Chen Zhixiong, Yang Jiong
Department of Geriatrics, People's Hospital of Wuhan University, Wuhan, 430060, China.
J Huazhong Univ Sci Technolog Med Sci. 2010 Apr;30(2):149-54. doi: 10.1007/s11596-010-0203-4. Epub 2010 Apr 21.
This study examined the effects of a recombinant adenovirus Ad-PTEN-EGFP on the proliferation of A549 cells, a human lung carcinoma cell line, in vitro and on the growth of the implanted tumors in the nude mice in vivo, explored the underlying mechanisms and evaluated the in vitro transfection efficiency of Ad-PTEN-EGFP into A549 cells. The expression of Ad-PTEN-EGFP in the A549 cells was determined. The proliferation and the apoptosis rates of the A549 cells with Ad-PTEN-EGFP transfection or not was detected by MTT and flow cytometry. Ad-PTEN-EGFP at different doses was injected intratumorally to the tumor-bearing mice induced by the A549 cells. Tumor sizes were measured on an alternate day. After all the mice were sacrificed, the implanted tumors were removed for routine histological examination, weight test, HE staining and immunohistochemical staining. The expressions of Bax, P16 and P53 in the tumor tissues and those of caspase-3, CD34 and VEGF in the mouse sera were detected. Tumor cell apoptosis was measured by TUNEL method. The results showed that the vitality of the A549 cells after transfection with Ad-PTEN-EGFP declined. The expression of green fluorescent protein was observed under fluorescent microscope. The transfection rate was in excess of 50%. The mRNA and protein expression of PTEN in the transfected cells was confirmed. The proliferation rate of the transfected cells was significantly decreased when compared with that of the non-transfected cells (P<0.05). The number of the apoptosis cells was increased in the transfected cells (P<0.05). The models of implanted tumors were successfully established by injection of the A549 cells in the flank of Balb/c nude mice. Administration of Ad-PTEN-EGFP to the tumor-bearing nude mice resulted in a suppression of tumor growth. There were statistically significant differences in the tumor weight and tumor volume between the Ad-PTEN-EGFP-treated group and the control groups (P<0.05). In contrast to those in the control groups, tumor tissues in the Ad-PTEN-EGFP-treated group were shown to have typical extensive vacuolar degeneration and massive hemorrhagic necrosis. Apoptotic bodies were also observed in the tumor cells. The expressions of Bax, caspase-3 and P16 were increased (P<0.05) while those of CD34, VEGF and P53 decreased (P<0.05) in the Ad-PTEN-EGFP-treated group. It is concluded that Ad-PTEN-EGFP could induce the apoptosis of the A549 cells and inhibit their proliferation. And it could also substantially suppress the tumor growth in the tumor-bearing nude mice and induce apoptosis of the tumor cells as well. These findings carry significant implications for adenovirus vector-based PTEN gene therapies for lung cancers.
本研究检测了重组腺病毒Ad-PTEN-EGFP对人肺癌细胞系A549细胞体外增殖及裸鼠体内移植瘤生长的影响,探讨其潜在机制,并评估Ad-PTEN-EGFP对A549细胞的体外转染效率。检测了Ad-PTEN-EGFP在A549细胞中的表达。采用MTT法和流式细胞术检测转染或未转染Ad-PTEN-EGFP的A549细胞的增殖及凋亡率。将不同剂量的Ad-PTEN-EGFP瘤内注射到由A549细胞诱导的荷瘤小鼠体内。隔天测量肿瘤大小。处死所有小鼠后,取出移植瘤进行常规组织学检查、称重、HE染色及免疫组化染色。检测肿瘤组织中Bax、P16和P53的表达以及小鼠血清中caspase-3、CD34和VEGF的表达。采用TUNEL法检测肿瘤细胞凋亡。结果显示,转染Ad-PTEN-EGFP后A549细胞活力下降。在荧光显微镜下观察到绿色荧光蛋白的表达。转染率超过50%。证实了转染细胞中PTEN的mRNA和蛋白表达。与未转染细胞相比,转染细胞的增殖率显著降低(P<0.05)。转染细胞中凋亡细胞数量增加(P<0.05)。通过将A549细胞注射到Balb/c裸鼠侧腹成功建立了移植瘤模型。给荷瘤裸鼠注射Ad-PTEN-EGFP可抑制肿瘤生长。Ad-PTEN-EGFP治疗组与对照组之间的肿瘤重量和肿瘤体积存在统计学显著差异(P<0.05)。与对照组相比,Ad-PTEN-EGFP治疗组的肿瘤组织表现出典型的广泛空泡变性和大量出血性坏死。在肿瘤细胞中也观察到凋亡小体。Ad-PTEN-EGFP治疗组中Bax、caspase-3和P16的表达增加(P<0.05),而CD34、VEGF和P53的表达降低(P<0.05)。结论是Ad-PTEN-EGFP可诱导A549细胞凋亡并抑制其增殖。它还可显著抑制荷瘤裸鼠的肿瘤生长并诱导肿瘤细胞凋亡。这些发现对基于腺病毒载体的PTEN基因治疗肺癌具有重要意义。