摘要
目的 研究结缔组织生长因子 (connectivetissuegrowthfactor,CTGF)的反义寡核苷酸 (antisenseoligonucleotide,AS)转染人肾小管上皮细胞 (humanrenaltubularepithelialcells,HKC)后对转化生长因子 β1 (TGFβ1 )诱导CTGF表达的抑制作用。方法 采用脂质体转染试剂DOTAP ,包裹针对CTGF的反义寡核苷酸转染HKC。用MTT比色法检测DOTAP对细胞增殖的影响 ,用荧光显微镜和荧光分光光度计检测 5′异硫氰酸荧光素 (5′FITC)标记的AS在细胞内的分布 ,用RT PCR和免疫组化分别检测TGFβ1 和AS对CTGFmRNA和蛋白表达的影响。结果 MTT比色 :不同浓度 (0、3.3、6 .7、1 0 μl/ml)的DOTAP对细胞增殖的影响无明显差异 (P >0 .0 5 )。荧光检测 :DOTAP能高效地介导AS转染入HKC。RT PCR和免疫组化 :浓度为 4 0 μg/ml的TGFβ1 能诱导HKC表达CTGFmRNA和蛋白 ,同时加入DOTAP AS复合物后 (DOTAP浓度为 6 .7μl/ml,AS浓度为 1 μg/ml) ,CTGFmRNA和蛋白的表达明显下降 (P <0 .0 1 )。结论 DOTAP能高效低毒地把CTGF反义寡核苷酸转染入HKC ,进而能有效地封闭TGFβ1
Objective To investigate the connective tissue growth factor (CTGF) antisense oligonucleotide(AS) suppresses the induction of CTGF by transforming growth factor β 1 (TGFβ 1) in human renal tubular epithelial cells(HKC) .Methods AS conjugated with 5′ fluorescent isothiocyanate(5′FITC) encapsulated with or without cationic liposome DOTAP were transfected into HKC. The influence of DOTAP to HKC proliferation was investigated by MTT method,the intracellular distribution of AS conjugated with 5′FITE was investigated by fluorescence microscopy and fluorescence spectrophotometer. The influence of TGFβ 1 and AS to the expression of CTGFmRNA and protein was investigated by immunohistochemistry and RT PCR assays. Results MTT method:the influence to the HKC proliferation by DOTAP(3.3μl/ml,6.7μl/ml,10μl/ml)have no significant diversity( P >0.05). Fluorescence investigation: DOTAP(6.7μl/ml) can promote the transfection of AS into HKC efficiently. Immunohistochemistry and RT PCR assays: TGFβ 1(40ug/ml) can induce, but DOTAP AS (include DOTAP 6.7μl/ml, AS 1μg/ml) can inhibit the CTGFmRNA and protein expression of HKC. Conclusions AS of CTGF can be transfected into HKC mediated by DOTAP by a high efficiency and low toxicity way, and inhibit the expression of CTGF induced by TGFβ 1.
出处
《重庆医学》
CAS
CSCD
2004年第5期732-734,共3页
Chongqing medicine
关键词
脂质体
结缔组织生长因子
反义寡核苷酸
liposome
connective tissue growth factor
antisense oligonucleotide
