摘要
目的:对于人脂肪来源干细胞的应用,目前已开始进行临床前期体内移植实验,细胞标记与示踪是证明移植细胞转归和生成组织来源的关键问题。实验观察了带有增强型绿色荧光蛋白的基因质粒转染人脂肪来源干细胞的效果及体内示踪效应。方法:实验于2007-03/12在沈阳军区总医院整形美容外科中心实验室(省级)、沈阳军区呼吸中心实验室(国家级)和全军神经外科中心实验室(国家级)完成。①细胞来源与动物:脂肪组织取自1例腹壁整形术女性患者,对治疗及实验均签署知情同意书,实验经医院医学伦理委员会批准。清洁级雄性裸鼠8只,实验过程中对动物的处置符合动物伦理学标准。大肠杆菌JM109由本室保存。转染质粒pEGFP-N3为美国Clontech公司产品。②实验方法:无菌条件下切取患者脂肪组织,采用酶消化法体外分离培养脂肪来源干细胞,传至第3代时以2.5×107L-1密度接种,分别加入200,300,400,500,600,700,800,900mg/LG418,确定G418杀死所有细胞的最低浓度(C0)。采用脂质体法对人脂肪来源干细胞进行pEGFP-N3质粒转染,C0浓度G418筛选,计数阳性细胞率。培养5代后制备单个细胞悬液,浓度为8×1010L-1,植入到裸鼠背部皮下,采用活体化学发光和荧光成像系统观察大鼠荧光表达情况。结果:①体外培养的人脂肪来源干细胞贴壁后为成纤维细胞样,细胞生长活性良好,传代后生长速度加快。②当G418浓度为600mg/L时细胞全部死亡,故将此浓度设定为C0。③pEGFP-N3质粒-脂质体复合物转化脂肪来源干细胞24h后,绿色荧光蛋白阳性细胞率为(13.0±3.5)%,经C0浓度G418筛选后高达(65.0±5.4)%。④裸鼠背部皮下注射脂肪来源干细胞8周后,可见移植区周围弥漫分布绿色荧光区,提示植入细胞存活并广泛分布。结论:增强型绿色荧光蛋白质粒可以有效转染人脂肪来源干细胞,并在裸鼠体内呈现良好的示踪效果。
AIM： The human adipose-derived stem cells （ADSCs） have been applied for pre-clinical transplantation experiments in vivo. Cell labeling and tracing are crucial problems to observe the transplanted cells＇ outcome and determine the newly formed tissues＇ source. This study is designed to observe the effect of the enhanced green fluorescent protein （EGFP） plasmid transfection for ADSCs and cell tracing. METHODS： The experiment was implemented from March to December in 2007 in three laboratories： the laboratory of Plastic and Aesthetic Surgery Center （provincial degree） in the General Hospital of Shenyang Military Area Command of Chinese PLA, the laboratory of Shenyang Military Area Command＇s Respiratory Center （national degree）, and the laboratory of Military Neurosurgery Center （national degree）. （1） Cell source and animal： The adipose tissue was harvested from a female patient underwent abdominoplasty. A detailed informed-consent form was signed by the patient. The Clinical Trial and Ethics Committee of the Hospital approved the protocol. Eight SPF-degree nude male mice were treated according to the animal ethics standards. E.coli JM109 was preserved by the laboratory. The transfection plasmid pEGFP-N3 was produced by Clontech Company （USA）.（2） Methods： The adipose tissues were harvested under aseptic condition. ADSCs were separated and cultured by the method of the collagenase digestion. The cells at passage 3 were seeded with the concentration 2.5 ×10^7 L^-1 and G418 were added under the concentration of 200, 300, 400, 500, 600, 700, 800, 900 mg/L to determine the lowest concentration of G418 （Co） to kill all the ADSCs. Human ADSCs underwent liposome-mediated pEGFP-N3 plasmid transfection. The transfected cells were screened by G418 with Co concentration and the ratio of positive cells was counted. After 5 passages of culture, the screened cells under the concentration of 8× 10^10 L^-1 were transplanted into nude mice subcutaneously. I.C.E Chemi ＆ Fluo System was used for the observation of fluorescence expression. RESULTS： （1）ADSCs cultured in vitro were shaped as fibroblast-like cells and proliferated well after adhesion. After subculture, ADSCs proliferated more rapidly.（2）The ADSCs all came to death under the G418 concentration of 600 mg/L, so it was set as Co.（3） At 24 hours after the pEGFP-N3 plasmid-liposome composites were transfected into ADSCs, the rate of positive ratio for green fluorescent protein was （13.0±3.5）%. With the screening of G418 under the concentration of Co, the ratio of GFP^＋ cells reached to （65.0±5.4）%.（4）In the nude mice, the green fluorescence was seen diffusely around the injection area at 8 weeks after ADSCs was transplanted into the back skin, proving the ADSCs＇ survival and widely distribution. CONCLUSION： EGFP plasmid can be transfected into human ADSCs effectively, and present good effect of cell tracing in nude mice.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2258-2262,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
解放军沈阳军区总医院博士后基金(2005-2007)
辽宁省自然科学基金(20071041)~~
