【作者】 李方；

【导师】 牛勃；

【作者基本信息】 山西医科大学 ， 生理学， 2010， 博士

【摘要】 目的1、构建靶向抑制Ndst1和Hs2st表达的RNA干扰载体2、观察抑制Ndst1和Hs2st表达对前列腺癌细胞系Myc-CaP生物学行为的影响3、初步探讨抑制Ndst1和Hs2st表达对前列腺癌细胞系Myc-CaP生物学行为影响的机制方法1、靶向抑制Ndst1和Hs2st表达的RNA干扰载体的构建、筛选和鉴定利用软件OligoEngine Workstation对Ndst1和Hs2st的mRNA序列进行分析,针对每个序列分别设计并合成3条备选干扰序列,与siRNA真核表达载体pSUPER.retro.neo+GFP进行重组,构建RNA干扰载体pSUPER-Ndst1和pSUPER-Hs2st。用干扰载体转染前列腺癌Myc-CaP细胞后,通过检测各转染组细胞中Ndst1和Hs2st的mRNA和蛋白表达情况,筛选出抑制效果最显著的干扰载体用于后续实验研究。2、靶向抑制Ndst1和Hs2st表达对前列腺癌细胞系Myc-CaP生物学行为影响的体外实验研究采用CCK-8法和细胞计数法绘制细胞生长曲线、软胶克隆形成实验和流式细胞仪检测细胞周期等实验方法,比较对照组和实验组Myc-CaP细胞的增殖能力；利用Transwell迁移实验、细胞划痕愈合实验和Transwell侵袭实验分别对对照组和实验组Myc-CaP细胞的迁移和侵袭能力进行观察；另外,采用Annexin V-FITC/PI双染法结合流式细胞仪检测了各转染组Myc-CaP细胞的凋亡。3、靶向抑制Ndst1和Hs2st表达对前列腺癌细胞系Myc-CaP生物学行为影响的体内实验研究将Ndst1和Hs2st敲低后的Myc-CaP细胞注射到裸鼠背部皮下,观察移植瘤的生成和生长情况。解剖出移植瘤组织,通过BrdU标记法检测瘤体细胞增殖情况,利用TUNEL法检测移植瘤细胞的凋亡情况。将Ndst1和Hs2st敲低后的Myc-CaP细胞用荧光染料染色,从尾静脉注射入裸鼠体内,3小时后解剖出肺组织进行冰冻切片,镜下观察短期肺转移情况。4、靶向抑制Ndst1和Hs2st表达对前列腺癌细胞系Myc-CaP生物学行为影响的机制初探将生长因子FGF-2与生物素结合后,加入Ndst1和Hs2st敲低后的Myc-CaP细胞中孵育,然后用R-藻红蛋白-抗生物素蛋白链菌素与生物素结合,用流式细胞仪检测FGF-2与Ndst1和Hs2st敲低后Myc-CaP细胞的结合能力。另外,我们还检测了FGF-2对Ndst1和Hs2st敲低前后的Myc-CaP细胞在生长、迁移和凋亡方面的作用变化。结果1、成功构建了靶向抑制Ndst1和Hs2st表达的RNA干扰载体：pSUPER-Ndst1和pSUPER-Hs2st,对Ndst1和Hs2st表达的抑制率分别为85.6%和80.2%。2、体外实验中,抑制Ndst1表达后的Myc-CaP细胞生长速度变慢；克隆形成能力降低46.2%迁移和侵袭能力分别降低30.0%和17.1%；凋亡率升高1.2倍。3、体外实验中,抑制Hs2st表达后的Myc-CaP细胞生长速度变慢；克隆形成能力降低81.2%；迁移和侵袭能力分别降低69.6%和69.5%。凋亡率升高2.9倍。4、裸鼠体内实验中,抑制Ndst1表达后的Myc-CaP细胞生长速度变慢,细胞增殖能力降低39.3%；短期肺转移能力降低40.8%；凋亡率升高1.6倍。5、裸鼠体内实验中,抑制Hs2st表达后的Myc-CaP细胞生长速度变慢,细胞增殖能力降低50.2%；短期肺转移能力降低65.6%；凋亡率升高5.9倍。6、抑制Ndst1或Hs2st表达后的Myc-CaP细胞与FGF-2的结合能力下降,分别为对照组Myc-CaP细胞的79.5%和38.8%。7、加入FGF-2 (20ng/ml)后,抑制Ndst1或Hs2st表达的Myc-CaP细胞生长速度加快,但明显比对照组慢；对照组、Ndst1组和Hs2st组Myc-CaP细胞迁移能力分别增加5.8、3.6和2.1倍；凋亡率分别下降了81.9%、51.4%和22.0%。结论1、抑制Ndst1或Hs2st表达能够降低Myc-CaP细胞在体内外的增殖、迁移和侵袭能力,促进其凋亡,其中抑制Hs2st表达对Myc-CaP细胞产生的影响更明显。2、抑制Ndst1或Hs2st表达后的Myc-CaP细胞对生长因子FGF-2的结合能力和反应能力下降,这可能是其生物学行为改变的机制之一。更多还原

【Abstract】 Objects1. To construct RNA interference vectors targeting Ndstl and Hs2st2. To observed effects of Ndst1 and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaP3. To investigate preliminarily the influence mechanism of Ndst1 and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPMethods1. Construction, screening and identification of RNA interference vectors targeting Ndst1 and Hs2stUsing OligoEngine Workstation 2.0 to analysis the mRNA sequences of Ndst1 and Hs2st, designed and synthesized three target sequences for each gene to recombined with the siRNA eukaryotic expression vector pSUPER.retro.neo+GFP, and obtain pSUPER-Ndst1 and pSUPER-Hs2st. After recombination interference vector was transfected into prostate cancer, Myc-Cap cells, we select the interference vector with the most significant inhibitory effect for follow-up experiments by detecting the mRNA and protein expression of Ndst1 and Hs2st in the transfected group cells2. In vitro effects of Ndstl and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPUsing drawing of the cell growth curve with CCK-8 method and cell counting method, soft colony forming assay and cell cycle detection by flow cytometry to detect cell proliferation; using Transwell chamber, cell scratch healing assay and Matrigel Invasion Chamber to detect migration and invasion capacity of Myc-CaP cells in the control group and experimental group; Using Annexin V-FITC/PI double staining method combined with flow cytometry to detect Myc-CaP cells apoptosis.3. In vivo effects of Ndst1 and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPInjected the Myc-CaP cells with Ndst1 and Hs2st suppression into nude mice subcutaneously, and observe the formation and growth of tumor. Detect tumor cell proliferation and apoptosis of transplanted tumor tissue by BrdU labeling and TUNEL method respectively. Injected the Myc-CaP cells stained with fluorochrome into nude mice through tail vein, and separate lung tissue for frozen section and observe of lung metastases under microscope4. Preliminary study on the influence mechanism of Ndstl and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPAdded biotin-FGF-2 into Ndstl and Hs2st knockdown Myc-CaP cells, Then used theR-phycoerythrin-streptavidin-biotin to combined with biotin, detected the binding capacity of FGF-2 with Ndstl and Hs2st knockdown Myc-CaP cells by flow cytometry. In addition, we examined effect of FGF-2 on Ndstl and Hs2st knockdown Myc-CaP cells in the growth, invasion and apoptosis.Results1. Constructed successfully RNA interference vectors, pSUPER-Ndstl and pSUPER-Hs2st, targeting Ndstl and Hs2st, inhibition rates were85.6% and 80.2% separately.2. In vitro studies:Ndstl suppression Myc-CaP cells growth slow down; colony-forming ability decreased by 46.2%; migration and invasive ability decreased by 30.0% and 17.1%; apoptosis rate increased 1.2 times.3. In vitro study:Hs2st suppression Myc-CaP cells growth slow down; colony-forming ability decreased by 81.2%; migration and invasive ability decreased by 69.6% and 69.5%; apoptosis rate increased 2.9 times.4. In nude mice:Ndstl suppression Myc-CaP cells growth slow down; proliferation rate reduced by 39.3%; capacity of short-term lung metastases by 40.8%; apoptosis rate increased 1.6 times.5. In nude mice:Hs2st suppression Myc-CaP cells growth slow down; proliferation rate reduced by50.2%; capacity of short-term lung metastases by 65.6%; apoptosis rate increased 5.9 times.6. Binding capacity of Ndstl and Hs2st suppression Myc-CaP cells with FGF-2 decreased by 79.5%和38.8% separately, compared with the control group.7. Adding the FGF-2 (20ng/ml), the growth of Ndstl and Hs2st suppression Myc-CaP cells accelerated, but obviously slower than the control group; the control group, Ndstl group and Hs2st group Myc-CaP cells migration capacity increased by 5.8,3.6 and 2.1 times; apoptosis rate decreased by 81.9%,51.4% and 22.0% respectively.Conclusions1. Suppression of Ndstl or Hs2st can inhibit Myc-CaP cells proliferation, migration and invasion and promote apoptosis, in which suppression of Hs2st has more pronounced effect.2. Changes in the biological behaviors of Ndstl or Hs2st suppression Myc-CaP cells are related to the binding capacity decline of FGF-2 with Myc-CaP cells.更多还原