminal of eight polyarginine chain, and prepare two Pro membrane peptides. It was purified and identified by high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The cytotoxicity of modified transmembrane peptides was evaluated by MTT test. The results showed that the cytotoxicity of the modified membrane peptides was relatively low and could be used as a carrier material.3. for delivery drugs. Preparation of modified membrane peptide nanoparticles and delivery of Si RNA, the modified membrane peptide has two affinity, which can form a self-assembly system with positive charge, and can form Si RNA loaded Si nanoparticles through electrostatic interaction and Si RNA. By the characterization of its properties, cell uptake and silencing target gene ability and so on, The modified transmembrane peptide nanoparticles with high efficiency, high protection ability and good delivery effect were selected for Si RNA. The results showed that when the charge ratio of OA-R8 (oleic acid modified eight arginine) nanoparticles and Si RNA was 1:1, the whole Si RNA could be combined with the highest binding efficiency, and the charge ratio was 4:1, with good serum and poly Yin. The average particle size of OA-R8 loaded Si RNA nanoparticles was 191.9 + 17.2 nm, the Zeta potential was 13.2 + 5.4 m V, and the particle size and potential distribution were uniform and stable. In vitro cell test results showed that OA-R8 loaded Si RNA nanoparticles could successfully transfect Si RNA into tumor cells through the endocytosis mediated by gridin and actin. The expression of survivin m RN A in Hep G2 and A549 cells was reduced to 30.2% and 38.9%, and the expression of survivin protein was reduced to 42.7% and 54.6%. It indicated that the modified membrane peptide OA-R8 could effectively pass through the cell membrane and send Si RNA into the cytoplasm of the tumor, and play an efficient RNAi action, and then inhibit the proliferation of tumor cells and block the cell cycle. As well as the preparation of.4. based multifunctional liposomes based on modified membrane peptide and the antitumor research in vivo and in vivo and in vivo, although the modified transmembrane peptide has small toxicity, good membrane effect and efficient delivery of Si RNA, the modified membrane peptide lacks cell specificity and can not target tumor tissue well, with a large number of positive charges and easy to be used. So in this paper, Transferrin (Tf) is used as a target ligand and modified membrane peptide OA-R8 is used as a cationic ligand, which is modified to the surface of liposome to make Tf and OA-R8 double modified multifunctional liposomes (TOLP). After loading survivin Si RNA, the Si RNA multi-functional liposome is made. The specificity of tumor cells and the strong membrane ability of OA-R8 can target the tumor cells and penetrate the cell membrane. It can be used to transport Si RNA in vivo and in vivo for anti-tumor research..s TOLP has a positive charge of about 3.4 + 2.3 m V, the average particle size is 150.5 + 14.6 nm, the particle size distribution is uniform, and the shape is mostly regular. In vitro cell test junction The results showed that the multifunctional liposomes had no effect on the activity of human normal cells and tumor cells, indicating that the introduction of modified membrane peptide OA-R8 in liposomes would not produce any cytotoxicity; the introduction of the OA-R8 loaded Si RNA liposomes (s TOLP and OA-R8 modified liposomes s OLP) and the unmodified liposomes (s LP) and the liposomes modified by Tf alone In contrast, cell uptake is better, and laser confocal observation of cell internalization is also the same. Therefore, the introduction of OA-R8 can improve the ability of liposomes to deliver Si RNA into cells in vitro, without any toxic and side effects. In the test of human liver cancer xenotransplantation, nude mice were injected with liposomes through the tail vein, by monitoring the swelling. The tumor volume, mouse weight and other changes showed that the growth of liver tumor in the s TOLP group was significantly inhibited, and the tumor inhibition rate was 61.7% compared with the normal saline group. The tissue distribution of Si RNA in the tumor bearing mice was observed by the living body imaging and laser confocal microscopy, and the Si RNA delivered by s TOLP and s TLP was mainly accumulated in the liver and swelling. The Si RNA delivered by s TOLP was more than that of s TLP. The pathological changes of tissues and organs were analyzed by pathological section, and the main organs of the s TOLP mice were found to have no obvious histological differences and pathological changes compared with the control group, indicating that s TOLP had no systemic toxicity in mice, and could be used in the delivery of Si RNA in the body. To sum up the growth of the tumor, the Si RNA sequence of the target survivin, synthesized in this paper, can play an efficient RNAi role in the tumor cells and significantly inhibit the expression of the survivin gene; the synthesized modified transmembrane peptide OA-R8 has low toxicity, and the self assembled nanoparticles have high binding efficiency to Si RNA, strong protection and good delivery effect. The stability and cell efficiency of Si RNA were improved; the Si RNA multifunctional liposomes, which were modified by OA-R8 and Tf, overcome the shortcomings of the modified transmembrane peptide specificity and the low membrane efficiency of the transferrin, which could be successfully delivered to the swelling tumor tissue across the barrier of the body, and the effective RNA interference was achieved. Finally, the inhibition of Si RNA was achieved. The effect of the growth of the tumor.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943
本文编号：2153626
[Abstract]:Cancer is a kind of disease that seriously threatens human health. According to the WHO, the number of new diagnosis and deaths in the world is increasing year by year. The treatment of cancer has attracted worldwide attention. The Small interfering RNA (Si RNA) is a RNA interference, RNAi effect. Messenger RNA (m RNA) is silent in cytoplasm excitation and complementation, and then regulates the expression of protein, which provides a promising means for cancer treatment. However, Si RNA itself is easily degraded by nuclease, so the stability is poor; meanwhile, Si RNA is highly hydrophilic and negative, so it is not easy to pass the negative charge. The cell membrane enters the cytoplasm and eventually leads to the difficult to play an efficient RNAi effect. Therefore, the lack of efficient Si RNA and the effective binding, and the protection and delivery of Si RNA carriers have severely restricted its application. This paper synthesizes a 2 '- methoxy Modified Si RNA sequence to evaluate the effect of RNA interference; at the same time, the membrane peptide is a membrane peptide. On the basis of eight polyarginine, a variety of fatty acid modified eight polyarginine was synthesized. After a series of experiments, a modified membrane peptide nanoparticle, which could be efficiently combined and protected and delivered Si RNA, was screened. In order to overcome the shortcomings of the modified membrane peptide specificity and the low membrane efficiency of the target ligand, the modified membrane peptide and target matching were used. The multifunctional liposomes were prepared to deliver Si RNA and investigate the effect of delivery of Si RNA in vivo and in vitro. The specific content of the paper is summarized as follows: 1.si RNA designed a Si RNA sequence of 2 '- methoxy modified by the target survivin gene in the tumor cells, and by real time fluorescent quantitative PCR, Western, and flow cytometry. The activity of Si RNA was detected by the method. The results showed that the inhibition rate of the expression of Si RNA of 20 N M to survivin m RN A was 90%, and the inhibition rate of protein expression reached 80%. 40 n can effectively inhibit the proliferation of tumor cells. The synthesis, identification and cytotoxicity evaluation of.2. modified transmembrane peptides that lead to the changes in the expression of a variety of apoptotic protein and mitochondrial membrane potential, combined with the method of solid phase peptide synthesis, combined 4 fatty acids (octanoic acid, stearic acid, oleic acid and linoleic acid) together to the free amino terminal of eight polyarginine chain, and prepare two Pro membrane peptides. It was purified and identified by high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The cytotoxicity of modified transmembrane peptides was evaluated by MTT test. The results showed that the cytotoxicity of the modified membrane peptides was relatively low and could be used as a carrier material.3. for delivery drugs. Preparation of modified membrane peptide nanoparticles and delivery of Si RNA, the modified membrane peptide has two affinity, which can form a self-assembly system with positive charge, and can form Si RNA loaded Si nanoparticles through electrostatic interaction and Si RNA. By the characterization of its properties, cell uptake and silencing target gene ability and so on, The modified transmembrane peptide nanoparticles with high efficiency, high protection ability and good delivery effect were selected for Si RNA. The results showed that when the charge ratio of OA-R8 (oleic acid modified eight arginine) nanoparticles and Si RNA was 1:1, the whole Si RNA could be combined with the highest binding efficiency, and the charge ratio was 4:1, with good serum and poly Yin. The average particle size of OA-R8 loaded Si RNA nanoparticles was 191.9 + 17.2 nm, the Zeta potential was 13.2 + 5.4 m V, and the particle size and potential distribution were uniform and stable. In vitro cell test results showed that OA-R8 loaded Si RNA nanoparticles could successfully transfect Si RNA into tumor cells through the endocytosis mediated by gridin and actin. The expression of survivin m RN A in Hep G2 and A549 cells was reduced to 30.2% and 38.9%, and the expression of survivin protein was reduced to 42.7% and 54.6%. It indicated that the modified membrane peptide OA-R8 could effectively pass through the cell membrane and send Si RNA into the cytoplasm of the tumor, and play an efficient RNAi action, and then inhibit the proliferation of tumor cells and block the cell cycle. As well as the preparation of.4. based multifunctional liposomes based on modified membrane peptide and the antitumor research in vivo and in vivo and in vivo, although the modified transmembrane peptide has small toxicity, good membrane effect and efficient delivery of Si RNA, the modified membrane peptide lacks cell specificity and can not target tumor tissue well, with a large number of positive charges and easy to be used. So in this paper, Transferrin (Tf) is used as a target ligand and modified membrane peptide OA-R8 is used as a cationic ligand, which is modified to the surface of liposome to make Tf and OA-R8 double modified multifunctional liposomes (TOLP). After loading survivin Si RNA, the Si RNA multi-functional liposome is made. The specificity of tumor cells and the strong membrane ability of OA-R8 can target the tumor cells and penetrate the cell membrane. It can be used to transport Si RNA in vivo and in vivo for anti-tumor research..s TOLP has a positive charge of about 3.4 + 2.3 m V, the average particle size is 150.5 + 14.6 nm, the particle size distribution is uniform, and the shape is mostly regular. In vitro cell test junction The results showed that the multifunctional liposomes had no effect on the activity of human normal cells and tumor cells, indicating that the introduction of modified membrane peptide OA-R8 in liposomes would not produce any cytotoxicity; the introduction of the OA-R8 loaded Si RNA liposomes (s TOLP and OA-R8 modified liposomes s OLP) and the unmodified liposomes (s LP) and the liposomes modified by Tf alone In contrast, cell uptake is better, and laser confocal observation of cell internalization is also the same. Therefore, the introduction of OA-R8 can improve the ability of liposomes to deliver Si RNA into cells in vitro, without any toxic and side effects. In the test of human liver cancer xenotransplantation, nude mice were injected with liposomes through the tail vein, by monitoring the swelling. The tumor volume, mouse weight and other changes showed that the growth of liver tumor in the s TOLP group was significantly inhibited, and the tumor inhibition rate was 61.7% compared with the normal saline group. The tissue distribution of Si RNA in the tumor bearing mice was observed by the living body imaging and laser confocal microscopy, and the Si RNA delivered by s TOLP and s TLP was mainly accumulated in the liver and swelling. The Si RNA delivered by s TOLP was more than that of s TLP. The pathological changes of tissues and organs were analyzed by pathological section, and the main organs of the s TOLP mice were found to have no obvious histological differences and pathological changes compared with the control group, indicating that s TOLP had no systemic toxicity in mice, and could be used in the delivery of Si RNA in the body. To sum up the growth of the tumor, the Si RNA sequence of the target survivin, synthesized in this paper, can play an efficient RNAi role in the tumor cells and significantly inhibit the expression of the survivin gene; the synthesized modified transmembrane peptide OA-R8 has low toxicity, and the self assembled nanoparticles have high binding efficiency to Si RNA, strong protection and good delivery effect. The stability and cell efficiency of Si RNA were improved; the Si RNA multifunctional liposomes, which were modified by OA-R8 and Tf, overcome the shortcomings of the modified transmembrane peptide specificity and the low membrane efficiency of the transferrin, which could be successfully delivered to the swelling tumor tissue across the barrier of the body, and the effective RNA interference was achieved. Finally, the inhibition of Si RNA was achieved. The effect of the growth of the tumor.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943
【相似文献】
相关期刊论文 前5条
1 庄炜东;阮世龙;杨旭中;王佳玮;王富军;赵健;;一种高效人源性穿膜肽的生物活性研究[J];食品与药品;2013年02期
2 李莉蓉;施用晖;乐国伟;;来自穿膜肽的新肽的抗菌活性及抑菌机制[J];微生物学报;2013年09期
3 张楠;白银;赵景壮;叶贤龙;王文飞;任桂萍;李德山;荆燕;;多聚精氨酸融合增强型绿色荧光蛋白制备方法及穿膜效果[J];生物工程学报;2013年11期
4 王军;雷楗勇;陈蕴;金坚;;细胞穿膜肽TAT与小鼠干细胞转录因子Oct4融合表达及纯化[J];食品与生物技术学报;2013年09期
5 ;[J];;年期
相关会议论文 前7条
1 王小波;周明;费浩;;一种基于环金属铱化合物与组氨酸结合的新型多肽标记方式及用于细胞穿膜肽的成像研究[A];中国化学会第27届学术年会第03分会场摘要集[C];2010年
2 王宏博;杨建海;王玮;刘文广;;富含精氨酸的细胞穿膜肽的研究进展(综述)[A];天津市生物医学工程学会第三十一届学术年会论文集[C];2011年
3 王怡;肖健;林海环;杨静;李校X;;新穿膜肽蛋白His-T1-GFP的跨膜效率和细胞毒性研究[A];2007年全国生化与生物技术药物学术年会论文集[C];2007年
4 王琥;柳长柏;黄宇彬;;不同条件下细胞穿膜肽TAT的穿膜效率的比较[A];第二届中国医学细胞生物学学术大会暨细胞生物学教学改革会议论文集[C];2008年
5 薛改青;祖莉莉;;穿膜肽Transportan10气相解离动力学的研究[A];第十三届全国化学动力学会议报告摘要集[C];2013年
6 马岩岩;荣靖;彭鑫磊;刘秋英;舒辉萍;陈海佳;王一飞;任哲;;穿膜肽KGF-2的构建及其对HaCaT增殖作用的影响[A];中国遗传学会“第十一届全国激光生物学学术会议”暨《激光生物学报》创刊廿周年庆祝会、第十届粤港生物物理研讨会暨2012年广东生物物理学术年会会议资料[C];2012年
7 张俊萍;孙青山;贾原;;穿膜肽介导p53多肽抗原致敏的DC的体外抗瘤效应研究[A];中国肿瘤内科进展 中国肿瘤医师教育（2014）[C];2014年
相关博士学位论文 前4条
1 马严;可在体实现胞内直接投递的新型细胞穿膜肽的构建、筛选和穿膜机制研究[D];华中科技大学;2012年
2 李玉环;靶向survivin的siRNA及其改性穿膜肽纳米给药系统的研究[D];吉林大学;2016年
3 严世荣;穿膜肽携带的目的蛋白穿膜效应的研究[D];华中科技大学;2006年
4 李珂;靶向toll样受体2多肽的筛选、鉴定及应用[D];北京协和医学院;2013年
相关硕士学位论文 前10条
1 荆鑫鑫;高效重组穿膜肽-vp3的筛选及其对Hela细胞的抑制作用[D];佳木斯大学;2015年
2 刘畅;穿膜肽介导的眼内基因递送系统[D];复旦大学;2014年
3 马德云;细胞穿膜肽Pep-1和人表皮生长因子在大肠杆菌中的重组表达[D];天津科技大学;2014年
4 庄炜东;新型人源性穿膜肽机理研究及性质比较[D];华东理工大学;2013年
5 杨冬梅;富含胍基的细胞穿膜肽合成[D];华中科技大学;2012年
6 张宏达;穿膜肽介导的新型基因转运载体的研究[D];中国人民解放军军事医学科学院;2008年
7 韦一凡;细胞穿膜肽在藻类细胞外源物质转导中的应用[D];中国科学院研究生院（海洋研究所）;2014年
8 刘云龙;穿膜肽-β-葡萄糖苷酶—西妥昔单抗偶联物的制备与鉴定[D];广西医科大学;2015年
9 吴强;穿膜肽/NF-κB拮抗多肽原核表达载体的构建、表达、纯化与功能检测[D];安徽农业大学;2006年
10 王光文;穿膜肽-β-葡萄糖苷酶偶联物制备鉴定及激活苦杏仁苷杀伤膀胱癌细胞的初步研究[D];广西医科大学;2014年
，本文编号：2153626
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2153626.html
最近更新
教材专著
