基础研究 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-03-15; 12(3): 642-645
在线出版日期: 2004-03-15. doi: 10.11569/wcjd.v12.i3.642
原代培养大鼠肝细胞的基因转染
何勇, 周峻, 窦科峰, 陈勇
何勇, 窦科峰, 陈勇, 中国人民解放军第四军医大学西京医院肝胆外科 陕西省西安市 710032
周峻, 中国人民解放军第四军医大学秦都医院病理科 陕西省西安市 710032
何勇, 男, 1968-09-27生, 辽宁省海城市人, 汉族, 医学博士, 讲师, 主治医师.
基金项目: 国家自然科学基金资助, No. 30170927, No. 30070210.
通讯作者: 何勇, 710032, 陕西省西安市长乐西路127号, 中国人民解放军第四军医大学西京医院肝胆外科. heyong007@yahoo.com
电话: 029-83375259 传真: 029-83244986
收稿日期: 2003-03-08
修回日期: 2003-03-25
接受日期: 2003-04-05
在线出版日期: 2004-03-15

目的: 研究原代培养大鼠肝细胞的基因转染的方法.

方法: 采用胶原酶灌注法获取原代培养大鼠肝细胞. 利用脂质体转染法将含有绿色荧光蛋白(GFP)和Neo基因的真核细胞表达载体(pEGFP-N3)转染原代培养大鼠肝细胞. 用荧光显微镜观察和Neo基因原位杂交染色方法检测肝细胞内基因表达情况.

结果: 获取的原代大鼠肝细胞活细胞率达95%. 荧光显微镜下观察可见转染基因的细胞可发出绿色荧光, 原位杂交显示有Neo基因的表达.

结论: pEGFP-N3基因可转入大鼠肝细胞并获得表达, 可用于标记原代培养的大鼠肝细胞, 有利于研究肝细胞移植后移植的肝细胞在体内的分布及功能.

关键词: N/A
引文著录: 何勇, 周峻, 窦科峰, 陈勇. 原代培养大鼠肝细胞的基因转染. 世界华人消化杂志 2004; 12(3): 642-645
Gene transfection for rat primary cultured hepatocytes
Yong He, Jun Zhou, Ke-Feng Dou, Yong Chen
Yong He, Ke-Feng Dou, Yong Chen, Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
Jun Zhou, Department of Pathology, Qindu Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
Supported by: the National Natural Science Foundation of China, No. 30170927, No. 30070210.
Correspondence to: Dr. Yong He, 127 Changle West Road, Xi'an 710032, Shaanxi Province, China. heyong007@yahoo.com
Received: March 8, 2003
Revised: March 25, 2003
Accepted: April 5, 2003
Published online: March 15, 2004

AIM: To study the efficient and stable gene transfection method of rat primary cultured hepatocytes by liposome.

METHODS: Rat hepatocytes were isolated by collegenase perfusion, and the pEGFP-N3 plasmid containing GFP and Neo gene was transfected into rat primary hepatocyte with liposome. The expression of GFP and Neo was observed by fluorescence microscopy and in situ hybridization.

RESULTS: The rate of live hepatocytes obtained was 95%. Under fluorescence microscope, hepatocyte transfected with pEGFP-N3 plasmid showed green fluorescence. By using in situ hybridization method, expression of Neo gene was observed in hetpatocyte transfected with pEGFP-N3.

CONCLUSION: Rat hepatocyte can be transfected by pEGFP-N3. pEGFP-N3 expression vector and makes it easy to assess the expression of target gene in transfected hepatocyte, and it can be used for localization of transplantated hepatocyte.

Key Words: N/A
0 引言

我们在建立一个肝细胞原代培养系统的基础上, 转染携带Neo基因和GFP基因的真核细胞表达载体, 并观察标记基因在肝细胞内的表达.

1 材料和方法
1.1 材料

♂幼龄Sprage-Dawley大鼠(第四军医大学实验动物中心), 体质量180-220 g, 普通块料喂养, 随意饮水, 室温18-22 ℃, 每日光照12-14 h, 术前禁食12 h. Ⅳ型胶原酶、地塞米松、胰岛素及HGF为Sigma公司产品; RPMI1640培养基为Gibco公司产品. 参照Seglen (Exp Cell Res 1972; 74: 450)的方法进行肝细胞的分离纯化并作了部分改进. 大鼠用20 g/L戊巴比妥钠溶液腹腔注射麻醉(30 mg/kg), 同时腹腔内注射肝素钠溶液200 U. 消毒皮肤后, 取正中切口进入腹腔. 显露门静脉后在距肝门2 cm处插入18号导管, 导管与一次性输血器相接, 后者连接输液瓶, 先以30 mL/min的速度输注无钙镁的Hanks液(含EDTA 1 mmol/L)200 mL, 夹闭肝上下腔静脉, 在肝下下腔静脉插入16号导管放出积血积液, 再输注含0.5 g/L ℃型胶原酶的Hanks液100 mL, 输注速度为15 mL/min, 取下肝脏, 置于含4 ℃ Hanks液的平皿中, 去除肝包膜及血管, 钝性撕裂肝组织, 多层纱布过滤, 制成细胞悬液, 将此悬液以100目及200目不锈钢网过滤, 以500 r/min离心2 min, 弃上清, 以4 ℃ Hank液清洗并离心3次, 用4 g/L台盼蓝进行染色判断细胞活率, 用血细胞计数板计数细胞浓度并计算细胞产量. 将分离、纯化的肝细胞用合成培养基(含RPMI1640 80 mL, 新生小牛血清20 mL, HGF 5 g/L, 地塞米松10-7 mol/L, 牛胰岛素10-8 mol/L, 青霉素100 ku/L, 链霉素100 ku/L)稀释成0.5×109/L, 在37 ℃, 50 mL/L CO2条件下培养, 每24 h更换培养基1次, 每天在倒置相差显微镜下观察细胞生长情况.

1.2 方法

采用脂质体介导的基因转染法, 脂质体为Gibco公司的lipofectamine2000, 真核细胞表达质粒pEGFP-N3含有Neo基因和GFP基因两个标记基因. 取pEGFP-HGF质粒2 g, lipofectamine 12 L, 与适量无血清无抗生素的RPMI1640在室温下混合摇匀成转染液. 取在35 mm培养皿中(培养皿中铺有盖玻片)培养生长3 d的肝细胞, 吸去培养液, 先用无血清无抗生素的RPMI1640将待转染细胞洗2遍, 再加入转染液, 转染6 h后更换成新鲜的含200 mL/L小牛血清的RPMI1640培养液继续培养. 1次/d, 重复转染4次.基因表达情况的检测: (1)GFP蛋白表达的检测用PBS洗细胞铺片2次, 40 g/L多聚甲醛(PBS配制)室温固定30 min, PBS振洗, 在荧光显微镜下, 488 nm激发波长下观察, 表达GFP的细胞发绿色荧光. (2)Neo基因表达检测细胞铺片经3 mL/L Triton X-100/PBS, 0.2 mol/L HCl及蛋白酶K处理, 2 mL/L甘氨酸终止反应, 40mL/L PFA后固定, 梯度酒精脱水干燥. 滴加热变性后经地高辛标记的Neo探针, 42℃杂交过夜, 分别用2×SSC, 1×SSC, 0.5×SSC充分漂洗, 20 ml/L正常羊血清封闭, 滴加标记碱性磷酸酶抗地高辛抗体(anti-Dig-AP, 1: 100), 37 ℃温育2 h, Buffer I, Buffer Ⅲ充分漂洗后NBT/BCIP暗处显色24 h, TE终止反应, 梯度酒精脱水, 二甲苯透明封片.

2 结果
2.1 肝细胞的分离和培养

采用胶原酶灌注法, 每只大鼠(体质量180-220 g)的肝脏大约可分离出2×108个肝细胞, 活细胞率在95%以上. 新鲜分离的肝细胞在显微镜下呈单个分散状态, 为具有立体感的圆球形, 晶莹透亮, 体积上大小一致, 整个细胞透明无色. 培养3 h后, 绝大多数细胞贴壁生长, 培养24 h后细胞由球形变扁变薄呈多边形, 胞质均匀, 核清晰, 以双核细胞为主, 并以多个细胞聚集成簇排列, 3 d后细胞逐渐生长融合形成单层膜状.

2.2 基因转染后标记基因的表达

经Dig标记的Neo探针原位杂交结果显示, 在转染基因的细胞中均有较强的紫蓝色颗粒样Neo基因阳性杂交信号(图1), 而对照组肝细胞中无Neo基因表达杂交信号, 即为阴性表达. 经基因转染的肝细胞爬片在荧光显微镜下观察, 可发现细胞表达GFP, 表现为细胞发出明亮的绿色荧光, 这些细胞多成对出现, 或数个发光细胞聚在一起(图2). 此外, 还可见到个别发出弱绿色荧光的细胞. 未转染基因的肝细胞中未见绿色荧光.

图1
图1 在基因转染的肝细胞中有紫蓝色颗粒样Neo基因阳性信号(原位杂交100×).
图2
图2 基因转染的肝细胞中可见绿色荧光(荧光显微镜100×).
3 讨论

目前有多种方法可用于分离肝细胞[1-10], 直接法是通过挤压、剪碎和振荡等机械手段, 使肝细胞从肝组织脱落而获得, 操作时间较长, 加之机械损伤, 其肝细胞收获量和细胞成活率均远远低于Seglen灌注法. 我们采用经过改进的灌注法获得的肝细胞纯度达95%以上, 减少了非实质细胞的污染, 有利于细胞在体外培养并进行各种研究.

肝细胞移植是替代肝移植治疗各种原因引起肝功能衰竭的疗法[11-18]. 肝细胞可经多途径注射至体内, 在合适的环境下植入肝细胞表达肝脏特异性功能. 由于缺乏移植的肝细胞标记, 因此无法判断移植后肝细胞在体内的分布, 无法确定植入后肝细胞功能与疗效的关系. 随着分子生物学技术的不断发展, 细胞标记基因的产生使肝细胞移植可采用基因标记肝细胞, 以便于追踪研究移植细胞的生物学特性. 肝脏活体外基因治疗的基本方法是首先获得原代肝细胞, 进行体外培养, 并选用靶基因进行体外转染, 再将表达靶基因的肝细胞移植到受体体内[19-26]. 另外肝细胞移植也需要带有标记基因的肝细胞以研究肝细胞在体内的分布情况等. 我们通过脂质体介导的基因转染法, 将含有Neo基因和GFP基因两个标记基因的真核细胞表达载体转导入原代培养的肝细胞. 通过Neo基因原位杂交、荧光显微镜观察的方法证实标记基因都获得了表达, 这也充分证实基因转染获得了成功. 我们所选择的pEGFP-N3载体中带有绿色荧光蛋白(GFP)序列, GFP可用与活体、原位、即时的检测基因表达及蛋白定位. GFP非常稳定, 不依赖于物种, 对活细胞没有伤害性. 而且GFP荧光在甲醛固定的细胞中能维持很长时间, 很适于和其他荧光标记进行双标实验. 这种新型的报告分子在暴露于紫外线后即可发出明亮的绿色荧光, 因而, pEGFP-N3常被用于检测细胞中GFP融合蛋白的表达情况及蛋白定位[27-40]. 本研究表明脂质体介导的基因转染法可用于体外肝细胞转染, 而可作为标记基因标记移植的肝细胞, 有利于研究肝细胞移植后移植的细胞在体内的分布、寿命和功能等情况, 为肝细胞移植[41-42]治疗急性肝衰[43-49]提供了实验基础.

编辑: N/A

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