Lu Yu-hua, Zhu Hui, Wang Zhi-wei, Fan Xiang-jun, Zhu Sha-jun, Li Xiao-hong, Wang Yao, Lu Jun-jie, Zhu Ming-yan
Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, China.
Zhonghua Yi Xue Za Zhi. 2011 Nov 29;91(44):3107-10.
To explore the altered expressions of embryonic stem-related genes Oct4 and Nanog in pancreatic cancer stem cells (CSCs).
The uni-cell suspension of human pancreatic cancer cell line PANC-1 was prepared and incubated with CD24 and CD44 antibodies. Flow cytometer was used to separate CD24(+)CD44(+) pancreatic cancer stem cells. Tumor cell spheres were observed under light microscope. Then CSCs were induced to differentiate with 10% fetal bovine serum and the expressions of CD24 and CD44 re-evaluated by flow cytometer. Finally the cells were divided into 2 groups, group 1: CD24(+)CD44(+) and group 2: non-separated group. RT-PCR (reverse transcription-polymerase chain reaction) and Q-PCR (quantitative-polymerase chain reaction) were used to examine the transcriptions of Oct4 and Nanog in CSCs. The immunofluorescence was employed to examine the expressions of Oct4 and Nanog. Chemo-sensitivity to gemcitabine was determined by CCK8 assay in each group.
About 1%-3% CD24(+)CD44(+) CSCs were separated from cell line PANC-1. The sorted cells were cultured in a stem cell culture medium to observe the spheroid-forming capacity. And they showed a higher colony-forming efficiency than the unsorted cells [(122 ± 6)‰, P < 0.05]. When cultured in medium with serum, these cells gradually returned to the status of parental cells with a low expression of CD24 and CD44. Both Oct4 and Nanog were highly expressed in CD24(+)CD44(+) stem cells. And the CD24(+)CD44(+) subgroup demonstrated a higher resistance to gemcitabine.
Subpopulation cells CD44(+)CD24(+) have the properties of tumor stem cells. The up-regulated levels of Oct4 and Nanog may be highly correlated with the multi-potency and a higher drug-resistance of pancreatic CSCs.
探讨胚胎干细胞相关基因Oct4和Nanog在胰腺癌干细胞（CSCs）中的表达变化。
制备人胰腺癌细胞系PANC-1的单细胞悬液，与CD24和CD44抗体孵育。采用流式细胞仪分离CD24(+)CD44(+)胰腺癌干细胞。在光学显微镜下观察肿瘤细胞球。然后用10%胎牛血清诱导CSCs分化，并用流式细胞仪重新评估CD24和CD44的表达。最后将细胞分为两组，第1组：CD24(+)CD44(+)组和第2组：未分离组。采用逆转录-聚合酶链反应（RT-PCR）和定量聚合酶链反应（Q-PCR）检测CSCs中Oct4和Nanog的转录情况。采用免疫荧光法检测Oct4和Nanog的表达。通过CCK8法测定每组对吉西他滨的化疗敏感性。
从细胞系PANC-1中分离出约1%-3%的CD24(+)CD44(+) CSCs。将分选后的细胞在干细胞培养基中培养，观察其成球能力。它们显示出比未分选细胞更高的集落形成效率[（122±6）‰，P<0.05]。当在含血清的培养基中培养时，这些细胞逐渐恢复到亲代细胞状态，CD24和CD44表达较低。Oct4和Nanog在CD24(+)CD44(+)干细胞中均高表达。并且CD24(+)CD44(+)亚组对吉西他滨表现出更高的耐药性。
CD44(+)CD24(+)亚群细胞具有肿瘤干细胞特性。Oct4和Nanog的上调水平可能与胰腺CSCs的多能性和更高的耐药性高度相关。
