靶向DF3的白喉毒素A链基因对乳腺癌细胞的杀伤作用

Killing effect of diphtheria toxin A-chain gene targeting DF3 on breast carcinoma cells

目的探讨白喉毒素A链（DTA）基因在DF3／MUC1启动子调控下的杀伤作用。方法构建含人乳腺癌DF3启动子和白喉毒素A链基因的表达载体pGL3-DF3-DTA，转染DF3阳性的乳癌细胞MCF-7，逆转录-聚合酶链反应（RT—PCR）检测DTA基因的表达；噻唑蓝（MTT）比色法检测细胞生长活性；免疫组织化学法和流式细胞术检测细胞凋亡。结果酶切和测序表明获得了与预期结果相一致的pGL3-DF3-DTA真核表达载体。转染MCF-7细胞后，RT—PCR检测到了249bp的DTA mRNA片段。与对照组比较，转染pGL3-DF3-DTA的MCF-7细胞生长受到了抑制，流式细胞术检测到的凋亡率达39．43％，而对照组仅有0．28％。结论pGL3-DF3-DTA可对DF3阳性的乳癌细胞产生特异性的杀伤，对乳腺癌的基因治疗有潜在的应用价值。

Objective To explore the specific killing effect of diphtheria toxin A-chain （DTA） gene under control of the transcriptional regulation sequence of DF3/MUC1 promoter. Methods Expression vector （pGL3-DF3-DTA） containing transcriptional regulation sequence of human breast carcinoma DF3/MUC1 promoter and diphtheria toxin A-chain gene was constructed by replacing the luciferase gene in pGL3-DF3 with DTA gene at the points of NcoI and Xba.I by molecular technique. The expression vector pGL3-DF3-DTA was transfected into human breast carcinoma ceils positive for DF3. The in vitro cellular growth activities were assayed by MTT colorimetry. The expression of DTA gene in ceils was detected by RT-PCR. Apoptosis was detected by immunohistochemistry and flow cytometry. Results Restrictive endonuclease identification and DNA sequencing revealed that the pGL3-DF3-DTA vector was in consistent with the vector we designed. After transient transfection into the DF3 positive breast cancer cells-MCF-7, the 249 bp base pares fragment of DTA mRNA was detected in transfected cells. Besides, the transfected MCF-7 ceils showed a retarded growth compared to untransfected cells. The transfected MCF-7 ceils showed a 39.43% apoptotic rate detected by flow cytometry, whereas the apoptotic rate of untransfected cells was only 0.28%. Conclusion pGL3-DF3-DTA could selectively kill human breast carcinoma cell line positive for DF3 and could inhibit the growth. It has potential value in gene therapy for human breast carcinoma.