CN110665006A - 一种顺铂类药物副作用抑制或治疗靶点 - Google Patents
一种顺铂类药物副作用抑制或治疗靶点 Download PDFInfo
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- CN110665006A CN110665006A CN201911104874.0A CN201911104874A CN110665006A CN 110665006 A CN110665006 A CN 110665006A CN 201911104874 A CN201911104874 A CN 201911104874A CN 110665006 A CN110665006 A CN 110665006A
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Abstract
本发明属于癌症药物治疗副作用抑制技术领域,涉及一种顺铂类药物副作用抑制或治疗靶点。本发明针对癌症治疗过程中存在的肾脏损伤问题,提出LncRNA母系表达基因3作为其治疗靶点,并提出丹皮酚对LncRNA母系表达基因3的抑制效果,为癌症治疗过程中副作用抑制提供了新思路。
Description
技术领域
本发明属于癌症药物治疗副作用抑制技术领域,涉及一种顺铂类药物副作用抑制或治疗靶点。
背景技术
顺铂(Cis-dischlorodiammine-platinum,CDDP)作为一种金属铂类络合物,是一种剂量依赖性化疗药,抗肿瘤效果与其药物剂量呈正相关。目前,顺铂在美国和加拿大被推荐为癌症治疗的首选药物之一,在我国以顺铂为主或顺铂参与的化疗方案占70-80%,顺铂对恶性肿瘤的治疗效果使得它的地位目前无可替代。然而,顺铂在人体内的主要聚集器官为肾脏,可发生累积性毒性损伤,对肾脏造成不可逆转的伤害。接受大剂量顺铂治疗的肿瘤患者,约1/3的患者发生肾功能障碍,这部分患者中的1/4可进展为急性肾功能衰竭,因此顺铂的临床应用受到极大的限制。到目前为止,尚未无毒副作用的常规防治顺铂肾毒性的药物。尽管临床常采用水化疗法辅以碱化和利尿手段进行防治,但其作用有限,且耗时长、易导致水电解质和酸碱平衡紊乱、加重心脏负荷等临床问题。其他如减少用药或给予谷胱甘肽等化学保护剂,在远期疗效等方面也不尽如意。因此,深入探讨顺铂肾毒性的发生机制,并寻找切实有效的防治方案具有重要的临床意义。
发明内容
本发明针对传统癌症治疗过程中存在的副作用大难以抑制的问题提出一种新型的顺铂类药物副作用抑制或治疗靶点。
为了达到上述目的,本发明是采用下述的技术方案实现的:
自噬(autophagy)是真核细胞维持自身稳定的重要方式,通过溶酶体降解衰老的细胞器和细胞内过期蛋白质。降低肾细胞内的自噬水平,能促进肾细胞调亡,加重肾脏损伤,而提高自噬水平则对肾细胞具有保护作用。
增强自噬启动阶段的关键调节因子哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂的活性能诱导自噬,延迟肾小管上皮细胞的凋亡,保护顺铂引起的急性肾损伤,抑制自噬能增加细胞凋亡,加重肾脏损伤,而促进自噬有利于肾小管的再生和修复,自噬活性的降低会导致顺铂诱导的肾毒性升高。
长链非编码RNA(Long non-coding RNA, LncRNA)是一类调控型大分子非编码RNA,广泛存在于哺乳动物体内,参与肾脏的发育,并在急性肾损伤及慢性肾脏疾病的发生、发展中起着重要作用。
LncRNA母系表达基因3(Materally expressed gene 3,MEG3)是一条定位于人类染色体14q32.3母系印记基因。
MEG3序列如下:
agcccctagc gcagacggcg gagagcagag agggagcgcg ccttggctcg ctggccttgg
cggcggctcc tcaggagagc tggggcgccc acgagaggat ccctcacccg ggtctctcct
cagggatgac atcatccgtc cacctccttg tcttcaagga ccacctcctc tccatgctga
gctgctgcca aggggcctgc tgcccatcta cacctcacga gggcactagg agcacggttt
cctggatccc accaacatac aaagcagcca ctcactgacc cccaggacca ggatggcaaa
ggatgaagag gaccggaact gaccagccag ctgtccctct tacctaaaga cttaaaccaa
tgccctagtg agggggcatt gggcattaag ccctgacctt tgctatgctc atactttgac
tctatgagta ctttcctata agtctttgct tgtgttcacc tgctagcaaa ctggagtgtt
tccctcccca agggggtgtc agtctttgtc gactgactct gtcatcaccc ttatgatgtc
ctgaatggaa ggatcccttt gggaaattct caggaggggg acctgggcca agggcttggc
cagcatcctg ctggcaactc caaggccctg ggtgggcttc tggaatgagc atgctactga
atcaccaaag gcacgcccga cctctctgaa gatcttccta tccttttctg ggggaatggg
gtcgatgaga gcaacctcct agggttgttg tgagaattaa atgagataaa agaggcctca
ggcaggatct ggcatagagg aggtgatcag caaatgtttg ttgaaaaggt ttgacaggtc
agtcccttcc cacccctctt gcttgtctta cttgtcttat ttattctcca acagcactcc
aggcagccct tgtccacggg ctctccttgc atcagccaag cttcttgaaa ggcctgtcta
cacttgctgt cttccttcct cacctccaat ttcctcttca acccactgct tcctgactcg
ctctactccg tggaagcacg ctcacaaagg cacgtgggcc gtggcccggc tgggtcggct
gaagaactgc ggatggaagc tgcggaagag gccctgatgg ggcccaccat cccggaccca
agtcttcttc ctggcgggcc tctcgtctcc ttcctggttt gggcggaagc catcacctgg
atgcctacgt gggaagggac ctcgaatgtg ggaccccagc ccctctccag ctcgaaatcc
ctccacagcc acggggacac cctgcaccta ttcccacggg acaggctgga cccagagact
ctggacccgg ggcctcccct tgagtagaga cccgccctct gactgatgga cgccgctgac
ctggggtcag acccgtgggc tggacccctg cccaccccgc aggaaccctg aggcctaggg
gagctgttga gccttcagtg tctgcatgtg ggaagtgggc tccttcacct acctcacagg
gctgttgtga ggggcgctgt gatgcggttc caaagcacag ggcttggcgc accccactgt
gctctcaata aatgtgtttc ctgtcttaac aaaaa。
MEG3在多种正常组织中均有表达,但在诸多肿瘤中表达水平下降或出现缺失。在肾癌等肿瘤细胞或组织中,过表达MEG3能促进肿瘤细胞凋亡。MEG3在顺铂耐药的人肺腺癌A549/DDP细胞中的表达与亲本人肺腺癌A549细胞相比有显著降低。因能抑制细胞增殖、促进细胞凋亡,故MEG3在A549/DDP细胞中的过表达能在体内外增强顺铂的化疗敏感性。相反地,顺铂对MEG3敲除的A549细胞的化疗敏感性有明显降低。MEG3表达低的病人也呈现出对顺铂化疗不敏感的反应。此外,MEG3在人肺腺癌中有明显下调,能通过p53和Bcl-xl等细胞凋亡途径调节顺铂对细胞的耐药性。我们前期预实验验证:在顺铂处理的HK-2肾小管上皮细胞中和顺铂治疗的C57BL/6肺癌小鼠的肾脏组织中,MEG3的表达明显高于对照组,并且随着顺铂剂量的增加和作用时间的延长,MEG3的表达量随之增多。而采用siRNA MEG3沉默HK-2细胞后,细胞凋亡明显减少,LncRNA-MEG3在顺铂肾毒性的发生、发展中起着重要的作用,可以作为治疗靶点。
具体作用机制为LncRNA-MEG3靶向miR-126调控mTOR介导的自噬。
LncRNA可作为一种竞争性内源性RNA(competing endogenous RNA,ceRNA)与微小核糖核酸(micro ribonucleic acid,miRNA)相互作用,参与靶基因的表达调控。MiR-126位于内皮细胞基因EGFl7的一个内含子上,能抑制mTOR活性从而诱导自噬。miR-126的过表达能够保护小鼠肾脏的缺血再灌注损伤。我们前期预实验验证,在顺铂处理的HK-1肾小管上皮细胞后,miR-126的表达处于较低水平,而沉默MEG3后,miR-126的表达显著增高,MEG3与miR-126有着密切的作用关系。
肾脏损伤机理:在顺铂肾毒性发生中,自噬水平有显著下降,提高自噬水平能够缓解肾损伤;顺铂作用后,MEG3的表达明显增多,通过调节mTOR和PI3K/AKT抑制自噬;MiR-126的表达则与MEG3呈负相关,且miR-126能够负调控PI3K/AKT/mTOR信号通路诱导自噬。顺铂能促进肾脏组织或细胞中LncRNA-MEG3的表达增高,伴随miR-126的表达降低,从而增强PIK3R2的活性,诱导PI3K/AKT/mTOR信号通路的活化,抑制细胞自噬,促进细胞凋亡,最终诱发肾脏损伤。
丹皮酚又称牡丹酚,是一种小分子的酚类化合物,是中药牡丹皮和徐长卿的主要有效成份,分子式为C9H10O3。
丹皮酚通过控制LncRNA-MEG3/miR-126/mTOR信号通路,影响细胞的自噬水平,抑制细胞凋亡,对顺铂诱导的肾脏损伤起到缓解作用,降低或逆转顺铂诱导的肾毒性。
与现有技术相比,本发明的优点和积极效果在于:
本发明针对癌症治疗过程中存在的肾脏损伤问题,提出LncRNA母系表达基因3作为其治疗靶点,并提出丹皮酚对LncRNA母系表达基因3的抑制效果,为癌症治疗过程中副作用抑制提供了新思路。
附图说明
图1为顺铂处理的HK-2肾小管上皮细胞中和顺铂治疗的C57BL/6肺癌小鼠的肾脏组织中,MEG3的表达结果。
图2为在顺铂处理HK-2肾小管上皮细胞中,SiRNA沉默 MEG3后,顺铂诱导的肾细胞凋亡情况以及miR-126的表达结果。
图3为加入自噬抑制剂3MA或沉默自噬关键蛋白ATG3后自噬结果和肾细胞凋亡结果。
图4为加入自噬诱导剂Rapamycin或沉默β-arrestins促进自噬后肾细胞凋亡情况。
图5为丹皮酚降低表柔比星诱导的肝脏损伤,减少表柔比星诱导的肝细胞凋亡情况。
图6显示了丹皮酚能降低PI3K, p-Akt蛋白的表达,抑制表柔比星诱导的PI3K/AKT信号通路的激活。
图7显示丹皮酚改善表柔比星诱导的肾功能改变及组织病理学损伤从而减少肾细胞凋亡情况。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合具体实施例对本发明做进一步说明。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开说明书的具体实施例的限制。
实施例1
LncRNA-MEG3靶向miR-126调控PI3K/AKT/mTOR信号通路对自噬的作用
1. MEG3在顺铂肾毒性中的表达
选用肾小管上皮细胞HK-2和TCMK-1细株,顺铂(20,40,60 μM)作用24 h或40 μM顺铂作用12、24和48 h后,RT-PCR检测细胞中MEG3的表达;C57BL/6正常/肺癌小鼠,在顺铂(10,20,30 mg/kg)作用3天或20 mg/kg顺铂作用1、3、5天后,RT-PCR检测小鼠肾脏组织中MEG3的表达。结果见附图1。
2. MEG3抑制肾小管上皮细胞自噬
2.1 在基因敲除或基因沉默体系中,检测MEG3缺失对细胞自噬的影响
1) SiRNA或shRNA转染HK-2细胞系沉默MEG3:选取生长状态良好的HK-2细胞,随机分为正常对照组,siRNA或shRNA MEG3组。铺板18 h待细胞融合度达到60-80%,无抗含10%血清的培养液换液。取若干无菌的EP管,分别加入200 μL转染缓冲液、4 μL脂质体和5 μLsiRNA,使siRNA终浓度为50 nM,室温静置10 min。将上述混合液转移至6孔板内,转染4-6 h后换含双抗含血清的培养基培养,转染48 h后收取细胞提取RNA,RT-PCR检测MEG3表达,测定转染效率。沉默效率>50%可继续试验。
2) Western blot、免疫荧光检测自噬标志物LC3Ⅱ/Ⅰ、Beclin 1、Atg5、Atg7和Atg12的表达。
3) 共聚焦显微镜检测自噬流形成情况:选取生长状态良好的HK-2细胞接种到细胞爬片上,随机分为三组:阴性对照组,siRNA或shRNA MEG3组和过表达MEG3组。将GLP-LC3的腺病毒转染至爬片HK-2细胞内,24 h后换液,转染至对应质粒培养24 h,细胞固定15min,清洗3次,防荧光淬灭剂封片,LSM780激光共聚焦显微镜观察自噬流。自噬形成时,GFP-LC3融合蛋白转位至自噬体膜,在荧光显微镜下形成多个明亮的绿色荧光斑点,一个斑点相当于一个自噬体,通过计数来评价自噬活性的高低。
4) 透射电镜检测自噬小体数目的变化:选取生长状态良好的HK-2细胞,随机分为三组:阴性对照组,siRNA或shRNA MEG3组和过表达MEG3组,转染24 h后,收集细胞离心(1000 rpm×30 min),弃上清加适量固定液,悬浮细胞团块确保固定完全。制备电镜切片,染色,透射电镜观察各组细胞内自噬小体的数目,典型的自噬小体是双层膜结构,内含胞浆成分,有的还能观测到未完全降解的细胞器。
2.2 利用过表达体系,进一步确定MEG3对细胞自噬的作用
1) 构建pcDNA3.0-MEG3真核表达质粒:选取生长状态良好的HK-2细胞,铺板18小时后细胞融合度达到60-80%,无抗含10%血清的培养液换液。取若干无菌的EP管,分别加入200 μL转染缓冲液、4 μL脂质体和2 μL pcDNA3.0-MEG3/ pcDNA3.0,吹打均匀,室温静置10 min。将上述混合液转移至6孔板内,转染4-6 h后换含双抗含血清的培养基培养,转染48 h后收取细胞。G418筛选MEG3稳定株后扩大培养。提取细胞总RNA,用MEG3的扩增引物PCR检测MEG3表达情况。
2) Western blot、免疫荧光检测自噬标志物LC3Ⅱ/Ⅰ、Beclin 1、Atg5、Atg7和Atg12的表达;共聚焦显微镜检测自噬流形成;透射电镜检测自噬小体数目的变化(方法参见2.1)。
2.3 加入Rapamycin诱导自噬或3MA阻断自噬,观察自噬的变化
选取生长状态良好的HK-2细胞接种于24孔板中培养24 h,加入50 nM Rapamycin孵育细胞4 h后诱导自噬,或加入5 nM 3MA作用30 min后阻断自噬,Western blot、免疫荧光检测自噬标志物LC3Ⅱ/Ⅰ、Beclin 1、Atg5、Atg7和Atg12的表达;共聚焦显微镜检测自噬流形成;透射电镜检测自噬小体数目的变化(方法参见2.1)。
3. MEG3靶向miR-126调控PI3K/AKT/mTOR信号通路
3.1 SiRNA沉默miR-126后,检测MEG3对miR-126及PI3K/AKT/mTOR信号通路的调控:
1) RT-PCR检测miR-126的表达水平。
2) 双荧光素酶报告基因、RNA原位杂交检测MEG3与miR-126结合。
双荧光素酶报告基因:选取生长状态良好的HK-2细胞接种于24孔板中培养24 h,将双荧光素酶报告基因载体与转录因子表达质粒共同转染细胞,培养48 h后裂解细胞提取蛋白。加入底物,测定萤火虫/海肾荧光素酶的活性。以海肾荧光素酶为内参,萤火虫荧光素酶活性/海肾荧光素酶活性作为萤火虫荧光素酶相对活性,每组设置3个复孔,重复3次。
3) RIP、RNA pulldown检测miR-126与PIK3R2结合。
RIP:收集HK-2细胞,用等体积的RIP裂解液重悬细胞,反复吹打混匀后冰上静置5min。标记不同的EP管,分别加入50 μL磁珠悬液、500 μL RIP wash Buffer涡旋震荡后置于磁力架上,待磁珠吸附成一条直线后弃上清。100 μL RIP wash Buffer重悬磁珠,各管加入相应抗体孵育30 min,重复上述步骤后每管加入900 μL RIP ImmunoprecipitationBuffer,再加入100 μL细胞裂解液于4℃过夜。离心弃上清,加入500 μL RIP wash Buffer涡旋震荡弃上清,重复5-6次。用150 μL Proteinase K Buffer重悬上述磁珠—抗体复合物,55℃孵育30 min,上清液移至新的EP管中,加入250 μL RIP wash Buffer、400 μL氯仿涡旋震荡离心(14000 rpm×10 min)后取上清至新EP管中,加入50 μL Salt SolutionⅠ、5μL Precipitate Enhancer、850 μL无水乙醇(无RNase)混匀,-80℃保持1 h以上。4℃离心(14000 rpm×30 min)弃上清,80%乙醇冲洗后,4℃离心(14000 rpm×15 min)弃上清,自然风干,10-20 μL DEPC水溶解,进行逆转录。SYBR Green染色,恒压80V下电泳20-30 min,根据marker确定目的基因所处位置,凝胶成像仪检测条带亮度。
RNA 下拉:
①质粒模板的线性化和纯化:根据构建的pcDNA3.0-MEG3和pcDNA3.0-MEG3(antisense)重组体信息,采用Xho Ⅰ限制性内切酶线性化质粒,反应体系包含Xho Ⅰ(10 μL)、10×Buffer 4(20 μL)、0.1%BSA(20 μL)、模板(10 μg)以及无菌水若干补至200 μL,37℃反应2 h。而后加入200 μL的Tris饱和酚/氯仿(25:24)混合液,涡旋震荡,静置后离心(13000rpm×5 min)。上清液移至新EP管中,加入等体积CHCl3,涡旋震荡后离心(13000rpm×5 min)。取150 μL上清加入15 μL的3M NaAc (Ph 5.2)和3倍体积的无水乙醇,-20℃放置60 min。4℃离心(12000 rpm×5 min)后,弃上清,75%乙醇洗涤沉淀,4℃离心(7500 rpm×5min),弃上清晾干,加无Rnase水溶解沉淀,调整浓度为1 μg/μL。
②体外转录:组装转录反应体系:Nuclease-free Water (20 μL)、5×Buffer (4μL)、100mM ATP (1.5 μL)、100 mM GTP (1.5 μL)、100 mM CTP (1.5 μL)、100 mM UTP(1.12 μL)、10 mM Bio-16-UTP (3.72 μL) 、模板(1 μg)和T7 RNA Polymerase (2 μL),37℃孵育4 h。按照每1 μg模板加入1U的RNase-free DNase以消化反应液中的DNA模板,37℃孵育15 min。抽提同步骤①,最后加入1 μL的糖原以标记沉淀位置,-20℃放置60 min。
③RNA纯化:采用Ambion公司的MEGAclearTMKit对体外转录得到的RNA纯化。在RNA样品中加入稀释液至100 μL,再加入350 μL的结合浓缩液和250 μL无水乙醇,混匀后移至立离心柱中,13000 rpm×1 min,弃滤液。加入500 μL洗液,13000 rpm×1 min,重复两次。取50μL洗脱液置于65-70℃水浴中孵育10 min,13000 rpm×1 min,重复两次,取洗液。
④Biotin-RNA下拉实验:取3 μg Biotin修饰的体外转录RNA,用RNA structurebuffer补至100 μL,90℃变性5 min,冰上2 min,随后室温放置20 min。提取细胞总蛋白与60 μL用裂解液清洗过的链亲和素包被的琼脂糖柱子室温孵育1 h后,与预处理的RNA室温缓慢旋转孵育1 h。再加入60 μL用裂解液清洗过的Streptavidin beads室温孵育1 h后,4℃离心(3000 rpm×1 min),弃上清,1 mL的低盐洗液清洗10 min×2次,而后高盐洗液清洗2次,弃上清。加入1×蛋白上样缓冲液30 μL,混匀后沸水加热10 min。将样品按照westernblot 步骤,检测目的条带。
4) Western blot检测PI3K/AKT/mTOR信号通路主要蛋白(p-PI3K,PI3K,p-AKT,AKT,p-mTOR,mTOR)表达。
3.2 Mimics过表达miR-126,检测MEG3对miR-126及PI3K/AKT/mTOR信号通路的调控:
选取生长状态良好的HK-2细胞接种于24孔板中培养24 h,将miR-126 mimics转染至细胞中孵育24 h,RT-PCR检测miR-126的表达水平;双荧光素酶报告基因或RNA原位杂交检测MEG3与miR-126结合;RIP或RNA pulldown检测miR-126与PIK3R2结合;Western blot检测PI3K/AKT/mTOR信号通路主要蛋白(p-PI3K,PI3K,p-AKT,AKT,p-mTOR,mTOR)表达。
以上结果见图2-图4。
实施例2
丹皮酚调控LncRNA-MEG3/miR-126/mTOR信号通路介导的自噬对顺铂肾毒性作用机制的体外实验
1. 顺铂肾毒性细胞模型建立及分组
肾小管上皮细胞HK-2和TCMK-1细胞株,40 μM顺铂分别作用24 h后,分为三组:阴性对照组、MEG3沉默组以及丹皮酚(40μM)干预组。
2. 丹皮酚对肾小管上皮细胞损伤的保护作用(图5-图7)
采用MTT、CCK-8检测细胞增殖能力;流式细胞术、TUNEL检测细胞凋亡;、Western blot、免疫荧光检测凋亡相关蛋白(P53、Bax、Bcl-2和caspase-3)的表达;ELISA检测炎性因子(IL-2、IL-6、TNF-α、IFN-γ)的变化。加入Rapamycin诱导自噬或3MA阻断自噬,观察丹皮酚对肾小管上皮细胞的保护作用。
3. 丹皮酚对MEG3/miR-126/mTOR相关基因和蛋白的表达影响
RT-PCR检测MEG3和miR-126的表达;western blot检测PIK3R2以及PI3K/AKT/mTOR信号通路(p-PI3K、PI3K、p-AKT、AKT、p-mTOR和mTOR)主要蛋白表达。加入Rapamycin诱导自噬或3MA阻断自噬,观察以上指标变化。
4. 丹皮酚对肾小管上皮细胞自噬的影响
Western blot、免疫荧光检测自噬标志物LC3Ⅱ/Ⅰ、Becline 1、Atg5、Atg7和Atg12的表达;共聚焦显微镜检测自噬流形成;透射电镜检测自噬小体数目的变化。加入Rapamycin诱导自噬或3MA阻断自噬,观察自噬的变化。
实施例3
丹皮酚调控LncRNA-MEG3/miR-126/mTOR信号通路介导的自噬对顺铂肾毒性作用机制的体内实验
1. 顺铂肾毒性动物模型建立
培养Lewis肺癌细胞,收集对数生长期的肿瘤细胞,调整细胞悬液浓度至1×107/mL,于C57BL/6小鼠右侧腋部皮下注射细胞悬液0.2 mL,建立Lewis肺癌小鼠皮下移植瘤动物模型。待接种后第8天(肿瘤体积约8mm)时,与正常小鼠同时给药:一次性腹腔注射20 mg/kg顺铂,一组为阴性对照组,一组每天灌胃丹皮酚(30 mg/kg),连续3天。第四天,取小鼠心脏血、肾脏组织后处死。RT-PCR检测小鼠肾脏组织中MEG3的表达。
2. 丹皮酚对小鼠肾脏组织损伤的保护作用
运用血生化分析肾功能指标BUN和SCR的变化;HE staining、TUNEL观察肾脏组织形态学变化;免疫组化检测凋亡蛋白caspase-3表达;肾组织匀浆后,用SOD、MDA、GSH和CAT试剂盒检测肾脏氧化应激水平;ELISA检测炎性因子(IL-2、IL-8、TNF-α、IFN-γ等)的变化;Western blot检测凋亡相关蛋白(P53、Bax、Bcl-2和caspase-3)的表达。加入Rapamycin诱导自噬或3MA阻断自噬,观察丹皮酚对小鼠肾脏组织损伤的保护作用。
3. 丹皮酚对MEG3/miR-126/mTOR相关基因和蛋白的表达影响 RT-PCR检测MEG3及miR-126的表达
Western blot检测PIK3R2以及PI3K/AKT/mTOR信号通路(p-PI3K、PI3K、p-AKT、AKT、p-mTOR、mTOR)主要蛋白表达。加入Rapamycin诱导自噬或3MA阻断自噬,观察以上指标的变化。
4. 丹皮酚对小鼠肾脏组织自噬的影响
免疫组化检测自噬蛋白LC3Ⅱ/Ⅰ表达;肾组织匀浆后,western blot检测自噬标志物(LC3Ⅱ/Ⅰ、Beclin 1、Atg5、Atg7和Atg12)的表达。加入Rapamycin诱导自噬或3MA阻断自噬,观察自噬的变化。
结果
从图1可以看出,顺铂处理的HK-2肾小管上皮细胞中和顺铂治疗的C57BL/6肺癌小鼠的肾脏组织中,MEG3的表达明显高于对照组,并且随着顺铂剂量的增加和作用时间的延长,MEG3的表达随之增多。
从图2可以看出,在顺铂处理的HK-2肾小管上皮细胞中,SiRNA沉默 MEG3后,顺铂诱导的肾细胞凋亡明显减少,而miR-126的表达显著升高。
从图3可以看出,加入自噬抑制剂3MA或沉默自噬关键蛋白ATG3抑制自噬能促进肾细胞凋亡。
从图4可以看出,加入自噬诱导剂Rapamycin或沉默β-arrestins促进自噬能减少肾细胞凋亡。
图3和图4说明抑制自噬能促进肾细胞凋亡,促进自噬则能减少肾细胞凋亡。
从图5可以看出,丹皮酚能降低表柔比星诱导的肝脏损伤,减少表柔比星诱导的肝细胞凋亡。
从图6可以看出,丹皮酚能降低PI3K, p-Akt蛋白的表达,抑制表柔比星诱导的PI3K/AKT信号通路的激活。
图5和图6说明丹皮酚通过抑制PI3K/AKT信号通路降低化疗药诱导的肝毒性。
从图7可以看出,丹皮酚改善表柔比星诱导的肾功能改变及组织病理学损伤,减少肾细胞凋亡。
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域,但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
SEQUENCE LISTING
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ggcaggatct ggcatagagg aggtgatcag caaatgtttg ttgaaaaggt ttgacaggtc 840
agtcccttcc cacccctctt gcttgtctta cttgtcttat ttattctcca acagcactcc 900
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Claims (7)
1.一种顺铂类药物副作用抑制或治疗靶点,其特征在于,所述药物靶点为LncRNA母系表达基因3, 其核苷酸序列如序列表SEQ ID NO:1所示。
2.根据权利要求1所述的顺铂类药物副作用抑制或治疗靶点,其特征在于,所述顺铂类药物副作用为肾功能障碍或急性肾功能障碍。
3.根据权利要求1所述的顺铂类药物副作用抑制或治疗靶点,其特征在于,所述LncRNA母系表达基因3能被丹皮酚抑制。
4.根据权利要求3所述的顺铂类药物副作用抑制或治疗靶点,其特征在于,所述丹皮酚通过中药牡丹皮提取得到。
5.根据权利要求3所述的顺铂类药物副作用抑制或治疗靶点,其特征在于,所述丹皮酚通过中药徐长卿提取得到。
6.抑制LncRNA母系表达基因3的物质在制备治疗顺铂类药物副作用抑制药物中的应用。
7.检测LncRNA母系表达基因3水平高低的物质在制备检测顺铂类药物副作用大小的试剂或试剂盒中的应用。
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XINLU PANG等: "Inhibition of lncRNA MEG3 protects renal tubular from hypoxia‐induced kidney injury in acute renal allografts by regulating miR‐181b/TNF‐α signaling pathway", 《J CELL BIOCHEM.》 * |
YANG XIA等: "Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/β-catenin signaling pathway", 《MOLECULAR MEDICINE REPORTS》 * |
匿名: "NCBI Reference Sequence: NR_002766.2", 《GENEBANK》 * |
汪明明等: "顺铂的肾毒性作用及其防治", 《癌症》 * |
董雅洁等: "bcl-2、 bax、 caspase-3 在细胞凋亡中的作用及其关系", 《中国老年学杂志》 * |
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