中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (30): 4879-4883.doi: 10.12307/2022.769

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

子宫内电穿孔转染胎鼠脑室管膜下区神经干细胞的条件优化

邹明明1，倪  莉2，周立宇2，李  迪3，赛吉拉夫2，韦善文2，张鹏飞1   

  1. 1 解放军总医院第一医学中心神经外科医学部派驻第七医学中心神经外科，北京市   100700；2苏州大学附属第一医院骨科，江苏省苏州市   215000；3 苏州大学附属张家港市第一人民医院，江苏省张家港市   215600
  • 收稿日期:2021-06-22 接受日期:2021-08-23 出版日期:2022-10-28 发布日期:2022-03-29
  • 通讯作者: 张鹏飞，硕士，主治医师，解放军总医院第一医学中心神经外科医学部派驻第七医学中心神经外科，北京市 100700 韦善文，硕士，主管技师，苏州大学附属第一医院骨科，江苏省苏州市 215000
  • 作者简介:邹明明，女，1981年生，河北省承德市人，满族，2016年中国人民解放军第三军医大学毕业，博士，主治医师，主要从事神经发育和神经损伤后的再生研究。 倪莉，女，1983年生，江苏省苏州市人，汉族，2019年苏州大学毕业，博士，助理研究员，主要从事骨质疏松的防御和治疗及神经再生的研究。
  • 基金资助:
    国家自然科学基金面上项目(81571189)，项目负责人：赛吉拉夫；国家自然科学基金青年项目(81801238)，项目负责人：李迪

Optimization of conditions for intrauterine electroporation transfection in neural stem cells from the subependymal region of fetal mice

Zou Mingming1, Ni Li2, Zhou Liyu2, Li Di3, Saijilafu2, Wei Shanwen2, Zhang Pengfei1   

  1. 1Department of Neurosurgery, First Medical Center of Chinese PLA General Hospital, Beijing 100700, China; 2Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China; 3Affiliated Zhangjiagang Hospital of Soochow University, Zhangjiagang 215600, Jiangsu Province, China
  • Received:2021-06-22 Accepted:2021-08-23 Online:2022-10-28 Published:2022-03-29
  • Contact: Zhang Pengfei, Master, Attending physician, Department of Neurosurgery, First Medical Center of Chinese PLA General Hospital, Beijing 100700, China Wei Shanwen, Master, Technologist-in-charge, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China
  • About author:Zou Mingming, PhD, Attending physician, Department of Neurosurgery, First Medical Center of Chinese PLA General Hospital, Beijing 100700, China Ni Li, MD, Assistant researcher, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China Zou Mingming and Ni Li contributed equally to this article.
  • Supported by:
    National Natural Science Foundation of China (General Project), No. 81571189 (to Saijilafu); National Natural Science Foundation of China (Youth Project), No. 81801238 (to LD)

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摘要：

文题释义：
神经前体细胞：能够进行自我增殖和分化的多潜能细胞，可通过增殖维持所需的神经前体细胞数目，也可分化为神经元、星形胶质细胞和少突胶质细胞。
子宫内电转染技术：子宫内电穿孔是研究神经发育过程中细胞增殖、分化、迁移和成熟分子机制的一项重要技术。利用电脉冲在细胞膜上产生瞬时孔隙，快速和靶向地将物质导入到细胞中。电穿孔在体外研究中的使用由来已久，但科学的发展将它的使用范围扩展到了研究完整的器官，如在子宫内发育的小鼠胚胎器官。

背景：神经干细胞是一群可以自我增殖并具有多向分化潜能的细胞。子宫内电穿孔转染可将RNA或质粒瞬时转入活体小鼠神经干细胞内对目的基因进行干扰或过表达，是目前活体研究大脑皮质发育的最直接可靠的研究方法。
目的：以胚胎小鼠脑室管膜下区的神经干细胞为目标，以增强型绿色荧光蛋白作为报告基因，优化子宫内电穿孔转染的条件，为研究大脑皮质的发育提供更加有效的方法。
方法：将成年ICR小鼠以1只雄鼠和3只雌鼠的比例进行交配，晚上8点将小鼠合笼，第2天早晨8点查验雌鼠阴栓，将有阴栓的雌鼠计为怀孕0.5 d。待怀孕14 d(E14)时腹腔注射Ketamin(120 mg/kg)和Xylazine(10 mg/kg)麻醉孕鼠，剃掉孕鼠腹部毛发，打开腹腔，不剪开孕鼠子宫，用ECM803电穿孔转染仪和BTX电极在不同的电压下将pEx-4-eGFP质粒在电场的作用下瞬时转入胎鼠脑室管膜下区的神经干细胞内。转染3 d后取出转染小鼠脑组织进行冰冻切片，观察增强型绿色荧光蛋白在大脑皮质上的表达，统计绿色荧光蛋白阳性细胞数来评估转染效率。
结果与结论：①电转电压在38，40，42，45 V时小鼠存活及转染效果不同，38 V 电压对胎鼠的致死性较小，但是转染效率较低，在42 V电压下电转的小鼠可存活，且电转的脑组织中绿色荧光蛋白荧光强度高于40 V电压，但是45 V电压对小鼠的致死性较大。因此，42 V电压是一个较好的电转条件。②在电压42 V条件下，脉冲数由5个改为8个后绿色荧光蛋白阳性细胞更多，荧光更亮。③改良后的电转条件对转染成功的细胞生长无影响。

https://orcid.org/0000-0002-4382-3483 (邹明明) 

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

关键词: 子宫, 电穿孔, 转染, 神经干细胞, 电压, 脉冲, 转染效率

Abstract: BACKGROUND: Neural stem cells are a group of cells that can self-proliferate and have multidirectional differentiation potential. In utero electroporation, which is the most direct and reliable method to study the development of cerebral cortex in vivo, can transduce RNA or plasmids into neural stem cells of mice to interfere with or overexpress target genes.
OBJECTIVE: To optimize the conditions of in utero electroporation by targeting neural stem cells in the subependymal region of embryonic mice and using enhanced green fluorescent protein as the reporter gene, so as to provide an effective method for studying the development of cerebral cortex.
METHODS: Adult ICR mice were mated at the ratio of one male to three female mice. The mice were caged at 8 p.m., and female mice with vaginal embolus were tested at 8 a.m. the next morning. The female mice with vaginal embolus were counted as 0.5-day pregnancy. On embryonic day 14 (E14), the pregnant mice were intraperitoneally anesthetized with ketamin (120 mg/kg) and xylazine (10 mg/kg). After shaving off the hair of the pregnant mouse’s abdomen and opening the abdominal cavity without cutting the pregnant mouse’s uterus, pEx-4-eGFP plasmid was transfected into neural stem cells of the subependymal region of fetal mice at different voltages using ECM803 electroporation transfection instrument and BTX electrode. After 3 days of transfection, the transfected mouse brain tissue was removed and frozen sections were performed to observe the expression of enhanced green fluorescent protein fluorescence on the cerebral cortex. The number of enhanced green fluorescent protein-positive cells was counted to evaluate the transfection efficiency.
RESULTS AND CONCLUSION: (1) The survival and transfection effect of mice were different at 38, 40, 42, and 45 V. The mortality of fetal mice was less at 38 V, but the transfection efficiency was lower. Mice treated at 42 V survived and the fluorescence intensity of enhanced green fluorescent protein was higher in the brain tissue treated at 42 V than in the brain treated at 40 V. However, 45 V was more lethal to mice. Therefore, 42 V was a better transformation condition. (2) Under the condition of 42 V, enhanced green fluorescent protein positive cells were more and the fluorescence was brighter after the pulse was changed from 5 to 8. (3) The improved conditions had no effect on the growth of transfected cortical cells.

Key words: uterus, electroporation, transfection, neural stem cells, voltage, impulse, transfection efficiency

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