摘要
目的：探讨15dPGJ2和PTEN质粒转染对人乳腺癌细胞株MCF-7体外培养的抑制作用，为临床靶向和基因治疗提供基础试验依据。方法：取生长良好的乳腺癌MCF-7细胞株，按5．0×10^6／L接种96孔板，按析因试验设置试验组，分别给予过氧化物酶体增殖物受体d然激动剂15dPGJ2，用野生型抑癌基因PTEN真核表达载体pcDNA3．0-PTEN质粒转染（脂质体转染法）MCF-7乳腺癌细胞株。采用MTT比色法观察15dPGJ2和野生型PTEN质粒转染对体外培养的MCF-7细胞系生长的抑制作用；用倒置光学显微镜观察MCF-7细胞用药前后的形态学变化；取生长良好对数生长期的MCF-7乳腺癌细胞株，调整细胞浓度至2．0×10^7／L接种于6孔板，设置3个复孔。连续培养3d；待细胞生长超过40％时，用15dPGJ240μmol／L，pcDNA3．0-PTEN质粒2μg／ml转染后连续培养3d常规消化收集细胞，离心，70％酒精固定，应用流式细胞仪检测凋亡细胞和细胞周期变化。结杲：15dPGJ2和PTEN质粒转染能有效的抑制体外培养的乳腺癌细胞株MCF-7的生长（P〈0．05），两者联合使用抑制效果更好（P〈0．05）。pcDNA3．0-PTEN质粒转染、15dPGJ240／μmol／L和两者联合干预在MCF-7乳腺癌细胞株连续72h时即达到有效抑制率，联合处理组处理MCF-7乳腺癌细胞株细胞生长抑制率最大（P〈0．05）。pcDNA3．0-PTEN质粒转染72h能将60．6％的MCF-7乳腺癌细胞阻滞在G0-1期（DNA合成前期）周期阻滞效果最为明显（P〈0．05）。与对照组相比pcDNA3．0-PTEN质粒转染、15d-PGJ2干预及联合用药均不能有效的诱导乳腺癌细胞MCF-7凋亡（P〈0．05））。联合用药细胞凋亡率低于单用15d-PGJ2（P〈0．05），pcDNA3．0-PTEN质粒转染可降低MCF-7乳腺癌细胞的凋亡率（P〈0．05）。结论：体外培养抑制试验证明pcDNA3．0-PTEN质粒转染、15dPGJ2 40μmol／L能有效的抑制乳腺癌细胞株MCF-7的生长，pcDNA3．0-PTEN质粒转染能抑制MCF-7乳腺癌细胞的恶性表型，pcDNA3．0-PTEN质粒转染抑制MCF-7乳腺癌生长的机制是周期阻滞，联合15d-PGJ2不增加周期阻滞作用。
Objective： To discuss MCF-7 in vitro developing resistant effect after 15dPGJ2 and pcDNA3.0- PTEN plasmid transfection interfered. In order to offer basical experimental data for clinical target and genetic therapy. Methods： We took some well developed MCF-7 cell line inoculated them in 96 well tissue culture plate by 5. 0 × 10^6/L. According factorial experimental design arrange groups. Experimental groups were given peroxisome proliferators-activated receptor）,crude activator 15dPGJ2 and wild type eukaryotic expression plasmid pcDNA3. 0-PTEN transfection MCF-7 breast cancer cell line by liposome transfection. The in vitro developing resistant function by 15dPGJ2 and wild type eukaryotic expression plasmid pcDNA3.0-PTEN transfection was observed by MTT. The morphological change was observed by converted optics microscope. Some well developed MCF-7 cell lines were inoculated them in 6 well tissue culture plate by 2.0× 10^7/L, repeated for 3 wells, cultured them 72 hours. When cell growth over 40%,interfering them by 15dPGJ2 40 μmol/L, pcDNA3. 0-PTEN plasmid 2 μg/ml transfection, developing them 72 hours, then digesting ,collecting and centrifugating the MCF-7 cell, then put them into 70% elthanol. Before testing, washing the under interfered MCF-7 cell by PBS buffer three times , then, suspended them by linkage buffer, after that, the cell apoptosis rate and cell generation cycle by flow cytometry were tested. Results. 15dPGJ2 and pcDNA3.0-PTEN plasmid transfection could resistant the MCF-7 growth in vitro effectively （P 〈0.05）. It is better by combined use （P 〈0.05）. It arrived efficient resistant rate within 72 hours by given pcDNA3.0-PTEN plasmid transfection, 15dPGJ2 40μmol/L and given them together on MCF-7 cell line. By combined treating with pcDNA3.0-PTEN plasmid transfeetion and 15dPGJ2 40μmol/L together,it will get the biggest resistant rate （P 〈0.05）, pcDNA3.0 -PTEN plasmid transfection 72 hours could block 60.6% MCF-7 cell in G0-1 stage （DNA pre-synthesis phase）. The cycle block effect much more isobviously than the other group （P 〈0.05）. Compared with control group pcDNA3.0- PTEN plasmid transfection, 15d-PGJ2 interfered and combined interfered all could not induce the MCF-7 cell apoptosis （P 〈0.05）. Combined interfere the cell apoptosis rate was lower than only use 15d-PGJ2 （P 〈0.05）. pcDNA3.0-PTEN plasmid transfection could lower the apoptosis rate of MCF-7 developing （P 〈0.05）. Conclusions： In vitro developing resistant experimentation shows that pcDNA3. 0-PTEN plasmid transfection, 15dPGJ2 40 pmol/L interfere can efficiently resistant the growth of MCF-7 human breast cancer cell line in vitro developing, pcDNA3.0-PTEN plasmid transfection can resistant the malignance behavior of MCF-7 human breast cancer cell. The mechanism of pcDNA3. 0-PTEN plasmid transfection resistant the growth of MCF-7 breast cancer cell is cell cycle block,and combined with 15d-PGJ2 cannot in- crease the cycle block function by pcDNA3.0-PTEN plasmid transfection.
出处
《新疆医科大学学报》
CAS
2009年第7期833-838,共6页
Journal of Xinjiang Medical University
基金
新疆少数民族科技人才特殊培养计划科研项目(编号:200823114)
新疆维吾尔自治区教育厅高校青年教师启动基金项目(编号:XJEDU2006525)
