• 1.南京醫(yī)科大學(xué)第一附屬醫(yī)院肝臟移植中心（南京210029）;;
  • 2.山東大學(xué)齊魯醫(yī)院（濟(jì)南250012）;;
  • 3.山東大學(xué)分子生物學(xué)實(shí)驗(yàn)中心（濟(jì)南250012）;

目的  用反轉(zhuǎn)錄PCR法，調(diào)取膽囊癌細(xì)胞內(nèi)的端粒酶RNA(hTR)基因，并針對其序列設(shè)計(jì)錘頭狀核酶切割基因序列，并將其構(gòu)建入真核表達(dá)載體pTriEx4內(nèi)。
方法  根據(jù)hTR cDNA序列，設(shè)計(jì)引物，經(jīng)RT-PCR法從體外培養(yǎng)的膽囊癌細(xì)胞內(nèi)調(diào)取hTR模板區(qū)基因，根據(jù)其測序結(jié)果，合成錘頭狀核酶基因，與經(jīng)相應(yīng)酶切的真核表達(dá)載體連接，酶切鑒定重組體的正確性。
結(jié)果 經(jīng)RT-PCR從細(xì)胞內(nèi)調(diào)出68 bp序列，與hTR cDNA比較，與模板區(qū)域堿基序列一致。核酶基因重組子經(jīng)酶切鑒定序列正確。
結(jié)論  RT-PCR法可調(diào)出膽囊癌hTR基因有效片段； 成功構(gòu)建了針對hTR模板區(qū)的核酶基因的真核表達(dá)載體，為膽囊癌的基因治療奠定了基礎(chǔ)。

引用本文： 靳斌,姜希宏,王學(xué)浩,張峰,王偉,劉賢錫. 膽囊癌hTR錘頭狀核酶基因真核表達(dá)載體的構(gòu)建. 中國普外基礎(chǔ)與臨床雜志, 2004, 11(5): 436-439. doi: 復(fù)制

1. Wan MS, Fell PL, Akhtar S. Synthetic 2’Omethylmodified hammerhead ribozymes targeted to the RNA component of telomerase as sequencespecific inhibitors of telomerase activity [Ｊ]. Antisense Nucleic Acid Drug Dev, 1998; 8(4)∶309.
2. Li QX, Robbins JM, Welch PJ, et al. A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation [Ｊ]. Nucleic Acids Res, 2000; 28(13)∶2605.
3. Kanazawa Y, Ohkawa K, Ueda K, et al. Hammerhead ribozymemediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells [Ｊ]. Biochem Biophys Res Commun, 1996; 225(2)∶570.
4. DeYoung M, Siwkowski AM, Lian Y, et al. Catalytic properties of hairpin ribozymes derived from Chicory yellow mottle virus and arabis mosaic virus satellite RNAs [Ｊ]. Biochemistry, 1995; 34(48)∶15785.
5. Feng J, Funk WD, Wang SS, et al. The RNA component of human telomerase [Ｊ]. Science, 1995; 269(5228)∶1236.
6. Greider CW. Telomerase activity, cell proliferation, and cancer [Ｊ]. Proc Natl Acad Sci USA, 1998; 95(1)∶90.
7. Yokoyama Y, Takahashi Y, Shinohara A, et al. Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells [Ｊ]. Cancer Res, 1998; 58(23)∶5406.
8. Cech TR. Ribozymes, the first 20 years [Ｊ]. Biochem Soc Trans, 2002; 30(6)∶1162.
  1. 1. Wan MS, Fell PL, Akhtar S. Synthetic 2’Omethylmodified hammerhead ribozymes targeted to the RNA component of telomerase as sequencespecific inhibitors of telomerase activity [Ｊ]. Antisense Nucleic Acid Drug Dev, 1998; 8(4)∶309.
  2. 2. Li QX, Robbins JM, Welch PJ, et al. A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation [Ｊ]. Nucleic Acids Res, 2000; 28(13)∶2605.
  3. 3. Kanazawa Y, Ohkawa K, Ueda K, et al. Hammerhead ribozymemediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells [Ｊ]. Biochem Biophys Res Commun, 1996; 225(2)∶570.
  4. 4. DeYoung M, Siwkowski AM, Lian Y, et al. Catalytic properties of hairpin ribozymes derived from Chicory yellow mottle virus and arabis mosaic virus satellite RNAs [Ｊ]. Biochemistry, 1995; 34(48)∶15785.
  5. 5. Feng J, Funk WD, Wang SS, et al. The RNA component of human telomerase [Ｊ]. Science, 1995; 269(5228)∶1236.
  6. 6. Greider CW. Telomerase activity, cell proliferation, and cancer [Ｊ]. Proc Natl Acad Sci USA, 1998; 95(1)∶90.
  7. 7. Yokoyama Y, Takahashi Y, Shinohara A, et al. Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells [Ｊ]. Cancer Res, 1998; 58(23)∶5406.
  8. 8. Cech TR. Ribozymes, the first 20 years [Ｊ]. Biochem Soc Trans, 2002; 30(6)∶1162.