新型非病毒基因给药载体聚阳离子脂质体的研究
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摘要
基因治疗为遗传性疾病,传染病和癌症等的治疗提供了良好的前景,而研制安全有效的基因传递系统将成为基因治疗成功的最重要因素之一。本课题结合阳离子聚合物和脂质体两种常用的非病毒基因载体的优点,构建了一种新型的聚阳离子脂质体,对其性质、细胞毒性、摄取和体外转染效率进行了探讨,并研究了该载体的细胞摄取机制,细胞摄取动力学、胞内分布以及携载端粒酶抑制剂后的体外细胞药效学。本课题在此研究基础上构建聚阳离子脂质体-鱼精蛋白-DNA复合载体,研究了鱼精蛋白对聚阳离子脂质体转染的促进作用和细胞核的靶向作用。最后考察了聚阳离子脂质体和复合载体介导的IL-12质粒对荷瘤小鼠的抑瘤作用。
本课题首先采用低分子量聚乙烯亚胺和酰氯胆固醇合成了PEI 800-Chol,经~1H-NMR和红外光谱鉴定,胆固醇分子共价结合到聚乙烯亚胺骨架上。PEI 800-Chol分子具有两亲性,亲水端为聚乙烯亚胺骨架,亲脂端是胆固醇分子,荧光探针法测得该化合物的临界胶团浓度为0.1012 mg/ml。这一性质为下一步的脂质体表面修饰提供了有利条件,胆固醇分子能够插入到脂质体双分子层,使得阳离子的聚乙烯亚胺覆盖于脂质体表面,从而使脂质体带正电荷。
本课题以大豆磷脂、胆固醇和PEI 800-Chol或DOPE和PEI 800-Chol为脂质膜材,采用薄膜分散法制备了两种聚阳离子脂质体(PCLs-S和PCLs-D)。聚阳离子脂质体的平均粒径为133 nm,表面电荷50.1±2.6 mV,具有较强的DNA压缩能力。两种聚阳离子脂质体在N/P大于2时都能结合大部分DNA,而PCLs-S的结合能力大于PCLs-D。PCLs-S的细胞毒性与普通脂质体相当,HeLa细胞的IC_(50)为603.19μg/ml,细胞毒性明显小于商品化试剂Lipofectamine~(TM)2000(48μg/ml)。PCLs-S表面的正电荷能够促进HeLa细胞对ODN的摄取,并保护其在细胞内不被降解。与普通脂质体相比,由于表面PEI分子的存在,使得PCLs-S保持了一定的缓冲能力,大大增加了脂质体的内体逃逸能力,有助于提高转染效率。当N/P为10时,PCLs-S/DNA复合物的转染效率最高,与Lipofectamine~(TM)2000转染能力相当。与Lipofectamine~(TM)2000不同的是在血清存在条件下,PCLs-S的转染效率不受影响,而更高N/P比时,血清的存在增加了绿色荧光蛋白的表达。亲水性的PEI层可能有助于保持复合物在血清中的稳定性,提高转染效率。利用DOPE和PEI800-Chol制备的PCLs-D具有膜融合及“质子泵”缓冲的双重内体逃逸能力,该聚阳离子脂质体介导的绿色荧光蛋白表达率高达68%,显著高于PCLs-S和Lipofectamine~(TM)2000(～44%)。
本课题初步研究了聚阳离子脂质体的细胞摄取机制、细胞内的分布和细胞摄取动力学。细胞摄取聚阳离子脂质体的能力和途径与最终的转染效率有一定的联系,而不同细胞株的摄取机制也存在差异。内吞抑制剂氯丙嗪(特异性抑制网格蛋白介导的内吞)作用A549细胞后,能够抑制聚阳离子脂质体的摄取和转染(大于97%),激光扫描共聚焦显微镜观察,FAM标记的ODN集中于细胞周围,PCLs-D/ODN复合物尤为明显,说明A549细胞摄取聚阳离子脂质体以网格蛋白介导的内吞途径占主导。MCF-7细胞的摄取方式相对复杂,除了非网格蛋白介导的内吞途径外还存在一些非能量依赖的内化方式。细胞摄取动力学研究表明,A549细胞对游离ODN、PCLs-S/ODN复合物和PCLs-D/ODN复合物的摄取在4 h后趋于稳态,PCLs-S和PCLs-D能够分别提高ODN的细胞摄取速度2.20倍和5.45倍,此外PCLs-D还能降低细胞对ODN的消除,增加ODN在胞内的滞留时间。
基于聚阳离子脂质体理化性质和转染能力的研究,本课题以hTERT为靶点的ASODN为治疗基因,比较了传统阳离子脂质体与聚阳离子脂质体的体外细胞药效学。CLs能够显著增加ASODN的肿瘤细胞抑制作用,ASODN经CLs介导后IC_(50)从153.9μmol/L下降至1.28μmol/L。CLs还能提高ASODN在胞内的稳定性,实现了一次给药后120 h内持续抑制肿瘤细胞的生长。PCLs同样能够提高ASODN的细胞抑制作用,而且细胞毒性更小,更有利于体内给药研究。
质粒DNA进入细胞核是限制基因转染的关键步骤之一,在成功解决载体内体逃逸之后,本课题又对PCLs-D进行了修饰,通过加入鱼精蛋白增加DNA的入核能力,构建聚阳离子脂质体-鱼精蛋白-DNA复合载体。采用“Pre-mixed”和“Post-mixed”两种方法制备的复合载体均显著提高了质粒DNA的转染效率,分别是PCLs-D/DNA复合物转染能力的85倍和12倍,而鱼精蛋白-DNA纳米粒没有转染能力;其他无细胞核靶向能力的阳离子聚合物如壳聚糖和PEI 25k经同样方法制备得到的复合载体没有表现出与鱼精蛋白相同的效果,说明鱼精蛋白的细胞核靶向能力对复合载体的转染起了很大的作用,而这一促进作用需要内体逃逸能力为前提条件。激光扫描共聚焦显微术也验证了复合载体传递的基因在细胞内的核靶向能力。鱼精蛋白不仅能增加质粒DNA进入细胞核的数量,经超声破坏实验显示,鱼精蛋白与DNA形成的纳米结构能够很好地保护质粒DNA的完整性,使质粒DNA在细胞内具有长时间转染能力,细胞转染72 h后仍有较高水平的基因表达。
经过一系列的体外研究,本课题最后选用IL-12质粒为治疗基因,考察了PCLs-D及其复合载体瘤内直接注射后对荷瘤小鼠的抑瘤效果。结果发现,实验浓度裸IL-12质粒没有抗肿瘤效果,且外周血清中IFN-γ水平较低。经两种载体介导后,均表现出对肿瘤生长的抑制作用,IFN-γ水平也进一步提高。IL-12质粒为5μg时,复合载体组的肿瘤抑制率达到65.8%,为PCLs-D组的两倍。通过PCLs-D和复合载体介导IL-12质粒治疗小鼠腹水肿瘤,在获得高效抑制率的同时也降低了体内直接应用重组IL-12蛋白所带来的副作用。本研究的结果为合理设计非病毒基因载体提供了新方法,也为进一步的体内外研究和应用奠定了一定的基础。
Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer.The development of efficient and safe gene transfer systems could be one of the most important factors for successful gene therapy.This study constructed a novel non-vrial gene delivery vector,polycation liposomes(PCLs),which combined the advantage of two widely used gene delivery systems,cationic polymers and cationic lipids.The character of PCLs,cytotoxicity,cellular uptake and transfection efficiency in vitro were studied.Then the mechanism of PCLs intemalization,kinetics of cellular uptake,intraceular distribution and the tumor cells inhibition by telomerase inhibitor transferred by PCLs were also investigated.Furthermore,we designed combined vectors,PCLs/protamine/DNA complexes,for increasing transfection efficiency,and then the tumor growth inhibition of PCLs-D/pCMV-IL-12 complexes and PCLs-D/protamine/pCMV-IL-12 complexes were studied.
In this work,the compound PEI 800-Chol was synthesized using PEI with low molecular weight and Cholesteryl chloroformate.The NMR and IR results indicated that cholesterol was covalently conjugated to the backbone of PEI.PEI 800-Chol was a amphiphilic compound with hydrophilic part PEI 800 and hydrophobic Chol.The apparent CMC of PEI 800-Chol was 0.1012 mg/ml,determined with a fluorescence probe technique using pyrene,which was very useful for PEI 800-Chol to modified the surface of liposomes.Chol part anchor to bilayer of liposomes and PEI 800 part bring the liposomes positive charge.
PCLs-S(SPL,Chol and PEI 800-Chol)and PCLs-D(DOPE and PEI 800-Chol)were prepared using film hydration method.The mean particle size of PCLs was 133 nm and the zeta potential was 50.1±2.6 mV which resulted to high DNA condensing ability. When the N/P ratio larger than 2,both PCLs-S and PCLs-D could bind most part DNA, while the former was prefer.Little difference was found between the cytotoxicity of PCLs-S and convention liposomes.The IC_(50)value of PCLs-S against HeLa cells was 603.19μg/ml which notablely higher than that of LipofectamineTM 2000(48μg/ml). Positive charge of PCLs-S could improve ODN internalization by HeLa cells and protect from degradation.Due to PEI anchord onto the surface of liposomes, considerable buffering capacity of PCLs-S was observed,which enhanced the endosomal escape ability of liposomes and increased the transfection efficiency.At N/P ratio 10,the highest transfection efficiency of PCLs-S/DNA complexes was obtained, having the equivalent transfection activity with Lipofectamine~(TM)2000.Interestingly,the transfection activity of PCLs-S was not influenced in the presence of serum,even improved GFP expression at highr N/P ratios.Hydrophilic PEI layer might be helpful for remaining stability of complexes in the presence of serum and increasing transfection efficiency.PCLs-D composed of DOPE and PEI 800-Chol has two endosomal escape abilitys,membrane destabilization and protonation.The GFP expression percentage of PCLs-D(68%)was significantly increased in comparison with that of PCLs-S and Lipofectamine~(TM)2000(～44%).
The mechanism of PCLs internalization,kinetics of cellular uptake and intraceular distribution were investigated.The relationship between internalization pathway and final transfection efficiency might be exited,while different internalization mechanism among various cell lines.When A549 cells were pre-treated by chloropromazine(CPZ), which could specifically inhibit clathin-dependent uptake,the ODN uptake and transfection activity were decreased and fluorescence was concentrated on cell membrane comparing with that CPZ non-treatd determined by laser scanning confocal microscope.The result indicated that the dominance internalization of PCLs in A549 cells was clathin-dependent uptake,while in MCF-7 cells the mean of internalization was more multiple,combining with nonclathin-dependent and nonengery-dependent process.The kinetics of cellular uptake results shown that the cellular uptake of free ODN,PCLs-S/ODN complexes and PCLs-D/ODN complexes trend to saturation after 4 h incubation.PCLs-S and PCLs-D could increase uptake rate by 2.20-fold and 5.45-fold, respectively.Additionally,the eliminate rate of ODN could be decreased by PCLs-D, resulting to longer intracellular retain time.
According to the study of PCLs character and transfection activity,tumor cells inhibition of ASODN against hTERT mediated by Cls and PCLs was compared.The results indicated that CLs could significantly increase the inhibition effect of ASODN and the IC_(50)value was decreased from 153.9μmol/L to 1.28μmol/L.Cls could also improve the intracellular stability of ASODN,achive continued tumor cell groth inhibition during 120 h.PCLs had the same enhancement effect to ASODN with the lower cytotoxicity,which might be more useful for in vivo study.
Plasmid DNA nuclear entry was one of the committed steps for gene transfection. This study increased the nuclear targeting ability of PCLs-D by adding protamin and formed PCLs-D/protamine/DNA complexes.The combined vectors prepared using“Pre-mixed”and“Post-mixed”methods could both improve transfection efficiency obviously,85-fold and 12-fold higher than that of PCLs-D/DNA complexes;however, no transfection activity was found when protamine-DNA nanpartilces used.Though using the same method to prepare combined vectors,other cationic polymers without nuclear targeting such as chitosan and PEI 25k,didn't shown the same effect as protamine.These results suggested nuclear targeting ability was very important in non-viral vectors,while endosomal escape was precondition.Laser scanning confocal microscope confirmed ODN could be carried into nucleus by combined vectors.The protamine-DNA nanoparticles could well protect the integrity of plasmid DNA under ultrasonic condition and might retain transcriptional activity.A549 cells transferred by combined vectors kept high transgene expression during 72 h.
At last,pCMV-IL-12 was used as therapic gene and the tumor growth inhibition of PCLs-D/pCMV-IL-12 complexes and PCLs-D/protamine/pCMV-IL-12 complexes were investigated.No antitumor effect was observed after intratumor injection of naked pCMV-IL-12 at different concentration and the IFN-γlevel in serum was low.When pCMV-IL-12 transferred by two vectors,tumor inhibition effect was appeared and IFN-γlevel increased too.Tumor inhibition rate of the group PCLs-D/protamine/pCMV-IL-12(5μg)reached to 65.8%,doubled than group PCLs-D/pCMV-IL-12(5μg).Intratumor injection of pCMV-IL-12 mediated by PCLs-D and combined vectors for antitumor treating in vivo could obtain high tumor inhibition rate as well as avoid the side effect of recombinate IL-12 protein direct incection.In conclusion,the results of our work provid new methods for reasonable design of non-viral gene vectors and some experimental proofs for further in vitro and in vivo application of non-viral gene delivery systems.
本课题首先采用低分子量聚乙烯亚胺和酰氯胆固醇合成了PEI 800-Chol,经~1H-NMR和红外光谱鉴定,胆固醇分子共价结合到聚乙烯亚胺骨架上。PEI 800-Chol分子具有两亲性,亲水端为聚乙烯亚胺骨架,亲脂端是胆固醇分子,荧光探针法测得该化合物的临界胶团浓度为0.1012 mg/ml。这一性质为下一步的脂质体表面修饰提供了有利条件,胆固醇分子能够插入到脂质体双分子层,使得阳离子的聚乙烯亚胺覆盖于脂质体表面,从而使脂质体带正电荷。
本课题以大豆磷脂、胆固醇和PEI 800-Chol或DOPE和PEI 800-Chol为脂质膜材,采用薄膜分散法制备了两种聚阳离子脂质体(PCLs-S和PCLs-D)。聚阳离子脂质体的平均粒径为133 nm,表面电荷50.1±2.6 mV,具有较强的DNA压缩能力。两种聚阳离子脂质体在N/P大于2时都能结合大部分DNA,而PCLs-S的结合能力大于PCLs-D。PCLs-S的细胞毒性与普通脂质体相当,HeLa细胞的IC_(50)为603.19μg/ml,细胞毒性明显小于商品化试剂Lipofectamine~(TM)2000(48μg/ml)。PCLs-S表面的正电荷能够促进HeLa细胞对ODN的摄取,并保护其在细胞内不被降解。与普通脂质体相比,由于表面PEI分子的存在,使得PCLs-S保持了一定的缓冲能力,大大增加了脂质体的内体逃逸能力,有助于提高转染效率。当N/P为10时,PCLs-S/DNA复合物的转染效率最高,与Lipofectamine~(TM)2000转染能力相当。与Lipofectamine~(TM)2000不同的是在血清存在条件下,PCLs-S的转染效率不受影响,而更高N/P比时,血清的存在增加了绿色荧光蛋白的表达。亲水性的PEI层可能有助于保持复合物在血清中的稳定性,提高转染效率。利用DOPE和PEI800-Chol制备的PCLs-D具有膜融合及“质子泵”缓冲的双重内体逃逸能力,该聚阳离子脂质体介导的绿色荧光蛋白表达率高达68%,显著高于PCLs-S和Lipofectamine~(TM)2000(～44%)。
本课题初步研究了聚阳离子脂质体的细胞摄取机制、细胞内的分布和细胞摄取动力学。细胞摄取聚阳离子脂质体的能力和途径与最终的转染效率有一定的联系,而不同细胞株的摄取机制也存在差异。内吞抑制剂氯丙嗪(特异性抑制网格蛋白介导的内吞)作用A549细胞后,能够抑制聚阳离子脂质体的摄取和转染(大于97%),激光扫描共聚焦显微镜观察,FAM标记的ODN集中于细胞周围,PCLs-D/ODN复合物尤为明显,说明A549细胞摄取聚阳离子脂质体以网格蛋白介导的内吞途径占主导。MCF-7细胞的摄取方式相对复杂,除了非网格蛋白介导的内吞途径外还存在一些非能量依赖的内化方式。细胞摄取动力学研究表明,A549细胞对游离ODN、PCLs-S/ODN复合物和PCLs-D/ODN复合物的摄取在4 h后趋于稳态,PCLs-S和PCLs-D能够分别提高ODN的细胞摄取速度2.20倍和5.45倍,此外PCLs-D还能降低细胞对ODN的消除,增加ODN在胞内的滞留时间。
基于聚阳离子脂质体理化性质和转染能力的研究,本课题以hTERT为靶点的ASODN为治疗基因,比较了传统阳离子脂质体与聚阳离子脂质体的体外细胞药效学。CLs能够显著增加ASODN的肿瘤细胞抑制作用,ASODN经CLs介导后IC_(50)从153.9μmol/L下降至1.28μmol/L。CLs还能提高ASODN在胞内的稳定性,实现了一次给药后120 h内持续抑制肿瘤细胞的生长。PCLs同样能够提高ASODN的细胞抑制作用,而且细胞毒性更小,更有利于体内给药研究。
质粒DNA进入细胞核是限制基因转染的关键步骤之一,在成功解决载体内体逃逸之后,本课题又对PCLs-D进行了修饰,通过加入鱼精蛋白增加DNA的入核能力,构建聚阳离子脂质体-鱼精蛋白-DNA复合载体。采用“Pre-mixed”和“Post-mixed”两种方法制备的复合载体均显著提高了质粒DNA的转染效率,分别是PCLs-D/DNA复合物转染能力的85倍和12倍,而鱼精蛋白-DNA纳米粒没有转染能力;其他无细胞核靶向能力的阳离子聚合物如壳聚糖和PEI 25k经同样方法制备得到的复合载体没有表现出与鱼精蛋白相同的效果,说明鱼精蛋白的细胞核靶向能力对复合载体的转染起了很大的作用,而这一促进作用需要内体逃逸能力为前提条件。激光扫描共聚焦显微术也验证了复合载体传递的基因在细胞内的核靶向能力。鱼精蛋白不仅能增加质粒DNA进入细胞核的数量,经超声破坏实验显示,鱼精蛋白与DNA形成的纳米结构能够很好地保护质粒DNA的完整性,使质粒DNA在细胞内具有长时间转染能力,细胞转染72 h后仍有较高水平的基因表达。
经过一系列的体外研究,本课题最后选用IL-12质粒为治疗基因,考察了PCLs-D及其复合载体瘤内直接注射后对荷瘤小鼠的抑瘤效果。结果发现,实验浓度裸IL-12质粒没有抗肿瘤效果,且外周血清中IFN-γ水平较低。经两种载体介导后,均表现出对肿瘤生长的抑制作用,IFN-γ水平也进一步提高。IL-12质粒为5μg时,复合载体组的肿瘤抑制率达到65.8%,为PCLs-D组的两倍。通过PCLs-D和复合载体介导IL-12质粒治疗小鼠腹水肿瘤,在获得高效抑制率的同时也降低了体内直接应用重组IL-12蛋白所带来的副作用。本研究的结果为合理设计非病毒基因载体提供了新方法,也为进一步的体内外研究和应用奠定了一定的基础。
Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer.The development of efficient and safe gene transfer systems could be one of the most important factors for successful gene therapy.This study constructed a novel non-vrial gene delivery vector,polycation liposomes(PCLs),which combined the advantage of two widely used gene delivery systems,cationic polymers and cationic lipids.The character of PCLs,cytotoxicity,cellular uptake and transfection efficiency in vitro were studied.Then the mechanism of PCLs intemalization,kinetics of cellular uptake,intraceular distribution and the tumor cells inhibition by telomerase inhibitor transferred by PCLs were also investigated.Furthermore,we designed combined vectors,PCLs/protamine/DNA complexes,for increasing transfection efficiency,and then the tumor growth inhibition of PCLs-D/pCMV-IL-12 complexes and PCLs-D/protamine/pCMV-IL-12 complexes were studied.
In this work,the compound PEI 800-Chol was synthesized using PEI with low molecular weight and Cholesteryl chloroformate.The NMR and IR results indicated that cholesterol was covalently conjugated to the backbone of PEI.PEI 800-Chol was a amphiphilic compound with hydrophilic part PEI 800 and hydrophobic Chol.The apparent CMC of PEI 800-Chol was 0.1012 mg/ml,determined with a fluorescence probe technique using pyrene,which was very useful for PEI 800-Chol to modified the surface of liposomes.Chol part anchor to bilayer of liposomes and PEI 800 part bring the liposomes positive charge.
PCLs-S(SPL,Chol and PEI 800-Chol)and PCLs-D(DOPE and PEI 800-Chol)were prepared using film hydration method.The mean particle size of PCLs was 133 nm and the zeta potential was 50.1±2.6 mV which resulted to high DNA condensing ability. When the N/P ratio larger than 2,both PCLs-S and PCLs-D could bind most part DNA, while the former was prefer.Little difference was found between the cytotoxicity of PCLs-S and convention liposomes.The IC_(50)value of PCLs-S against HeLa cells was 603.19μg/ml which notablely higher than that of LipofectamineTM 2000(48μg/ml). Positive charge of PCLs-S could improve ODN internalization by HeLa cells and protect from degradation.Due to PEI anchord onto the surface of liposomes, considerable buffering capacity of PCLs-S was observed,which enhanced the endosomal escape ability of liposomes and increased the transfection efficiency.At N/P ratio 10,the highest transfection efficiency of PCLs-S/DNA complexes was obtained, having the equivalent transfection activity with Lipofectamine~(TM)2000.Interestingly,the transfection activity of PCLs-S was not influenced in the presence of serum,even improved GFP expression at highr N/P ratios.Hydrophilic PEI layer might be helpful for remaining stability of complexes in the presence of serum and increasing transfection efficiency.PCLs-D composed of DOPE and PEI 800-Chol has two endosomal escape abilitys,membrane destabilization and protonation.The GFP expression percentage of PCLs-D(68%)was significantly increased in comparison with that of PCLs-S and Lipofectamine~(TM)2000(～44%).
The mechanism of PCLs internalization,kinetics of cellular uptake and intraceular distribution were investigated.The relationship between internalization pathway and final transfection efficiency might be exited,while different internalization mechanism among various cell lines.When A549 cells were pre-treated by chloropromazine(CPZ), which could specifically inhibit clathin-dependent uptake,the ODN uptake and transfection activity were decreased and fluorescence was concentrated on cell membrane comparing with that CPZ non-treatd determined by laser scanning confocal microscope.The result indicated that the dominance internalization of PCLs in A549 cells was clathin-dependent uptake,while in MCF-7 cells the mean of internalization was more multiple,combining with nonclathin-dependent and nonengery-dependent process.The kinetics of cellular uptake results shown that the cellular uptake of free ODN,PCLs-S/ODN complexes and PCLs-D/ODN complexes trend to saturation after 4 h incubation.PCLs-S and PCLs-D could increase uptake rate by 2.20-fold and 5.45-fold, respectively.Additionally,the eliminate rate of ODN could be decreased by PCLs-D, resulting to longer intracellular retain time.
According to the study of PCLs character and transfection activity,tumor cells inhibition of ASODN against hTERT mediated by Cls and PCLs was compared.The results indicated that CLs could significantly increase the inhibition effect of ASODN and the IC_(50)value was decreased from 153.9μmol/L to 1.28μmol/L.Cls could also improve the intracellular stability of ASODN,achive continued tumor cell groth inhibition during 120 h.PCLs had the same enhancement effect to ASODN with the lower cytotoxicity,which might be more useful for in vivo study.
Plasmid DNA nuclear entry was one of the committed steps for gene transfection. This study increased the nuclear targeting ability of PCLs-D by adding protamin and formed PCLs-D/protamine/DNA complexes.The combined vectors prepared using“Pre-mixed”and“Post-mixed”methods could both improve transfection efficiency obviously,85-fold and 12-fold higher than that of PCLs-D/DNA complexes;however, no transfection activity was found when protamine-DNA nanpartilces used.Though using the same method to prepare combined vectors,other cationic polymers without nuclear targeting such as chitosan and PEI 25k,didn't shown the same effect as protamine.These results suggested nuclear targeting ability was very important in non-viral vectors,while endosomal escape was precondition.Laser scanning confocal microscope confirmed ODN could be carried into nucleus by combined vectors.The protamine-DNA nanoparticles could well protect the integrity of plasmid DNA under ultrasonic condition and might retain transcriptional activity.A549 cells transferred by combined vectors kept high transgene expression during 72 h.
At last,pCMV-IL-12 was used as therapic gene and the tumor growth inhibition of PCLs-D/pCMV-IL-12 complexes and PCLs-D/protamine/pCMV-IL-12 complexes were investigated.No antitumor effect was observed after intratumor injection of naked pCMV-IL-12 at different concentration and the IFN-γlevel in serum was low.When pCMV-IL-12 transferred by two vectors,tumor inhibition effect was appeared and IFN-γlevel increased too.Tumor inhibition rate of the group PCLs-D/protamine/pCMV-IL-12(5μg)reached to 65.8%,doubled than group PCLs-D/pCMV-IL-12(5μg).Intratumor injection of pCMV-IL-12 mediated by PCLs-D and combined vectors for antitumor treating in vivo could obtain high tumor inhibition rate as well as avoid the side effect of recombinate IL-12 protein direct incection.In conclusion,the results of our work provid new methods for reasonable design of non-viral gene vectors and some experimental proofs for further in vitro and in vivo application of non-viral gene delivery systems.
引文
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