CN113373210A - 巨噬细胞中Act1作为牙周炎疾病标志物的应用 - Google Patents
巨噬细胞中Act1作为牙周炎疾病标志物的应用 Download PDFInfo
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Abstract
本发明涉及生物医疗领域,具体而言,涉及巨噬细胞中Act1作为牙周炎疾病标志物的应用。本发明首次进行特异性巨噬细胞中Act1在牙周炎中的作用研究,所并首次发现了特异性抑制巨噬细胞Act1表达使牙周组织出现炎症加重、骨吸收增加、炎症因子表达上调等改变,表明巨噬细胞Act1参与牙周炎发生发展。
Description
技术领域
本发明涉及生物医疗领域,具体而言,涉及巨噬细胞中Act1作为牙周炎疾病标志物的应用。
背景技术
牙周炎是最常见的口腔炎症性疾病之一,全球重度牙周炎患病率约为10%,是患者缺牙最主要的原因之一。目前主要采用抗生素辅助下定期进行牙周洁治、根面平整等非手术治疗,存在疗效不稳定的问题。由于免疫调节在牙周炎的发生发展中起重要的调控作用,寻找免疫治疗靶点进而对牙周炎进行精准有效治疗,是未来牙周炎治疗的目标。然而,在牙周炎中免疫调控机制仍不清楚。所以深入研究牙周炎免疫调节机制,探索新的免疫靶点以精准治疗牙周炎是当前的科学前沿问题。
发明内容
本发明的第一方面涉及用于检测巨噬细胞中Act1表达量的检测剂在制备诊断牙周炎的试剂盒中的应用。
可选的,如上所述的应用,所述检测剂于转录水平对Act1表达量进行检测。
可选的,如上所述的应用,所述检测剂用于执行如下方法中的至少一种:
PCR法、共振光散射法和生物质谱法。
可选的,如上所述的应用,所述PCR法为实时荧光定量PCR和/或数字PCR。
可选的,如上所述的应用,所述检测剂包括核苷酸序列如SEQ ID NO:1和2所示的引物。
可选的,如上所述的应用,所述试剂盒还含有探针、dNTP、DNA聚合酶、双链特异性荧光染料、内参引物以及水中的一种或多种。
可选的,如上所述的应用,所述检测剂于翻译水平对Act1表达量进行检测。
可选的,如上所述的应用,所述检测剂用于执行如下任一种方法:
生物质谱法、电泳法、色谱法、酶联免疫吸附试验、免疫荧光法、免疫化学发光法、免疫比浊法、免疫印迹法以及斑点印迹。
可选的,如上所述的应用,所述检测剂为抗体。
本发明的第二方面涉及巨噬细胞中Act1表达量降低的非人动物在制备牙周炎非人动物模型中的应用。
本发明的第三方面涉及巨噬细胞中Act1表达量降低的非人动物在鉴别和/或测试药物中的用途;
所述药物用于预防和/或治疗的适应症为牙周炎和/或治疗与牙周炎相关的并发症。
Act1是NF-κB激活蛋白,在巨噬细胞中高表达并参与免疫调节,但在牙周炎的作用未见报道。本发明首次进行特异性巨噬细胞中Act1在牙周炎中的作用研究,所并首次发现了特异性抑制巨噬细胞Act1表达使牙周组织出现炎症加重、骨吸收增加、炎症因子表达上调等改变,表明巨噬细胞Act1参与牙周炎发生发展。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明一个实施例中anti-Act1基因工程小鼠中Act1 mRNA表达水平的检测结果;
图2为本发明一个实施例中Micro CT扫描分析结果和HE染色结果;
图3为本发明一个实施例中anti-Act1小鼠牙周局部组织中炎症因子和野生型C57BL/6小鼠的表达水平比较。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明涉及用于检测巨噬细胞中Act1表达量的检测剂在制备诊断牙周炎的试剂盒中的应用。
本发明发现了一种新的牙周炎标志物:Act1。
在本发明中,Act1单独出现时可同时指代其mRNA和蛋白。Act1,即NF-κB激活剂1,亦称为CIKS(connection to IKK and SAPK/JNK)。
本文使用的术语“标志物”或“生化标志物”指要用作分析患者实验样品的靶标的分子。本领域技术人员公知对某分子表达量的检测可从转录或翻译水平进行,因此,这样的分子靶标的实例是mRNA或蛋白。如本领域技术人员显而易见的,mRNA的检测不应当解释为限于上述基因直接转录得到的特定mRNA序列,例如,作为替代性的mRNA变体、短链或前-mRNA处理的结果。变体的核苷酸序列与对应的标志物序列具有95%、96%、97%、98%、99%或更高的同一性;短链则只要能代表全长mRNA本身的特异性序列都可胜任。显然地,mRNA水平的检测也可通过检测cDNA以进行。在本发明中用作标志物的蛋白预期包括所述蛋白的天然存在的变体以及所述蛋白或所述变体的片段,特别是免疫学上可检测的片段。免疫学上可检测的片段优选地包含所述标志物多肽的至少5、6、7、8、9、10、11、12、15或20个连续氨基酸。本领域的技术人员可认识到,由细胞释放的蛋白或存在于胞外基质中的蛋白可能受到损害(例如,在炎症过程中),且可被降解或切割成这样的片段。某些标志物以无活性形式合成,其可以随后通过蛋白酶解来激活。如熟练的技术人员将明白的,蛋白或其片段也可以作为复合物的部分而存在。这样的复合物也可以用作本发明意义上的标志物。另外,或在替代方案中,标志物多肽或其变体可以携带翻译后修饰。翻译后修饰的非限制性实例是糖基化、酰化和/或磷酸化。
在一些实施方式中,所述检测剂于转录水平对Act1表达量进行检测。
在一些实施方式中,所述检测剂用于执行如下方法中的至少一种:
PCR法、共振光散射法和生物质谱法。
在一些实施方式中,所述PCR法为实时荧光定量PCR和/或数字PCR。
在一些实施方式中,所述检测剂包括核苷酸序列如SEQ ID NO:1和2所示的引物。
在一些实施方式中,所述试剂盒还含有探针、dNTP、DNA聚合酶、双链特异性荧光染料、内参引物以及水中的一种或多种。
在一些实施方式中,所述双链特异性荧光染料定量选自溴化乙锭、SYBR Green、PicoGreen、RiboGreen中的任一种。
在一些实施方式中,所述水通常为核酸和/或无核酸酶的水。水可以为蒸馏水(Distilled Water)、去离子水(Deionized Water)或反渗水(Reverse osmosis Water)。
在一些实施方式中,所述DNA聚合酶选自Taq、Bst、Vent、Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、Pab、Mth、Pho、ES4 DNA聚合酶、Klenow片段中的任一种。
内参引物即为用于检测参考标准品的引物,还可以理解为进一步包括mRNA测量标准化所需的样本和/或试剂。例如,RNA测量可通过一种或多种“持家”mRNA作为标准化的基准,这对于本领域技术人员是熟悉的。
在一些实施方式中,所述检测剂于翻译水平对Act1表达量进行检测。
在一些实施方式中,所述检测剂用于执行如下任一种方法:
生物质谱法、电泳法、色谱法、酶联免疫吸附试验、免疫荧光法、免疫化学发光法、免疫比浊法、免疫印迹法以及斑点印迹。
翻译水平的检测剂通常是特异性地检测Act1蛋白的试剂,例如,特异性结合Act1蛋白的凝集素、特异性结合Act1蛋白的适配体或特异性结合Act1蛋白的抗体及抗体片段。特异性的结合剂对其相应的靶分子具有至少107l/mol的亲和力。特异性的结合剂优选对其靶分子具有108l/mol、或更优选109l/mol的亲和力。技术人员将理解,使用术语“特异性的”表示,样品中存在的其它生物分子不与Act1蛋白的检测剂发生显著的结合。
在一些实施方式中,所述检测剂为抗体。
在一些实施方式中,所述抗体为单克隆抗体或多克隆抗体。
根据本发明的再一方面,本发明还提供了一种用于牙周炎的诊断、辅助诊断或预后分析的方法,所述方法包括:使用如上所述的检测剂/试剂盒测量巨噬细胞中Act1的含量。
检测的样本优选可以为受试者的血液样品,例如外周血样品。
受试者通常为哺乳动物,优选为灵长类动物,再优选为人。
根据本发明的再一方面,还涉及巨噬细胞中Act1表达量降低的非人动物在制备牙周炎非人动物模型中的应用。
Act1表达量降低的非人动物可通过本领域常规的基因沉默技术制备得到,这对本领域技术人员是熟悉的。例如,Cre-LoxP、TALEN、CRISPR等技术介导的基因敲除,miRNA,siRNA等介导的干扰基因表达的技术等。
非人动物通常是哺乳动物,优选啮齿动物,再优选小鼠(Mus musculus)。
小鼠品系可以选择BALB/c、C57BL、C3H/He、昆明小鼠、ICR、NIH、CFW、LACA、nude小鼠或Scid小鼠等,优选C57BL小鼠。
根据本发明的再一方面,本发明还涉及如上所述方法得到的动物在鉴别和/或测试药物中的用途;
所述药物用于预防和/或治疗的适应症为牙周炎和/或治疗与牙周炎相关的并发症。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1
一、实验材料
1、实验动物
C57BL/6小鼠:雄性,8周龄,50只。
anti-Act1小鼠:雄性,8周龄,50只。本发明中anti-Act1小鼠通过如下方法构建:将表达cre酶的基因插入到CD68基因启动子的下游,CD68为巨噬细胞的特异性marker,因而能保证cre酶特异性地在巨噬细胞中表达。LoxP位点位于act1基因启动子的两侧,可配合cre酶将act1基因的启动子敲除,从而降低act1基因的表达。
2试剂
2.1主要试剂
4%多聚甲醛固定液 Biosharp
PBS磷酸盐缓冲液(粉剂,0.01M,pH 7.2-7.4) Biosharp
Rabbit Anti-CIKS antibody Bioss
琼脂糖Biowest
TRAP染色试剂盒(组织)GENMED
β-硫基乙醇Gibco
PureLinkTM RNA Mini Kit Invivogen
BSA Solarbio
中性树胶 Solarbio
Triton X-100 Solarbio
逆转录试剂盒 TAKARA
SYBR Green试剂盒 TAKARA
PCR Master Mix Thermo
苏木素、伊红染色液 康龙生物
EDTA脱钙液 生工
动物全蛋白提取试剂盒 生工
辣根酶标记山羊抗小鼠IgG 中杉金桥
2.2主要耗材及器械
2mL离心管 Axygen
1.5mL离心管 Axygen
0.2mL透明平盖PCR薄壁管 Axygen
200μL、10μL枪头(吸头) Axygen
96孔PCR反应板 Axygen
15mL离心管 Corning
50mL离心管 Corning
冻存管 Corning
1000mL枪头(吸头) KIRGEN
刀片 Leica
眼科剪 雷奥巴赫豹牌手术器械
显微镊 雷奥巴赫豹牌手术器械
5-0丝线 强生
组织包埋盒 世泰
盖玻片 世泰
粘附性载玻片 世泰
2.3主要引物
引物名称 序列(5'-3')
Act1
上游引物TCCCGTGGAGGTTGATGAATC(SEQ ID NO:1)
下游引物TCAGGGTGCCTTCTAAAGAAACT(SEQ ID NO:2)
IL-6
上游引物CTGCAAGAGACTTCCATCCAG
下游引物AGTGGTATAGACAGGTCTGTTGG
IL-1β
上游引物GAAATGCCACCTTTTGACAGTG
下游引物TGGATGCTCTCATCAGGACAG
TNFα
上游引物TGTCTCAGCCTCTTCTCATT
下游引物TGATCTGAGTGTGAGGGTCT
MCP-1
上游引物TTAAAAACCTGGATCGGAACCAA
下游引物GCATTAGCTTCAGATTTACGGGT
MIP1α
上游引物TTCTCTGTACCATGACACTCTGC
下游引物CGTGGAATCTTCCGGCTGTAG
MIP-1β
上游引物TTCCTGCTGTTTCTCTTACACCT
下游引物CTGTCTGCCTCTTTTGGTCAG
GAPDH
上游引物GTGAAGGTCGGTGTGAACGG
下游引物TCCTGGAAGATGGTGATGGG
2.4实验仪器
凝胶成像仪 Bio-Rad
电泳仪 Bio-Rad
荧光定量PCR仪 Bio-Rad
PCR仪 Bio-Rad
高速低温离心机 Eppendorf
倒置显微镜 Leica
电子分析天平 Mettler-Toledo
酶标仪 Thermo
切片机 Thermo
包埋机 Thermo
制冰机 宁波新芝
自动生物组织脱水机 日本樱花
纯水仪 上海力康
高压锅 上海申安
干燥箱 上海一恒
Micro-CT Bruker
二、实验方法
1.基因工程小鼠饲养管理与鉴定
小鼠的饲养管理:
(1)C57BL/6小鼠和anti-Act1小鼠均饲养于SPF级环境中。
(2)饲养室:温度维持在22-28℃之间,相对湿度稳定在40%-60%之内,噪音小于60dB,饲养室内光照明暗交替时间为各12h,紫外灯照射每天2h,氨浓度不超过20ppm,饲养室换气次数达到10-20次/h,饲养器具定期更换并清洗消毒。
(3)饲料、垫料及饮用水:所有小鼠的饲料均从广东省动物中心购买,为无菌全价营养颗粒饲料。笼架内持续供应新鲜、干燥饲料,每周固定时间添加饲料;小鼠饮用水经高温高压灭菌后的水瓶分装蒸馏水后供给,每周3d换水一次,更换后的水瓶及时清洗消毒;笼架内的垫料均为经高压灭菌的锯木花或玉米芯;所有笼架经灭菌后使用,鼠笼和垫料每周更换两次。
(4)日常登记和观察:每天检查并记录动物房环境数据并密切观察小鼠饮食、活动及全身情况,若发现异常状况需及时登记并处理。小鼠健康的标准是:①体毛光滑浓密,肌肉丰满,行动能力正常;②眼睛有神,反应敏捷;③食欲旺盛,进食波动不大;④身无破损及畸形;⑤粪便黑色干燥呈麦粒状。
(5)基因工程小鼠保种:小鼠在6-8周龄性成熟,此为适宜的配种周龄,一般雌鼠比雄鼠早成熟;尽量挑取状态好的小鼠用做繁殖。判断状态的标准包括小鼠的大小、毛色等。如果繁殖笼内小鼠长时间不生育,需及时更换新鼠。将8周龄以上符合配种要求的成熟雄性和6周龄以上符合配种要求的雌性以1:2的比例合笼饲养,每笼最多不超过三只。
anti-Act1基因工程小鼠的鉴定:
(1)剪尾:用酒精消毒后的剪刀剪下小鼠尾尖部尾巴约1cm,置于1.5mL EP管中。剪尾后用棉球捏紧尾端伤口进行止血。剪尾后的小鼠需编号处理,以便鉴定辨认。
(2)抽提DNA:向每只EP管中加入180μL浓度为50mmol/L的NaOH溶液,100℃金属浴裂解60min。从金属浴中拿出碱裂解好的鼠尾组织,加入20μL浓度为1mol/L的HCl溶液,10000rpm离心10s,得到上清即为DNA样本。4℃保存待用。
(3)使用qPCR方法鉴定Act1的表达水平,所用引物为SEQ ID NO:1和2所示。
图1所示为anti-Act1基因工程小鼠中Act1 mRNA表达水平的检测结果。
2、小鼠牙周炎模型建立
取8周龄的雄性鼠为实验小鼠,按照动物伦理要求对小鼠进行麻醉后,俯卧固定在动物专用固定板上,酒精消毒口腔。围绕左上颌第二磨牙放置5-0丝线,并用外科结固定丝线,所有线结置于腭侧。结扎完成后将小鼠舌头稍微牵拉出口外,置于较为温暖的场所,直至小鼠苏醒,继续普通饲料喂养及自由饮水。结扎当天记为0d,于7d对实验小鼠进行安乐死。术后每天检查丝线固定情况,取材前若丝线松脱或线结不完整则排除在实验之外。
3、动物标本取材
小鼠牙周炎组织样品取材:将小鼠放入盛有异氟烷的干燥器内,待小鼠麻醉晕倒后约10s后立即取出,脊椎脱臼处死小鼠,将其腹部朝上固定在解剖板上,手持镊子、剪刀将小鼠上下颌分开暴露小鼠的左侧上颌骨,轻轻剪开并取下结扎线,分离小鼠左上颌骨,用PBS将血液冲洗干净后根据后续实验需要进行进一步处理;
Micro CT扫描实验样本处理:解剖得到的小鼠左侧上颌骨马上浸入4%多聚甲醛固定液在室温中固定16h,4%多聚甲醛固定液体积与样本体积比例至少5:1,必须完全浸没样品表面;
组织总RNA及蛋白提取实验样本处理:解剖得到的小鼠的左侧上颌骨立刻装入无菌干燥的冻存管内并置于液氮中,稍后可转移至负80℃冰箱中保存;
切片制作的样本处理:
(1)将上述取得的小鼠左上颌骨标本放入包埋盒内,浸入4%多聚甲醛固定在室温中固定16h后转入EDTA脱钙液中缓慢脱钙约24d,至上颌骨组织完全脱钙后,取出进行下一步操作;
(2)自动生物组织脱水机进行脱水:70%乙醇、80%乙醇、90%乙醇、95%乙醇依次各浸泡30min,无水乙醇浸泡60min(重复两次),二甲苯60min(重复两次);
(3)浸蜡:于低熔点蜡缸浸泡60min(重复两次),高熔点蜡缸浸泡60min;
(4)浸蜡后的组织块进行石蜡包埋,用石蜡切片机沿着左上颌后牙中央窝连线切割小鼠左上颌骨样品,切成3μm的标准切片,用APES(3-氨基丙基三乙氧基硅烷)处理过的载玻片收集组织切片,晾干,暂未使用的切片分类置于4℃待用。
4、Micro CT扫描分析
Micro CT扫描分析软件:为了检测牙槽骨吸收高度,对所有实验小鼠左上颌骨样本通过skyscan1172软件进行扫描,通过CT vox软件获取样品三维图像并进行骨密度(BMD)分析,利用Dataviewer软件测量数据及选择感兴趣区域
小鼠牙槽骨吸收高度测量方法:扫描后通过三维重建测量上颌第二磨牙颊、腭侧各三处的骨吸收高度,骨吸收高度为从釉牙骨质界到牙槽嵴顶的距离(如图1-3A)。在上颌第二磨牙根分叉处选择大小一致的感性趣区域进行骨密度分析,最后在软件中导出数据,将所得数据进行统计分析。
5、病理相关实验
HE染色实验:
(1)脱蜡水化:将组织玻片置于烤箱内65℃烘片60min;将玻片依次浸入二甲苯1(20min);二甲苯2(20min);无水乙醇1(20min);无水乙醇2(20min);95%乙醇(15min);90%乙醇(15min);80%乙醇(10min);75%乙醇(10min);双蒸水浸洗后稍晾干;
(2)染色及脱水透明:苏木素浸泡约3min,盐酸酒精返蓝液约5s,自来水冲洗20min,伊红染色液浸泡40s;接着75%乙醇,85%乙醇,90%乙醇,95%乙醇,无水乙醇1,无水乙醇2,二甲苯1,二甲苯2中各15s,过程中轻微震荡;最后在通风橱中晾干;
(3)封片:在载破片上组织中央滴入适量中性树胶盖上盖破片进行封片。
TRAP染色实验:按照TRAP染色试剂盒(组织)说明书对切片中抗酒石酸酸性磷酸酶阳性细胞进行染色。
免疫组化实验:
(1)制备pH 6.0的柠檬酸抗原修复液:
称量柠檬酸粉末10.505g及柠檬酸三钠粉末14.705g,加入双蒸水定容至1000mL,调节pH值至6.0,现配现用。
(2)配置3%过氧化氢/甲醇溶液:
量筒量取30%过氧化氢溶液30mL及甲醇溶液270mL,两种溶液于室温下混匀,避光保存,现配现用,使用前放置于37℃恒温箱中预热。
(3)脱蜡:如HE染色法中所示预备对石蜡切片进行脱蜡处理;随后将切片放入PBS中轻轻洗5min,重复三次;
(4)抗原修复:然后将切片放入装有pH 6.0的柠檬酸抗原修复液容器中,将其放入有水的高压锅内,当高压锅中的水开始沸腾时计时约10min后停止加热;取出装有切片机修复液的容器,室温中自然恢复室温后,将切片放入PBS液中5min,重复三次;
(5)接着将组织玻片浸泡在预热的3%过氧化氢/甲醇溶液中并放置在37℃恒温箱内,30min后取出切片再次放入PBS液中5min,重复三次;
(6)封闭非特异抗原:擦干切片上组织周围的溶液,然后在组织上滴上10%BSA使其完全覆盖组织,轻轻放入装有双蒸水的湿盒中,于37℃恒温箱中放置60min;(7)一抗孵育:吸干组织上的10%BSA,在组织上加上按照一定比例稀释的一抗溶液,再次放回湿盒中,4℃过夜;
(8)二抗孵育:从4℃冰箱中取出湿盒后,放置在室温中回温30min,然后从湿盒中取出切片,放入PBS液中5min,重复三次。吸干组织周围的液体,往组织上滴加稀释过的二抗,放回湿盒中,37℃恒温箱中孵育60min,从湿盒中取出玻片,放入PBS液中5min,重复三次;
(9)显色及复染细胞核:轻轻擦干组织周围溶液,DAB溶液显色,显微镜下观察显色情况,待阳性显色可见后,马上用自来水冲洗停止显色;苏木素复染细胞核30s,自来水冲洗返蓝60min;
(10)脱水透明及封片步骤同HE染色法。
病理切片统计分析:
(1)对于HE染色切片:在显微镜放大倍数相同的情况下拍摄每个切片样品中小鼠左上第二磨牙及其近中牙槽骨,在Imag J软件中测量釉质牙骨质界到近中牙槽嵴顶的高度,得出牙槽骨吸收高度数值,然后按照图中比例尺转换为以μm为单位的数据进行统计分析。
(2)对于TRAP染色切片:每组样本最少来自六只不同小鼠的上颌骨。用Image-ProPlus 6.0软件计数40×物镜下拍摄的至少五个不重复视野中抗酒石酸酸性磷酸酶阳性细胞的总和,重复至少三次,得到的数据进行统计分析。
6、实时荧光定量PCR
提取组织总RNA:取新鲜或冻存于负80℃冰箱中的小鼠组织倒入经过液氮预冷的研钵中,同时加入1mL含有1%β硫基乙醇的裂解液,迅速研磨成液状;后续步骤根据试剂盒说明书进行。最后得到组织总RNA溶液后,用超微量分光光度计检测RNA浓度。
用两步法试剂盒并根据试剂盒说明书进行反转录PCR:
(1)第一步反应条件及反应体系:反应条件为42℃变性2min,4℃储存降温;反应体系如下:
1)总RNA样品含1μg RNA的样品体积
2)5×gDNA Eraser Buffer 2μL
3)gDNA Eraser 1μL
4)RNase Free dH2O补足至总体积10μL
(2)第二步反应条件及反应体系:反应条件为37℃(15min),85℃(5s),4℃储存降温,获得的cDNA储存于-20℃,使用时进行相应稀释。反应体系如下:
1)第一步反应产物10μL
2)5×Prime Script Buffer2 4μL
3)RT Enzyme Mix 1μL
4)RT Prime Mix 1μL
5)RNase Free dH2O 4μL
采用Takara的SYBR Green试剂盒进行实时荧光定量PCR检测:
(1)反应程序:
1)预变性95℃5min
2)变性95℃30s
3)退火60℃30s
4)延伸72℃30s
(以上步骤按顺序重复40个循环)
5)溶解曲线分析
(2)反应体系
1)RT2 SYBR Green Master Mix 12.5μL
2)ddH2O 8.5μL
3)cDNA样品2μL
4)上游引物1μL
5)下游引物1μL
(3)测得的阈循环(threshold cycle,Ct值),根据公式△Ct=[Ct(目的基因)]-[Ct(GAPDH)]和△△Ct=[△Ct(实验组)]-[△Ct(对照组)],计算出2△△Ct,反应目的基因表达水平。
7、统计分析
所有实验数据采用Graph Pad Prism 7统计软件对数据进行统计分析。两组间比较采用t检验进行统计分析,显著性水平设定为p<0.05,p<0.01,p<0.001。
三、实验结果
为了观察对比C57BL/6小鼠及anti-Act1小鼠的牙周炎表型,本发明通过运用micro CT扫描分析技术及HE染色法对比牙周炎时C57BL/6小鼠及anti-Act1小鼠的牙槽骨吸收情况及病理改变的差异。Micro CT扫描分析结果(图2的A~C)显示anti-Act1小鼠牙周炎时左上颌第二磨牙周围牙槽骨吸收较C57BL/6小鼠更明显且骨密度明显降低;而HE染色结果(图2的D~E)显示anti-Act1小鼠具有更明显的炎症反应,其主要表现为anti-Act1小鼠牙龈上皮变薄、断裂甚至消失,大量炎症细胞堆积于上皮断裂处,附着丧失至根中1/3,而C57BL/6小鼠只表现轻微附着丧失,且anti-Act1小鼠左上颌第二磨牙近中釉质-牙骨质界水平至牙槽嵴顶水平距离明显大于C57BL/6小鼠。从上述实验结果可以发现特异性抑制巨噬细胞Act1表达强烈影响小鼠牙周炎,对牙周炎的发生发展起促进作用。
此外,本研究通过实时定量PCR检测牙周炎局部组织中炎症因子(IL-6、IL-1β、TNFα)的表达情况。结果显示anti-Act1小鼠牙周局部组织中炎症因子的表达均高于C57BL/6小鼠(图3)。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 广州医科大学附属口腔医院(广州医科大学羊城医院)
<120> 巨噬细胞中Act1作为牙周炎疾病标志物的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
tcccgtggag gttgatgaat c 21
<210> 2
<211> 23
<212> DNA
<213> artificial sequence
<400> 2
tcagggtgcc ttctaaagaa act 23
Claims (11)
1.用于检测巨噬细胞中Act1表达量的检测剂在制备诊断牙周炎的试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,所述检测剂于转录水平对Act1表达量进行检测。
3.根据权利要求2所述的应用,其特征在于,所述检测剂用于执行如下方法中的至少一种:
PCR法、共振光散射法和生物质谱法。
4.根据权利要求3所述的应用,其特征在于,所述PCR法为实时荧光定量PCR和/或数字PCR。
5.根据权利要求4所述的应用,其特征在于,所述检测剂包括核苷酸序列如SEQ ID NO:1和2所示的引物。
6.根据权利要求5所述的应用,其特征在于,所述试剂盒还含有探针、dNTP、DNA聚合酶、双链特异性荧光染料、内参引物以及水中的一种或多种。
7.根据权利要求1所述的应用,其特征在于,所述检测剂于翻译水平对Act1表达量进行检测。
8.根据权利要求7所述的应用,其特征在于,所述检测剂用于执行如下任一种方法:
生物质谱法、电泳法、色谱法、酶联免疫吸附试验、免疫荧光法、免疫化学发光法、免疫比浊法、免疫印迹法以及斑点印迹。
9.根据权利要求8所述的应用,其特征在于,所述检测剂为抗体。
10.巨噬细胞中Act1表达量降低的非人动物在制备牙周炎非人动物模型中的应用。
11.巨噬细胞中Act1表达量降低的非人动物在鉴别和/或测试药物中的用途;
所述药物用于预防和/或治疗的适应症为牙周炎和/或治疗与牙周炎相关的并发症。
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