CN107119133A - 非疾病诊断和治疗用途的检测瘦素基因多态性的方法及试剂盒 - Google Patents
非疾病诊断和治疗用途的检测瘦素基因多态性的方法及试剂盒 Download PDFInfo
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Abstract
本发明涉及分子生物学技术领域,公开了一种非疾病诊断和治疗用途的检测瘦素基因多态性的方法及试剂盒。该方法包括下述步骤:(1)以待检测基因组DNA为模板,进行PCR扩增;得到PCR扩增产物;(2)利用限制性内切酶对PCR扩增产物进行酶切,得到酶切产物;(3)对酶切产物进行琼脂糖胶电泳,然后进行胶回收;得到胶回收产物;(4)对胶回收产物进行基因型分析,确定瘦素基因的多态性;其中,步骤(1)中的PCR扩增,使用SEQ ID NO:1所示的上游引物和SEQ ID NO:2所示的下游引物进行。本实施方式通过设计特异性引物对,使得瘦素基因的多态性位点的基因型能够快速、准确的检测,降低他汀类降脂药物导致的肌病副作用的风险,提高了高血脂症患者的降脂疗效。
Description
技术领域
本发明涉及分子生物学技术领域,特别涉及一种非疾病诊断和治疗用途的检测瘦素基因多态性的方法及试剂盒。
背景技术
高血脂症是一种常见的代谢性疾病,它是引起心脑血管疾病的重要风险因素,与动脉硬化、肾病、糖尿病、肥胖、脂肪肝等疾病有密切关联,尤其是与高血压和心脑血管疾病等相关,严重地危害了人们的健康。他汀类药物是目前临床上应用得最广泛的一类降脂药物,但由于遗传和环境因素导致的个体化差异,他汀类药物的降脂疗效在不同个体中也表现得各不相同。同时,他汀类药物具有严重的肌病副作用很大程度上限制了他汀类药物临床的广泛使用。
3-羟基-3-甲基戊二酰辅酶A还原酶抑制剂(HMG-CoA)即是临床他汀类药物,由于它可以抑制胆固醇的合成,因此广泛用于降低胆固醇治疗高血脂症患者。HMG-CoA还原酶是胆固醇合成限速酶,他汀类药物能阻断HMG-CoA还原酶的活性。该抑制可导致低密度脂蛋白胆固醇受体水平上调,进而增加低密度脂蛋白的摄入和降解、减少胆固醇累积、减少脂蛋白分泌和胆固醇的合成。尽管HMG-CoA还原酶能有效降低胆固醇水平、降低临床心血管事件达20%~50%,且风险降低获益显著,但是临床在使用时需要谨慎,因为他汀类药物能导致明确的副作用,主要包括从非特异性的肌炎到横纹肌溶解。研究发现临床上使用他汀类药物可导致约10.5%患者患有肌病症状。血清肌酐激酶(CK)通常是反映他汀类药物诱导肌毒性的指标。而且携带超过CK上限3倍的患者CK水平与肌损伤显著相关。他汀类药物导致的横纹肌溶解症是患者体内迅速增加的CK水平超过上限的10倍导致肌肉纤维断裂。尽管市场上他汀类药物导致的横纹肌溶解症并不常见,临床数据显示阿托伐他汀会导致副反应占12%,而辛伐他汀导致的副反应占25%。遗传变异可能提供证据来解释服用辛伐他汀降脂药物的患者产生肌毒性的个体差异性。
具体地,CK是由两种亚单位组成,一种是M型(肌肉型),另一种是B型(脑型)。这两种亚单位可以组成3种不同的同工酶形式:MM-CK(主要存在于各种肌肉细胞中),MB-CK(主要存在于心肌细胞中),BB-CK(主要存在于大脑中)。CK和AMP依赖的蛋白激酶(AMPK)系统可以共同维持身体的能量稳态。不过,CK系统可以迅速地反应来改变能量状态,而AMPK系统更倾向于中长期的能量调控。AMPK是由细胞内升高的AMP:ATP和Cr:PCr的比率来驱动的,这两种比率的升高也反应出了身体内能量的缺乏。AMPK一旦被激活,将磷酸化并失活肌肉型MM-CK。PCr系统的功能主要是作为能量供给过程中ATP快速补充的缓冲剂。而PCr的消耗又可以升高AMP:ATP的比率,进而提高AMPK的活性。
CK水平在不同肌病个体中都有着差异,并且会被多种因素影响,比如劳作或体育锻炼等。但升高的CK水平仍然反应了一定程度的肌肉损伤。肌肉损伤的特点是受损的横纹肌细胞且细胞内的酶类释放到体内参与循环,特别是CK和myoglobin。在某些罕见的情况下,他汀类的治疗会导致肌病的发生,同时伴随着CK水平的异常升高,特别是在大剂量地服用他汀类或其他类药物的时候,并且,他汀类偶尔还会导致肌肉分解和肌红蛋白的释放(比如横纹肌溶解),甚至会有肾衰竭和死亡的风险。证据显示瘦素基因(LEP)可以通过中央和外周机制来磷酸化并激活AMPK,而CK和AMPK系统可以共同平衡能量稳态。
进一步地,LEP基因,位于染色体的7q31.3位,由3个外显子和两个内含子组成,编码一段16KDa大小的蛋白质,这种蛋白质已被证明和内分泌相关机制有关。LEP通过在下丘脑中结合并激活其受体(LEPR,一种单跨膜转运蛋白,分布于多种组织中,基因位于染色体1p31位)来发挥它的生理作用。已有多个比较常见的LEP基因多态性被报道和人类肥胖的病理生理学有关。LEP基因的一个比较常见的SNP G2548A已被报道和肥胖病人的血清瘦素水平与BMI的变化有关,并且LEP G2548A可能在转录水平上影响瘦素的表达和脂肪中荷尔蒙的分泌。
举例来说:中国专利公开号为CN105018338A(申请号为201410178891.X)公开了一种预测他汀类药物疗效和安全性的基因突变检测芯片,该检测芯片包括固相基片和点载在承载基片上的基因探针阵列,所述基因探针是与他汀类药物疗效和安全性相关基因突变位点互补的寡核苷酸序列。虽然上述检测芯片可以预测他汀类药物对患者的疗效,但是却不能降低他汀类药物诱导肌病的风险。综上所述,目前亟需提供一种能够降低他汀类药物诱导肌病的风险的检测方法。
发明内容
本发明的目的在于提供一种非疾病诊断和治疗用途的检测瘦素基因多态性的方法及试剂盒,降低他汀类药物带来的肌病的风险,提高高血脂症患者的治疗效果。
为解决上述技术问题,本发明的实施方式提供了一种非疾病诊断和治疗用途的检测瘦素基因多态性预测他汀类降脂药物诱导肌病风险的方法,该方法包括下述步骤:(1)以待检测基因组DNA为模板,进行PCR扩增;得到PCR扩增产物;(2)利用限制性内切酶对PCR扩增产物进行酶切,得到酶切产物;(3)对酶切产物进行琼脂糖胶电泳,然后进行胶回收;得到胶回收产物;(4)对胶回收产物进行基因型分析,确定瘦素基因的多态性;其中,步骤(1)中的PCR扩增,使用SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物进行:
SEQ ID NO: 1:5’- AGC CAA GGC AAA ATT GAG G -3’
SEQ ID NO: 2:5’- GGA GAC TGA GGC GGG AGG A -3’。
本发明的实施方式还提供了一种检测瘦素基因多态性预测他汀类降脂药物诱导肌病风险的的试剂盒,该试剂盒包括特异性引物对,其中,该特异性引物对包括:
SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物:
SEQ ID NO: 1:5’- AGC CAA GGC AAA ATT GAG G -3’
SEQ ID NO: 2:5’- GGA GAC TGA GGC GGG AGG A -3’。
本发明实施方式相对于现有技术而言,通过设计特异性引物对(即SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物),使得瘦素基因(LEP)的多态性位点的基因型能够快速、准确的检测出来,有效降低了他汀类降脂药物导致的肌病副作用的风险,提高了高血脂症患者的降脂疗效。
另外,步骤(1)中进行PCR扩增的反应体系为:待检测基因组DNA模板 45ng;上游引物20umol/L 0.15ul;下游引物20umol/L 0.15ul;dNTPs 2.0 mmol/l 4ul;10×缓冲液1.0ul 2.5ul;Taq DNA聚合酶3U 0.15ul;加ddH2O补足至25ul。
另外,步骤(1)中进行PCR扩增的反应程序为:预变性:95℃10分钟;变性:94℃ 30秒;退火:59℃ 45秒;延伸:68℃ 45秒,进行35个循环;终延伸:68℃ 7min;得到PCR扩增产物。
另外,步骤(2)中限制性内切酶为HhaI内切酶。
另外,步骤(2)中酶切的反应体系为:PCR扩增产物10ul,10×缓冲液 1.5ul,HhaI内切酶0.4ul,ddH2O 3.1ul;总15ul。
另外,步骤(3)中对酶切产物进行琼脂糖胶电泳的反应步骤为:将酶切产物点样在质量百分含量为2.5%的琼脂糖胶上,200V电压下电泳1小时。
另外,步骤(4)中的确定瘦素基因的多态性是指:确定待检测基因组DNA中瘦素基因的G2548A位点的多态性。
附图说明
图1是本发明第一实施方式中LEP G2548A(rs7799039)基因PCR产物酶切电泳图,其中:
从右至左分别为泳道1~11:泳道1~11分别表示:1:Marker(标准);2:GA;3:GA;4:GG;5:GG;6:GG;7:GG;8:GG;9:AA;10:GA;11:GA。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明的各实施方式进行详细的阐述。然而,本领域的普通技术人员可以理解,在本发明各实施方式中,为了使读者更好地理解本申请而提出了许多技术细节。但是,即使没有这些技术细节和基于以下各实施方式的种种变化和修改,也可以实现本申请所要求保护的技术方案。
本发明的第一实施方式涉及一种非疾病诊断和治疗用途的检测瘦素基因多态性预测他汀类降脂药物诱导肌病风险的方法,该方法包括下述步骤:
(1)以待检测基因组DNA为模板,进行PCR扩增;得到PCR扩增产物;
(2)利用限制性内切酶对PCR扩增产物进行酶切,得到酶切产物;
(3)对酶切产物进行琼脂糖胶电泳,然后进行胶回收;得到胶回收产物;
(4)对胶回收产物进行基因型分析,确定瘦素基因的多态性;其中,步骤(1)中的PCR扩增,使用SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物进行:
SEQ ID NO: 1:5’- AGC CAA GGC AAA ATT GAG G -3’
SEQ ID NO: 2:5’- GGA GAC TGA GGC GGG AGG A -3’。
值得注意的是,步骤(1)中的待检测基因组DNA的提取步骤如下:
(a)抽取734个受检者的全血,在全血中加入30ml红细胞裂解液,缓慢摇匀,室温静置10分钟,期间,摇动数次,彻底裂解红细胞;
(b)于4℃、2000转/分离心10分钟,去上清,将沉淀的白细胞在旋转振荡器上打散,加蛋白酶40ul、RNA酶50ul,摇匀,加白细胞裂解液置15ml,混匀37℃水浴20分钟后取出,置冷水中;
(c)加冷的蛋白沉淀液4ml,混匀后放在-20℃冰箱5分钟,取出于4℃、3000转/分离心10分钟,将上清液倒入已加好15ml异丙醇的50ml离心管中缓慢摇动数次,至DNA絮状物析出;
(d)将析出的DNA絮状物移至另一个1.5ml离心管中,75%乙醇1ml洗DNA絮状物后,室温干燥;
(e)加DNA水化液1.0ml,置摇床,摇动过夜,备用;
(f)DNA浓度的测定采用紫外分光光度法,分别测定260nm及280nm两个波长下的OD值,以OD 260nm * 50所得值为DNA浓度。并以OD 260nm/280nm比值估计DNA纯度。
优选地,步骤(1)中进行PCR扩增的反应体系为:待检测基因组DNA模板 45ng;上游引物20umol/L 0.15ul;下游引物20umol/L 0.15ul;dNTPs 2.0 mmol/l 4ul;10×缓冲液1.0ul 2.5ul;Taq DNA聚合酶3U 0.15ul;加ddH2O补足至25ul。
进一步地,步骤(1)中进行PCR扩增的反应程序为:预变性:95℃10分钟;变性:94℃30秒;退火:59℃ 45秒;延伸:68℃ 45秒,进行35个循环;终延伸:68℃ 7min;得到274bp的PCR扩增产物。
优选地,步骤(2)中限制性内切酶为HhaI内切酶。
具体地,步骤(2)中酶切的反应体系和反应条件为:PCR扩增产物10ul,10×缓冲液1.5ul,HhaI内切酶0.4ul,ddH2O 3.1ul;总15ul,37℃过夜,得到酶切产物。
优选地,步骤(3)中对酶切产物进行琼脂糖胶电泳的反应步骤为:将酶切产物点样在2.5%的琼脂糖胶上,200V电压下电泳1小时。
另外,步骤(4)对胶回收产物进行基因型分析的步骤是在紫外灯下读取胶回收产物的胶图(如图1所示)并进行基因型分析。其中,步骤(4)中的确定瘦素基因的多态性是指:确定待检测基因组DNA中瘦素基因的G2548A位点的多态性。
本实施方式的实验结果如下:具体如图1所示;
酶切片段为242bp,LEP基因型为2548GG;
酶切片段为242+181+61bp,LEP基因型为2548GA;
酶切片段为181+61bp,LEP基因型为2548AA。
由于人群中(非个人)有三种基因型,即GG、GA和AA,它们的区别是通过酶切位点的不同,产生不同的片段跑胶后进行分型。本实施方式统计分析并计算了不同基因型(即不同人的DNA)对他汀类药物诱导肌病的风险进行评估,最后得到的结果如下:LEP多态性位点基因型为2548AA纯合突变型时,预测他汀类药物诱导CK升高风险较高,并且肌病风险升高可能性较大;LEP多态性位点基因型为2548GG野生型或2548GA杂合型时,预测他汀类药物诱导CK升高风险较低,并且肌病风险升高可能性较小。
表1. LEP G2548A多态性对辛伐他汀治疗29和57天后CK水平改变的影响
表1中的校正变量:年龄、性别、BMI、抽烟和饮酒;加粗字体表示显著性结果。
为了调查清楚他汀类治疗后,LEP G2548A多态性对肌酸激酶水平变化的影响,我们研究了这些多态性和辛伐他汀治疗29天和57天后的CK变化之间的关联。结果如表1所示。在经过29天的辛伐他汀治疗后,CK水平有了显著的升高。在总人群中,AA基因型携带者CK水平的升高大于GA(P=0.005)和GA+GG(P=0.009)基因型携带者CK水平的上升。在经过57天的辛伐他汀治疗后,我们同样发现在总人群中,AA基因型携带者CK水平的升高大于GA(P<0.001)和GA+GG(P<0.001)基因型携带者CK水平的升高。
如表2所示,基于CK水平将高血脂人群进行三分位,进一步评估LEP基因型与肌病风险的相关性。他汀类药物治疗29天后,与低三分位人群相比,适度增加CK水平的人群和高增加CK水平的人群AA基因型频率显著增加,肌病风险显著增加(P=0.027和P=0.002)。他汀类药物治疗57天后,与低三分位人群相比,适度增加CK水平的人群和高增加CK水平的人群AA基因型频率显著增加,肌病风险进一步显著上升(P=0.013和P<0.001)。
表2. LEP G2548A基因型增加肌病风险的多元logistic回归模型
表2中的校正变量:年龄、性别、BMI、抽烟和饮酒;加粗字体表示显著性结果。
与现有技术相比,本实施方式提供了一种用于测定瘦素基因多态性位点基因型位点的引物序列,并且根据该引物序列和靶序列特点确定了扩增效率高、特异性好和省时的检测方法,方便本领域的普通技术人员掌握和使用,有很好的实用价值。另外,使用该引物序列测定LEP基因多态性位点基因型,以用于预测他汀类降脂药物诱导肌病风险升高。因此能够更早地预见降脂药的副作用,同时更好地预防和干预高脂血症的进程,降低医疗成本。
本发明的第二实施方式涉及一种检测瘦素基因多态性预测他汀类降脂药物诱导肌病风险的的试剂盒,该试剂盒包括特异性引物对,其中,特异性引物对包括:
SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物:
SEQ ID NO: 1:5’- AGC CAA GGC AAA ATT GAG G -3’
SEQ ID NO: 2:5’- GGA GAC TGA GGC GGG AGG A -3’。
本领域的普通技术人员可以理解,上述各实施方式是实现本发明的具体实施例,而在实际应用中,可以在形式上和细节上对其作各种改变,而不偏离本发明的精神和范围。
SEQUENCE LISTING
<110> 安徽大学
<120> 非疾病诊断和治疗用途的检测瘦素基因多态性的方法及试剂盒
<130> 2017
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<213> 人工序列
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Claims (8)
1.一种非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,包括下述步骤:
(1)以待检测基因组DNA为模板,进行PCR扩增;得到PCR扩增产物;
(2)利用限制性内切酶对所述PCR扩增产物进行酶切,得到酶切产物;
(3)对所述酶切产物进行琼脂糖胶电泳,然后进行胶回收;得到胶回收产物;
(4)对所述胶回收产物进行基因型分析,确定瘦素基因的多态性;
其中,所述步骤(1)中的PCR扩增,使用SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物进行:
SEQ ID NO: 1:5’- AGC CAA GGC AAA ATT GAG G -3’
SEQ ID NO: 2:5’- GGA GAC TGA GGC GGG AGG A -3’。
2.根据权利要求1所述的非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,所述步骤(1)中进行PCR扩增的反应体系为:
待检测基因组DNA模板 45ng;上游引物20umol/L 0.15ul;下游引物20umol/L 0.15ul;dNTPs 2.0 mmol/l 4ul;10×缓冲液 1.0ul 2.5ul;Taq DNA聚合酶3U 0.15ul;加ddH2O补足至25ul。
3.根据权利要求1所述的非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,所述步骤(1)中进行PCR扩增的反应程序为:
预变性:95℃10分钟;变性:94℃ 30秒;退火:59℃ 45秒;延伸:68℃ 45秒,进行35个循环;终延伸:68℃ 7min;得到PCR扩增产物。
4.根据权利要求1所述的非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,所述步骤(2)中限制性内切酶为HhaI内切酶。
5.根据权利要求4所述的非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,所述步骤(2)中酶切的反应体系为:PCR扩增产物10ul,10×缓冲液 1.5ul,HhaI内切酶0.4ul,ddH2O 3.1ul;总15ul。
6.根据权利要求1所述的非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,所述步骤(3)中对酶切产物进行琼脂糖胶电泳的反应步骤为:
将所述酶切产物点样在质量百分含量为2.5%的琼脂糖胶上,200V电压下电泳1小时。
7.根据权利要求1所述的非疾病诊断和治疗用途的检测瘦素基因多态性的方法,其特征在于,所述步骤(4)中的确定瘦素基因的多态性是指:确定待检测基因组DNA中瘦素基因的G2548A位点的多态性。
8.一种检测瘦素基因多态性预测他汀类降脂药物诱导肌病风险的的试剂盒,其特征在于,该试剂盒包括特异性引物对,其中,所述的特异性引物对包括:
SEQ ID NO: 1所示的上游引物和SEQ ID NO: 2所示的下游引物:
SEQ ID NO: 1:5’- AGC CAA GGC AAA ATT GAG G -3’
SEQ ID NO: 2:5’- GGA GAC TGA GGC GGG AGG A -3’。
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