国际泌尿系统杂志
國際泌尿繫統雜誌
국제비뇨계통잡지
INTERNATIONAL JOURNAL OF UROLOGY AND NEPHROLOGY
2010年
2期
148-153
,共6页
前列腺肿瘤%低氧
前列腺腫瘤%低氧
전렬선종류%저양
Prostatic Neoplasms%Anoxia
目的 构建2种抑制HIF-1α的siRNA表达载体.方法 根据Gen Bank中HIF-1α序列信息并结合2种pSUPER载体的共同的酶切位点结构.化学合成2对编码短发夹RNA序列的靶向HIF-1α基因的寡核苷酸链,各60个碱基、退火、克隆到经Bgl Ⅰ、Hind Ⅲ双酶切的2种pSUPER载体上,获得的2种重组RNAi质粒转化感受态大肠杆菌.挑选阳性克隆,抽取重组质粒,EcoR Ⅰ、HindⅢ双酶切电泳、测序鉴定,培养前列腺癌PC-3细胞通过Western blot检测2种重组BNAi载体干扰效果.结果 将重组构建的2种pSUPER质粒经双酶切电泳分析及插入基因片段进行序列分析,目的 基因片段成功插人到设计位点,并且序列完全一致.2种重组质粒可显著降低前列腺癌PC-3细胞蛋白的表达.结论 2种重组RNAi载体的成功构建,为进一步研究HIF-1α在前列腺癌发病机理及增殖、转移中的功能奠定了基础,同时可以用于其在其他肿瘤的功能研究.
目的 構建2種抑製HIF-1α的siRNA錶達載體.方法 根據Gen Bank中HIF-1α序列信息併結閤2種pSUPER載體的共同的酶切位點結構.化學閤成2對編碼短髮夾RNA序列的靶嚮HIF-1α基因的寡覈苷痠鏈,各60箇堿基、退火、剋隆到經Bgl Ⅰ、Hind Ⅲ雙酶切的2種pSUPER載體上,穫得的2種重組RNAi質粒轉化感受態大腸桿菌.挑選暘性剋隆,抽取重組質粒,EcoR Ⅰ、HindⅢ雙酶切電泳、測序鑒定,培養前列腺癌PC-3細胞通過Western blot檢測2種重組BNAi載體榦擾效果.結果 將重組構建的2種pSUPER質粒經雙酶切電泳分析及插入基因片段進行序列分析,目的 基因片段成功插人到設計位點,併且序列完全一緻.2種重組質粒可顯著降低前列腺癌PC-3細胞蛋白的錶達.結論 2種重組RNAi載體的成功構建,為進一步研究HIF-1α在前列腺癌髮病機理及增殖、轉移中的功能奠定瞭基礎,同時可以用于其在其他腫瘤的功能研究.
목적 구건2충억제HIF-1α적siRNA표체재체.방법 근거Gen Bank중HIF-1α서렬신식병결합2충pSUPER재체적공동적매절위점결구.화학합성2대편마단발협RNA서렬적파향HIF-1α기인적과핵감산련,각60개감기、퇴화、극륭도경Bgl Ⅰ、Hind Ⅲ쌍매절적2충pSUPER재체상,획득적2충중조RNAi질립전화감수태대장간균.도선양성극륭,추취중조질립,EcoR Ⅰ、HindⅢ쌍매절전영、측서감정,배양전렬선암PC-3세포통과Western blot검측2충중조BNAi재체간우효과.결과 장중조구건적2충pSUPER질립경쌍매절전영분석급삽입기인편단진행서렬분석,목적 기인편단성공삽인도설계위점,병차서렬완전일치.2충중조질립가현저강저전렬선암PC-3세포단백적표체.결론 2충중조RNAi재체적성공구건,위진일보연구HIF-1α재전렬선암발병궤리급증식、전이중적공능전정료기출,동시가이용우기재기타종류적공능연구.
Objectives To construct expressing vector of two siRNA in order to inhibit HIF -1αactivity. Methods According to the H1F -1α sequence and the same constmcture of two pSUPER vectors, Sixty base -pair oligos for hairp in RNA expression which targeted HIF -1α gene were chemically synthesized and annealed. The two recombinant RNAi vectors were then transformed into competent Ecoli ,the positive clones were selected and re-combinant. The two pSUPER RNAi plnsmids were extracted. The two pSUPER RNAi plasmids were digested with EcoR Ⅰ and Hind ⅠⅢ, and loaded in agurose gel electrophoresis. The recombinant vectors were transfected into the Prostate Cancer PC -3 cells , and detechted the expression levels of HIF -1α by Western blot. Results Recom-binant the two pSUPER RNAi plasmids were identified by digestion with EcoR Ⅰ and Hind Ⅲ, and confirmed by se-quencing analysis, we knew the annealed oligos were inserted into the two pSUPER vectors,and the insertion se-quence were exactly correct. The levels of the whole Prostate Cancer PC -3 cells proteins extremely down -regulated by the two recombinant vectors. Conclusions The two pSUPEH HNAi systems have been constructed successful-ly. These will facilitate the studying of HIF -1α activity inhibition and function in the pathogenesis, infiltration, pro-liferation and transformation of the prostate cancer, meantime, they can help us to study the functions of HIF -1α in other carcinomas.
