目的探讨建立转座子载体过表达外源肿瘤坏死因子相关凋亡诱导配体(TRAIL)的人脐带来源间充质干细胞(hUC-MSCs)转基因细胞系及其应用于脑胶质瘤裸鼠模型的疗效。方法自主专门设计的PiggyBac转座子过表达系统,通过嘌呤霉素(puromycin)抗...目的探讨建立转座子载体过表达外源肿瘤坏死因子相关凋亡诱导配体(TRAIL)的人脐带来源间充质干细胞(hUC-MSCs)转基因细胞系及其应用于脑胶质瘤裸鼠模型的疗效。方法自主专门设计的PiggyBac转座子过表达系统,通过嘌呤霉素(puromycin)抗性筛选制备稳定表达TRAIL基因的靶向HER2型和非靶向型的2种转基因细胞。将带有萤火虫荧光素酶标记的脑胶质瘤细胞(U87MG-FLUC)接种于免疫缺陷性小鼠(BALB/c-nu/nu)颅内,接种剂量为1×10^(6)个/只,接种7 d后利用小动物活体成像仪对植瘤小鼠脑颅检测,确定颅内肿瘤的大小和位置,然后将胶质瘤裸鼠分为4组(n=8),分别注射2种表达TRAIL转基因hUC-MSCs(分别命名为target-TRAIL和untarget-TRAIL组),以及只注射非转基因hUC-MSCs(接种剂量均为1×10^(6)/只)或PBS(分别命名为WT-MSCs和PBS组,作为阴性对照),每周用小动物活体成像仪监测肿瘤信号的变化情况,约检测34周。结果2种转基因细胞经过6次传代扩增后能稳定表达TRAIL基因,流式检测:绿色荧光蛋白(GFP)阳性(共表达TRAIL基因)的细胞比例达到93%97%,且经过6次传代后均为MSC相关表面标志物CD34、CD45、HLA-DR阳性率<0.1%,CD90阳性率>99%,CD73阳性率>98%,CD105阳性率>60%。中位生存期(d):胶质瘤裸鼠经颅内注射untarget-TRAIL、target-TRAIL、WT-MSCs或PBS组分别为41 vs 39 vs 24 vs 23(P<0.05)。结论过表达TRAIL转基因的hUC-MSCs-TRAIL细胞明显延长移植了脑胶质瘤的裸鼠的生存时间。展开更多
The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PC...The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PCR. Results showed that 65.4% of the T-DNA was integrated in different locations of rice genome, and some T-DNA flanking sequences were located on certain chromosomes. A number of T-DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac x Ds and Ds x Ac were constructed. The excision frequency of Ds element trans-activated by Ac transposase was 22.7% in the F-2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to 'cut-paste' mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.展开更多
文摘目的探讨建立转座子载体过表达外源肿瘤坏死因子相关凋亡诱导配体(TRAIL)的人脐带来源间充质干细胞(hUC-MSCs)转基因细胞系及其应用于脑胶质瘤裸鼠模型的疗效。方法自主专门设计的PiggyBac转座子过表达系统,通过嘌呤霉素(puromycin)抗性筛选制备稳定表达TRAIL基因的靶向HER2型和非靶向型的2种转基因细胞。将带有萤火虫荧光素酶标记的脑胶质瘤细胞(U87MG-FLUC)接种于免疫缺陷性小鼠(BALB/c-nu/nu)颅内,接种剂量为1×10^(6)个/只,接种7 d后利用小动物活体成像仪对植瘤小鼠脑颅检测,确定颅内肿瘤的大小和位置,然后将胶质瘤裸鼠分为4组(n=8),分别注射2种表达TRAIL转基因hUC-MSCs(分别命名为target-TRAIL和untarget-TRAIL组),以及只注射非转基因hUC-MSCs(接种剂量均为1×10^(6)/只)或PBS(分别命名为WT-MSCs和PBS组,作为阴性对照),每周用小动物活体成像仪监测肿瘤信号的变化情况,约检测34周。结果2种转基因细胞经过6次传代扩增后能稳定表达TRAIL基因,流式检测:绿色荧光蛋白(GFP)阳性(共表达TRAIL基因)的细胞比例达到93%97%,且经过6次传代后均为MSC相关表面标志物CD34、CD45、HLA-DR阳性率<0.1%,CD90阳性率>99%,CD73阳性率>98%,CD105阳性率>60%。中位生存期(d):胶质瘤裸鼠经颅内注射untarget-TRAIL、target-TRAIL、WT-MSCs或PBS组分别为41 vs 39 vs 24 vs 23(P<0.05)。结论过表达TRAIL转基因的hUC-MSCs-TRAIL细胞明显延长移植了脑胶质瘤的裸鼠的生存时间。
文摘The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PCR. Results showed that 65.4% of the T-DNA was integrated in different locations of rice genome, and some T-DNA flanking sequences were located on certain chromosomes. A number of T-DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac x Ds and Ds x Ac were constructed. The excision frequency of Ds element trans-activated by Ac transposase was 22.7% in the F-2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to 'cut-paste' mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.
