脐带间充质干细胞通过类胰岛素样生长因子-Ⅰ介导的Janus激酶/信号转导及转录活化因子信号通路抑制奶牛乳腺上皮细胞凋亡的体外研究
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摘要
本试验旨在探究Janus激酶/信号转导及转录活化因子(JAK/STAT)信号通路是否参与脐带间充质干细胞(UC-MSCs)通过类胰岛素样生长因子-Ⅰ(IGF-Ⅰ)抑制奶牛乳腺上皮细胞(BMECs)凋亡的调节。将UC-MSCs和BMECs利用TranswellTM小室双层共培养,以BMECs单纯培养为对照,给予类胰岛素样生长因子-Ⅰ受体(IGF-ⅠR)抑制剂AG1024进行干预,并用信号阻断剂AG490处理细胞,24 h后采用实时荧光定量PCR检测各组细胞B细胞淋巴瘤/白血病-2(Bcl-2)、B细胞淋巴瘤/白血病基因伴随蛋白x(Bax)、半胱氨酸蛋白酶3(Caspase-3)基因的相对表达丰度,流式细胞仪检测细胞凋亡情况。结果表明:UC-MSCs和BMECs共培养组BMECs的凋亡率极显著低于其他各组(P<0.01);UC-MSCs和BMECs共培养组Bcl-2基因的相对表达丰度较BMECs组极显著上调(P<0.01),Caspase-3、Bax基因的相对表达丰度则显著或极显著下调(P<0.05或P<0.01);AG1024和AG490单独处理或二者共同处理升高了单独培养的BMECs和与UC-MSCs共培养的BMECs的凋亡率,并上调了Bax、Caspase-3基因的相对表达丰度,下调了Bcl-2基因的相对表达丰度,均具有统计学意义(P<0.05或P<0.01)。由此得出,UC-MSCs能够通过IGF-Ⅰ介导JAK/STAT信号通路调节BMECs凋亡相关基因的表达,降低BMECs的凋亡率。
This experiment was conducted to explore whether Janus kinase( JAK)/signal transducer and activator of transcription( STAT) signaling pathway was involved in regulation of umbilical cord mesenchymal stem cells( UC-MSCs) by insulin like growth factor-Ⅰ( IGF-Ⅰ) inhibited bovine mammary gland epithelial cells( BMECs) apoptosis.UC-MSCs and BMECs were co-cultured by TranswellT Mdouble chamber,BMECs cultured alone as control,cells was treated with insulin like growth factor-Ⅰ receptor( IGF-Ⅰ R) inhibitor AG1024 and signal blocking agent AG490.After 24 h,the relative expression abundances of B-cell lymphoma/leukemia-2( Bcl-2),Bcl-associated x protein( Bax),cysteine aspartic acid specific protease( Caspase-3)were detected by real-time quantitative PCR( RT-q PCR),and flowcytometry was adopted to detect apoptosis changes of cells.The results showed as follows: the apoptosis rate of BMECs in UC-MSCs and BMECs co-culture group was significantly lower than that in other groups( P < 0.01); compared with BMECs group,the relative expression abundance of Bcl-2 gene in UC-MSCs and BMECs co-culture group was significantly increased( P < 0.01),while the relative expression abundances of Caspase-3 and Bax genes were significantly decreased( P < 0.05 or P < 0.01).Treated by AG1024 or/and AG490,the apoptosis rate of single cultured BMSCs and co-cultured BMSCs with UC-MSCs was raised,and the relative expression abundances of Bax and Caspase-3 genes were up-regulated,while the relative expression abundance of Bcl-2 gene was down-regulated,and they were statistically significant( P < 0.05 or P < 0.01).In conclusion,UC-MSCs can regulate the expression of apoptosis relation genes in BMECs by IGF-Ⅰ mediated JAK/STAT signaling pathway,and reduce the apoptosis rate of BMECs.
This experiment was conducted to explore whether Janus kinase( JAK)/signal transducer and activator of transcription( STAT) signaling pathway was involved in regulation of umbilical cord mesenchymal stem cells( UC-MSCs) by insulin like growth factor-Ⅰ( IGF-Ⅰ) inhibited bovine mammary gland epithelial cells( BMECs) apoptosis.UC-MSCs and BMECs were co-cultured by TranswellT Mdouble chamber,BMECs cultured alone as control,cells was treated with insulin like growth factor-Ⅰ receptor( IGF-Ⅰ R) inhibitor AG1024 and signal blocking agent AG490.After 24 h,the relative expression abundances of B-cell lymphoma/leukemia-2( Bcl-2),Bcl-associated x protein( Bax),cysteine aspartic acid specific protease( Caspase-3)were detected by real-time quantitative PCR( RT-q PCR),and flowcytometry was adopted to detect apoptosis changes of cells.The results showed as follows: the apoptosis rate of BMECs in UC-MSCs and BMECs co-culture group was significantly lower than that in other groups( P < 0.01); compared with BMECs group,the relative expression abundance of Bcl-2 gene in UC-MSCs and BMECs co-culture group was significantly increased( P < 0.01),while the relative expression abundances of Caspase-3 and Bax genes were significantly decreased( P < 0.05 or P < 0.01).Treated by AG1024 or/and AG490,the apoptosis rate of single cultured BMSCs and co-cultured BMSCs with UC-MSCs was raised,and the relative expression abundances of Bax and Caspase-3 genes were up-regulated,while the relative expression abundance of Bcl-2 gene was down-regulated,and they were statistically significant( P < 0.05 or P < 0.01).In conclusion,UC-MSCs can regulate the expression of apoptosis relation genes in BMECs by IGF-Ⅰ mediated JAK/STAT signaling pathway,and reduce the apoptosis rate of BMECs.
引文
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[19]GAO H N,HU H,ZHENG N,et al.Leucine and histidine independently regulate m ilk protein synthesis in bovine m am m ary epithelial cells via m TOR signaling pathw ay[J].Journal of Zhejiang University Science B,2015,16(6):560-572.
[20]ZHANG X K,DARNELL J E.Functional importance of STAT3 tetram erization in activation of theα2-m acroglobulin gene[J].Journal of Biological Chem istry,2001,276(36):33576-33581.
[21]HEBENSTREIT D,HOREJS-HOECK J,DUSCHL A.JAK/STAT-dependent gene regulation by cytokines[J].Drug New s&Perspectives,2005,18(4):243-249.
[22]黄田英,李庆章,侯晓明.乳腺中JAK-STAT信号通路的研究进展[J].中国乳品工业,2010,38(2):41-44.
[23]肖阳,张莉,高学军,等.奶牛乳腺上皮细胞凋亡周期性规律及机理研究[J].畜牧与兽医,2012,44(增刊1):229-230.
[24]XIONG H,ZHANG Z G,TIAN X Q,et al.Inhibition of JAK1,2/STAT3 signaling induces apoptosis,cell cycle arrest,and reduces tum or cell invasion in colorectal cancer cells[J].Neoplasia,2008,10(3):287-297.
[25]田青,季昀,庞学燕,等.胰岛素对奶牛乳腺上皮细胞酪蛋白合成调节机理的研究[J].动物营养学报,2013,25(3):550-560.
[26]邢媛媛,李大彪,李红磊,等.催乳素对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响[J].动物营养学报,2016,28(8):2439-2447.
[27]KE Y H,LESPERANCE J,ZHANG E E,et al.Conditional deletion of Shp2 in the m am m ary gland leads to im paired lobulo-alveolar outgrow th and attenuated Stat5 activation[J].Journal of Biological Chem istry,2006,281(45):34374-34380.
[2]牛朝诗.IGF-ⅠR与细胞增殖、凋亡和肿瘤[J].国外医学:生理、病理科学与临床分册,2000,20(3):212-215.
[3]黄露麒,王万铭.IGF-Ⅰ通过PI3K/Akt通路抑制M PP+诱导PC12细胞凋亡机制的研究[J].中风与神经疾病杂志,2014,31(10):897-899.
[4]FLEMING J M,BRANDIMARTO J A,COHICK W S.The m itogen-activated protein kinase pathw ay tonically inhibits both basal and IGF-Ⅰ-stim ulated IGFbinding protein-5 production in m am m ary epithelial cells[J].Journal of Endocrinology,2007,194(2):349-359.
[5]GUO C,YANG L,WAN C X,et al.Anti-neuroinflam m atory effect of sophoraflavanone G from Sophora alopecuroides in LPS-activated BV2 m icroglia by M APK,JAK/STAT and Nrf2/HO-1 signaling pathw ays[J].Phytom edicine,2016,23(13):1629-1637.
[6]王俊宏.胰岛素样生长因子Ⅰ受体与细胞凋亡信号转导[J].国外医学:生理、病理科学与临床分册,2002,22(5):445-447.
[7]HACHIM I Y,SHAMS A,LEBRUN J J,et al.A favorable role of prolactin in hum an breast cancer reveals novel pathw ay-based gene signatures indicative of tum or differentiation and favorable patient outcom e[J].Human Pathology,2016,53:142-152.
[8]王立文.奶牛乳腺上皮细胞和奶牛脐带间充质干细胞共培养的试验研究[D].硕士学位论文.乌鲁木齐:新疆农业大学,2014:35-38.
[9]董雅洁,高维娟.Bcl-2、Bax、Caspase-3在细胞凋亡中的作用及其关系[J].中国老年学杂志,2012,32(21):4828-4830.
[10]袁长青,丁振华.Caspase的结构与功能[J].国外医学分子生物学分册,2002,24(3):146-151.
[11]CHEN K,WANG D,DU W T,et al.Human umbilical cord m esenchym al stem cells h UC-M SCs exert imm unosuppressive activities through a PGE2-dependent m echanism[J].Clinical Im m unology,2010,135(3):448-458.
[12]DENG Y,ZHANG Y,YE L,et al.Umbilical cord-derived m esenchym al stem cells instruct m onocytes tow ards an IL10-producing phenotype by secreting IL6and HGF[J].Science Reports,2016,5(6):37566.
[13]HA W T,JEONG H Y,LEE S Y,et al.Effects of the insulin-like grow th factor pathw ay on the regulation of m am m ary gland developm ent[J].Developm ent&Reproduction,2016,20(3):179-185.
[14]TONNER E,ALLAN G,SHKRETA L,et al.Insulinlike grow th factor binding protein-5(IGFBP-5)potentially regulates program m ed cell death and plasm inogen activation in the m am m ary gland[J].Advances in Experim ental M edicine and Biology,2000,480:45-53.
[15]HADSELL D L,BONNETTE S G,LEE A V.Genetic m anipulation of the IGF-Ⅰaxis to regulate m am m ary gland developm ent and function[J].Journal of Dairy Science,2002,85(2):365-377.
[16]高玉红,李庆章.IGFs对小鼠乳腺IGFBP-5和Cyclin D1表达的影响[J].中国乳品工业,2007,35(11):41-43.
[17]WARESKI P,MOTYL T,RYNIEWICZ Z,et al.Expression of apoptosis-related proteins in m am m ary gland of goat[J].Sm all Rum inant Research,2001,40(3):279-289.
[18]黄鑫华.泌乳期牛乳腺上皮细胞凋亡的研究[D].硕士学位论文.大庆:黑龙江八一农垦大学,2007.
[19]GAO H N,HU H,ZHENG N,et al.Leucine and histidine independently regulate m ilk protein synthesis in bovine m am m ary epithelial cells via m TOR signaling pathw ay[J].Journal of Zhejiang University Science B,2015,16(6):560-572.
[20]ZHANG X K,DARNELL J E.Functional importance of STAT3 tetram erization in activation of theα2-m acroglobulin gene[J].Journal of Biological Chem istry,2001,276(36):33576-33581.
[21]HEBENSTREIT D,HOREJS-HOECK J,DUSCHL A.JAK/STAT-dependent gene regulation by cytokines[J].Drug New s&Perspectives,2005,18(4):243-249.
[22]黄田英,李庆章,侯晓明.乳腺中JAK-STAT信号通路的研究进展[J].中国乳品工业,2010,38(2):41-44.
[23]肖阳,张莉,高学军,等.奶牛乳腺上皮细胞凋亡周期性规律及机理研究[J].畜牧与兽医,2012,44(增刊1):229-230.
[24]XIONG H,ZHANG Z G,TIAN X Q,et al.Inhibition of JAK1,2/STAT3 signaling induces apoptosis,cell cycle arrest,and reduces tum or cell invasion in colorectal cancer cells[J].Neoplasia,2008,10(3):287-297.
[25]田青,季昀,庞学燕,等.胰岛素对奶牛乳腺上皮细胞酪蛋白合成调节机理的研究[J].动物营养学报,2013,25(3):550-560.
[26]邢媛媛,李大彪,李红磊,等.催乳素对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响[J].动物营养学报,2016,28(8):2439-2447.
[27]KE Y H,LESPERANCE J,ZHANG E E,et al.Conditional deletion of Shp2 in the m am m ary gland leads to im paired lobulo-alveolar outgrow th and attenuated Stat5 activation[J].Journal of Biological Chem istry,2006,281(45):34374-34380.