CN110468195A - 一种肾脏疾病诊断用分子标志物及其应用 - Google Patents
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Abstract
本发明公开了一种肾脏疾病诊断用分子标志物及其应用，属于医学技术领域，检测动物组织样本中miR‑30a‑5p表达水平的试剂在制备诊断肾脏疾病产品中的应用，所述miR‑30a‑5p的序列如SEQ：ID：NO：1所示。本发明检测miR‑30a‑5p在HHcy致肾损伤过程中的表达水平。本发明在体内和体外合成过表达和沉默miR‑30a‑5p病毒，研究其对肾损伤的影响，阐明了在HHcy致肾损伤过程中miR‑30a‑5p的作用。本发明涉及的miR‑30a‑5p在HHcy致肾损伤过程中具有重要作用，为以后从分子水平诊断肾脏疾病提供了理论依据，具有重大的理论意义和潜在的实用价值。该方法简单易行，容易推广。
Description
技术领域
本发明属于医学技术领域，涉及一种肾脏疾病诊断用分子标志物及其应用。
背景技术
同型半胱氨酸(homocysteine，Hcy)是一种含硫氨基酸，是体内甲硫氨酸的代谢产物。美国心脏病协会明确将血浆总Hcy水平高于10μmol/L定义为高Hcy血症(Hyperhomocysteinemia，HHcy)。随着有关Hcy研究的发展和深入，大量的研究表明，HHcy不仅可以作为动脉粥样硬化和高血压等心血管疾病的独立危险因素，也与多种肾脏疾病的发生发展有关，是导致肾小球损伤及滤过率下降等肾功能损伤的独立危险因子。肾小球滤过屏障是由最内层的肾小球内皮、中间层肾小球基底膜(glomerular basement membrane，GBM)以及最外层的足细胞组成。Hcy可直接作用于肾小球足细胞，使肾小球正常功能受限，加速尿蛋白的形成进而导致肾损伤。但HHcy致肾损伤的分子机制迄今尚不明了，因此需要寻找潜在的早期诊断并采用更加有效的治疗途径。
微小RNA(microRNA，miRNA)是一类长度为18-25nt的非编码RNA分子，可以通过靶向结合mRNA的3'-UTR中的互补序列，在转录后翻译水平抑制或降解mRNA，使靶基因在基因的转录后水平或表观遗传水平通过序列的互补配对，从而发挥调控基因表达的作用。作为一种调节基因表达的小分子RNA，miRNA在许多疾病中扮演着重要的角色，已经成为多种疾病的生物标志物和潜在的治疗靶点。研究表明，miRNA同时参与了急性肾损伤(AKI)、慢性肾损伤(CKD)、肾移植和肾脏遗传疾病等肾脏疾病的发生和预防，可以作为肾脏疾病的风险评估、早期诊断、评估预后和分类损伤严重程度的生物标志物。因此，检测和验证miRNA在肾脏疾病中的具体作用和功能能够有效的帮助我们了解肾脏疾病的发生发展，并且还可能产生新的治疗方法。此外，在定量检测方面，miRNA的定量精度和灵敏度非常高，使用qRT-PCR检测miRNA的表达操作简单、特异性强、快速准确，拓宽了其在实验研究和临床中的作用，如能检测并验证其作用，将为今后临床上预防和治疗HHcy诱导的肾损伤提供新的理论依据。
发明内容
本发明的目的在于提供一种肾脏疾病诊断用分子标志物及其应用，即miR-30a-5p。验证其在HHcy肾损伤过程中的作用，进一步了解HHcy致肾损伤的分子机制，为今后临床预防和治疗肾脏疾病提供理论依据。
其具体技术方案为：
检测动物组织样本中miR-30a-5p表达水平的试剂在制备诊断肾脏疾病产品中的应用，所述miR-30a-5p的序列如SEQ：ID：NO：1所示。
进一步，所述试剂包括：通过qRT-PCR法检测miR-30a-5p表达水平的试剂。miR-30a-5p的引物由广州锐博生物科技公司合成，逆转录试剂盒购自Thermo FisherScientific，SYBR Green Mix由DBI提供。
进一步，验证miR-30a-5p作用的步骤包括：①首先建立HHcy致肾损伤动物模型；②qRT-PCR检测miR-30a-5p在Cbs+/+和Cbs+/-小鼠肾脏中的表达；③在体内和体外分别验证miR-30a-5p在HHcy致肾损伤过程中的作用。
与现有技术相比，本发明的有益效果：
(1)本发明检测miR-30a-5p在HHcy致肾损伤过程中的表达水平。
(2)本发明构建miR-30a-5p前体(miR-30a-5p mimic)和miR-30a-5p抑制物(miR-30a-5pinhibitor)慢病毒，在细胞水平验证miR-30a-5p对肾小球足细胞超微结构的影响；并且合成miR-30a-5p过表达腺相关病毒(血清型为AAV9)，在体内研究miR-30a-5p对肾损伤的影响；进而阐明miR-30a-5p在HHcy致肾损伤过程中的作用。
(3)本发明涉及的miR-30a-5p在HHcy致肾损伤过程中具有重要作用，为以后从分子水平诊断肾脏疾病提供了理论依据，具有重大的理论意义和潜在的实用价值。
(4)本发明所涉及的方法简单易行，容易推广。
附图说明
图1为Cbs+/+和Cbs+/-组小鼠血清Hcy，肌酐(SCr)，尿素氮(BUN)及胱抑素C(CytC)的水平。A.Cbs+/+和Cbs+/-组小鼠血清Hcy水平；B.Cbs+/+和Cbs+/-组小鼠血清肌酐(SCr)，尿素氮(BUN)及胱抑素C(CytC)的水平。与Cbs+/+组比较，*P<0.05，**P<0.01。
图2为透射电镜下观察Cbs+/+和Cbs+/-组小鼠肾小球超微结构的改变(Scale bar,5μm，2μm)，A为Cbs+/+组小鼠肾小球超微结构，B为A的局部放大图；C为Cbs+/-组小鼠肾小球超微结构，D为C的局部放大图。
图3为糖原染色观察Cbs+/+和Cbs+/-组小鼠肾小球病理学改变(Scale bar,170μm)，其中A为Cbs+/+组，B为Cbs+/-组。
图4为miR-30a-5p在Cbs+/+和Cbs+/-组小鼠肾脏中的表达水平。与Cbs+/+组比较，**P<0.01。
图5为miR-30a-5p过表达和干扰稳定细胞系构建。A.分别用miRNA阴性对照(miNC)、miR-30a-5p前体(miR-30a-5p mimic)和miR-30a-5p抑制物(miR-30a-5pinhibitor)慢病毒感染足细胞，荧光显微镜下观察病毒转染效率(40×)；B.qRT-PCR检测miR-30a-5p的表达。与mi-NC组比较，*P<0.05，**P<0.01。
图6为体外观察分别感染miNC、miR-30a-5p mimic和miR-30a-5p inhibitor后miR-30a-5p对肾小球足细胞超微结构的影响，其中，A为miNC组，B为miR-30a-5p mimic组，C为miR-30a-5p inhibitor组。
图7为体内观察miR-30a-5p过表达腺相关病毒原位注射小鼠肾皮质后对Cbs+/-组小鼠肾脏功能的影响。A：注射AAV9-miR-30a-5p后小鼠血清SCr和BUN的改变；B：注射AAV9-miR-30a-5p后TUNEL染色检测肾小球内细胞凋亡情况。与AAV9-miNC组比较，**P<0.01。
具体实施方式
下面结合附图和实施例对本发明的技术方案作进一步详细地说明。
本发明的具体技术方案为：
(1)建立高蛋氨酸饮食诱导HHcy动物模型：选择Cbs+/+和Cbs+/-小鼠，两组小鼠均给予2％的高蛋氨酸饮食，喂养8周后，进行后续实验。
(2)眼球取血，分离小鼠血清，全自动生化分析仪检测血清Hcy水平，肌酐(SCr)、尿素氮(BUN)、胱抑素C(Cyt C)水平；透射电镜下观察肾小球超微结构的改变；糖原染色观察肾脏组织病理学改变。
(3)检测miR-30a-5p在小鼠肾组织中的表达，并构建miR-30a-5p过表达和干扰稳定细胞系，透射电镜观察其对足细胞超微结构的影响。
(4)将过表达AAV9-miR-30a-5p通过原位注射，注入给予8周高蛋氨酸饮食后的Cbs+/-小鼠肾皮质内，2周后观察其对肾损伤相关指标及肾小球内细胞凋亡的影响。
本发明所述miR-30a-5p在HHcy致肾损伤过程中的作用，进一步阐明了HHcy致肾损伤的分子机制。
1实验对象
1.1实验动物
雄性Cbs+/+小鼠和雄性Cbs+/-小鼠
1.2细胞株
小鼠肾小球足细胞(MPC-5)
2仪器设备与实验试剂
2.1主要试剂
无菌细胞培养瓶、吸管、滤器(Corning公司，美国)；胎牛血清、DMEM高糖培养基、磷酸缓冲液(Gibco公司，美国)；miR-30a-5p引物(广州锐博生物科技公司，中国)；血液/细胞/组织基因RNA提取试剂(北京天根生化科技有限公司，中国)；逆转录试剂盒(Thermo fisher公司，美国)；TUNEL染色试剂盒(BD公司，美国)；miR-30a-5p腺相关病毒委托上海吉凯公司合成，血清型为AAV9。
2.2主要仪器
恒温细胞培养箱(Thermo Fisher Scientific，美国)；超净工作台(安泰技术有限公司，中国苏州)；激光共聚焦荧光显微镜(Olympus，日本)；5415D型微量台式离心机(Eppendorf，德国)；精密电子天平(Sartorius，德国)；制冰机(AF10SCOTSMAN，美国)；荧光定量PCR仪(Funglyn，上海)；普通PCR仪(Bio-Rad，美国；透射电子显微镜(日本，JEOL)；微量移液器(Eppendorf，德国)。
3方法
3.1血清Hcy、SCr、BUN、CytC水平的测定
各组小鼠眼球取血后，将血液在室温静置20min，4℃3000rpm离心15min，取上清，全自动生化分析仪检测Hcy、SCr、BUN、CytC水平。
3.2透射电镜和糖原染色检测肾小球损伤程度
分离出各组小鼠双侧肾脏并摘除，先在生理盐水中清洗，在液氮中速右侧肾脏，而后保存于-80℃冰箱，将左侧肾脏分割为两部分，一部分快速去除肾脏表面的包膜并且分离肾皮质，每组小鼠取大约1mm×1mm×1mm的肾脏组织标本置于3％的戊二醛中，用饿酸固定，分别经乙醇、丙酮脱水后，包埋剂浸透，37℃恒温箱中聚合，经超薄切片机切片，经枸橼酸铅染色，然后在透射电镜下观察肾小球超微结构；另一部分肾脏置于分别置于4％多聚甲醛溶液中，按照自动脱水机设定的既定程序进行脱水，透明处理。然后经浸蜡、包埋、蜡块整修后，通过切片机将蜡块切成厚度约4um左右的石蜡切片，将石蜡切片脱蜡至蒸馏水，0.5％过碘酸水溶液氧化10min，蒸馏水洗，入Schiff试剂在37℃培养箱内作用30min，流水冲洗2min，苏木素复染3min，水洗，梯度酒精脱水，二甲苯透明，中性树胶封片，光镜下观察肾小球病理学改变。
3.3荧光定量PCR(qRT-PCR)检测miR-30a-5p的表达
3.3.1肾脏组织RNA的提取
按照试剂盒说明书提取肾脏组织RNA，将冻存的肾脏组织取出置于冰上，称取80mg肾脏组织并加入1ml裂解液RZ，用匀浆器进行匀浆处理，整个过程在冰上操作，将裂解样品转移到1.5ml无RNase离心管中；在超净台(经紫外照射杀毒20～30min)内静置裂解样品5min左右，根据样品裂解情况，可适当延长时间，使核酸-蛋白复合物分离完全；4℃12，000rpm高速离心5min除去组织中的蛋白、脂肪等，将上清转入到一个新的1.5ml无RNase离心管中；加入氯仿200μl/样品，漩涡器震荡仪振荡15sec后室温静置3min，4℃12，000rpm高速离心10min，RNA包含在无色的水相中，小心吸取400μl无色的水相转移到新的1.5ml无RNase离心管中；缓慢加入200μl无水乙醇，轻轻颠倒离心管将无色水相和无水乙醇混匀，转入到吸附柱中后12，000rpm离心30sec(4℃)，倒掉废液；向吸附柱中加入500μl RD去蛋白液，4℃12，000rpm离心30sec，倒掉废液；垂直加入700μl RW漂洗液到吸附柱中，室温条件下静置2min，4℃12，000rpm离心30sec，弃掉废液，此步操作共进行2次；再次离心2min(4℃12，000rpm)后，将吸附柱从收集管中取出，置于常温静置5～10min，去除遗留的漂洗液；准备新的1.5ml无RNase的离心管，做好标记，将已经晾干的吸附柱放到离心管中，垂直悬空在吸附膜中央滴加无RNase的水70μl，室温放置等待2min，4℃12，000rpm离心2min，即得到RNA，为防止RNA降解，将其应保存在-80℃。
3.3.2 miR-30a-5p的逆转录
此步操作在紫外灯照过的超净工作台内完成，操作过程中均使用无RNase的枪头，将200μl无RNase的离心管置于冰上，然后依次分别加入以下表1中的成分：
表1
以上体系混匀后，瞬时离心，进行RT反应，反应程序为：42℃60min，70℃10min。反应后的产物短时间内保存在-20℃，长时间则保存在-80℃，切忌反复冻融。
3.3.3qRT-PCR检测miR-30a-5p的表达
将以下表2中体系依次分别加到200μl无RNase离心管中：
表2
所有样品加样完成后，涡旋震荡混匀，瞬时离心后将样品放到荧光定量PCR仪进行反应，反应程序：37℃20s，95℃10min，95℃2s，60℃20s，72℃10s，反应进行45个循环，将U6作为内参对照。根据目的基因的相对量＝2-△△Ct计算结果，miR-30a-5p引物由广州锐博公司设计合成。
3.4病毒感染
感染前24小时，将细胞按一定浓度重新接种于新的培养瓶中，确保细胞汇合度在第二天可以达到60％～80％左右；吸掉培养瓶中原来的培养基，加入PBS清洗两次；再次加入2ml不含血清的培养基逐滴缓慢加入miRNA阴性对照(miNC)、miR-30a-5p前体(miR-30a-5p mimic)和miR-30a-5p抑制物(miR-30a-5pinhibitor)慢病毒各15μl(病毒感染条件在前期已经通过实验确定)，轻轻晃动培养瓶以充分混匀，置于恒温37℃、含5％CO2的细胞孵育箱中培养6小时后，换液；48小时后在倒置荧光显微镜下观察感染效率。经嘌呤霉素筛选后，镜下观察感染效率达到要求后，收集细胞用于后续实验操作。
3.5靶向性病毒注射及处理
Cbs+/-小鼠成模后，将小鼠分为3组：PBS，AAV9-miR-30a-5p和AAV9-miNC组。通过吸入的异氟烷进行麻醉，待麻醉后，以俯卧位固定于鼠手术台上，术前常规备皮，以左侧背部肋脊角处为手术入口，碘伏消毒皮肤，暴露小鼠左侧肾脏，微量进样器抽取10μl腺相关病毒，用等量的PBS稀释，选取肾脏皮质上中下三个部位，向皮质部注射PBS，AAV9-miR-30a-5p和AAV9-miNC，注射过程应缓慢，防止液体外漏。用棉签按压，待注射部位不再出血，缝合肌肉及皮肤。术后密切观察生命体征，预防性抗感染。2周后进行后续实验。
3.6 TUNEL染色
取出样品石蜡切片，65℃烤片1小时，然后放入二甲苯脱蜡15min，重复进行2次；切片按照100％、100％、95％、85％、75％、50％梯度酒精顺序依次浸洗，每次5min；PBS漂洗3次，每次5min，然后37℃条件下Proteinase K工作液封闭处理组织30min；准备TUNEL反应混合液，处理组用50μl TdT+450μl荧光素标记的dUTP液混匀，而阴性对照组仅加50μl荧光素标记的dUTP液，阳性对照组先加入100μl DNase 1，在15～25℃反应10min；PBS漂洗3次，每次5min，然后在玻片上滴加50μlTUNEL反应混合液(阴性对照组仅加50μl荧光素标记的dUTP液)封闭标本，避光条件下，放入湿盒中37℃反应1小时；PBS漂洗3次，每次5min，之后滴加PBS在激光共聚焦显微镜下观察凋亡细胞(激发光波长为450～500nm，检测波长为515～565nm)，选取合适的地方进行拍照取片。
4.统计学处理
所有数据类型均为定量资料，统计结果以均数±标准差表示，两样本均数间的比较采用Student's t检验，多样本均数间比较采用One-way ANOVA检验，组间的两两比较采用Student-Newman-Keuls检验，检验水准取α＝0.05。
5结果
5.1两组小鼠血清Hcy、SCr、BUN及CytC的水平
全自动生化分析仪检测血清Hcy、BUN、SCr、CytC的浓度，对其结果进行分析显示：与Cbs+/+组小鼠比较，Cbs+/-组小鼠Hcy水平明显升高(P<0.01)，同时肾损伤相关指标BUN、SCr、CytC水平也明显升高(P<0.01，P<0.01，P<0.05)，差异具有显著性。
5.2肾小球电镜微结构改变
为了进一步证实Hcy对肾脏的损伤作用，利用透射电子显微镜对肾小球的基底膜及足细胞的超微结构进行了观察。结果显示：Cbs+/+组肾小球上的基底膜清晰，足突规则，系膜区细胞无增生；Cbs+/-组肾小球上的基底膜局灶性增厚，内皮细胞内染色质聚集，足突不规则融合。
5.3肾皮质糖原染色
肾皮质糖原染色结果可见Cbs+/+组小鼠肾小球上的基底膜清楚，粗细均匀，肾小球血管袢薄而清晰，而Cbs+/-组小鼠肾小球基底膜则呈现不同程度的粗细不均，系膜增生。
5.4两组小鼠肾脏中miR-30a-5p的表达
采用qRT-PCR法检测两组小鼠肾脏组织中miR-30a-5p的表达，结果显示：与Cbs+/+组小鼠比较，Cbs+/-组小鼠miR-30a-5p表达明显降低(P<0.01)，差异具有显著性。
5.5miR-30a-5p过表达和干扰的细胞系的建立
分别用miRNA阴性对照(miNC)、miR-30a-5p前体(miR-30a-5p mimic)和miR-30a-5p抑制物(miR-30a-5pinhibitor)慢病毒感染足细胞，荧光倒置显微镜观察慢病毒的感染效率，qRT-PCR验证细胞模型是否构建成功。荧光显微镜下观察细胞感染效率，结果显示：各组细胞荧光效率均达到了80％以上，符合实验要求。qRT-PCR结果显示：与miNC组相比，miR-30a-5p mimic细胞系miR-30a-5p表达升高(P<0.01)，miR-30a-5pinhibitor细胞系miR-30a-5p表达明显降低(P<0.05)，说明细胞系构建成功。
5.6体外观察miR-30a-5p对足细胞微结构的影响
为了确定miR-30a-5p对足细胞微结构的影响，用miNC、miR-30a-5p mimic和miR-30a-5p miR-30a-5pinhibitor慢病毒感染足细胞，结果可见：感染mi-NC后内质网丰富，线粒体结构明显，细胞形态正常；感染miR-30a mimic后细胞表面有较多的突起，细胞大小较一致，多为圆形或椭圆形，细胞膜完整，细胞核一般居中，核膜清楚，染色质丰富；而感染miR-30ainhibitor后，细胞体积缩小、细胞核膜皱缩，细胞质内可见小空泡，少数内质网扩张，可见少量的细胞核碎裂、溶解，呈现出凋亡的现象。
5.7体内观察miR-30a-5p对Cbs+/-组小鼠的影响
为了确定miR-30a-5p对肾脏损伤的作用，在Cbs+/-组注射过表达AAV9-miR-30a-5p，2周后，眼球取血，全自动生化分析仪检测BUN和SCr浓度，结果显示：与AAV9-miNC组相比，AAV9-miR-30a-5p组BUN和SCr水平明显降低，同时TUNEL染色结果显示AAV9-miR-30a-5p组肾小球内凋亡细胞的数量明显减少，提示miR-30a-5p在HHcy致肾损伤过程中起保护作用。
6结论
HHcy对肾小球基底膜有损伤作用，并且miR-30a-5p在HHcy致肾损伤过程中发挥着保护作用。
本发明用Cbs小鼠复制HHcy肾损伤模型，观察肾损伤过程中miR-30a-5p的作用，为今后临床上预防和治疗肾脏疾病提供新的理论依据，具有十分重要的意义。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 宁夏医科大学
<120> 一种肾脏疾病诊断用分子标志物及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
uguaaacauc cucgacugga ag 22
Claims (3)
1.检测动物组织样本中miR-30a-5p表达水平的试剂在制备诊断肾脏疾病产品中的应用，所述miR-30a-5p的序列如SEQ：ID：NO：1所示。
2.根据权利要求1所述的应用，其特征在于，所述试剂包括：通过qRT-PCR法检测miR-30a-5p表达水平的试剂，miR-30a-5p的引物由广州锐博生物科技公司合成，逆转录试剂盒购自Thermo Fisher Scientific，SYBR Green Mix由DBI提供。
3.根据权利要求1所述的应用，其特征在于，应用过程中，验证miR-30a-5p作用的步骤包括：①首先建立高同型半胱氨酸血症致肾损伤动物模型；②qRT-PCR检测miR-30a-5p在Cbs+/+和Cbs+/-小鼠中的表达；③在体内和体外分别验证miR-30a-5p在高同型半胱氨酸血症致肾损伤过程中的作用。
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