超声微泡造影剂联合脂质体介导pNogo-R shRNA转染神经干细胞的体外实验研究
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摘要
目的:探讨超声微泡介导pGPU6/Neo质粒转染大鼠NSCs的最优参数。
方法:以一定能量的超声介导质粒转染大鼠NSCs,分为:空白组、pGPU6/Neo质粒组、pGPU6/Neo质粒+微泡组、pGPU6/Neo质粒+超声组、pGPU6/Neo质粒+微泡+超声组,并按不同转染条件分为亚组。转染48 h后荧光显微镜观察转染效率,台盼蓝染色法检测细胞活性。
结果:最优参数为声强1.0 W/cm~2,辐照时间30 s时,超声微泡介导质粒转染率最高,且对细胞活性无明显影响。超声微泡介导质粒对NSCs的转染效率,明显高于其他实验组(P<0.05)。
结论:在超声声强为1.0 W/cm~2,辐照时间30 s的条件下,超声微泡造影剂能够安全、有效地介导基因转染NSCs。
目的:探讨超声微泡介导pNogo-R shRNA转染大鼠NSCs的有效率及可行性,同时评估UMMD联合脂质体提高基因转染效率的协同作用。
方法:NSCs自清洁级SD新生大鼠分离获得,以不同的方式转染培养至第三代的NSCs,分为:A.空白组、B.阴性质粒+超声微泡组、C. pNogo-R shRNA质粒+超声微泡组、D. pNogo-R shRNA质粒+脂质体组、E. pNogo-R shRNA质粒+超声微泡+脂质体组。
结果:荧光显微镜观测结果显示,E组转染效率明显高于其他实验组,而C组与D组间无显著差异;同时,台盼蓝染色法显示,与其他组相比较,超声微泡联合脂质体组对细胞活性影响无显著差异;转染24小时后,E组Nogo-R的蛋白及mRNA表达水平明显低于其他组别(P<0.05),差异具有统计学意义。
结论:本研究结果表明,超声微泡联合脂质体的基因转染为一非侵袭性转染方法,对细胞活性影响小,具有一定可行性。超声微泡和脂质体的联合作用能增强pNogo-R shRNA质粒转染NSCs,这种联合转染方式为NSCs转基因治疗提供了一种新方法。
Objective: To investigate the parameters of ultrasound-mediated microbubble destruction of pGPU6/Neo plasmid transfected into NSCs of rats by ultrasound.
Methods: The plasmid was used to transfect into NSCs by ultrasound-mediated microbubble destruction. The NSCs were divided into 5 groups: blank control, pGPU6/Neo plasmid, pGPU6/Neo plasmid-microbubble, pGPU6/Neo plasmid-ultrasound and pGPU6/Neo plasmid-microbubble-ultrasound. The last group was further divided into subgroups according to different conditions of ultrasound. After 48 h, the expression of GFP was detected by fluorescent microscope. The cell vitality was measured by typan-blue stainning.
Results: The transfection efficiency was the highest and the cell vitality was not different from other subgroups when ultrasound was radiated at frequency of 300 kHz and sound intensity of 1.0 W/cm2 for 30 s. The transfection efficiency of plasmid-microbubble-ultrasound group was statistically higher than those of other groups (P<0.05).
Conclusions: Under sound intensity 1.0 W/cm~2 and exposure time 30 s, ultrasound-mediated microbubble destruction could enhance gene transfection in NSCs.
Objective: To explore the feasibility and efficiency of transfection into NSCs with pNogo-R shRNA by ultrasound-mediated microbubble destruction (UMMD), and to assess synergistic effect of UMMD together with liposome -plasmid DNA in increasing transfection efficiency.
Methods: NSCs was sterilely obtained from neonatal Sprague Dawley rats. The third passaged cells were treated transgene following by different transfection conditions with (A) blank, (B) negative control+ultrasound microbubble, (C) pNogo-R shRNA+ ultrasound microbubble, (D) pNogo-R shRNA+Liposome, and (E) pNogo-R shRNA+Liposome+ultrasound microbubble, respectively.
Results: In vitro cell experiments, no significant difference between C and D with fluorescence microscope was found, while transfection efficiency in group E was the highest compared with those of the other groups. More importantly, Liposome+ultrasound microbubble-mediated group did not affect the cell vitality comparing with the other groups by trypan-blue staining. In addition, Nogo-R mRNA and protein expression was dramatically decreased in group E (P<0.05), compared with other groups after 24 h transfection.
Conclusion: Taken together, the present data demonstrates that UM+Lip-mediated transtection could be an efficiently noninvasive mean in transfection of NSCs. Ultrasound-microbubble technique could increase Nogo-R gene transfer into NSCs together with liposome, suggesting that the combination transfection provides a new method for transgene therapy of NSCs
方法:以一定能量的超声介导质粒转染大鼠NSCs,分为:空白组、pGPU6/Neo质粒组、pGPU6/Neo质粒+微泡组、pGPU6/Neo质粒+超声组、pGPU6/Neo质粒+微泡+超声组,并按不同转染条件分为亚组。转染48 h后荧光显微镜观察转染效率,台盼蓝染色法检测细胞活性。
结果:最优参数为声强1.0 W/cm~2,辐照时间30 s时,超声微泡介导质粒转染率最高,且对细胞活性无明显影响。超声微泡介导质粒对NSCs的转染效率,明显高于其他实验组(P<0.05)。
结论:在超声声强为1.0 W/cm~2,辐照时间30 s的条件下,超声微泡造影剂能够安全、有效地介导基因转染NSCs。
目的:探讨超声微泡介导pNogo-R shRNA转染大鼠NSCs的有效率及可行性,同时评估UMMD联合脂质体提高基因转染效率的协同作用。
方法:NSCs自清洁级SD新生大鼠分离获得,以不同的方式转染培养至第三代的NSCs,分为:A.空白组、B.阴性质粒+超声微泡组、C. pNogo-R shRNA质粒+超声微泡组、D. pNogo-R shRNA质粒+脂质体组、E. pNogo-R shRNA质粒+超声微泡+脂质体组。
结果:荧光显微镜观测结果显示,E组转染效率明显高于其他实验组,而C组与D组间无显著差异;同时,台盼蓝染色法显示,与其他组相比较,超声微泡联合脂质体组对细胞活性影响无显著差异;转染24小时后,E组Nogo-R的蛋白及mRNA表达水平明显低于其他组别(P<0.05),差异具有统计学意义。
结论:本研究结果表明,超声微泡联合脂质体的基因转染为一非侵袭性转染方法,对细胞活性影响小,具有一定可行性。超声微泡和脂质体的联合作用能增强pNogo-R shRNA质粒转染NSCs,这种联合转染方式为NSCs转基因治疗提供了一种新方法。
Objective: To investigate the parameters of ultrasound-mediated microbubble destruction of pGPU6/Neo plasmid transfected into NSCs of rats by ultrasound.
Methods: The plasmid was used to transfect into NSCs by ultrasound-mediated microbubble destruction. The NSCs were divided into 5 groups: blank control, pGPU6/Neo plasmid, pGPU6/Neo plasmid-microbubble, pGPU6/Neo plasmid-ultrasound and pGPU6/Neo plasmid-microbubble-ultrasound. The last group was further divided into subgroups according to different conditions of ultrasound. After 48 h, the expression of GFP was detected by fluorescent microscope. The cell vitality was measured by typan-blue stainning.
Results: The transfection efficiency was the highest and the cell vitality was not different from other subgroups when ultrasound was radiated at frequency of 300 kHz and sound intensity of 1.0 W/cm2 for 30 s. The transfection efficiency of plasmid-microbubble-ultrasound group was statistically higher than those of other groups (P<0.05).
Conclusions: Under sound intensity 1.0 W/cm~2 and exposure time 30 s, ultrasound-mediated microbubble destruction could enhance gene transfection in NSCs.
Objective: To explore the feasibility and efficiency of transfection into NSCs with pNogo-R shRNA by ultrasound-mediated microbubble destruction (UMMD), and to assess synergistic effect of UMMD together with liposome -plasmid DNA in increasing transfection efficiency.
Methods: NSCs was sterilely obtained from neonatal Sprague Dawley rats. The third passaged cells were treated transgene following by different transfection conditions with (A) blank, (B) negative control+ultrasound microbubble, (C) pNogo-R shRNA+ ultrasound microbubble, (D) pNogo-R shRNA+Liposome, and (E) pNogo-R shRNA+Liposome+ultrasound microbubble, respectively.
Results: In vitro cell experiments, no significant difference between C and D with fluorescence microscope was found, while transfection efficiency in group E was the highest compared with those of the other groups. More importantly, Liposome+ultrasound microbubble-mediated group did not affect the cell vitality comparing with the other groups by trypan-blue staining. In addition, Nogo-R mRNA and protein expression was dramatically decreased in group E (P<0.05), compared with other groups after 24 h transfection.
Conclusion: Taken together, the present data demonstrates that UM+Lip-mediated transtection could be an efficiently noninvasive mean in transfection of NSCs. Ultrasound-microbubble technique could increase Nogo-R gene transfer into NSCs together with liposome, suggesting that the combination transfection provides a new method for transgene therapy of NSCs
引文
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[4] Eftekharpour E, Karimi-Abdolrezaee S, Fehlings MG. Current status of experimental cell replacement approaches to spinal cord injury [J]. Neurosurg Focus. 2008,24(3-4):E19
[5] Wei Li, Su Liu, Jianli Ren,et al. Gene Transfection to Retinal Ganglion Cells Mediated by Ultrasound Microbubbles in vitro [J]. Acad Radiol. 2009,16(9): 1086- 1094
[6] Akowuah EF, Gray C, Lawrie A, et al. Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model [J]. Gene Ther. 2005;12(14):1154-1157
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[20]王丰,朱悦.RNA干扰沉默神经干细胞Nogo-66受体基因阻断Nogo-66抑制神经突生长作用的研究[J].中国修复重建外科杂志. 2007,21(6):642-647
[21]王东,张建军,杨忠旭.神经干细胞NgR基因沉默移植治疗大鼠脊髓损伤[J].中国危重病急救医学. 2010,22(1):28-31
[2] Webb AA, Ngan S, Fowler JD. Spinal cord injury I: A synopsis of the basic science [J]. Can Vet J. 2010,51(5):485-492
[3] Bottai D, Madaschi L, Di Giulio AM, et al. Viability-dependent promoting action of adult neural precursors in spinal cord injury [J]. Mol Med. 2008, 14(9-10):634-644
[4] Eftekharpour E, Karimi-Abdolrezaee S, Fehlings MG. Current status of experimental cell replacement approaches to spinal cord injury [J]. Neurosurg Focus. 2008,24(3-4):E19
[5] Wei Li, Su Liu, Jianli Ren,et al. Gene Transfection to Retinal Ganglion Cells Mediated by Ultrasound Microbubbles in vitro [J]. Acad Radiol. 2009,16(9): 1086- 1094
[6] Akowuah EF, Gray C, Lawrie A, et al. Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model [J]. Gene Ther. 2005;12(14):1154-1157
[7]聂芳,徐辉雄,吕明德,等.超声介导微泡造影剂促进HSV-TK基因转染抑制小鼠肝癌移植瘤的实验研究[J].中国超声医学杂志. 2008,24(5):399-402
[8]李红丽,杜联芳,郑孝志,等.超声辐照微泡介导脂质体小干扰RNA转染视网膜色素上皮细胞[J].中国医学影像技术. 2010,26(1):22-24
[9]廖沁,周希瑗,王志刚,等.超声微泡造影剂介导PEDF质粒转染大鼠视网膜的实验研究[J].中国医学影像技术. 2008,24(8):1149-1152
[10] Yu P, Huang L, Zou J,et al.Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats [J]. Neurobiol Dis.2008;32(3):535-542
[11] Wang F, Zhu Y.The interaction of Nogo-66 receptor with Nogo-p4 inhibits the neuronal differentiation of neural stem cells [J]. Neuroscience. 2008;151(1):74-81
[12] Kabatas S, Teng YD.Potential roles of the neural stem cell in the restoration of the injured spinal cord: review of the literature [J].Turk Neurosurg.2010,20(2):103-110
[13] Kim SU, de Vellis J. Stem cell-based cell therapy in neurological diseases: a review [J]. J Neurosci Res. 2009,87(10):2183-2200
[14] Mayer CR, Geis NA, Katus HA, et al.Ultrasound targeted microbubble destruction for drug and gene delivery [J].Expert Opin Drug Deliv. 2008,5(10):1121-1138
[15] Dijkmans PA, Juffermans LJ, Musters RJ,et al.Microbubbles and ultrasound:from diagnosis to therapy [J].Eur J Echocardiogr. 2004,5(4):245-256
[16] Yong FU, Shen-qing WANG, Ying-peng LIU, et al. Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein [J]. Zhejiang Univ Sci B. 2008;9(4):299-305
[17]湛彦强,王芙蓉,黎逢光,等.不同方法及载体转染神经干细胞的实验研究[J].卒中与神经疾病. 2007,14(5):259-262
[18] Zhou BJ, Zha DG, DU RS, et al.Impact of ultrasound-mediated microbubbles on myocardial vascular permeability in rats [J].Nan Fang Yi Ke Da Xue Xue Bao. 2010,30(2):239-241
[19] Mayer CR,Bekeredjian R.Ultrasonic gene and drug delivery to the cardiovascular system [J].Adv Drug Deliv Rev. 2008,60(10):1177-1192
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