ADAM17-shRNA转染的骨髓间充质干细胞对裸鼠乳腺癌移植瘤生长的影响
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摘要
目的:探讨转染解聚素-金属蛋白酶17-shRNA(a disintegrin and metalloprotease 17-shRNA,ADAM17-shRNA)的骨髓间充质干细胞(bone marrow mesenehymal stem cells,BMMSC)对乳腺癌MCF-7细胞裸鼠移植瘤的抑制效果。方法:全骨髓贴壁法分离并培养3周雄性SD大鼠的BMMSC,利用慢病毒介导的ADAM17-shRNA转染BMMSC。30只裸鼠建立MCF-7乳腺癌移植瘤模型,种植肿瘤细胞14 d后建模成功。按照数字表法随机分成对照组(注射等量PBS)、BMMSC组(注射1×10~6/ml BMMSC)和转染组(注射1×10~6/ml转染ADAM17-shRNA的BMMSC),每组10只。在种植细胞第15天开始经尾静脉注射BMMSC进行抑瘤实验(0.1 ml/只,每3 d给药1次,共计5次),观察裸小鼠移植瘤的生长情况;抑瘤实验16 d后处死裸鼠。利用Real-time PCR法检测移植瘤组织ADAM17 mRNA表达,Western blotting法检测移植瘤组织ADAM17蛋白表达。结果:抑瘤实验16 d时,对照组、BMMSC组移植瘤体积明显高于转染组[(787.15±25.95)、(767.02±28.98)vs(361.89±19.75)mm~3,均P<0.01];BMMSC组、转染组抑瘤率明显高于对照组(2.57%、53.89%vs 0.00%,均P<0.05)。对照组、BMMSC组ADAM17 mRNA的表达水平明显高于转染组(1.00±0.01、0.97±0.08 vs 0.30±0.09,均P<0.05);对照组、BMMSC组ADAM17蛋白表达水平明显高于转染组(0.70±0.09、0.68±0.02 vs 0.45±0.05,均P<0.05)。结论:ADAM17-shRNA通过BMMSC介导可将ADAM17靶向归巢至裸鼠乳腺癌移植瘤并发挥抑瘤作用。
Objective: To investigate the inhibitory effect of bone marrow mesenehymal stem cells(BMMSCs)transfected with ADAM17-shRNA(adisintegrin and metalloprotease 17-shRNA) on the growth of implanted breast cancer MCF-7 cell xenograft in nude mice. Methods: BMMSCs from 3-week-old male SD rats were isolated and cultured with the whole bone marrow adherence method. BMMSCs were transfected with Lentivirus-mediated ADAM17-shRNA. Breast cancer MCF-7 cell xenograft model was successfully established in 30 nude mice after 14 days implantation of tumor cells. According to the random number table, nude mice were randomly divided into control group(equal volume PBS), BMMSCs group(1×10~6/ml BMMSCs) and transfection group(1×10~6/ml BMMSCs transfected with ADAM17-shRNA) with 10 nude mouses in each group. The tumor inhibition test was carried out on the 15 thday by injecting BMMSCs into tail vein(0.1 ml/each, administration was carried every 3 days with a total of 5 times). The growth of implanted tumor was observed every day. All the nude mice were sacrificed on 16 th day after treatment. The expressions of ADAM17 mRNA and ADAM17 protein in tumor tissues were detected by Real-time PCR and Western blotting, respectively. Results:The volume of implanted tumor in control group,BMMSCs group was significantly larger than that of transfection group([787.15 ± 25.95], [767.02 ± 28.98] vs[361.89±19.75] mm~3, all P<0.01) on D 30. The tumor inhibition rate of BMMSCs group and transfection group was significantly higher than that of control group(2.57%, 53.89% vs 0.00%, all P<0.05). The expression of ADAM17 mRNA in control group, BMMSCs group was significantly higher than that of transfection group(1.00±0.01, 0.97±0.08 vs 0.30±0.09, P<0.05). The expression of ADAM17 protein in control group, BMMSCs group was significantly higher than that of transfection group(0.70±0.09, 0.68±0.02 vs 0.45±0.05, all P<0.05). Conclusion: The tropism of ADAM17-shRNA to breast cancer xenograft in nude mice was accomplished by BMMSCs mediation, which may play an anti-tumor effect.
Objective: To investigate the inhibitory effect of bone marrow mesenehymal stem cells(BMMSCs)transfected with ADAM17-shRNA(adisintegrin and metalloprotease 17-shRNA) on the growth of implanted breast cancer MCF-7 cell xenograft in nude mice. Methods: BMMSCs from 3-week-old male SD rats were isolated and cultured with the whole bone marrow adherence method. BMMSCs were transfected with Lentivirus-mediated ADAM17-shRNA. Breast cancer MCF-7 cell xenograft model was successfully established in 30 nude mice after 14 days implantation of tumor cells. According to the random number table, nude mice were randomly divided into control group(equal volume PBS), BMMSCs group(1×10~6/ml BMMSCs) and transfection group(1×10~6/ml BMMSCs transfected with ADAM17-shRNA) with 10 nude mouses in each group. The tumor inhibition test was carried out on the 15 thday by injecting BMMSCs into tail vein(0.1 ml/each, administration was carried every 3 days with a total of 5 times). The growth of implanted tumor was observed every day. All the nude mice were sacrificed on 16 th day after treatment. The expressions of ADAM17 mRNA and ADAM17 protein in tumor tissues were detected by Real-time PCR and Western blotting, respectively. Results:The volume of implanted tumor in control group,BMMSCs group was significantly larger than that of transfection group([787.15 ± 25.95], [767.02 ± 28.98] vs[361.89±19.75] mm~3, all P<0.01) on D 30. The tumor inhibition rate of BMMSCs group and transfection group was significantly higher than that of control group(2.57%, 53.89% vs 0.00%, all P<0.05). The expression of ADAM17 mRNA in control group, BMMSCs group was significantly higher than that of transfection group(1.00±0.01, 0.97±0.08 vs 0.30±0.09, P<0.05). The expression of ADAM17 protein in control group, BMMSCs group was significantly higher than that of transfection group(0.70±0.09, 0.68±0.02 vs 0.45±0.05, all P<0.05). Conclusion: The tropism of ADAM17-shRNA to breast cancer xenograft in nude mice was accomplished by BMMSCs mediation, which may play an anti-tumor effect.
引文
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[22]张艳丽,傅晋翔,张宏,等.骨髓间充质干细胞对多发性骨髓瘤细胞迁移和归巢特性的影响[J].中国实验血液学杂志,2016,24(2):469-473.DOI:10.7534/j.issn.1009-2137.2016.02.030.
[23]LIU N,TIAN J,CHENG J,et al.Migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the acute injured kidney[J].J Cell Biochem,2013,114(12):2677-2689.DOI:10.1002/jcb.24615.
[24]蔡准,陈国福,吴丽君,等.sh RNA沉默ADAM17基因对人乳腺癌细胞MCF-7增殖的影响[J].大连医科大学学报,2016,38(6):542-545.DOI:10.11724/jdmu.2016.06.05.
[25]彭晓兵,孙影,张雪鹏,等.转染ADAM17-sh RNA的骨髓间充质干细胞对MCF-7乳腺癌细胞增殖能力的影响[J].广东医学,2015,25(19):2955-2958.DOI:10.13820/j.cnki.gdyx.2015.19.006.
[2]MILLER K D,SIEGEL R L,LIN C C,et al.Cancer treatment and survivorship statistics,2016[J].CA Cancer J Clin,2016,66(4):271-289.DOI:10.3322/caac.21349.
[3]帖鹏,赵文彩,梁亮,等.骨髓间充质干细胞对裸鼠肾癌细胞系A498生物学特性影响的研究[J].临床和实验医学杂志,2016,15(17):1657-1661.DOI:10.3969/j.issn.1671-4695.2016.17.001.
[4]MITCHELL R C,FLORIANE M I,SARAH K B.Mesenchymal stem cells inhibit breast cancer cell migration and invasion through secretion of tissue inhibito of metallo-proteinase-1and-2[J].Mol Carcinog,2015(10):56-57.DOI:10.1002/mc.22178.
[5]丁华,孙影,张雪鹏,等.转染ADAM17-sh RNA的大鼠骨髓间质干细胞对MCF-7人乳腺癌细胞侵袭能力的影响[J].广东医学,2014,35(14):2154-2156.DOI:10.13820/j.cnki.gdyx.2014.14.004.
[6]孟祥潮,蔡准,陈国福,等.ADAM17-sh RNA对人乳腺癌MCF-7细胞增殖的影响及机制[J].山东医药,2017,57(4):21-24.DOI:10.3969/j.issn.1002-266X.2017.04.004.
[7]陈国福,吴丽君,张雪鹏,等.缺氧条件下沉默解聚素-金属蛋白酶17基因对人乳腺癌MCF-7细胞增殖的影响[J].中国现代医学杂志,2016,26(15):22-27.DOI:10.3963/j.issn.1005-8982.2016.15.004.
[8]SUN J,JIANG J,LU K,et al.Therapeutic potential of ADAM17modulation in gastric cancer through regulation of the EGFR and TNF-αsignalling pathways[J].Mol Cell Biochem,2017,426(1/2):17-26.DOI:10.13820/j.cnki.gdyx.2015.19.006.
[9]WOLF C,QIAN Y,BROOKE M A,et al.ADAM17/EGFR axis promotes transglutaminase-dependent skin barrier formation through phosholipase Cγ1 and protein kinase C pathways[J].Sci Rep,2016,6(125):39780-39788.DOI:10.1038/srep39780.
[10]GROOT A J,COBZARU C,WEBER S,et al.Epidermal ADAM17is dispensable for notch activation[J].J Invest Dermatol,2013,133(9):2286-2288.DOI:10.1038/jid.2013.162.
[11]JIA S,QU T,WANG X,et al.KIAA1199 promotes migration and invasion by Wnt/β-catenin pathway and MMPs mediated EMT progression and serves as a poor prognosis marker in gastric cancer[J/OL].PLo S One,2017,12(4):e0175058[2017-05-03].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397282/.DOI:10.1371/journal.pone.0175058.
[12]WANG H P,WANG X,GONG L F,et al.Nox1 promotes colon cancer cell metastasis via activation of the ADAM17 pathway[J].Eur Rev Med Pharmacol Sci,2016,20(21):4474-4478.
[13]KéRAMIDAS M,DE FRAIPONT F,KARAGEORGIS A,et al.The dual effect of mesenchymal stem cells on tumour growth and tumour angiogenesis[J].Stem Cell Res Ther,2013,4(2):41-47.DOI:10.1186/scrt195.
[14]WANG A,ZHOU X,ZHAO J,et al.Therapeutic effects of bone marrow mesenchymal stem cells expressing interleukin-12 in mice bearing malignant ascites tumor[J].Int J Clin Exp Med,2015,8(9):15840-15845.
[15]YUE W,YAN F,ZHANG Y L,et al.Differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells in vitro and cocultured with biological scaffold as transplantation carrier[J/OL].Med Sci Monit,2016,22:1766-1772[2017-05-03].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917310/.[16 ZHANG Y,QIU B,WANG J,et al.Effects of BDNF-transfected BMMSC on neural functional recovery and synaptophysin expression in rats with cerebral infarction[J].Mol Neurobiol,2017,54(5):3813-3824.DOI:10.1007/s12035-016-9946-7.
[17]HAN C,JIANG C,YU C,et al.Differentiation of transforming growth factorβ1-induced mesenchymal stem cells into nucleus pulposus-like cells under simulated microgravity conditions[J].Cell Mol Biol,2015,61(2):50-55.
[18]RITTER E,PERRY A,YU J,et al.Breast cancer cell-derived i broblast growth factor 2 and vascular endothelial growth factor are chemoattractants for bone marrow stromal stem cells[J].Ann Surg,2008,247(2):310-314.DOI:10.1097/SLA.0b013e31816401d5.
[19]WU D,GUO X,SU J,et al.CD138-negative myeloma cells regulate mechanical properties of bone marrow stromal cells through SDF-1/CXCR4/AKT signaling pathway[J].Biochim Biophys Acta,2015,1853(2):338-347.DOI:10.1016/j.bbamcr.2014.11.019.
[20]杨芳,于雁.肿瘤微环境——肿瘤转移的关键因素[J].中国肺癌杂志,2015,18(1):48-54.DOI:10.3779/j.issn.1009-3419.2015.01.08.
[21]JUNTTILA M R,DE SAUVAGE F J.Influence of tumour micro-environment heterogeneity on therapeutic response[J].Nature,2013,501(7467):346-354.DOI:10.1038/nature 12626.
[22]张艳丽,傅晋翔,张宏,等.骨髓间充质干细胞对多发性骨髓瘤细胞迁移和归巢特性的影响[J].中国实验血液学杂志,2016,24(2):469-473.DOI:10.7534/j.issn.1009-2137.2016.02.030.
[23]LIU N,TIAN J,CHENG J,et al.Migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the acute injured kidney[J].J Cell Biochem,2013,114(12):2677-2689.DOI:10.1002/jcb.24615.
[24]蔡准,陈国福,吴丽君,等.sh RNA沉默ADAM17基因对人乳腺癌细胞MCF-7增殖的影响[J].大连医科大学学报,2016,38(6):542-545.DOI:10.11724/jdmu.2016.06.05.
[25]彭晓兵,孙影,张雪鹏,等.转染ADAM17-sh RNA的骨髓间充质干细胞对MCF-7乳腺癌细胞增殖能力的影响[J].广东医学,2015,25(19):2955-2958.DOI:10.13820/j.cnki.gdyx.2015.19.006.