CN111592585A - Ul16蛋白在制备促进细胞线粒体功能相关药物中的应用 - Google Patents
Ul16蛋白在制备促进细胞线粒体功能相关药物中的应用 Download PDFInfo
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Abstract
本发明公开了UL16蛋白在制备促进胚胎干细胞体外分化的小分子药物,以及干细胞移植治疗神经***退行性疾病的应用。本发明通过构建UL16质粒转染宿主细胞,发现UL16能够促进葡萄糖的有氧氧化和氧化磷酸化,激活细胞产能途经,从而提升胞内的ATP含量,改善线粒体的功能。最后通过构建UL16表达质粒转染小鼠胚胎干细胞,发现线粒体功能状态增强和能量代谢增强,可以促进干细胞分化。实现对分化缺陷状态的细胞和低分化率细胞的临床治疗,以及用于治疗细胞线粒体功能障碍或低下的相关疾病或病理、生理过程。
Description
技术领域
本发明蛋白筛选和应用机制领域,更具体地说,本发明涉及一种蛋白UL16具有增强线粒体功能状态并增加干细胞分化效率的功能及其应用。
背景技术
胚胎干细胞( embryonic stem cells,ESCs) 由于具有自我更新、高度增殖和多向分化的能力, 为多种代谢性肝病的细胞替代治疗提供了种子细胞来源,使它成为研宄的热点,但是其诱导分化的低效率一直是难以解决的关键问题。在体外,小鼠细胞能定向分化成几乎所有类型的成体细胞;人细胞也能成功分化为神经元、肝细胞、内皮细胞、心肌细胞、胰腺细胞和造血细胞等多种类型细胞。体外研究表明,不同来源的干细胞都能向肝脏细胞方向分化,表达肝细胞相关的表面标志,具有一些肝细胞功能,但是分化效率低。近几年研究显示,干细胞分化与能量代谢有重要联系,能量代谢和细胞命运紧密相关. 许多正常细胞在细胞***期增加氧耗和能耗,在此期间如果 ATP合成不足, 细胞将在没有任何 DNA损伤的情况下静止于G1 期。有报导指出线粒体氧化磷酸化的下调会造成体细胞和小鼠的早衰;而更有趣的是,最近众多研究者对成体/胚胎干细胞(embryonic stem cells,ESCs)的线粒体及能量代谢的研究发现线粒体的功能可能影响正常干细胞的增殖、分化潜能及生存期等。实验证明给衰老小鼠损伤组织植入人胚胎干细胞,胚胎干细胞有固有的抗衰老作用,可以产生可溶性的蛋白这种物质是MAPK路径中抗衰老的信号。
线粒体参与多种细胞信号通路和代谢途径,在细胞代谢和抗病毒信号途径中发挥了几个关键功能,包括其在ATP产生中的核心作用,同时有许多报导阐述了线粒体在干细胞多能性维持的作用。因而线粒体功能发生障碍可加速哺乳动物衰老并引发许多相关生理病理缺陷。病毒也不例外的依赖宿主的线粒体相关的代谢通路,比如HSV-1依赖于宿主细胞的代谢网络为病毒复制提供能量和大分子前体。能够通过增加果糖-6-磷酸激酶(PFK-1)的活性来激活宿主细胞的糖酵解途径,使宿主细胞的葡糖糖摄取量增加、细胞的胞内ATP含量增加。由此可知,病毒蛋白与线粒体关系密切。
我们发现这样一种线粒体定位的病毒蛋白UL16作为线粒体刺激因子,在抵抗线粒体功能障碍引发的衰老中发挥重要作用,并且应用到改善与线粒体功能障碍相关的生理病理进程中去。这为病毒蛋白作为有益于胚胎干细胞临床治疗的后期研究打开了新篇章。因此探讨UL16蛋白的免疫原性和诱导机体能量稳态的能力、预防或治疗新药作用的靶点等成为今后的重要研究方向之一。
发明内容
本发明发现一种疱疹病毒蛋白UL16在线粒体的功能作用并研究其作用机制,使其在疾病治疗中发挥作用。
本发明发现病毒蛋白UL16可应用于制备治疗抗衰老、线粒体功能障碍或代谢综合征的药物。
本发明还发现UL16可以应用于胚胎干细胞分化过程中,促进线粒体能量代谢。
本章成功构建了UL16基因的原核表达质粒pET-31b(+)-UL16,转化入(DE3)表达宿主菌中。Western blotting实验显示重组蛋白与预测的分子量大小一致,约为,主要以不溶的包涵体形式表达;与兔抗进行免疫印迹,能得到特异性的条带;纯化复性后的重组蛋白免疫新西南大白兔,制备兔抗多克隆抗体,制备的多克隆抗体可与重组蛋白发生特异性识别,表明重组蛋白具有良好的免疫原性和反应原性,制备的多克隆抗体可用于后续基因功能的研究。相对于现有技术,本发明经实验发现,增强细胞葡萄糖有氧氧化,提高线粒体呼吸,对ATP能量代谢通路进行调控,因此,可实现对细胞衰老、线粒体功能障碍和代谢综合征等多种代谢相关疾病的治疗和预防。
附图说明
图1为实施例1UL16基因鉴定结果。
图2为实施例1UL16蛋白电泳图。
图3为实施例2重组质粒转染HEK293T细胞后的ATP水平。
图4为实施例2免疫共沉淀结果UL16基因对葡萄糖代谢酶活力的影响。
图5为实施例2UL16基因乳酸水平的影响。
图6为实施例2UL16转染对葡萄糖消耗水平的测定。
图7为实施例2重组质粒转染对线粒体数量的影响测定
图8为实施例2重组质粒转染胚胎干细胞后的mtDNA水平。
具体实施方式
以下结合实施例,对本发明进行进一步详细说明。
实施例1
1.实验方法和材料
HSV-1 的UL16表达质粒pET-31b(+)-UL16,表达载体pET-31b(+)购自Invitrogen公司,质粒pFLAG-CMV-2购自Sigma公司。单纯疱疹病毒F株(HSV-1 F)、菌株DH5α受体菌和细胞均为本实验室保存。
引物设计及合成
根据NCBI上查询到的HSV-1 UL16基因的序列,利用Primers5.0软件设计引物UL16-for和UL16-rev用于HSV-1 UL16的扩增,引物由上海生工公司合成。
表1-1构建所用引物
1.2病毒扩增及模板制备
取单层HEK293T细胞用于HSV-1病毒培养,当约有75%细胞发生病变时, 收集并处理病毒液,最终获得HSV-1基因组并作为目的基因扩增模板。具体操 作步骤如下:
(1)取单层HEK293T细胞,弃去原细胞培养液,用PBS缓冲液反复冲洗3次,充分去除死亡或者未贴壁的细胞。
(2)从-80℃冰箱中取出HSV-1病毒,并溶解。
(3)取200 μl病毒加入到细胞中,均匀混合,置于培养箱中。每隔15-20 min摇匀一次,使病毒完全吸附细胞。
(4)约2 h后病毒可完成侵染细胞,加入4 ml新鲜细胞培养液,置于37℃、5%CO2培养箱中培养过夜。
(5)第二天早,在倒置显微镜下观察细胞形态,约有75%的细胞变成圆形并且折光率增加,即说明病毒已成功侵染细胞。
(6)用吸管充分吹打均匀后,每管200 μl分装于冻存管中,-80℃保存备用。
(7)取适量病毒液,在100℃水浴煮10 min,然后12000 rpm离心5 min,离心沉淀即为目的基因扩增模板。
基因的克隆
利用引物UL16-for和UL16-rev(如表1-1)扩增HSV-1UL16目的基因,并 纯化回收PCR产物。具体操作步骤如下:
(1)PCR反应体系如下:
10×PCR Buffer | 5.00μl |
DNA聚合酶 | 0.25μl |
dNTP(2.5mM) | 2.00μl |
MgCl<sub>2</sub> | 1.50μl |
UL16-for(10μM) | 1.00μl |
UL16-rev(10μM) | 1.00μl |
模板 | 4.00μl |
ddH<sub>2</sub>O | 12.75μl |
总计 | 25.00μl |
PCR条件设置:94℃5min,94℃1min,55℃1min,72℃1min30s,30个 循环;72℃10min。PCR结束后,将PCR产物置于4℃保存,1%琼脂糖凝胶电 泳分离鉴定。
产物的回收及纯化
使用浓度为1%的琼脂糖凝胶进行核酸电泳验证PCR的产物。利用胶回收试剂盒对目的片段进行回收、纯化。具体操作步骤如下:
(1)电泳结束后,在紫外照射下用已灭菌的干净的切胶刀将含有DNA片段的亮条带的琼脂糖胶块切下。切胶时,尽可能的将空琼脂块切除,且切胶的速度要快,防止DNA损失的过多。
(2)按每1 mg凝胶约为1 μl凝胶体积来换算,加入3倍体积量的Buffer W1于EP管,65℃水浴10 min,均与摇动至凝胶完全溶解。
(3)再向EP管中加入Buffer W2 体积为Buffer W1用量的一半,均匀混合,12000rpm离心30 s,弃上清,重复操作一次。
(4)用洗涤液漂洗两次,12000 rpm离心1 min,弃上清。
(5)加入35 μl Eluent静止5 min,12000 rpm离心2 min,洗脱DNA,即可得到纯化的目的DNA。
制备表达质粒pET-31b(+)-UL16
1.5.1 目的片段和克隆载体的体外重组
(1)以UL16-for和UL16-rev为上下游引物,分别以pHUL16质粒为模板,分别进行独立的PCR过程,PCR产物进行1%琼脂糖凝胶电泳鉴定,回收纯化DNA。
(2)同时,EcoRⅠ和BamHⅠ双酶切pET-31b(+)空质粒,制备pET-31b(+) 载体大片段。
(4)将UL16和突变UL16双酶切后回收产物分别与pET-31b(+)载体大片段混合,在T4 DNA连接酶的作用下,获得pET-31b(+)-UL16表达质粒。
感受态细胞制备
(1)取实验室冻存的DH5α菌种接种于LB平板中,37℃培养16 h。
(2)挑取单菌落接种于50 ml LB液体培养基中,37℃培养至OD值为0.5左右(范围0.4-0.6)。
(3)将25 ml菌液移至预冷的50 ml EP管中,在冰上放置30 min,使培养物冷却到0℃,4℃,4000 rpm低温离心10 min,弃上清,回收菌体。
(4)加入5 ml预冷的0.1 mol/L的CaCl2,重悬每份沉淀,放置于冰浴上30 min。4℃,4000 rpm低温离心10 min,弃上清,回收菌体。
(5)加入1 ml 用冰预冷的0.1 mol/L的CaCl2(含15%甘油)重悬菌体沉淀。
(6)在冰上将细胞分装成小份,100 ul/份,-80℃冻存备用。
重组质粒的转化
(1)取适量DH5α感受态细胞,向其中加入9 μl连接反应物后的产物, 混匀, 冰浴 30min;
(2)放入42℃水浴,热休克 2 min;立即转移到冰水浴中冷却1 min;
(3)冷却后向其中加入400 μl LB液体培养液,37℃, 180 rpm, 震荡培养1 h,使其活化
(4)涂布于含X-gal, IPT, Amp的LB固体培养基上,置于37℃生化培养箱15 min,待菌液吸收完全后,倒置培养16-20 h。
质粒的鉴定与提取
挑取白色菌落接种于5 ml LB液体培养基(含氨苄抗性)中,过夜培养,根据质粒小提取试剂盒,操作如下:
(1)取2 ml细菌培养物,12000 rpm离心1 min,弃上清,并加入250 μl溶液1(含有RNaseA),震荡,使沉淀彻底悬浮。
(2)向EP管中加入溶液2,上下翻转6-8次。同样加入溶液3,上下翻转6-8次,充分混匀。12000 rpm离心10 min,将上清移至另一EP管中。
(3)将上清加入吸附柱中,室温放置2 min,12000 rpm 离心1 min,弃废液,将吸附柱重新放回收集管中。
(4)向洗脱柱中加入 750 μl 的洗柱液,离心1 min (12000 r/min),弃液,向其中倒入 250 μl的洗柱液, 离心 5 min (12000 r/min) 后,弃液。
(5)向柱子中加入50 μL的去离子水,静置 2 min,离心收集液体,-20℃保存备用。
脂质体转染
根据Lipofectamine 2000脂质体转染试剂盒,具体操作如下:
(1)在24孔板中每孔0.5-2×105个细胞接种于500 μl不含抗生素和血清的DMEM培养基中,在转染时细胞可长至90-95%融合。
(2)用50 μl Opti-ME I无血清培养基稀释质粒DNA,轻轻混匀。取适量Lipofectamine 2000在50 μl Opti-ME I培养基中稀释,室温孵育5分钟。
(3)将前两步所稀释的DNA和Lipofectamine 2000混合(使总体积为100 μl),轻轻混匀,室温放置20分钟。
(4)每孔细胞中加入100 μl转染液,轻轻摇匀。
(5)37℃,5%CO2培养转染4-6 h后可更换培养基,24 h后裂解细胞,以备检测基因表达。
目的蛋白的鉴定
通过Western blotting来检测HSV-1 UL16蛋白的表达情况,具体操作步骤如下:
(1)制胶:配置12%的分离胶并注入玻璃板间隙至离上边缘1.5 cm处,上层加适量75%乙醇;待凝固,倒掉上层液体,注入5%浓缩胶,***梳子,自然晾干。
(2)上样:将蛋白质样品100℃水浴3 min,在电泳槽中倒入新配置的电泳液,分别在两边泳道加入8 μl和5 μl蛋白maker,用1×蛋白上样缓冲液补足体积到10 μl,其余泳道各加10 μl样品,50 V恒压跑电泳。
(3)转膜:电泳结束后,剪取11 cm×8 cm滤纸和合适大小的0.22 μm的PVDF膜,用甲醇活化5 min,按照“海绵-6层滤纸-凝胶-PVDF膜-6层滤纸-海绵”组装转印夹层,夹板组装后转移至转膜槽,200 mA恒流转移60 min。
(4)孵育抗体:转膜结束后,TBS液洗膜5 min,5%的脱脂奶粉封闭1 h,TBST液洗膜3次,每次5 min,加一抗稀释液4℃过夜孵育;TBST液洗膜3次,每次5 min,加二抗稀释液室温孵育1 h。
(5)发光检测:TBST液洗膜,加发光液孵育3 min,用滤纸吸干发光液,将胶片和PVDF膜放入压片盒中压片1 min,取出胶片定影30 s,用水清洗,烘干,扫描。
实施例2 pET-31b(+)-UL16转染细胞及胞内功能研究
1. 实验材料
HEK293T细胞也是由本实验室冻存。
实验方法
1.2.1 ATP含量测定
根据ATP含量测定试剂盒说明书操作:
(1)在冰上溶解待用的试剂,利用ATP测定裂解液将ATP标准溶液稀释成0.1、1、10 μM/L的浓度,制备标准曲线。
(2)细胞培养皿中加入200 μl裂解液,反复吹打至充***解,4℃下12000 rmp离心10 min,取上清。
(3)稀释ATP检测工作液,每个样品重复3次,每个监测孔中加100 μl ATP检测工作液,室温放置5 min,消耗掉本地ATP,在检测孔中加上100 μl待测样品或标准样品,充分混匀,间隔2 s,立即用生物发光仪检测CMP值。根据计算公式利用测出的标准曲线计算待测样品中ATP浓度。
1.2.2丙酮酸激酶活力检测
根据丙酮酸激酶试剂盒说明书操作:
(1)取400μl细胞提取液,超声波破碎细胞(功率20%,超声3 s,间隔10 s,重复30次),8000 g,4℃离心10 min,保存上清待用。
(2)将试剂盒中试剂四与试剂五混匀,37℃水浴5 min,加入30μl样本,开始计时,在340 nm波长下记录 20 s时初始吸光度A1。
(3)之后快速将比色皿水浴37℃水浴中准确反应2 min,快速取出比色皿后用擦镜纸擦干,340 nm下比色,记录2 min20 s时的吸光度A2,计算△A=A1-A2。
(4)PK酶活力计算公式:PK(U/104 cell) =反应总体积÷样本体积÷反应时间÷NADPH消光系数×△A÷活细胞密度。
1.2.3 果糖-6-磷酸激酶活力检测
试剂盒的说明书操作如下:
(1)细胞处理方法同3.3.7中步骤一。
(2)PFK工作液800μl、样本30μl、试剂六5μl、试剂七5μl依次加入到 1 mL比色皿中,340 nm波长纪录20 s时的吸光度为A1。
(3)然后37℃水浴10 min,取出用擦镜纸擦干后,再在340 nm下比色,测定10min20 s时的吸光度为A2,计算△A=A1-A2。
(4)PFK酶活力计算公式:同4.3.3中计算公式。
1.2.4葡萄糖消耗量的检测
(1)取同一数量级 1×105、无血清单层贴壁细胞置于 6 孔板,加 2ml无血清干细胞培养液中培养,取同一数量级1×105 A549 细胞系细胞置于6 孔板,分别加 2ml 无血清干细胞培养液中和 10%胎牛血清的 DMEM 培养基,放 37℃,5%CO2,5%大气,饱和湿度条件下分化培养;
(2)培养 24 小时和 48 小时后,测量起始和 24 小时、48 小时时各组细胞培养液的血糖浓度;
(3)采用葡萄糖氧化酶法测定培养液葡萄糖浓度(根据葡萄糖测定试剂盒说明):
将 96孔板中每孔加入试剂盒中试剂 R1 和 R2 各 100µl;
加待测培养液、标准品(葡萄糖浓度 5.5mmol/L)、蒸馏水(空白管)各 2µl 于孔中,充分混匀,37℃、15min;于酶标仪中于 505nm 波长处,空白管调零,读取样本管和标准管吸光值(A); 样本葡萄糖浓度=样本管 A/标准管 A×标准管葡萄糖浓度;
(4)以每组细胞培养液各时间点葡萄糖浓度减去起始葡萄糖浓度,差值为每组细胞的葡萄糖消耗。
1.2.5 线粒体数量检测
(1) ESCs细胞 1×105置于含盖玻片六孔板中,加以含 10%胎牛血清的 DMEM 培养基,放 37℃,5%CO2,5%大气,饱和湿度条件下培养 3 天,细胞爬片;
(2)去处培养液,加入含为 5μg/ml Hoechst 33342(染核)的 DMEM 培养基 2ml,37℃避光孵育 100min;
(3)吸去培养基,PBS 冲洗 3 次;
(4)加入含 100nM Mito-Tracker Green (线粒体非依赖线粒体膜电位绿色荧光探针)的 DMEM 培养基 2ml,37℃避光孵育 30min;
(5)弃染色液,用 PBS 或培养液洗涤 2-3 次即可进行荧光检测;
(6)取出盖玻片,用抗荧光淬灭封片液封片;
(7)激光共聚焦显微镜观察。检测时 Mito-Tracker Green 最大激发波长为 488nm,最大发射波长为 525nm;Hoechst 33342 最大激发波长为 352nm,最大发射波长为 530 nm(蓝色)和 630 nm(红色);
(8) A549细胞根据细胞核淡染情况分为质粒转染细胞和非转染细胞,比较两种细胞中线粒体各分布形态比例和线粒体荧光强度(代表线粒体数量)。
1.2.6
2 .实验结果
2.1 质粒pET-31b(+)-UL16构建结果及鉴定
经NCBI查询可知正常HSV-1 UL16基因大小为1122bp,利用引物UL16-for和UL16-rev,以构建的HSV-1 UL16真核表达载体质粒pHUL16为模板,PCR扩增产物,通过电泳均可以观察到扩增出的两条大约为1122 bp左右的特异性目的条带(如图1),与实验前预期结果一致。
2.2 Western blot 验证重组蛋白表达
将转化后含有阳性重组质粒pET-31b(+)-UL16的表达菌接种在含的培养基中,进行诱导表达,空载体pET-31b(+)转化菌诱导表达。结果显示:空载体转化菌诱导表达出现一个约且大小与标签蛋白一致的蛋白条带,含重组表达质粒pET-31b(+)-UL16的菌发现在41 kDa左右有目的条带。如图2所示,与实际相符,从蛋白水平特异性的鉴定了质粒pET-31b(+)-UL16可以在细胞中正常表达。
2.3 pET-31b(+)-UL16对细胞葡萄糖有氧氧化途径的影响
糖酵解和TCA循环是哺乳动物细胞中心碳代谢的中枢。通过这两种途径,葡萄糖被氧化,以NADH和ATP的形式产生能量,或者转化为氨基酸、脂类和核苷酸的前体。
2.3.1 pET-31b(+)-UL16转染对细胞产生ATP的影响
pET-31b(+)-UL16转染HEK293T细胞后,胞内ATP水平明显增加。pET-31b(+)-UL16转染细胞约有2倍的更高水平的ATP比WT和空载体转染的细胞在转染48 h后,24 h后略有增加(图3)。
2.3.2 pET-31b(+)-UL16转染对细胞葡萄糖代谢酶活性的影响
糖酵解途径调控的关键限速酶是果糖-6-磷酸激酶(PFK-1)和丙酮酸激酶(PK)。pET-31b(+)-UL16显著增加PFK-1和PK活性在转染48 h后(图4)。
2.3.3. pET-31b(+)-UL16转染对细胞乳酸生成的影响
上面的结果证明了pET-31b(+)-UL16增强葡萄糖代谢,为了进一步证明葡萄糖有氧氧化增加,通过试剂盒对乳酸水平进行检测。结果表明,pET-31b(+)-UL16转染细胞显示乳酸水平显著低于空载体转染和野生型细胞(图5)。
2.3.4 pET-31b(+)-UL16转染对细胞葡萄糖摄取的影响
在无血清培养液中培养转染pET-31b(+)-UL16的 HEK293T细胞,测定48 小时葡萄糖消耗低于无血清单层贴壁细胞和 A549 细胞系通过检测重组质粒转染后,葡萄糖的消耗量低于对照组(图6)。
2.4 pET-31b(+)-UL16转染对细胞线粒体数量的影响
通过激光共聚焦显微镜观察,转染pET-31b(+)-UL16质粒的胚胎干细胞和对照组细胞线粒体Mito Tracker Green 荧光强度无显著差异。表明细胞转染前后细胞间线粒体数量无明显区别(图7)。
2.5 pET-31b(+)-UL16转染对ESCs 的 mt DNA 拷贝数影响
尽管pET-31b(+)-UL16转染前后细胞间线粒体数量无明显区别,但 ESCs细胞的mt DNA拷贝数(Cox I/β-actin 值)较转染前增加(图8)。
成功构建了pET-31b(+)-UL16真核表达载体,并转染HEK293T细胞;荧光显微镜和Western blot结果显示,UL16蛋白在HEK293T细胞中稳定表达。接着通过利用相关的试剂盒分别检测了葡萄糖有氧氧化过程中细胞中ATP含量、PFK-1、PK、LDH的酶活力,事实表明UL16增加细胞葡萄糖代谢及ATP含量;另外对UL16转染细胞耗氧量及乳酸水平进行检测,发现细胞耗氧量增加,乳酸水平降低。UL16转染胚胎干细胞之后发现线粒体数量无明显差异,且转染之后线粒体DNA的拷贝数增加,表现在COX1等酶的活性增加。以上的这些结果表明,UL16转染HEK293T细胞增加细胞葡萄糖有氧氧化,促进胚胎干细胞的分化过程中能量代谢。
Claims (7)
1.一种疱疹病毒UL16基因的表达质粒以及与UL16基因活性片段或其相关核酸、蛋白。
2.权利要求1所述表达UL16蛋白的质粒在制备治疗线粒体功能障碍或促进干细胞分化的药物中的应用。
3.权利要求1所述表达UL16蛋白的质粒可用于制备一种预防细胞线粒体功能障碍的药物,其特征在于,UL16基因的转染可以促进线粒体的能量代谢。
4.权利要求1所述表达UL16蛋白的质粒可用于制备一种干细胞促分化药物,其特征在于,包括有效剂量的UL16蛋白作为活性成分来促进干细胞体外诱导分化方案的完善,以促进干细胞移植的临床应用。
5.权利要求1所述表达UL16蛋白的质粒,其特征在于可通过加强分化中的线粒体功能来促进干细胞分化的效率。
6.权利要求1所述表达UL16蛋白的质粒,其特征在于,可通过影响细胞线粒体功能调控肿瘤的发生、增殖和转移等过程药物中的应用。
7.权利要求1所述表达UL16蛋白的质粒,其特征在于,可实现对细胞衰老、线粒体功能障碍和代谢综合征等多种代谢相关疾病的治疗和预防。
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