摘要
目的:明确非受体酪氨酸激酶Fyn在胰腺癌侵袭转移中的作用;探究Fyn是否参与Bcl-X基因的选择性剪切的调控,并阐明其分子机制。方法:对三种侵袭转移能力不同的胰腺癌细胞进行Fyn激酶活性测定,明确Fyn活性与胰腺癌侵袭转移能力之间的关系。将携带激酶活性缺失的KD-Fyn(Kinase Dead-Fyn)的腺病毒转染Bx PC3胰腺癌细胞,Transwell侵袭实验、TUNEL法分别检测抑制Fyn活性前后Bx PC3侵袭能力以及凋亡水平的改变。RT-PCR检测Bcl-X基因两种剪切体比例的变化。结果:转染KD-Fyn以后,Fyn激酶活性明显下降,降低至正常水平的1/2以下。抑制Fyn活性,转染组穿膜细胞数由原来的108±14降为54±7,明显降低了Bx PC3胰腺癌细胞侵袭运动能力,促进凋亡。RT-PCR证实,抑制Fyn活性明显改变了凋亡相关基因Bcl-X的选择性剪切方式。Bcl-X(s)/Bcl-X(L)的剪切比例由0.57±0.08改变为2.12±0.15。在裸鼠成瘤实验中,转染Fyn的裸鼠成瘤能力为100%(12/12),而转染KD-Fyn的裸鼠成瘤能力降低为16.7%(2/12),抑制Fyn活性可以明显抑制Bx PC3胰腺癌细胞在裸鼠中的成瘤能力。结论:抑制Fyn的激酶活性,可以下调胰腺癌细胞的侵袭转移能力,而这种作用是通过影响Bcl-X基因选择性剪切方式改变,进而影响胰腺癌细胞凋亡实现的。
Objective： To explore the roles of non- receptor kinase Fyn in pancreatic cancer metastasis and investigate the molecular mechanism of Fyn in Bcl- X alternative splicing. Methods：Fyn kinase activity assay was performed in three pancreatic cancer cell lines with different metastatic ability to investigate the relationship between Fyn kinase activity and pancreatic cancer metastasis.Invasiveness and apoptosis was investigated by Transwell and TUNEL assay after KD- Fyn adenovirus transfection in Bx PC3 pancreatic cancer cells. Bcl- X alternative splicing was assayed by RT- PCR. Results： KD- Fyn transfection decreases Fyn kinase activity into 50% of the standard level in pancreatic cancer cells. The down- regulation of Fyn activity decreases transwell cells from108 ±14 into 54 ±7, which inhibited pancreatic cancer invasion and promoted apoptosis. RT- PCR revealed that the inhibition of Fyn activity modulated Bcl- X alternative splicing. The ratio of Bcl- X（s）/Bcl- X（L）increased from 0.57±0.08 into 2.12±0.15 in pancreatic cancer cells after KD- Fyn transfection. In vivo study indicated that the down- regulation of Fyn activity inhibited pancreatic cancer metastasis from 100%（12/12）into 16.7%（2/12）. RT- PCR showed that Fyn kinase activity regulates Bcl- X alternative splicing.
出处
《中国现代普通外科进展》
CAS
2015年第5期337-342,共6页
Chinese Journal of Current Advances in General Surgery
基金
国家自然科学基金(81472775)
