微流体数字化细胞显微注射实验研究
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摘要
生物技术的发展以及细胞注射技术的广泛应用,对实验手段和实验设备提出了越来越高的要求。比如,设备操作的容易程度、设备对微操作对象的影响大小、微注射量的控制精度等问题。在细胞注射过程中,对微操作要求迅速准确,注射速度快;细胞刺膜过程对细胞的变形影响小、微注射量一般要求达到pL级。目前还没有相关设备和手段来完全解决这些问题,南京理工大学微系统研究室提出的“微流体数字化技术”在这方面取得了突破性进展。
本文是针对数字化细胞微注射仪的细胞注射实验研究,该注射仪是基于“微流体数字化技术”研发的。利用微系统研究室研发的细胞工程用微针制备仪器,在大量制备微针实验的基础上,制作出符合细胞注射要求的微针。对微针进行表面疏水化以及清洗处理,可以提高微注射针的注射性能;利用图像处理技术可以精确测出其物理参数。此外,对数字化微注射仪的进针、退针性能进行测试;重点研究了注射仪的注射性能,得到不同参数下的注射量,并对其影响因素进行分析。在此基础上,对三种具有代表性的卵细胞进行微注射操作,通过实验探索注射驱动参数,得出注射成功率较高的参数范围。最后对注射过荧光标记物的细胞进行荧光检测,证实微注射仪确实将注射药液注入细胞内。
With the development of biotechnology and the wider application of cell injection technology, the necessity of qualified experimental means and facilities closes behind. For example, the facility of operation and fidelity of microinjection dose, and so on. Generally, quick and accurate micro operations, velocity of injection, small transfiguration of cell and microinjection dose to the degree of pL are required in executing cell injection. There are no good equipment and means to resolve these issues yet. So it's very important to research on this respect. "Digitalization of micro fluids technology" from micro system lab of Nanjing University of Science and Technology has a breakthrough at this respect.
This paper is for experimental research of cell injection by using digital micro-injection device which is based on "digitalization of micro fluids technology". The micro needle preparation devices which are used in Cell Engineering and researched by micro-system lab can create micro-injection needles. The micro-injection needles are fabricated by a large number of micro-needle preparation experiments. The performances of injecting droplets are enhanced after the surface hydrophobic and wash treatment. The micro-needles' precise physical parameters are measured by image processing technology. Then, some tests are made to detect the performance about advance and retreat of digital micro-injection device. Its injection performance are researched to get different parameters of the injection, and then the factors of the injection quantity are analyzed. On this condition, author carry out micro injection experiment on three typical and different cells. Search after drive parameter of injection, and then get the parameter range of the high ration of successful injecting. Finally, confirmed that micro-injection device can inject drug into the cell by fluorescence detecting on the cell which is injected fluorescent marker.
本文是针对数字化细胞微注射仪的细胞注射实验研究,该注射仪是基于“微流体数字化技术”研发的。利用微系统研究室研发的细胞工程用微针制备仪器,在大量制备微针实验的基础上,制作出符合细胞注射要求的微针。对微针进行表面疏水化以及清洗处理,可以提高微注射针的注射性能;利用图像处理技术可以精确测出其物理参数。此外,对数字化微注射仪的进针、退针性能进行测试;重点研究了注射仪的注射性能,得到不同参数下的注射量,并对其影响因素进行分析。在此基础上,对三种具有代表性的卵细胞进行微注射操作,通过实验探索注射驱动参数,得出注射成功率较高的参数范围。最后对注射过荧光标记物的细胞进行荧光检测,证实微注射仪确实将注射药液注入细胞内。
With the development of biotechnology and the wider application of cell injection technology, the necessity of qualified experimental means and facilities closes behind. For example, the facility of operation and fidelity of microinjection dose, and so on. Generally, quick and accurate micro operations, velocity of injection, small transfiguration of cell and microinjection dose to the degree of pL are required in executing cell injection. There are no good equipment and means to resolve these issues yet. So it's very important to research on this respect. "Digitalization of micro fluids technology" from micro system lab of Nanjing University of Science and Technology has a breakthrough at this respect.
This paper is for experimental research of cell injection by using digital micro-injection device which is based on "digitalization of micro fluids technology". The micro needle preparation devices which are used in Cell Engineering and researched by micro-system lab can create micro-injection needles. The micro-injection needles are fabricated by a large number of micro-needle preparation experiments. The performances of injecting droplets are enhanced after the surface hydrophobic and wash treatment. The micro-needles' precise physical parameters are measured by image processing technology. Then, some tests are made to detect the performance about advance and retreat of digital micro-injection device. Its injection performance are researched to get different parameters of the injection, and then the factors of the injection quantity are analyzed. On this condition, author carry out micro injection experiment on three typical and different cells. Search after drive parameter of injection, and then get the parameter range of the high ration of successful injecting. Finally, confirmed that micro-injection device can inject drug into the cell by fluorescence detecting on the cell which is injected fluorescent marker.
引文
[1]李志勇.细胞工程[M].第1版.北京:科学出版社,2003
[2]安利国.细胞工程[M].第1版.北京:科学出版社,2004
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[5]Graessmann A,Graessmann M,Muller C.Microinjection of early SV40 DNA fragments and Tantigen[J].Meth Enzymol.1980,65:816-825
[6]Stacey,DW Allfrey VG,Hanafusa H.Microinjection analysis of envelope glycoprotein messenger activities of avian leucosis viral RNAs[J].Proc Natl Acad Sci.1977,74:1614-1618
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[42]张灵,章云,杨宜民等.转基因微注射系统中注射量的控制技术[J].中国机械工程.2003,14(24):2146-2148
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[2]安利国.细胞工程[M].第1版.北京:科学出版社,2004
[3]罗九甫,李志勇.生物工程原理与技术[M].第1版.北京:科学出版社,2006
[4]Graessmann.Mikrochirurgische Zellkerntransplantion bei Saugetierzellen[J].Exp Cell Res.1970,60:373-382
[5]Graessmann A,Graessmann M,Muller C.Microinjection of early SV40 DNA fragments and Tantigen[J].Meth Enzymol.1980,65:816-825
[6]Stacey,DW Allfrey VG,Hanafusa H.Microinjection analysis of envelope glycoprotein messenger activities of avian leucosis viral RNAs[J].Proc Natl Acad Sci.1977,74:1614-1618
[7]Mabuchi I,Okuno M.The effect of myosin antibody on the division of starfish blastomeres[J].Cell Biol.1977,74:251-263
[8]西德-阿里吉尔,A.加西亚-卡冉卡,张玉静等.微注射和转基因实验指南[M].第1版.北京:科学出版社,2002
[9]Palmiter R D,Brmster R L,Hammer RE,et al.Dramatic growth of mice that develop from eggs microinjection with metallothionein growth hormonegene[J].Nature.1982,300(5893):611-615
[10]程炜中,刘应伯,余其兴.转基因动物的遗传修饰与应用[J].遗传.1995,17(2):42-48
[11]Tong TC,Wu SC.Nuclear Transplantation in Fishes[J].Scientia Sinica.1963,14(8):1244-1247
[12]钟红梅,刘越华,徐海山.转基因动物研究进展及应用[J].西南民族学院学报.2000,28(3):334-337
[13]崔文涛,单同领,李奎.转基因猪的研究现状及应用前景[J].中国畜牧兽医.2007,34(4):58-61
[14]Jun Sasaki,Sachihiro Youoku,Kazuo Tamamushi,Akio Ito.Development of microchannel device for automated microinjection[J].SPIE.2005,5718:186-196
[15]殷红,杨淑慎,郝建国.细胞工程[M].第一版.北京:化学工业出版社,2006
[16]Gurdon,J.B.Nuclear transplantation and the control of gene activity in animal development[J].Proc.R.Soc.Lond.Ser.B.1970.176:303-314
[17]张惠展.基因工程[M].第1版.上海:华东理工大学出版社,2005
[18]Gordon J W,Scangos G A,Plotkin D J,et al.Genetic transformation of mouse embryo by microinjection of purified DNA[J].Proc Natl Acad Sci USA.1980,77:7280-7384
[19]Brinster RL,Chen HY,Trumbauer M,Senear A.W,Warren R,Palmiter R.D.Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs[J].Proc.of SPIE.1982,27:223-231
[20]Kiyoshi Taninaka,A.Yabuki,A.Ito,T.Harada.Precise Microinjection into Living Cells by Summation of Fluorescence Intensity[J].Proc.of SPIE.2000,Vol6441:64410Z-1-64410Z-10
[21]Ali Eroglu,Joel A.Lawitts,Mehmet Toner,Thomas L.Toth.Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development[J].Cryobiology.2003,46:121-134
[22]Kim JH,Jung-Ha Hs,Lee HT et al.Development of positive method for male stem cell-mediated gene transfer in mouse and pig[J].Mol Reprod dev.2005,48..153-161
[23]科技导报编辑部.2004年中国重大科学、技术与工程进展[J].科技导报,2005,23(2):58—62
[24]章维一,侯丽雅.微流体数字化的科学与技术问题(Ⅰ):概念、方法和效果[J].科技导报,2005,23(8):4-9
[25]章维一,侯丽雅.微流体数字化的科学与技术问题(Ⅱ):物质数字化及物质能量信息统一数字化概念研究[J].科技导报,2006,24(3):41-47
[26]章维一,侯丽雅.影响流体流动的方法及其装置和应用[P].中国:ZL 03152948.8,2006年5月24日授权公告
[27]章维一,侯丽雅.微系统及其商品化[J].中国机械工程.2000,3:348-352
[28]王承遇.仪器玻璃的成分和原料处理[M].第一版.北京:轻工业出版社,1960
[29]初相臣.我国硼硅酸盐玻璃的应用与发展前景研究[J].中国科技信息.2005,13:67-70
[30]万军鹏,程金树,汤李缨.浅谈硼硅酸盐玻璃的应用现状和发展趋势[J].全国性建材科技期刊.2004,5:21-25
[31]徐伟.石英玻璃管拉制过程的微机检测与控制[J].数采与监测.2003,3:33-35
[32]马育芳,吕自力,王小宁等.微操作针的制作[J].黑龙江动物繁殖.2004,12(1):46-47
[33]王文亭,桑润滋.哺乳动物胚胎细胞核移植微操作用针的制备与应用[J].黑龙江动物繁殖.2006,14(2):17-19
[34]廉莉,李光鹏,廉颍等.实验用微操作用针的制备与应用[J].生殖与避孕.2003,23(1):56-58
[35]宁佳,姚爱华,付海罗等.硼硅酸盐生物玻璃的生物活性测试及其细胞相容性[J].中国组织工程研究与临床康复.2007,2(35):7041—704
[36]王承遇,陶瑛.玻璃表面处理技术[M].第2版.北京:化学工业出版社,2004
[37]王福荣.生物工程分析与检验[M].第1版.北京:中国轻工业出版社,2005
[38]穆莉莉,章维一,侯丽雅等.疏水化工艺在数字化超微量注射中的应用[J].化工学报.2004,55(11):1773-1776
[39]Sun Yu,Bradley J.Nelson B.J.Microrobotic cell injection[J].Proceedings-IEEE International Conference on Robotics and Automation.2001,5(1)620-625
[40]邱晨曦.数字化量可控细胞微注射仪实验研究[D].南京:南京理工大学,2005
[41]穆莉莉.数字化细胞注射中的改性与检测技术[D].安徽:安徽理工大学.2005
[42]张灵,章云,杨宜民等.转基因微注射系统中注射量的控制技术[J].中国机械工程.2003,14(24):2146-2148
[43]Ron C.Hogg,Florence Bandelier,Audrey Benoit et al.An automated system for intracellular and intranuclear injection[J].Journal of Neuroscience Methods.2008,169:65-75
[44]刘天军,侯丽雅,章维一,任志俊.细胞注射微针的流动特性研究[J].中国机械工程.2004,15(8):731-734
[45]李智,俞生等.转基因微注射的剂量控制与效果分析[J].中国实验动物学杂志.1998,8(2):65-69
[46]Ernst L K,et al.Theriogenology[J].Journal of Neuroscience Methods.1991,35:1257-1268
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