CN105339384A - 作为鉴别潜在味觉调节剂的模型的细胞 - Google Patents
作为鉴别潜在味觉调节剂的模型的细胞 Download PDFInfo
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Abstract
T1R2和T1R3的共同表达形成响应甜味刺激物(包括天然产生的和人造甜味剂)的味觉感受器。表达功能性甜味感受器的细胞(如U2-OS)可以用于基于细胞的试验中以检测对呈味剂的细胞响应。
Description
本公开内容中引用的每篇参考文献将其全部并入本文中。
本申请按引用并入2014年5月5日形成的并且名称为“056943.01231sequencelisting.txt”的13.7kb文本文件,这是用于本申请的序列表。
技术领域
本公开内容总地涉及可以用于鉴别甜味感受器调节剂的细胞系和试验。
附图简述
图1A-B.对甜味化合物的Ca(2+)响应。图1A,使用(钻石)和未用(三角)T1R3/T1R2/Gα15激活的JUMP-INTMT-REXTMU2-OS中Ca(2+)-释放的剂量响应曲线(n=3次实验)。图1B,lactisole和U73122对JUMP-INTMT-REXTMU2-OS细胞中的Ca(2+)响应的作用(平均±SD)。
图2.显示了Gα15蛋白对U2-OS响应100mM三氯蔗糖的影响的图。用3、6和12μl/40,000细胞的编码G蛋白,味蛋白(gustducin)(Gα15)的BacMam在24小时(左)或48小时(右)过程中瞬时转染U2-OS细胞。
图3.显示了100mM的天然糖(葡萄糖、果糖和蔗糖)对用0或6μl/40,000细胞的编码Gα15的BacMam转染的U2-OS的影响的图。
图4.显示了用100mM葡萄糖处理时U2-OS细胞中甜味感受器激活pERK1/2的图。用lactisole(20mM,左条)处理U2-OS细胞阻断了D-葡萄糖介导的pERK1/2的激活。
图5.证明了用Gα15(6μl/40,000细胞)转染U2-OS细胞显著提高了pERK1/2激活,其被PLCβ2抑制剂U73122(10μM)阻断。
图6.显示了用Gα15(6μl/40,000细胞)转染的U2-OS细胞中用D-葡萄糖和麦芽糖处理时pERK1/2激活的图。
图7.显示了用Gα15(6μl/40,000细胞)转染的U2-OS细胞中用不同的糖处理时pERK1/2激活的图。
图8A-B.Transfluor试验。图8A,用编码Gα15的BacMam转染的TransfluorU2-OS细胞中绿色荧光泡囊的形成。用Hoechst33342将核复染成蓝色。图8B,Transfluor模型中T1R2/T1R3内化的定量(平均±SD)。
图9A-B.U2-OS细胞表达内源性甜味感受器T1R2/T1R3。图9A,将U2-OS细胞固定并处理,用于使用对抗甜味感受器T1R2和T1R3的抗体的间接免疫荧光。使用ImageXpressMicro自动化显微镜,使用20x物镜,获取所有图像。Hoechst33342染色是假着色的蓝色,并且抗体特异性染色是假着色的绿色。图9B.U2-OS和NCI-H716细胞中的T1R2的Western印迹。将细胞在RIPA缓冲液(50mMTris-HClpH7.5,150mMNaCl,1%NP-40,5mMEDTA,0.1%SDS和10μg/ml蛋白酶抑制剂)中裂解。冻-融循环后,加入终浓度2x的加样缓冲液,将样品在95℃下加热10分钟并且在最高速度下在RT下在微离心机中离心10分钟。将上清液加载于凝胶上,用于通过免疫印迹分析。用于T1R2和GAPDH的一抗来自ThermoFisher。
详述
T1R2和T1R3的共同表达形成响应甜味刺激物(包括天然产生的和人造甜味剂)的味觉感受器。甜味配体结合T1R2/T1R3感受器并激活G-蛋白途径转导,其诱导感受器内化和钙动员,以及下游靶标的诱导,如ERK1/2的磷酸化(Jang等,“Gut-expressedgustducinandtastereceptorsregulatesecretionofglucagon-likepeptide-1,”ProcNatlAcadSciUSA.2007Sep18;104(38):15069-74)。
任何包含或可以工程化以包含功能性调味感受器的细胞可以用于所公开的试验中。在一些实施方案中,细胞表达一种或多种甜味感受器。在一些实施方案中,细胞被遗传修饰,以表达或超表达甜味感受器。例如,可以通过引入编码T1R2和T1R3的外源性核酸或两个分开的分别编码T1R2和T1R3的外源性核酸来遗传修饰细胞。用于这些蛋白质的氨基酸序列是已知的;例如,GenBank登记号NP_689418.2(T1R2)和GenBank登记号NP_689414.1(T1R3)。
可以使用本领域公知的方法,将细胞工程化来表达T2R14、T1R2/T1R3、α-gustducin和/或GLUT4。其中,GLUT4描述于美国专利7,799,538和其中引用的参考文献中。对于α-gustducin的描述,参见美国专利8,338,115和其中引用的参考文献以及Adler等,Cell100,693-702,2000。
在一些实施方案中,将细胞遗传修饰来超表达α-gustducin(例如,Gα16、Gα15、Gα16gust25、Gα15gust25、Gα16gust44、Gα15gust44、Gα15-i/3-5),其提高试验的灵敏度。SEQIDNO:l是Gα15的氨基酸序列。序列表中提供了可以用于表达Gα16(SEQIDNO:2)、Gα16gust25(SEQIDNO:3)、Gα15(SEQIDNO:4)、Gα15gust25(SEQIDNO:5)、Gα16gust44(SEQIDNO:6)、Gα15gust44(SEQIDNO:7)和Gα15-i/3-5(SEQIDNO:8);然而,编码这些α-gustducin的任何核苷酸序列可以用于表达它们。
在一些实施方案中,使用U2-OS细胞,如下所述。人U2-OS细胞(ATCC目录#HTB-96)表达功能性甜味感受器(图9A-B)。因此,通过将U2-OS细胞接触测试化合物并测量甜味感受器活性,U2-OS细胞可以用于检测对潜在甜味呈味剂(tastant)的细胞响应。可以使用各种功能性试验,包括胞内钙浓度变化的测量(例如,基于荧光计成像平板阅读器的Ca2+动员试验,FLIPR)、pERK1/2激活(例如,高含量成像,HCI)和感受器内化(例如,transfluor)。在测试化合物的存在下通过甜味剂的甜味感受器活性的变化表明甜味感受器受到测试化合物的调节,因此鉴别甜味调节剂(例如,其自身提供甜味的分子、阻断一些或全部苦味的分子、增强另一种分子的甜味的分子)。甜味感受器活性的提高表明测试化合物是甜味调节剂。甜味调节剂可以包括在各种消耗品,包括食品、饮料和药物中。
可以用于所公开的试验中的其他细胞包括,但不限于,1A2,ARH-77,RWPE-1,WI-38,EJM,NCI-H1155,L-1236,NCI-H526,JM1,SHP-77,SNU-878,NCI-H2196,C3A,CA46,SNU-466,KS-1,SNU-738,MOLP-2,HDLM-2,Pfeiffer,HCC-15,Alexandercells,L-540,KMS-12-BM,JK-1,NCI-H1092,SW1990,NCI-H1184,SU-DHL-1,Hep3B2.1-7,P3HR-1,NCI-H2029,SU-DHL-5,SNU-1,MOLP-8,SUP-M2,MONO-MAC-1,SNU-1040,KYM-1,HEC-59,HCC1569,OCI-LY3,Hs819.T,DU4475,CI-1,S-117,OVCAR-8,SNU-626,HL-60,SUIT-2,T3M-4,RKO,MOR/CPR,DK-MG,GA-10,OCUM-1,HCT-15,HT,MONO-MAC-6,G-402,Toledo,COV362,SU-DHL-8,Daoy,NCI-H1435,LS513,Hs839.T,Hs172.T,BT-483,KMS-21BM,AGS,NCI-H2172,LC-1/sq-SF,SNU-201,NUGC-4,SK-HEP-1,SUP-B15,SNU-5,HT-1197,SUP-T1,AMO-1,KU812,AN3CA,AML-193,VMRC-RCW,HLE,HuH28,Hs751.T,NCI-H2110,MEG-01,MV-4-11,HepG2,KYSE-30,KALS-1,BICR6,RMUG-S,JHH-6,Ki-JK,IST-MES1,HCC-95,HPB-ALL,HSC-3,697,LOU-NH91,KARPAS-299,GI-1,COLO792,SK-N-FI,D341Med,HGC-27,SR-786,COLO-818,MHH-CALL-2,SF126,NCI-H322,A-253,NCI-H1623,MCF7,HCC-44,FU97,OCI-LY-19,Hs766T,NCI-H522,RL,HCC1428,RPMI6666,U-937,NCI-H460,SW1088,NCI-H1792,NCI-H1693,UACC-257,JHUEM-2,HuT78,UACC-893,NCI-H929,A-704,OV56,LN-229,OE19,SK-MEL-24,RD-ES,NCI-H211,KCI-MOH1,NCI-H1963,Hs706.T,ChaGo-K-1,EPLC-272H,OPM-2,KHM-1B,A549,HuG1-N,NCI-H508,MHH-CALL-3,SNU-1076,A3/KAW,MEL-HO,TO175.T,Caki-1,Hs936.T,SK-LU-1,WM-983B,K-562,EFE-184,SNU-520,NCI-H2291,HCC-1195,ABC-1,KE-39,NH-6,HCC2218,CMK,RS4;11,KYSE-450,OV7,KYSE-510,SK-UT-1,SNU-C1,OE33,P12-ICHIKAWA,DLD-1,COV434,HuNS1,SNU-899,SW480,COLO-678,LU99,KOPN-8,NCI-H2227,SW1463,Hs675.T,JHH-4,NCI-H1703,HEC-1-A,BDCM,MIAPaCa-2,PC-3,TE-15,PK-45H,MKN-45,HCC-366,CAL-29,HEC-50B,CPC-N,KMRC-20,SW1116,EOL-1,COLO205,EHEB,YD-38,MC116,SK-N-BE(2),BV-173,NCI-H2347,LU65,RT4,U-87MG,LK-2,KP-N-YN,HEC-251,NCI-H1651,GP2d,RERF-LC-MS,NB-4,NCI-H2286,SNU-61,T-47D,huH-1,KYSE-180,ST486,SW1353,M-07e,KASUMI-1,YH-13,NCI-H28,GAMG,JeKo-1,GOS-3,SNU-324,PA-TU-8902,MFE-280,SNU-245,NALM-1,RERF-LC-Sq1,BICR22,ZR-75-1,COR-L23,SW579,COR-L88,KM12,Hs611.T,OUMS-23,RERF-LC-Ad1,NCI-H1385,SK-LMS-1,COLO-320,BL-70,GRANTA-519,MCAS,Panc08.13,AM-38,KMS-11,SIG-M5,SNU-407,JHOS-2,OVCAR-4,Set-2,OV-90,MeWo,HEL,HT-29,MDA-MB-231,TOV-21G,NCI-H1355,KMS-27,NALM-6,KMS-26,Caov-4,KASUMI-2,UACC-62,U266B1,Hs695T,HT55,BICR31,TCC-PAN2,KMS-20,Hs578T,RI-1,Hs606.T,NCI-H1341,THP-1,BCP-1,Hs737.T,SW1417,MOLT-4,Raji,ESS-1,MEL-JUSO,SH-10-TC,Hs683,ME-1,EB2,PLC/PRF/5,NCI-H1339,A4/Fuk,SEM,HEC-265,IST-MES2,KE-97,NCI-H1437,COLO-704,NCI-H1915,TE-5,NCI-H2023,NCI-H82,T1-73,SNU-840,HuT102,NCI-H1944,KYSE-520,Kasumi-6,1321N1,Hs742.T,IM95,PL45,CL-40,WM1799,KMM-1,SNU-449,JHUEM-1,KARPAS-620,Loucy,SNU-1079,Daudi,HCC-56,HSC-2,COR-L47,PA-TU-8988S,OAW28,COR-L311,L-363,Malme-3M,NOMO-1,Hs870.T,SU-DHL-10,Hs229.T,NCI-H810,KYSE-410,RPMI-8402,SNU-175,EBC-1,RVH-421,K029AX,PA-TU-8988T,LXF-289,OVSAHO,CAL-12T,Hs940.T,MM1-S,SUP-HD1,LNCaPcloneFGC,HSC-4,NU-DHL-1,NCI-H2228,BEN,CAL-78,Sq-1,NCI-H1793,SNU-C2A,MDA-MB-134-VI,COV318,KE-37,TYK-nu,MOTN-1,T98G,SW837,EB1,Becker,PE/CA-PJ34(cloneC12),Hs616.T,NCI-H446,WM-88,CHP-126,Calu-1,SNU-283,NCI-H1573,SW1271,SNU-16,JHOS-4,ACHN,Calu-3,KMRC-1,SW1783,TE-11,TE-9,HuH-6,P31/FUJ,HT-1376,NCI-H520,786-O,KNS-60,Caki-2,OVK18,PL-21,NCI-H2452,JURL-MK1,TEN,JHH-7,MDA-MB-157,Calu-6,RKN,NUGC-2,ONS-76,J82,OUMS-27,SNU-1196,Hs739.T,RPMI-7951,NCI-H854,JHH-5,JVM-2,Hey-A8,5637,KYSE-140,Capan-2,KYSE-150,HEC-1-B,BICR16,HEL92.1.7,MHH-NB-11,SNU-387,SK-OV-3,SK-MEL-28,IGROV1,ML-1,HLF-a,CHL-1,YKG1,A-204,OCI-M1,8505C,JVM-3,NCI-H647,DB,COLO-800,PK-59,FaDu,HLF,OVMANA,EFO-27,PF-382,NCI-H747,LS123,SU-DHL-6,SJRH30,PANC-1,NCI-H2342,KM-H2,DND-41,HH,HuCCT1,F-36P,DMS454,Hs274.T,AU565,NCI-H1666,EN,RH-41,NCI-H1373,NCI-H838,SK-MEL-30,MOLM-6,DEL,NCI-H226,NCI-H1648,NCI-H661,143B,Mino,C32,KMS-34,NCI-H1694,SK-ES-1,UACC-812,GDM-1,NCI-H23,Panc02.03,CCF-STTG1,LOXIMVI,SJSA-1,MDST8,PK-1,NCI-H716,SU-DHL-4,MPP89,MJ,COLO829,PE/CA-PJ15,HD-MY-Z,BxPC-3,WM-793,COLO668,T84,JHOM-1,PEER,LS411N,GMS-10,KMBC-2,RMG-I,KELLY,SNU-761,NALM-19,HEC-151,G-361,OVTOKO,A-498,SW900,LCLC-103H,FTC-133,QGP-1,Reh,CMK-11-5,NU-DUL-1,BT-20,Hs600.T,Hs604.T,KATOIII,SNU-410,NCI-H2126,SK-MEL-5,MDA-MB-468,AsPC-1,HUP-T3,KP-N-SI9s,L-428,SNU-1105,HUP-T4,769-P,LMSU,NCI-H1869,NCO2,MOLM-16,CAL27,HCC70,NCI-H1930,COV644,Hs863.T,HCC-2279,D283Med,Hs944.T,HCC1599,MDA-MB-415,HCC2157,NCI-H1618,SNU-308,HCC1954,DMS153,HPAF-II,T24,CJM,VM-CUB1,UM-UC-3,LAMA-84,NCI-H1734,JHH-2,VMRC-RCZ,MFE-319,MDA-MB-453,SNU-503,TOV-112D,B-CPAP,GSU,HCC-78,NCI-H2171,CAMA-1,HEC-108,HCC4006,CAL-85-1,NCI-H2122,COLO-699,NCI-H196,LUDLU-1,SW780,RPMI8226,LP-1,PC-14,HuTu80,T.T,SW948,22Rv1,HARA,NCI-H596,IPC-298,SCaBER,NCI-H1838,NB-1,Hs934.T,Hs895.T,DMS114,KYSE-70,KP-3,KP4,DAN-G,NCI-H2009,OC316,SCC-25,U-138MG,RCC10RGB,MFE-296,NCI-H1755,RERF-LC-KJ,8305C,WSU-DLCL2,ES-2,MSTO-211H,SCC-15,ZR-75-30,PSN1,SNU-423,NCI-H2106,TE-1,UT-7,KMS-28BM,NCI-H2081,SK-MM-2,COLO741,OC314,HCC1395,MOLT-13,LN-18,Panc10.05,PE/CA-PJ41(cloneD2),Hs746T,CW-2,SKM-1,NUGC-3,TE-10,NCI-H358,NCI-H69,BFTC-909,HOS,BICR18,NCI-H1395,OVKATE,Hs698.T,EFM-19,COLO-783,MHH-CALL-4,ACC-MESO-1,NCI-H1436,KP-N-RT-BM-1,SK-MEL-31,NCI-H1105,CAL-51,YD-15,NCI-H2085,NCI-H2444,HCC1187,Hs939.T,CAL-120,SCC-9,TUHR14TKB,KMRC-2,KG-1-C,ECC10,CGTH-W-1,NCI-H841,C2BBe1,SUP-T11,RCH-ACV,CADO-ES1,JURKAT,647-V,SK-MEL-2,MDA-MB-175-VII,MKN74,SNU-C4,LCLC-97TM1,SCC-4,BHY,IGR-37,KYO-1,Hs281.T,TT,TUHR4TKB,HT-1080,NCI-H660,TE441.T,LS1034,KNS-42,Panc04.03,HCC1419,AZ-521,SNG-M,NCI-N87,G-292,cloneA141B1,KPL-1,MDA-MB-361,CL-14,NCI-H2170,HuH-7,RD,NCI-H2066,IGR-1,TE-14,VCaP,BL-41,SNU-620,SK-MES-1,MEC-2,NCI-H1299,IGR-39,RT112/84,SF-295,DV-90,A2780,BICR56,NCI-H510,NCI-H2141,YD-8,NCI-H2405,TF-1,MEC-1,CCK-81,NCI-H1048,Hs822.T,NCI-H2052,KO52,CAL-54,Hs840.T,SW620,SK-CO-1,BT-474,CL-11,KNS-62,NCI-H1650,G-401,MOLT-16,SNU-398,COLO-680N,EM-2,Hs294T,CAL-62,KMRC-3,A101D,KG-1,BT-549,HT115,A-375,SW-1710,WM-115,KLE,JHUEM-3,MKN7,CHP-212,HCC202,BC-3C,NCI-H1568,KMS-18,PE/CA-PJ49,COLO-849,SIMA,OCI-AML3,GSS,EC-GI-10,EFO-21,RCM-1,DMS273,KU-19-19,RERF-GC-1B,SH-4,SK-MEL-3,RERF-LC-Ad2,M059K,JHOM-2B,MDAPCa2b,Hs852.T,RL95-2,Panc03.27,SNU-216,Panc02.13,CFPAC-1,SK-N-SH,OCI-AML2,LoVo,SBC-5,NCI-H1876,NCI-H441,SK-N-AS,COR-L24,HCC38,NCI-H1781,DOHH-2,NCI-H1563,U-251MG,HPAC,JIMT-1,U-2OS,A-673,TC-71,NCI-H650,NIH:OVCAR-3,CAS-1,JL-1,SK-MEL-1,MDA-MB-435S,Ishikawa(Heraklio)02ER-,TE617.T,SU.86.86,RERF-LC-AI,TT2609-C02,LS180,YAPC,HDQ-P1,KNS-81,FU-OV-1,KP-2,DMS53,SNU-1272,Detroit562,42-MG-BA,L3.3,COLO-679,NCI-H2087,NCI-H2030,GCT,NCI-H889,Caov-3,MDA-MB-436,NCI-H524,MKN1,KCL-22,Capan-1,CML-T1,H4,NCI-H727,Hs343.T,MHH-ES-1,NMC-G1,HCC-1171,REC-1,Hs618.T,A172,YD-10B,SW48,MUTZ-5,TE-6,JHH-1,HCT116,TE-4,IA-LM,MG-63,NCI-H1975,TALL-1,HCC1806,HMCB,SCLC-21H,HCC1500,CL-34,Panc05.04,SW403,TM-31,HCC1937,JMSU-1,DMS79,SNB-19,NCI-H1836,Li-7,HCC827,639-V,MOLM-13,SK-BR-3,IMR-32,TUHR10TKB,OAW42,SK-N-MC,TGBC11TKB,NCI-H1581,EFM-192A,YMB-1,HCC2935,ECC12,HCC-33,DU145,NCI-H146,SNU-1214,SNU-1077,23132/87,HT-144,SNU-182,Hs888.T,SNU-475,GCIY,Hs729,JHOC-5,SW1573,HEC-6,OCI-AML5,Hs688(A).T,Hs821.T,PCM6,RT-112,SK-N-DZ,SNU-478,SNU-119,HCC1143,NCI-H209,8-MG-BA,COR-L105,COR-L95,SNU-46,COV504,CAL-148,SNU-C5,DBTRG-05MG,BHT-101,WM-266-4,BFTC-905,KYSE-270,TE-8,SNU-213和SH-SY5Y。
测试化合物
测试化合物可以是天然产生的或合成产生的。蛋白质、多肽、肽、多糖、小分子和无机盐是可以使用本文中公开的方法筛选的测试化合物的实例。
可以将编码这些蛋白质的任何核酸分子引入细胞中。核酸可以被稳定地或瞬时地引入细胞中。外源性核酸可以在包含可操作地连接核酸编码部分的启动子的构建体或载体中。
细胞可以在合适的基质,如多孔平板、组织培养皿、烧瓶等上生长;参见以下的实施例1和2。
筛选方法
在一些实施方案中,鉴别甜味调节剂的方法包括使细胞与测试化合物接触并测量甜味感受器活性。甜味感受器活性因测试化合物的变化表明甜味感受器受到测试化合物的调节,由此将测试化合物鉴别为甜味调节剂。
可以通过本领域标准的任何方式来定量或定性地测量甜味感受器活性,包括用于G-蛋白耦合感受器活性的试验、细胞中第二信使水平的变化、通过磷脂酰肌醇的磷脂酶C-介导的水解的三磷酸肌醇(IP3)的形成、细胞质钙离子水平的变化、β-拘留蛋白募集等。在以下特定的实施例中提供了这样的试验的实例。
结合活性还可以用于测量味觉感受器活性,例如,通过竞争性结合试验或表面等离波子共振。如本领域已知的,还可以测量感受器内化和/或感受器脱敏。还可以测量基因转录的感受器依赖性激活,以评估味觉感受器活性。可以通过使用本领域技术人员已知的任一种方法来测量转录的量。例如,可以使用PCR技术、微阵列或Northern印迹来检测目标蛋白的mRNA表达。可以通过本领域中标准的用于定量细胞中的蛋白质的方法,如Western印迹、ELISA、ELISPOT、免疫沉淀、免疫荧光(例如,FACS)、免疫组织化学、免疫细胞化学等,以及现在已知的或之后为定量细胞中或由细胞产生的蛋白质研发的任何其他方法来测定由mRNA产生的多肽的量。
还可以测量细胞的物理变化,例如,通过显微镜评价由味觉感受器激活介导的大小、形状、密度或任何其他物理变化。流式细胞术也可以用于评估物理变化和/或确定细胞标志物的存在或不存在。
在一些实施方案中,通过检测细胞中胞内第二信使(例如,cAMP、cAMP、cGMP、NO、CO、H2ScGMP、DAG、IP3)的水平来测量甜味感受器活性。在一些实施方案中,通过检测胞内钙的水平来测量甜味感受器活性。在一些实施方案中,甜味感受器活性是结合活性。
在这些实施方案的任何一个中,细胞可以被修饰以超表达甜味感受器和或Gα15,如下所述。
本领域技术人员将认识到存在各种落入所附权利要求范围内的上述实施方案的变化和排列。
实施例1
JUMP-INTMT-REXTMU2-OS细胞系和可靶向JUMP-INTMU2-OS细胞系的产生
JUMP-INTMT-REXTMU2-OS细胞系的产生。使用LIPOFECTAMINETMLTX试剂(LifeTechnologies#15338-100),用pLentiTRpuro载体转染JUMP-INTMU2-OS细胞。用1μg嘌呤霉素(LifeTechnologies#A1113803)选择了转染的细胞,并且将未转染的亲本细胞用作抗生素选择阴性对照。
将选定的克隆扩增并用pJTITM-R4EXPCMVTOGFPpA载体(LifeTechnologies)瞬时转染。然后测试细胞的tet-抑制和tet-诱导性,使用或未用1μg强力霉素,并且用SafireII微平板阅读器定量GFP表达水平。
在FACS分选后八十四个克隆存活。将每个克隆重复三份涂布于96-孔平板中。一个孔用于维持,而另两个孔用于瞬时转染,使用编码绿色荧光蛋白(GFP)的pJTITMR4EXPCMV-TOpA表达载体(LifeTechnologies),持续24小时。然后使用或未用1μg强力霉素处理细胞24小时,接着GFP定量。按照
计算响应比。
进一步扩增具有超过2的响应比的十二个JUMP-INTMT-REXTMU2-OS克隆。用R4整合酶构建体和pJTITM-R4EXPCMV-TOGFP载体(提供诱导型GFP表达)共同转染这些克隆中的五个。将用CMV-GFP构建体(提供组成型GFP表达)和R4整合酶构建体共同转染的细胞用作用于转染和抗生素选择的阳性对照。用5μg/mL杀稻瘟菌素选择28天后,用1μg/mL强力霉素诱导用pJTITM-R4EXPCMV-TOGFP载体重新靶向的细胞诱导20小时,并随后通过FACS分析。
可靶向JUMP-INTMU2-OS细胞系的产生。使用JUMP-INTMTITM平台试剂盒(LifeTechnologies)将可靶向JUMP-INTM细胞系工程化。使用pJTITM/杀稻瘟菌素靶向载体(其含有ATT位点,其与哺乳动物宿主基因组中的假-ATT位点互补),将pJITMPhiC31Int载体共同转染至U2-OS宿主细胞中。通过phiC31整合酶促进R4载体的ATT位点与宿主基因组之间的同源重组。pJITM靶向载体含有潮霉素抗性基因,其允许选择含有稳定基因组整合的细胞。然后通过流式细胞术克隆这些细胞并测试,以通过Southern印迹分析测定宿主细胞基因组中存在的R4位点的数量。
确认具有验证过的单个R4整合位点的克隆,用于通过使用pJTITM-R4EXPCMV-TO-EmGFP-pA和JTITMR4整合酶载体转染的重新靶向,接着抗生素选择4周。将克隆以60-80%汇合涂布于6-孔培养皿中的不含抗生素的生长培养基(McCoy’s5A加10%渗析的胎牛血清,HEPES,非必需氨基酸和丙酮酸钠)中,并用1:1比例的pJTITM-R4EXPCMV-TO-EmGFP-pA和JTITMR4整合酶载体(总的2.5μgDNA)转染,使用LIPOFECTAMINETMLTX(6.25μl)和PLUSTM试剂(2.5μl)。将CMV-GFP阳性克隆平行地引入细胞中。过夜孵育后,用生长培养基(McCoy’s5A加10%渗析的FBS,HEPES,非必需氨基酸,丙酮酸钠和青霉素/链霉素)中的5μg/mL杀稻瘟菌素,将细胞选择28天。抗生素选择28天后,通过FACS分析了选定的集合。
选择五个克隆来测试重新靶向效率和可诱导潜能。所有克隆成功重新靶向。进一步测试具有最佳重新靶向效率的两个克隆通过强力霉素的诱导潜能。一个克隆在GFP重新靶向的对照细胞中证明了~88%GFP细胞,并且对强力霉素诱导响应非常好,在诱导的培养物中具有>99%GFP阳性群,而在非诱导的培养物中仅有~4%GFP阳性细胞。另一个克隆也很好地响应了强力霉素诱导,在诱导的培养物中具有>99%GFP阳性细胞,而在非诱导的培养物中仅有~8%GFP阳性细胞;在GFP重新靶向的对照细胞中64%的细胞是GFP阳性。
细胞培养基。下表列出了用于JUMP-INTMT-REXTMU2-OS细胞培养物(LifeTechnologies)的培养基的组分:
细胞培养基和试剂 | 量 | 目录号 |
McCoy’s 5A培养基(改良)(1x),液体 | 500mL | 16600-082 |
胎牛血清(FBS),渗析的 | 100mL | 26400-036 |
青霉素/链霉素,10,000U/10,000pg | 100mL | 15140-122 |
潮霉素B(50mg/mL) | 20mL | 10687-010 |
二盐酸嘌呤霉素(10mg/mL) | 1mL | A11138-03 |
不含钙或镁的磷酸盐缓冲盐水 | 1000mL | 14190-136 |
实施例2
方法
细胞培养物和化合物处理。将U2-OS细胞生长在补充了10%胎牛血清的McCoy培养基中。将细胞以5,000细胞/孔的密度接种于PDL覆盖的384-孔平板上。
BacMam转染。在24-48小时期间用0.5-12μl编码G蛋白的BacMam瞬时转染U2-OS细胞,gustducin(Gα15,Ga16gust44)/40,000细胞。将转染的细胞以5,000细胞/孔的密度接种于PDL-覆盖的384-孔平板上。
功能性表达。用pJTITMR4EXP-CMV-TO-T1R3/T1R2/Gα15-pA和JTITMR4整合酶载体(LifeTechnology)转染JUMP-INTMT-REXTMU2-OS细胞。通过RT-PCR检测强力霉素诱导的T1R3/T1R2/Gα15的表达。
pERK1/2试验。对于pERK1/2研究,用不含血清的培养基替代生长培养基2小时。用100mM甜味化合物处理U2-OS细胞,使用或未用U73122(10μM)和使用或未用lactisole(20mM)。对照组细胞还接受了培养基中的DMSO(0.1%)。用来自CellSignalingTechnologies的兔抗-磷-p44/p42MAPK(pERK1/2)抗体,然后用Alexa488-缀合的二抗(LifeTechnology)和Hoechst33342,将固定的细胞染色。在ImageXpressMicro(MDC)上获取图像并用MWCS模块分析。
钙试验。在FLIPRTetra(MDC)中记录随着时间的Fluo-4AM加载的细胞中的钙痕迹,使用λEX=470-495nm和λEM=515-575nm。测定超过基线的荧光的峰提高并且表达为相对荧光单位(RFU)。
Transfluor模型。用味觉G蛋白,gustducin(Gα15或Gα16gust44)(LifeTechnologies)瞬时转染稳定表达β-拘留蛋白-GFP的TransfluorU2-OS细胞或用甜味感受器T1R2/T1R3和味觉G蛋白瞬时转染。用甜味化合物处理细胞诱导了荧光泡囊的形成。通过MetaXpress软件的TransfluorApplication模块(MDC)定量甜味感受器内化。
实施例3
ERK1/2的磷酸化
ERK1/2的磷酸化是通过PLCβ2-IP3途径的GPCR激活后的共同下游事件。用不含血清的Dulbecco’s磷酸盐缓冲盐水(DPBS)替代生长培养基2小时。用100mMD-葡萄糖处理U2-OS细胞,使用或未用20mMlactisole,持续5、15和30分钟,然后固定并用抗-pERK1/2和Hoechst33342染色,接着成像。使用MolecularDevices’Multiwaves细胞分选分析算法定量pERK1/2表达。对于每个数据点,从一式四份数据点计算平均细胞累积强度的平均和标准偏差。D-葡萄糖在亲本U2-OS(图4,左)和超表达Gα15的U2-OS细胞(图5,左)中以时间依赖性方式诱导pERK1/2激活。重要地,通过甜味感受器拮抗剂,lactisole(图4,右)或PLCβ2抑制剂U73122(图5,右)阻断pERK1/2活性。
实施例4
Gα15的超表达
U2-OS细胞中Gα15的超表达显著提高了响应甜味调节剂的信号传输。三氯蔗糖以浓度依赖性方式诱导了用Gα15转染的U2-OS细胞系中的Ca(2+)响应(图2)。我们观察到用12μl/40,000细胞转染的U2-OS细胞中最高的Ca(2+)响应(图2)。在用天然糖,如葡萄糖、果糖和蔗糖处理时,用6μl/40,000细胞转染U2-OS显著提高了Ca(2+)响应(图3)。此外,在U2-OS细胞中用葡萄糖或麦芽糖处理时,Gα15的超表达提高了pERK1/2活性(图6)。重要地,pERK1/2激活被PLCβ2抑制剂U73122阻断了(图7)。
实施例5
Ca(2+)响应
我们已经发现了单糖和双糖诱导了具有甜味感受器T1R2/T1R3/Gα15的强力霉素诱导表达的JUMP-INTMT-REXTMU2-OS细胞系中的浓度依赖性Ca(2+)响应(图1A)。T1R2/T1R3-介导的Ca(2+)释放受到lactisole抑制(图1B)。此外,用D-葡萄糖和D-果糖处理时,U73122消除了Ca(2+)释放并且在用蔗糖和三氯蔗糖刺激后对Ca(2+)释放无作用(图1B)。
总之,这些数据证明了糖的分子结构和T1R2/T1R3甜味感受器之间的关联。
实施例6
人造甜味剂
使用人造甜味剂,Ace-K、阿斯巴甜和糖精进行了一些惊人的观察。Ace-K和阿斯巴甜没有诱导JUMP-INTMT-REXTMU2-OS细胞中的T1R2/T1R3-介导的Ca(2+)响应(图1A)。用糖精处理时发生了T1R2/T1R3-依赖性Ca(2+)释放(图1A),表明人造甜味剂Ace-K、阿斯巴甜和糖精靶向尚未鉴别的共同感受器。
实施例7
甜味化合物对募集β-拘留蛋白至甜味感受器的作用
多个系的证据表明GPCR配体选择性地激活β-拘留蛋白信号传输途径。为了评估甜味化合物对募集β-拘留蛋白至甜味感受器的作用,我们使用了Transfluor模型(MDC),其对β-拘留蛋白重新分配是高度敏感的。用甜味化合物处理细胞诱导了荧光泡囊的形成,表明T1R2/T1R3-β-拘留蛋白复合物内化至内体中(图8A)。通过MetaXpress软件的TransfluorApplication模块(MDC)定量甜味感受器内化。D-葡萄糖、D-果糖和Ace-K略微提高了甜味感受器内化,而蔗糖、三氯蔗糖和阿斯巴甜对T1R2/T1R3内化具有强烈作用(图8B)。相反,甚至用糖精处理时没有发生最小内化(图8B)。
Claims (19)
1.鉴别甜味调节剂的方法,包括:
a)使U2-OS细胞与测试化合物接触;和
b)测量甜味感受器活性,其中甜味感受器活性因测试化合物的变化表明甜味感受器受到测试化合物的调节,由此将测试化合物鉴别为甜味调节剂。
2.权利要求1的方法,其中U2-OS细胞被修饰以超表达甜味感受器。
3.权利要求1的方法,其中通过检测U2-OS细胞中胞内第二信使的水平来测量甜味感受器活性。
4.权利要求3的方法,其中第二信使是cAMP、cGMP、NO、CO或H2S。
5.权利要求3的方法,其中第二信使是DAG或IP3。
6.权利要求1的方法,其中通过检测细胞中的胞内钙的水平来测量甜味感受器活性。
7.权利要求1的方法,其中所述甜味感受器活性是结合活性。
8.权利要求7的方法,其中通过竞争性结合试验来检测结合活性的变化。
9.权利要求7的方法,其中通过表面等离波子共振来检测结合活性的变化。
10.权利要求1的方法,其中通过检测ERK1/2的磷酸化来测量甜味感受器活性。
11.权利要求1的方法,其中通过检测感受器的内化来测量甜味感受器活性。
12.权利要求1-11任一项的方法,其中U2-OS细胞进一步包括编码Gα15、Gα16、Gα16gust25、Gα15gust25、Gα16gust44、Gα15gust44或Gα15-i/3-5的外源性核酸。
13.权利要求1-12任一项的方法,其中U2-OS细胞包括Tet-阻遏蛋白表达构建体。
14.分离的U2-OS细胞,包括编码Gα15、Gα16、Gα16gust25、Gα15gust25、Gα16gust44、Gα15gust44或Gα15-i/3-5的外源性核酸。
15.分离的U2-OS细胞,包括一个或多个编码甜味感受器的外源性核酸。
16.分离的U2-OS细胞,包括编码检测标记的外源性核酸。
17.权利要求16的分离的U2-OS细胞,其中所述检测标记是β-拘留蛋白GFP。
18.权利要求14或15的分离的U2-OS细胞,进一步包括编码检测标记的外源性核酸。
19.权利要求14-16任一项的分离的U2-OS细胞,进一步包括编码Gα15、Gα16、Gα16gust25、Gα15gust25、Gα16gust44、Gα15gust44或Gα15-i/3-5的外源性核酸。
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HK1218301A1 (zh) | 2017-02-10 |
JP6367315B2 (ja) | 2018-08-01 |
CA2912023A1 (en) | 2014-11-13 |
US10107794B2 (en) | 2018-10-23 |
AU2014262543B2 (en) | 2017-07-20 |
AU2014262543A1 (en) | 2015-11-26 |
EP2994477A4 (en) | 2017-03-22 |
WO2014183044A1 (en) | 2014-11-13 |
US20160091488A1 (en) | 2016-03-31 |
RU2644222C2 (ru) | 2018-02-08 |
RU2015152840A (ru) | 2017-06-16 |
JP2016519931A (ja) | 2016-07-11 |
MX2015015451A (es) | 2016-09-09 |
CA2912023C (en) | 2018-10-23 |
RU2018100104A (ru) | 2019-02-20 |
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