自噬基因Beclinl对宫颈癌HeLa细胞生长影响的体内外研究

Effect of autophagy gene Beclin 1 on the growth of cervical cancer HeLa cells in vitro and vivo

目的研究自噬基因Beclinl在宫颈癌细胞株HeLa中过表达对HeLa细胞体内外生长的影响。方法构建自噬基因Beclinl的真核表达载体pcDNA3．1（＋）-Beclinl，转染HeLa细胞[pcDNA3．1（＋）．Beclinl组]，同时设空载体转染组[pcDNA3．1（＋）组]和空白对照组。采用荧光定量PCR和Westernblot法检测3组HeLa细胞中BeclinlmRNA和蛋白的表达变化，采用四甲基偶氮唑蓝（MTF）法检测3组HeLa细胞生长的变化，采用流式细胞术检测3组HeLa细胞的凋亡情况，采用单丹磺酰尸胺染色检测3组HeLa细胞的自噬情况。将3组HeLa细胞分别接种于裸鼠皮下，观察体内成瘤性和生长情况。结果pcDNA3．1（＋）-Beclinl组、pcDNA3．1（＋）组和空白对照组HeLa细胞中BeclinlmRNA的表达水平分别为994．718±468．764、0．570±0．121和0．736±0．251。pcDNA3．1（＋）-Beclinl组HeLa细胞中BeclinlmRNA的表达水平较pcDNA3．1（＋）组和空白对照组明显增高（P〈0．05），而pcDNA3．1（＋）组与空白对照组间的差异无统计学意义（P〉0．05）；3组HeLa细胞中Beclinl蛋白的表达情况与mRNA相似。pcDNA3．1（＋）-Beclinl组的自噬细胞率为10．9％，明显高于空白对照组和pcDNA3．I（＋）组（分别为2．5％和3．1％，P〈0．05）。pcDNA3．1（＋）-Beclinl组HeLa细胞的凋亡率为（28．2±2．3）％，明显高于peDNA3．1（＋）组和空白对照组[分别为（14．6±4．6）％和（11．2±3．0）％，P〈0．05]。Beclinl在HeLa细胞中过表达可抑制肿瘤细胞在体外的生长，抑制率为58．7％；并可使HeLa细胞在裸鼠体内成瘤时间延长，瘤体体积和重量明显小于pcDNA3．1（＋）组和HeLa组（P〈0．05），抑瘤率为52．2％。结论自噬基因Beclinl过表达可抑制HeLa细胞在体内外的增殖，促进细胞的自噬与凋亡，为宫颈癌基因治疗提供了新的选择途径。

Objective To investigate the effects of autophagy gene Beclin 1 on growth of cervical cancer HeLa cells in vitro and vivo. Methods The eukaryotic expression vector of Beclinl was constructed and transfected via lipofectamine into HeLa cells. The experimental cells were classified into 3 groups： pcDNA3.1 （ ＋ ）-Beclinl group, pcDNA3. 1 （ ＋ ） group and HeLa group. Real time-ploymerase chain reaction and Western blot were used for detecting expression of Beclinl mRNA and protein in the transfected ceils. Flow cytometry （FCM） was employed to observe the effect of transfection on the apoptosis of HeLa cells, and proliferation was analyzed by MrlT assay. The formation of autophagic vacuoles was measured by MDC staining. HeLa cells transfected with plasmid pcDNA3. 1 （ ＋ ）-Beclinl and pcDNA3. 1 （ ＋ ） were inoculated subcutaneously in nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed. Results Eukaryotic expression vector pcDNA3.1 （ ＋ ）-Beclinl was constructed successfully. It significantly improved the expression of Beclinl mRNA and protein in HeLa cells. The proliferation of HeLa cells was inhibited, and the inhibition rate was 58.7%. FCM investigation showed that the apoptotic rate was （28.22 ± 2.34） % of pcDNA3.1 （ ＋ ） -Beclinl group, significantly higher than the （ 14.6± 4.6 ） % in the pcDNA3.1 （ ＋ ） group and （ 11.2±3.0） % in the HeLa group （ P 〈 0.05 ）. The monodansylcadaverin （MDC） staining showed significantly more autophagic vacuoles in the pcDNA3. 1 （ ＋ ）-Beclinl group （ 10.9% ） than that in the pcDNA 3.1 （ ＋ ） group （3.1% ） and HeLa group （2.5%） （ P 〈 0. 05 ）. After transfected with vector pcDNA3.1 （ ＋ ）-Beclinl, the carcinogenic activity of HeLa cells was decreased in nude mice, and the inhibition rate of tumor growth was 52.2%. Conclusions Autophagy gene Beclin 1 overexpression can inhibit the proliferation and growth of HeLa cells in vitro and vivo, while promote autophagy and apoptosis of HeLa cells. So it might be one of new gene therapy strategies for cervical carcinoma.