Chinese Journal of Tissue Engineering Research ›› 2017, Vol. 21 ›› Issue (13): 2009-2014.doi: 10.3969/j.issn.2095-4344.2017.13.008
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Zhang Xia, Du Zhi-xiang, Wang Na, Meng Ya-li
Revised:
2017-03-22
Online:
2017-05-08
Published:
2017-06-09
Contact:
Meng Ya-li, Chief physician, the First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China
About author:
Zhang Xia, Master, Attending physician, the First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China
Supported by:
the Key Project of Medical Science Research in Hebei Province in 2014, No. ZD20140325
CLC Number:
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.
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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