骨髓间充质干细胞分泌的外泌体治疗胰腺癌机制的研究

The molecular mechanisms of exosomes derived from human bone marrow mesenchymal stem cells for pancreatic cancer in vivo

目的探讨人骨髓间充质干细胞(BMSC)分泌的外泌体对胰腺癌(PC)细胞调节控制的分子机制。方法应用PANC-1和AsPC-1细胞系建立胰腺癌裸鼠模型,采用随机数字法将每细胞系各自分为3组,每组10只。空白对照组[鼠尾静脉注射磷酸盐缓冲液(PBS)],治疗组(经尾静脉进行注射外泌体PBS悬液),抑制剂组(尾静脉注射靶向抑制剂LY294002,随后尾静脉注射含外泌体的PBS悬液),8周后处死裸鼠,采用蛋白质印迹法(Western blot)法检测肿瘤组织细胞中磷脂酰肌醇3-激酶(PI3K)以及蛋白激酶B(Akt)的蛋白、生存素(Survivin)、基质金属蛋白酶(MMP)-9以及CD31的表达表达。使用酶联免疫吸附试验(ELISA)法对B7-H4和CA199的表达进行检测。多组间比较采用单因素方差分析,两组数据间比较采用t检验。结果提取BMSC分泌的外泌体成功。给予外泌体治疗后,两细胞系中治疗组磷酸化PI3K(p-PI3K)以及磷酸化Akt(p-Akt)的蛋白表达均高于空白对照组[PANC-1(p-PI3K:8.5±2.7比17.9±5.6,F=19.645,p-Akt:9.3±2.9比19.4±6.2,F=26.813),AsPC-1(p-PI3K:9.7±3.2比19.7±6.4,F=18.578,p-Akt:8.4±2.6比18.7±6.3,F=19.817),P<0.05],而抑制剂组p-PI3K以及p-Akt与空白对照组相比差异无统计学意义(P>0.05);给予外泌体治疗后,两细胞系中治疗组胰腺癌标志物的水平均低于空白对照组,同时CD31水平降低[PANC-1(B7-H4:15.9±4.8比6.8±2.3,F=23.467,CA199:17.5±5.8比5.8±1.7,F=29.284,Survivin:12.4±4.0比4.7±1.4,F=28.714,MMP-9:10.3±3.4比3.9±1.3,F=28.927,CD31:16.9±5.6比6.1±1.9,F=30.168),AsPC-1(B7-H4:14.7±4.8比3.1±0.9,F=18.736,CA199:16.2±5.7比3.9±1.2,F=26.948,Survivin:14.4±4.7比4.8±1.5,F=29.841,MMP-9:11.3±3.8比4.7±1.5,F=29.798,CD31:17.1±5.7比5.7±1.8,F=32.864),P<0.05],而抑制剂组两细胞系中上述指标与空白对照组相比差异无统计学意义(P>0.05)。结论BMSC分泌的外泌体可通过激活PI3K/Akt信号通路抑制PC的增殖、侵袭和转移能力,同时抑制PC血管形成,达到抑癌作用。

Objective To investigate the molecular mechanism of exosomes secreted from human bone marrow mesenchymal stem cells(BMSC)for pancreatic cancer(PC)cells.Methods The nude mouse model of PC was established using PANC-1 and AsPC-1 cell lines respectively,and each cell line was divided into 3 groups:control group[mice were injected with phosphate buffer saline(PBS)via the tail vein],treatment group(PBS suspension containing exosomes was injected into the tail vein),inhibitor group[phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway inhibitor LY294002 was added,followed by exosomes].After 8 weeks,the mice were killed,tumor inhibition rate and tumor volume in situ were measured,tumor metastasis and ascites were observed,and tumor weight and liver weight were observed.The expression of PI3K and Akt in tumor cells and the expression of Survivin,matrix metalloproteinase(MMP)-9 and CD31 were detected by Western blotting.The expression of CA199 and B7-H4 was detected by ELISA.Significant differences among groups were determined by the Student t test for 2-group comparisons and One Way ANOVA followed by post hoc analysis for multiple-group comparisons.Results The exosomes secreted by BMSC were extracted successfully.After exosome treatment,the expression of phosphorylated PI3K(p-PI3K)and phosphorylated Akt(p-Akt)was up-regulated in the two cell lines[PANC-1(p-PI3K:8.5±2.7 vs.17.9±5.6,F=19.645,p-Akt:9.3±2.9 vs.19.4±6.2,F=26.813),AsPC-1(p-PI3K:9.7±3.2 vs.19.7±6.4,F=18.578,p-Akt:8.4±2.6 vs.18.7±6.3,F=19.817),P<0.05],but there was no significant differernce between the control group and the inhibitor group for the p-PI3K and p-Akt(P>0.05).After exosome treatment,the levels of pancreatic cancer markers and CD31 in the two cell lines were lower than those in the blank control group[PANC-1(B7-H4:15.9±4.8 vs.6.8±2.3,F=23.467,CA199:17.5±5.8 vs.5.8±1.7,F=29.284,Survivin:12.4±4.0 vs.4.7±1.4,F=28.714,MMP-9:10.3±3.4 vs.3.9±1.3,F=28.927,CD31:16.9±5.6 vs.6.1±1.9 F=30.168),AsPC-1(B7-H4:14.7±4.8 vs.3.1±0.9,F=18.736,CA199:16.2±5.7 vs.3.9±1.2,F=26.948,Survivin:14.4±4.7 vs.4.8±1.5,F=29.841,MMP-9:11.3±3.8 vs.4.7±1.5,F=29.798,CD3:17.1±5.7 vs.5.7±1.8,F=32.864),P<0.05],while there was no statistically significant difference between the control group and the inhibitor group for the above level(P>0.05).Conclusion Exosomes secreted by BMSCs can inhibit the proliferation,invasion and metastasis of PC by activating the PI3K/Akt signaling pathway,and inhibit the angiogenesis of PC.