CN105602904B - Cd133质粒的新用途 - Google Patents
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Abstract
本发明提供了CD133质粒的新用途，所述CD133质粒可有效用于增强DC‑CIK对乳腺癌干细胞的体外杀伤作用。
Description
技术领域
本发明涉及CD133质粒的新用途。
背景技术
乳腺癌是世界范围内女性最常见的恶性肿瘤。大量证据表明正是由于乳腺癌干细胞(Breast Cancer Stem Cell,BCSC)的存在导致其容易转移，复发且对放化疗抵抗。而近年来兴起的过继细胞免疫疗法(adoptive cell immunotherapy,ACI)是一种针对放化疗不敏感肿瘤的新疗法，临床前景广阔，获得越来越多的重视。
CSC的存在是影响乳腺癌治疗效果和预后最重要的因素之一，目前临床上应用的放化疗通常只能消除普通乳腺癌细胞，而对放化疗抵抗的残存BCSC仍能不断增殖，导致乳腺癌复发转移，所以寻找一种行之有效的杀灭CSC的方法成为乳腺癌治疗的新方向。ACI是一种依赖自体效应性免疫细胞的体外扩增进行肿瘤细胞有效杀灭的新型疗法，其首先应用于恶性白血病的治疗，后逐步推广至包括乳腺癌、乳腺癌、肺癌等在内的实体肿瘤。ACI的效应细胞主要包括细胞因子诱导的杀伤细胞(CIKs)，淋巴因子激活的杀伤细胞(lymphokine-activated killer cells,LAKs)，肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TILs)、细胞毒性T淋巴细胞(cytotoxic T lymphocyte cells,CTLs)及新近提出的嵌合抗原受体的T细胞(chimeric antigen receptor T cells,CAR-T)等。而作为体内功能最强大的专职抗原提呈的DC也是ACI重要的组成部分。
CIK细胞具有增殖快速、杀瘤力强、杀瘤谱广、对正常细胞无杀伤作用、对耐药肿瘤敏感等优点，是目前已知的活性最高的非特异性杀伤免疫效应细胞。DC能够将肿瘤抗原提呈给免疫活性细胞，促进其增殖活化，增强对肿瘤细胞的杀伤活性。有研究表明，负载肿瘤干细胞抗原的DC免疫表型较前成熟，而将其与CIK细胞共同培养后，DC-CIK免疫表型进一步成熟且分泌细胞因子水平相应提高。
CD133是乳腺癌干细胞最重要的分子标记，它是一种单跨膜糖蛋白，通过与多种配体的结合在细胞粘附和迁移等行为中发挥关键作用。
发明内容
本发明所要解决的技术问题在于提供CD133质粒的新用途。
为解决上述技术问题，本发明的技术方案是：
CD133质粒在增强DC-CIK对乳腺癌干细胞的体外杀伤作用方面的应用。
优选的，上述CD133质粒的应用，以CD133质粒冲击DC(dendritic cell)后与CIK(cytokine-induced killer)细胞联合培养使DC-CIK对乳腺癌干细胞的体外杀伤效果增强。
优选的，上述CD133质粒的应用，所述CD133质粒的部分核苷酸序列为SEQ ID NO：1所示序列。
本发明的有益效果是：
CD133质粒在增强DC-CIK对乳腺癌干细胞的体外杀伤作用方面具有重要应用价值。
附图说明
图1CSC-RNA诱导的树突状细胞；
图2CSC-裂解物抗原诱导的树突状细胞；
图3辅助质粒和目的质粒共转染293T 24h和48h的绿色荧光强度；
图4用浓缩的慢病毒去侵染293T细胞，12h换液，48后的荧光强度。
具体实施方式
为了使本领域的技术人员更好的理解本发明的技术方案，下面结合具体实施方式对本发明所述技术方案作进一步的详细说明。
实施例1
一、乳腺癌MCF-7细胞干细胞全细胞裂解物的制备
收集乳腺癌MCF-7细胞及其干细胞细胞、分别调整细胞浓度至1×107(MCF-7细胞及其干细胞细胞：DC细胞＝10:1)，转至冻存管中，将冻存管放入液氮15min，取出后立即放入37℃水浴锅中15min，反复4次，裂解后，4℃离心，10000g离心10min，收集上清，0.22um滤膜过滤除菌，存于-80℃备用。用BCA法测定上述裂物蛋白浓度的，按BCA试剂盒说明书进行蛋白浓度测定。
乳腺癌MCF-7细胞干细胞总RNA的提取
按照Qiagen Rneasy mini kit试剂盒说明书操作。收集对数期生长的MCF-7细胞及磁珠分选的MCF-7干细胞，计数不少于1×107个，移至无RNase的1.5ml EP管中，加入600μl Buffer RLT，用1ml无菌注射器反复吹打5-8次至细胞完全裂解，然后，加入600μl 70％乙醇混匀，8 000g离心15s，弃流穿液，加入700μl RW1Buffer至收集柱中，8 000g离心15s洗涤收集柱，再加入含有10μl DNase的Buffer RDD 80μl于收集柱中20-30℃孵育15min彻底消化基因组DNA。加入350μl RW1 Buffer至收集柱中，8 000g离心15s洗涤收集柱，然后加入500μl RPE Buffer至收集柱中，8 000g离心2min彻底洗涤收集柱，弃流穿液。将收集柱置于新的1.5ml EP管中，加入30-50μl无RNase的去离子水，离心8 000g离心1min，收集洗脱下来的RNA。Nanodrop 2000分光光度计检测提取的RNA浓度。
CD133慢病毒表达体系的构建
1.1.1.1引物设计
以pCDNA3.1+CD133(s16-s24)为模板
引物：CD133F:5-CGGAATTCATGGGTGCAAATGTGGA-3′(序列表<400>2所示序列)
CD133R:5-ATAAGAATGCGGCCGCTCACAAGGGGTCGATAATGTAGC-3
(序列表<400>3所示序列)
1.1.1.2PCR扩增
反应体系：按顺序加入以下7种成分，混匀：
反应程序：
PCR产物胶回收，用EcoRI和NotI双酶切后与我们提供的过表达对照载体相连接。
1.1.1.3酶切
片段酶切：
37℃消化2-3h。电泳切胶回收(酶切回收试剂盒，TaRaKa)
载体酶切：
37℃消化2-3h。电泳切胶回收(琼脂糖凝胶DNA回收试剂盒，BPI)
1.1.1.4连接、转化
(1)连接：1μl载体，3μl酶切回收产物，1μl连接酶(BPI)，5μl 2×Rapid Buffer，混匀，室温反应30min。
(2)转化：取出－80℃保存的100μl感受态细胞(BL21)，放在冰上缓慢解冻。将感受态细胞加入连接产物中混匀，冰上放置30min。42℃热激90s。冰浴2min后，加入800μl无抗性的LB培养基。37℃培养45min。5000rpm离心3min，弃大部分上清，留约100-150μl，重悬菌体，选择有相应抗性的LB平板，涂板。晾干，于37℃培养箱中倒置培养过夜。筛选阳性克隆，质粒提取，并测序。
1.1.1.5质粒提取(去内毒素)
将带有质粒的E.coli接种于200ml LB/抗生素培养液中，37℃搖床培养12h；取200ml的菌液，室温下5000g离心10min收集细菌。倒弃培养基。加入10ml Solution I/RNaseA混和液，漩涡振荡使细胞完全悬浮。往重悬混和液中加入10ml Solution II，轻轻颠倒混匀7-10次后可将混合液室温放置2min以提高产量。避免剧烈混和裂解液且裂解反应不要超过5min。加入5ml冰浴Buffer N3，并温和颠倒离心管10次至形成白色絮狀沉淀。准备好过滤器。把HiBind中量柱套在收集管中，加入3ml Buffer GPS至柱子中，静置3-5min。室温5000g离心5min。去滤液，把柱子重新放回收集管中。加入10ml无菌水，5000g离心5min。将裂解液倒进预先准备好的针筒过滤器中(针筒开口朝上，下面出口处接收集管)，静置2min。插入并轻推活塞使裂解液流进下面的收集管中。在过滤澄清的裂解液中加入1/10体积的ETR，颠倒7-10次后溶液变得浑浊，冰浴20min。42℃水浴5min后，25℃，5000g离心5min，ETR溶液将在管底形成蓝色分层。离心后，如果溶液中有大量的液滴悬浮，静置10min让液滴自然沉降。转移上清液移至离心管中，加入0.5倍体积无水乙醇(室温)，混匀后室温静置2min。将HiBind大量柱装在50ml收集管中。转移20ml混合液至柱子內，室温下5000g离心5min，倒去滤液。重复该步骤，把剩余的过滤液转移至柱子，直至所有的溶液都从柱子滤出。把柱子重新装回收集管，加入10ml HB Buffer，按上述条件离心，弃滤液。把柱子重新装回收集管，加入15ml DNA Wash Buffer，按上述条件离心，弃滤液，重复一次。把柱子重新装回收集管，最大速度(<6000g)离心15min以甩干柱子基质。把柱子装在干净的50ml离心管上，加入70℃预热3mlEndotoxin free Elution Buffer到柱子基质上(所加的量取决于预期终产物浓度)，室温下静置5min后4000g速度离心5min以洗脱DNA。取10μl用微量核酸定量仪定量。
1.1.1.6慢病毒包装
用明胶(0.2％)包被10cm dish在4℃下孵育过夜。胰酶消化前期培养的293T细胞，均匀铺板待细胞融合度达到70％-90％方可进行包装。小心吸取细胞培养基上清液，加入6mL无血清无双抗DMEM，放入培养箱待用。制备转染试剂与DNA复合物，取无菌无酶EP管2只，其中1只分别加入1号辅助质粒6.5μg，2号辅助质粒2.5μg，3号辅助质粒3.5μg，目的质粒15μg(若包装对照慢病毒将目的质粒更换为对照质粒即可)另一只EP管加入55μl转染试剂(1mg/ml)。使用无菌过滤PBS或DMED分别将两只EP稀释至500μl，吸取转染试剂500μl加入4质粒中轻柔吹打混匀(注：切记不可涡旋，不可将加入顺序颠倒)。静止放置20min，快速逐滴加入待用的培养皿中，8字混匀培养皿后放入培养箱。6h后换新鲜完全培养基12mL。换液后48h收集上清，1000g 10min离心或0.45μm无菌滤器过滤。
1.1.1.7慢病毒浓缩PEG-8000：
5倍浓度PEG8000，121℃30min湿热灭绝30min；保存在4℃；使用0.45μm滤头过滤慢病毒上清液；每30ml过滤后的病毒初始液，加入5倍浓度PEG-8000加NaCl母液7.5ml；每30min混合一次，共进行3-5次；4℃放置过夜；4℃，4000g，离心20min；吸弃上清，静置管子2min，吸走残余液体；加入适量的(10ml对应100μl)慢病毒溶解液(PBS)溶解慢病毒沉淀；浓缩后的病毒悬液分装成50μl每份，保存在成品管中，用碎干冰速冻后储存在-80℃。
1.1.1.8病毒低度检测：利用GFP荧光细胞数进行病毒滴度测定
滴度单位：TU/ml，指每毫升中含有的具有生物活性的病毒颗粒数。“TU”为“transducing units”的缩写，中文为“转导单位”，表示可以感染并进入到靶细胞中的病毒基因组数。
将生长状态良好的293T细胞消化,按照一传六的稀释比例加入6孔板，2ml/孔。放入37℃，5％CO2培养箱中培养。加病毒，汇合度达到20％-50％，加入稀释病毒，稀释比例(1:500)。用完全培养基稀释，每孔培养基体积1ml。同时选取3个空白孔，进行细胞计数。追加培养液，12h后吸去带有病毒的培养液，在每个孔中再加入2ml新鲜完全培养液，以利于细胞的生长。流式测阳性率并计算滴度，48h-72h后消化细胞，PBS清洗两次，流式测定GFP阳性率，MOI指感染复数(批一个细胞被几个病毒感染)
滴度(TU/ml)＝细胞数×荧光百分比×MOI×病毒稀释倍数
1.1.1.9DC的制备与鉴定
DC细胞培养：分离健康人的白细胞50ml，收集贴壁细胞，加入含有IL-41 000U/ml、GM-CSF 1 000U/ml及10％小牛血清1640培养基中培养。于培养的第5天用不同冲击物冲击DC，实验组为MCF-7细胞干细胞(CSC)构建肿瘤特异性生物标记CD133重组质粒转染CD133-DC-CIK(MOI＝80)、MCF-7细胞干细胞CSC-RNA(RNA浓度分别为20ug)及CSC-裂解物(CSC：DC细胞为10:1)，以及MCF-7细胞MCF-7-RNA提取物(RNA浓度分别为20ug)、MCF-7细胞裂解物(MCF-7细胞：DC细胞分别为10:1)；对照组为直接培养的DC细胞。培养的第6天，分别向实验组和对照组中加入TNF-α10ng/ml，再继续培养2-3d得到成熟DC细胞。
DC细胞鉴定：分别收集培养第0、5、8天DC细胞，200g/min离心3min；用PBS洗细胞2遍，计数；PBS调整细胞浓度至1×105/100μl，设定空白管和实验管，每组设3个复管；分别加入鼠抗人CD40抗体、鼠抗人CD80抗体、鼠抗人HLA-DR抗体、鼠抗人CD86抗体，充分混匀，4℃避光孵育30min；孵育结束后，用1-2ml PBS重悬细胞，218g/min离心3min，重复1次；弃上清，用1ml PBS重悬细胞，充分混匀，置于4℃冰盒中；流式细胞仪检测，记录结果。
表1DC免疫表型分析(n＝3,％,)
*P<0.01
CIK细胞的制备与鉴定
1.1.1.10CIK细胞培养
收集上述非贴壁细胞，加入含有IFN-γ50ng/m的10％小牛血清1640培养基中。24h后加入IL-2500U/m、IL-1α5ng/ml、抗体(如，CD3)50ng/ml每2-3天半量换液。于培养的第8天将CIK细胞于DC细胞混合培养，共培养过程每2天半量换液，并补加IL-2，至14天CIK细胞成熟。
表2CIK细胞免疫表型分析(n＝3,％,)
*P<0.01
二、CD133质粒的序列(DNA序列)，部分核苷酸序列为SEQ ID NO：1所示序列：
ATGGGTGCAAATGTGGAAAAACTGATCTGTGAACCTTACACGAGCAAGGAATTATTCCGGGTTTTGGATACACCCTACTTACTAAATGAAGACTGGGAATACTATCTCTCTGGGAAGCTATTTAATAAATCAAAAATGAAGCTCACTTTTGAACAAGTTTACAGTGACTGCAAAAAAAATAGAGGCACTTACGGCACTCTTCACCTGCAGAACAGCTTCAATATCAGTGAACATCTCAACATTAATGAGCATACTGGAAGCATAAGCAGTGAATTGGAAAGTCTGAAGGTAAATCTTAATATCTTTCTGTTGGGTGCAGCAGGAAGAAAAAACCTTCAGGATTTTGCTGCTTGTGGAATAGACAGAATGAATTATGACAGCTACTTGGCTCAGACTGGTAAATCCCCCGCAGGAGTGAATCTTTTATCATTTGCATATGATCTAGAAGCAAAAGCAAACAGTTTGCCCCCAGGAAATTTGAGGAACTCCCTGAAAAGAGATGCACAAACTATTAAAACAATTCACCAGCAACGAGTCCTTCCTATAGAACAATCACTGAGCACTCTATACCAAAGCGTCAAGATACTTCAACGCACAGGGAATGGATTGTTGGAGAGAGTAACTAGGATTCTAGCTTCTCTGGATTTTGCTCAGAACTTCATCACAAACAATACTTCCTCTGTTATTATTGAGGAAACTAAGAAGTATGGGAGAACAATAATAGGATATTTTGAACATTATCTGCAGTGGATCGAGTTCTCTATCAGTGAGAAAGTGGCATCGTGCAAACCTGTGGCCACCGCTCTAGATACTGCTGTTGATGTCTTTCTGTGTAGCTACATTATCGACCCCTTGTAA
三、结果
DC细胞的形态
培养第5-7天DC细胞，体积增大，出现贴壁细胞，数量增多，多形性细胞增多。第5天分别用本研究制备的抗原进行冲击，培养5d后大部分细胞聚集成团，胞体较大，形状由开始时的圆形变成不规则形，培养7d后细胞具有树突状突起的细胞增多且突起更为明显。电镜下呈现典型的DC形态，比淋巴细胞体积大，胞浆突起明显，呈树突样生长，见图1、图2。
CD133慢病毒感染结果
CD133慢病毒感染后，pLenti-CMV-CD133-EF1-copGFP的阳性率为69.7％(图3、4)，pLenti-CMV-CD133-EF1-copGFP的滴度范围为5.12×108TU/ml-15.37×108TU/ml。
四、不同DC-CIK细胞体系对乳腺癌MCF-7细胞及其干细胞的体外杀伤实验
实验组为CD133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组三组，对照组为CIK和DC-CIK组，选择上述细胞作为效应细胞，乳腺癌MCF-7细胞及其干细胞为靶细胞，采用乳酸脱氢酶(LDH)释放法进行杀伤实验。
乳酸脱氢酶释放法：调整效应细胞浓度为1×106/mL，靶细胞浓度为1×105/mL。按照效靶比10:1、20:1、40:1、80:1比例加于96孔板中，每一效靶比下，设3个复孔。同时设效应细胞自然释放孔、靶细胞自然释放孔、靶细胞最大释放孔、培养液背景对照孔、裂解液背景对照孔，每组均复设3孔，混匀细胞，在37℃，5％二氧化碳孵箱中培养4h，收集上清液，用酶标仪在490nm波长下检测其吸光度(OD)值。按以下公式计算：
实验数据采用SPSS 18.0进行统计处理,各组内及组间相同指标进行配对t检验及方差分析，所测数据用均数±标准差表示，P<0.05表示结果有统计学意义。
不同细胞对乳腺癌MCF-7细胞的杀伤作用
将CIK、DC-CIK、CSC133-DC-CIK、CSC-RNA-DC-CIK、CSC-裂解物-DC-CIK、MCF-7-RNA-DC-CIK和MCF-7-裂解物-DC-CIK作为效应细胞，MCF-7细胞为靶细胞，采用LDH释放法检测，随着效靶比的增加，细胞杀伤作用增强，效靶比为80:1时，对MCF-7细胞的杀伤率最高。效靶比为10:1、20:1、40：1、80：1时，CSC133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组的杀伤率要高于CIK组和DC-CIK组，差异有统计学意义(P<0.05)；DC-CIK组要高于CIK组，差异有统计学意义(P<0.05)；但CSC133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组之间，差异无统计学意义(P>0.05)，见表3。
表3LDH法检测不同细胞对乳腺癌MCF-7细胞的杀伤活性(％)
注：与CIK组比，*P<0.05；与DC-CIK组比，△P<0.05；
不同细胞对乳腺癌MCF-7细胞干细胞(CSC)的杀伤作用
将CIK、DC-CIK、CSC133-DC-CIK、CSC-RNA-DC-CIK、CSC-裂解物-DC-CIK、MCF-7-RNA-DC-CIK和MCF-7-裂解物-DC-CIK作为效应细胞，MCF-7细胞的CSC细胞为靶细胞，采用LDH释放法检测，随着效靶比的增加，细胞杀伤作用增强，效靶比为80:1时，对MCF-7细胞CSC细胞的杀伤率最高。效靶比为10:1、20:1、40：1、80：1时，CSC133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组的杀伤率要高于CIK组和DC-CIK组，差异有统计学意义(P<0.05)；DC-CIK组要高于CIK组，差异有统计学意义(P<0.05)；但CSC133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组之间，差异无统计学意义(P>0.05)，见表4。
表4LDH法检测不同细胞对CSC细胞的杀伤活性(％)
注：与CIK组比，*P<0.05；与DC-CIK组比，△P<0.05；
可见，经乳腺癌MCF-7细胞干细胞(CSC)构建肿瘤特异性生物标记CD133重组质粒转染、RNA转染DC细胞或全细胞裂解诱导DC细胞，与CIK细胞共培养，对乳腺癌MCF-7细胞及其干细胞的杀伤作用，结果显示CD133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组的CIK免疫表型(CD3、CD8、CD56)和DC免疫表型(CD40、CD80、CD86、HLA-DR)成熟表达率均明显高于CIK组和DC-CIK组，差异均有统计学意义(P<0.05)。但CD133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组之间，差异无统计学意义(P>0.05)。随着效靶比的增加，CIK组、DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组、CD133-DC-CIK组作为效应细胞对MCF-7细胞及其干细胞的杀伤作用也逐渐增强，各效靶比中，CD133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组的杀伤率高于CIK组和DC-CIK组，差异均有统计学意义(P<0.05)；DC-CIK组高于CIK组，差异有统计学意义(P<0.05)；但CD133-DC-CIK组、CSC-RNA-DC-CIK组、CSC-裂解物-DC-CIK组组之间，差异无统计学意义(P>0.05)。
上述参照具体实施方式对该CD133质粒的新用途进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Claims (2)
1.CD133在增强DC-CIK对乳腺癌干细胞的体外杀伤作用方面的应用，所述CD133的核苷酸序列为SEQ ID NO：1所示序列。
2.根据权利要求1所述的CD133的应用，其特征在于：以含有CD133的重组质粒冲击DC细胞后，将成熟的DC细胞与CIK细胞联合培养使DC-CIK对乳腺癌干细胞的体外杀伤效果增强，所述CD133的核苷酸序列为SEQ ID NO：1所示序列。
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| CN102727524A (zh) * | 2012-07-10 | 2012-10-17 | 黄建华 | 一种抗cd3/抗cd133双特异性抗体装载的cik细胞的应用 |
| CN104781417A (zh) * | 2012-08-02 | 2015-07-15 | 迪肯大学 | 用于检测癌症干细胞的cd133适配子 |
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2016
- 2016-02-19 CN CN201610092181.4A patent/CN105602904B/zh not_active Expired - Fee Related
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|---|---|---|---|---|
| CN102727524A (zh) * | 2012-07-10 | 2012-10-17 | 黄建华 | 一种抗cd3/抗cd133双特异性抗体装载的cik细胞的应用 |
| CN104781417A (zh) * | 2012-08-02 | 2015-07-15 | 迪肯大学 | 用于检测癌症干细胞的cd133适配子 |
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| 抗原负载的DC诱导CIK对MDA-MB-231细胞的杀伤活性研究;王妍柏等;《现代免疫学》;20111231;第31卷(第6期);第486-489页 |
| 树突状细胞肿瘤疫苗抗肿瘤研究进展;孟冉冉等;《中华肿瘤防治杂志》;20121028;第19卷(第20期);第1597-1600页 |
| 登录号：XM_006713974.2;佚名;《GenBank》;20150312;第1-3544位 |
| 负载干细胞抗原的DC联合CIK细胞对乳腺癌荷瘤鼠肿瘤杀伤研究;庞春淼等;《天津医药》;20140630;第42卷(第6期);第554-557页 |
| 负载抗原的DC与CIK共培养对耐药乳腺癌细胞的杀伤作用;岳玲玲等;《中国肿瘤生物治疗杂志》;20120831;第19卷(第4期);第437-441页 |
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