中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (50): 8151-8155.doi: 10.3969/j.issn.2095-4344.2014.50.020

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

超声微泡介导pEGFP-N1转染大鼠牙囊细胞：细胞生物学性质相对稳定

冉  玲1，李晓倩2，蒋欣益3，邓  锋1，宋锦璘1，曹  礼1   

  1. 1重庆医科大学附属口腔医院，口腔疾病与生物医学重庆市重点实验室，重庆市  401147；2重庆三峡中心医院口腔分院，重庆市  404199；3四川省绵阳市中心医院口腔科，四川省绵阳市  621000
  • 收稿日期:2014-11-18 出版日期:2014-12-03 发布日期:2014-12-03
  • 通讯作者: 宋锦璘，博士，教授，主任医师，硕士生导师，重庆医科大学附属口腔医院，口腔疾病与生物医学重庆市重点实验室，重庆市 401147
  • 作者简介:冉玲，女，1985年生，四川省内江市人，汉族，重庆医科大学在读硕士，主要从事牙周组织工程再生研究。
  • 基金资助:

    国家自然科学基金资助项目(30870754)；重庆市教委第二批优秀专业技术人才资助项目(渝教人2010[72])

pEGFP-N1 transfection of rat dental follicle cells under ultrasound-mediated lipid microbubble: transfected cells have a relatively stable biological property

Ran Ling1, Li Xiao-qian2, Jiang Xin-yi3, Deng Feng1, Song Jin-lin1, Cao Li1   

  1. 1Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing 401147, China; 2the Stomatological Hospital of Chongqing Three Gorges Central Hospital, Chongqing 404199, China; 3Department of Stomatology, Mianyang Central Hospital, Mianyang 621000, Sichuan Province, China
  • Received:2014-11-18 Online:2014-12-03 Published:2014-12-03
  • Contact: Song Jin-lin, M.D., Professor, Chief physician, Master’s supervisor, Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing 401147, China
  • About author:Ran Ling, Studying for master’s degree, Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing 401147, China
  • Supported by:

    the National Natural Science Foundation of China, No. 30870754; the Second Batch of Outstanding Technical Personnel of Chongqing Municipal Education Commission, No. 2010[72]

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摘要：

背景：利用超声波和微泡对比剂相互作用，产生空化效应和机械效应，破坏细胞膜的完整性，产生暂时性、可逆性的小孔，增加细胞膜的通透性，增强微泡载体对基因的转移，提高基因转染率。
目的：探讨在超声波辐照下微泡对比剂介导pEGFP-N1质粒转染SD大鼠乳鼠牙囊细胞的效率及安全性。
方法：体外原代培养新生SD大鼠牙囊细胞并传至第4代，在不同条件下采用pEGFP-N1质粒转染乳鼠牙囊细胞。以不同的超声辐照时间(15，30，45，60 s)和辐照强度(0.5，1 W/cm2)两两组合进行辐照，筛选较高转染效率的参数组合并应用于后续实验。实验分组为质粒组、微泡+质粒组、超声+质粒组、超声+微泡+质粒组和脂质体+质粒组。转染48 h后倒置荧光显微镜观察pEGFP表达，MTT法检测转染后的乳鼠牙囊细胞增殖抑制率。
结果与结论：超声强度为0.5 W/cm2且辐照时间为30 s时转染率明显高于其他超声参数组合。该条件下超声微泡介导pEGFP-N1质粒对乳鼠牙囊细胞的转染率高于传统脂质体介导的转染率，且对细胞活力无明显影响。提示超声微泡能安全、高效介导pEGFP-N1质粒转染大鼠牙囊细胞，其细胞生物学性质相对稳定，可为牙周组织工程提供一种较理想的基因转染方法。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

关键词: 干细胞, 培养, 超声辐照, 微泡, 大鼠牙囊细胞, 基因转染

Abstract:

BACKGROUND: With the interaction of ultrasound and microbubbles, cavitation and mechanical effects undermine the integrity of the cell membrane, resulting in temporary and reversible holes, increasing the permeability of cell membranes, enhancing gene transfer and improving gene transfection efficiency.
OBJECTIVE: To investigate the efficiency and safety of rat dental follicle cells transfected with pEGFP-N1 plasmid mediated by microbubble under ultrasonic irradiation.
METHODS: The primary dental follicle cells from newborn rats were cultured in vitro and passaged to the 4th generation. Under different conditions, pEGFP-N1 was used to transfect rat dental follicle cells. By combining the ultrasonic intensity (0.5, 1 W/cm2) with the irradiation time (15, 30, 45, 60 seconds), we got the best conditions of ultrasonic irradiation for the next experiment. There were five groups: plasmid, microbubble+plasmid, ultrasound+plasmid, ultrasound+microbubble+plasmid, and liposomes+plasmid groups. The expression of pEGFP was observed by inverted fluorescence microscope 48 hours after transfection, and meanwhile, the proliferation inhibition rate of rat dental follicle cells was determined by MTT method. 
RESULTS AND CONCLUSION: Under the 0.5 W/cm2 ultrasound for 30 seconds, the transfection efficiency was obviously higher than that under the other combinations. Under the above-mentioned condition, the transfection efficiency of rat dental follicle cells with pEGFP-N1 plasmid was higher than that mediated by the traditional liposome, and the cell viability had no obvious changes. Under suitable conditions, ultrasound microbubble technology can safely and effecitively mediate the transfection of rat dental follicle cells with pEGFP-N1 plasmid, and transfected cells also have a stable biological property as normal dental follicle cells. Therefore, ultrasound microbubble technology can provide an ideal method of gene transfection in periodontal tissue engineering.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

Key words: dental sac, high-energy shock waves, genes, transfection

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