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・ 研究原著 ・ ??щ???:

10002 2790 (2007)

112 10142

04 Galecti n2

3 shRNA??????????????δ?? lovo?????? 程变巧

1 ,姜泊1,包 杰2(南方医科大学附属南方医院 :

1 消化病研究所 ,

2 检验医学中心 ,广东 广州 510515) ????:

20062 122 13;

????:

20072 012

25 ????:姜泊.

Tel: (020)

61641541 Email: drjiang@163. com ????:程变巧. 博士生 (导师姜 泊),讲师. Tel: (020)

61365653 Email: nyd0129@ tom. com Construction and expression of galectin2

3 shRNA recom binant vector and its effect on proliferation rate of colorectal cancer lovo cells CHENG B ian2 Q iao

1 , J IANG B o

1 , BAO J ie

2 1 Institute of D igestive D iseases,

2 Center of Laboratory Medicine, Nanfang Hosp ital, Southern Medical University, Guangzhou 510515, China 【Abstract】A I M : To construct the eukaryotic vector exp ressing shRNA of galectin2

3 and study its effect on the proliferation rate in lovo cells . M ETHOD S: Lovo cells transferred with empty PRNAT2U6.

1 or nothing were regarded as negative or positive control groups, respectively . Two different

140 bp shRNA targe2 ting the coding sequence of galectin2

3 were designed, and inserted to the p las m id vector PRNAT2 U6.

1 for getting PRNAT2 U6.

1 /ga2 lectin2

3 shRNA. The human colorectal cancer lovo cells were transferred by two PRNAT2 U6.

1 /galectin2

3 shRNA. The transfec2 tion efficiency was detected by i mmunofluorescence;

the expres2 sion levels of galectin2

3 mRNA and protein were detected by RT2 PCR and W estern B lot, respectively;

the proliferation rate was assayed byMTT assay . RESULTS: It was verified by restriction endonuclease digestion that the constructed vector expressing ga2 lectin2

3 shRNA was correct . The transfection efficiency was 62% at

72 h. The expression of galectin2

3 was significantly supp ressed in lovo cells transfected by two PRNAT2 U6.

1 /galectin2

3 shRNA compared with positive and negative groups on the levels ofmRNA and protern;

there were significant differences on t wo levels ( F = 2. 214, 148. 566, P = 0. 000, 0. 000) , but no difference between positive and negative group s (P = 0. 448, 0. 263). The lovo cells transfected with PRNAT2 U6.

1 /galectin2

3 shRNA had lower cell p roliferation rate ( F = 20. 830, P = 0.

000 vs before transfection;

F = 149. 710, P = 0. 000). There was significant difference as compared with positive and negative group s ( P = 0. 000, 0. 000) , but there was no difference between the latter t wo groups ( P >

0. 05). CO NCLUS I O N: The galectin2

3 shRNA vector has been successfully constructed. The expression levels of galectin2

3 mRNA and protein are significantly depressed. The dep ressed ex2 pression of galectin2

3 m ight decrease the p roliferation rate of lovo cells . 【Keywords】galectin2 3;

shRNA;

cell p roliferation }? ?~??: 构建 galectin2

3 shRNA真核表达载体 ,观察该 基因的抑制对大肠癌细胞系 lovo细胞增殖率的影响. ????: 以未转染 lovo细胞作为阳性对照组 ,转染空质粒的 lovo细胞 作为阴性对照组 ,将针对人 galectin2 3基因的不同部位设计的 长度约

140 bp的shRNA,插入到真核表达载体 PRNAT2 U6.

1 并转染人大肠癌细胞 lovo,双酶切鉴定重组载体 ,免疫荧光检 测转染效果 , RT2PCR和Western2 B lot检测其对该基因 mRNA 和蛋白水平的抑制 ,MTT测定干扰后对 lovo细胞增殖率的影 响. ????: 双酶切出一条约

120 bp的DNA片断 ,与插入片断 长度相符. PRNAT2 U6.