Effects of recombinant adenovirus vector expressing human lactoferrin on proliferation and apoptosis of cervical cancer stem-like cells

doi:10.3969/j.issn.2095-4344.2017.13.008

Abstract

Abstract:

BACKGROUND: Gene engineering plays an important role in the process of human malignant tumor treatment, and adenovirus vectors for gene therapy are commonly used. In recent years, lactoferrin is found to exert important effects on the occurrence, development and metastasis of a variety of tumors. However, little is reported on the in vivo expression of adenovirus vector mediated lactoferrin in the human body.
OBJECTIVE: To investigate the effect of adenovirus mediated human lactoferrin (Ad-hLF) on proliferation and apoptosis of cervical cancer stem-like cells.
METHODS: Primary cervical cancer cells from mice were cultured in vitro to sort cervical cancer stem-like cells using SP method. Afterwards, the stem-like cells were divided into three groups, blank control group, empty vector group, and Ad-hLF group, followed by transfection with nothing, Ad-GFP and Ad-hLF, respectively. After transfection, MTT assay was used to detect the proliferation of cervical cancer stem-like cells; scratch-wound assay was employed to detect the migration of cervical cancer stem-like cells; western blot assay was used to determine the expression of lactoferrin in cervical cancer stem-like cells; and Annexin V-FITC/PI was used to detect cell apoptosis.
RESULTS AND CONCLUSION: Compared with the blank control and empty vector groups, Ad-hLF significantly inhibited the proliferation of cervical cancer stem-like cells and reduced the number of scratched cells, while the cell apoptosis increased in the Ad-hLF group (P < 0.05 or P < 0.01). These findings indicate that the recombinant adenovirus vector mediated lactoferrin Ad-hLF for transfection of cervical cancer stem-like cells can be stably expressed in cervical cancer stem-like cells, and can inhibit the proliferation, increase apoptosis and reduce migration of cervical cancer stem-like cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Cell Proliferation

CLC Number:

R394.2

Zhang Xia, Du Zhi-xiang, Wang Na, Meng Ya-li. Effects of recombinant adenovirus vector expressing human lactoferrin on proliferation and apoptosis of cervical cancer stem-like cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 2009-2014.

Figures/Tables 2

2.1 SP法从宫颈癌细胞中分选宫颈癌干样细胞的鉴定结果在宫颈癌原代细胞中采用流式细胞仪进行分析，采用Hoechst 33342荧光染料进行染色，可见多数细胞聚集在图的中央及偏右部分，为非SP细胞，将其他部分对Hoechst 33342荧光染料显示阴性结果的细胞分选出来即为宫颈癌干样细胞，见图1。 2.2 Ad-GFP对宫颈癌干样细胞的感染效率由表1可见，Ad-GFP对宫颈癌干样细胞的感染效率随着MOI的升高而增加，但在显微镜下观察到当MOI=200时，细胞状态较差，虽然感染率较高，但出现细胞脱落不贴壁的状况，所以采用MOI=100在后续实验中感染细胞。 2.3 MTT法检测各组宫颈癌干样细胞增殖能力由表2可见Ad-hLF感染宫颈癌干样细胞后，培养24 h检测时各组细胞增殖未见显著性差异，在36，48，72 h检测结果显示Ad-hLF组宫颈癌干样细胞的增殖情况受到明显抑制，比空白对照组及空病毒组明显降低(P < 0.05，P < 0.01)，而对照组与空病毒组相比宫颈癌干样细胞增殖情况均无明显差异。 2.4 宫颈癌干样细胞凋亡情况 Annexin V-FITC/PI染色后，荧光显微镜下可以观察到细胞发出荧光信号，计算出200个细胞视野中阳性细胞数，空白对照组中宫颈癌干样细胞凋亡率为(20.11±3.25)%，空病毒组中宫颈癌干样细胞凋亡率为(23.43±2.55)%，Ad-hLF组中宫颈癌干样细胞凋亡率为(55.57±3.41)%。与对照组及空病毒组相比，Ad-hLF组阳性细胞数明显增多，宫颈癌干样细胞的凋亡数量明显增加(P < 0.05，P < 0.01)，而对照组与空病毒组相比宫颈癌干样细胞凋亡情况差异无显著性意义。 2.5 宫颈癌干样细胞感染后划痕实验结果从图2可见，细胞划痕实验后经过24 h培养，空白对照组及空病毒组细胞增殖明显增加，划痕处可见细胞迁移，基本能使划痕处汇合，而重组腺病毒Ad-hLF感染组划痕处仍可清晰辨认，细胞迁移明显减少。 2.6 Western blot检测Ad-hLF感染后乳铁蛋白在宫颈癌干样细胞中的表达由图3可见，对照组及空病毒GFP感染组均未见乳铁蛋白的表达，而重组腺病毒Ad-hLF感染组可见乳铁蛋白在宫颈癌干样细胞中的表达显著升高(P < 0.01)，其中空白对照组中乳铁蛋白相对灰度值为8.8±3.1，空病毒组灰度值为30.9±1.4，Ad-hLF组为110.2±2.4，3组比较差异有显著性意义(P < 0.01)。"

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