摘要
目的 筛选经辐射诱导后小鼠肠上皮细胞的差异或特异表达基因。方法 通过基因库检索和计算机分析 ,设计出较通用的 5’端武断引物在基因库中其呈现几率要高出数倍到数十倍的 4条 5′端高同源mRNA差示引物 ,应用DDRT PCR ,测序凝胶电泳 ,放射自显影 ,分子杂交等方法 ,分析并筛选小鼠受 12Gyγ射线全身照射后 3h(R1组 )和 96h(R2组 )肠上皮细胞差异或特异表达基因。结果 5′端高同源性引物mRNA差异结果显示 ,与正常比较 ,小鼠全身 12Gy照射后不同时相的肠上皮细胞基因表达存在差异 ,从近 80 0 0条PCR扩增产物中 ,比较筛选出 12个差异表达基因片段。RNA blot杂交对 11个辐射损伤情况下差异表达基因片段进行了鉴定。R1S1和R1S2差异基因片段在辐射损伤后 3h的肠上皮细胞中呈特异性表达 ;R2S2和R2S5基因片段在辐射损伤后 96h的肠上皮细胞中呈特异性表达 ;R2S1、R2S6和R2S8基因片段是假阳性差异表达基因片段 ;而R2S1、R2S4及R2S8用RNA blot法检测不到表达。序列分析结果显示R1S1和R2S2在基因库中未见高同源的基因序列 ,与R1S1同源性最高 (60 0 0 % )的是一个源于大鼠输卵管特异性糖蛋白基因 ,R2S5与一种小鼠肌细胞钙离子信号传导分子有 67 2 1%的同源性 ;R2S2高度同源于鼠源性肿瘤诱导蛋白p3 2 (91 5 2 % )
Objective To screen the differentially expressed intestinal epithelial cells (IECs) derived from mice with mild intestinal type of radiation sickness by high homological primer DDRT PCR. Methods A set of 4 highly homologenic 5' primer was designed through computer analysis and Genbank searching, and used to replace the 20 5' arbitrary primers in the mRNA differential display. The higher display rate of mRNA was available after the modified DDRT PCR had been taken in comparison with the routine 5' arbitrary primer's display. By means of the modified DDRT PCR and comparison with normal control, 12 differentially expressed genes of IECs derived from mouse mild intestinal type of radiation sickness were isolated from 10 000 DDRT PCR products displaying on the sequencing gels. Eleven differentially expressed genes were identified with RNA blotting hybridization. Results The results showed that R1S1 and R1S2 were highly specifically expressed at the IEC's early damage phase (3 h) after 12 Gy γ ray radiation, and that R2S2 and R2S5 were specifically expressed in the IEC's regeneration phase (96 h) after 12 Gy γ ray radiation. R2S1, R2S6 and R2S8 were false differentially expressed genes because the expressions of R2S1, R2S4 and R2S8 were measured with RNA blotting hybridization. The highest score homological gene of R1S1 in the GenBank was a specific mouse oviduct glycoprotein. There was no homological gene to R1S2 in the GenBnak. R1S5 was 67.21% homological to a murine calcium signal molecule and R2S5 was highly homological (91.52%) to a tumor induced protein gene (p32). p32 which was high homological to R2S2 was a member of the tumor induced protein BRCA gene family. BRCA 2, one member of the BRCA gene family had also been found in our cDNA microarray experiment, was specifically expressed in the IECs of the regeneration phase after radiation injury. Conclusion The function of R2S2 and R2S5 may be associated with the repair of intestinal epithelium. BRCA gene family maybe plays a very important role in t
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2002年第6期705-708,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目 ( 39770 2 37)
国家教育部高等学校骨干教师资助计划资助项目
