CN110575540B - Pdgf抑制剂用于制备治疗肠道炎症疾病的药物方面的用途 - Google Patents
Pdgf抑制剂用于制备治疗肠道炎症疾病的药物方面的用途 Download PDFInfo
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Abstract
本发明属于生物医药领域,涉及PDGF抑制剂用于制备治疗肠道炎症疾病的药物方面的用途,尤其涉及PDGFR信号通路抑制剂在制备治疗放射性肠病的药物中的应用。本发明首次发现了在放射性肠病的肠组织中,PDGFR信号通路的配体和受体均高表达。进一步地,通过抑制PDGFR信号通路,有效地缓解放疗引起的肠道损伤。本发明为治疗放射性肠病提供了一种全新的思路。
Description
技术领域
本发明属于生物医药领域,涉及PDGF抑制剂用于制备治疗肠道炎症疾病的药物方面的用途,尤其涉及PDGFR抑制剂在制备治疗放射性肠病的药物中的应用。
背景技术
放射性肠病(Radiation Enteropathy,RE)是指盆腹腔肿瘤患者接受放疗后出现的迁延反复的放射性肠道损伤,其常见症状包括腹泻、便秘、腹胀、腹痛、肛门疼痛、直肠出血和大便失禁等,少数患者症状迁延不愈,甚至会出现肠梗阻、穿孔、肠瘘和顽固性肠道出血等严重并发症。RE的主要病理改变包括粘膜血管异常增生、粘膜下层纤维化及阻塞性动脉内膜炎,与RE常见症状如直肠出血、肠梗阻的发生发展密切相关1。随着放疗的普及应用,RE 的发生率也随之升高,因此,探寻及研究治疗放射性肠病(Radiation Enteropathy RE)的靶点及机制越来越受到人们的关注。
然而,疾病的发病机制千变万化,各个器官和组织的疾病发生各有不同,往往同一个分子在不同组织中的作用都不一样甚至会相反,不同分子在不同组织间的作用不能简单推论。虽然放射性肠病是以纤维化损伤为重要病理表现,但是具体机制尚不清楚。放射性肠病的治疗仍然是医学界的一大难题。
现已被公开的纤维化通路有多个,例如:
(1)CCL2-CCR2通路:CCL2(monocyte chemoattractant protein-1,MCP-1)能够吸引 CCR2阳性的单核细胞、T细胞及成纤维细胞等聚集,并诱导单核细胞分化为分泌促炎症因子为主的M1单核细胞及分泌促纤维化因子为主的M2单核细胞,在糖尿病诱导的肾纤维化疾病中,抑制CC2-CCR2信号轴能够减轻肾脏纤维化病变。
(2)TNFα-TNFR通路:TNFα能够刺激TNFR阳性细胞分泌促纤维化因子如IL-1β、CCL2以及TGF-β1,进而促进纤维化。
(3)TGF-β1/Smad通路(经典通路):TGF-β1与细胞膜表面的I型及II型丝/苏氨酸激酶受体(TGFR1、TGFR2)结合,诱导细胞质内Smad因子磷酸化,进而入核调节纤维化相关因子的转录,TGF-β1/Smad通路作为机体损伤修复纤维化过程的经典通路,在大部分生理及病理纤维化过程中都起到重要作用。
(4)TGF-β1非经典通路:TGF-β1通过激活非Smad依赖的信号通路来调节纤维化进程,如已有报道在慢性放射性小肠病中,TGF-β1通过早期激活Rho/ROCK通路上调 CTGF,而较高浓度的CTGF能够通过自激活过程将RE-SMC细胞长期维持在分泌状态,进而引起放射性小肠病慢性进行性纤维化。
(5)EGF-EGFR通路:EGF与EGFR阳性细胞结合能够刺激胶原纤维、CTGF以及 TGF-β的表达,已有报道显示抑制EGF-EGFR通路能够减轻慢性肾病纤维化2。
(6)PDGF-PDGFR通路:PDGF(Platelet derived growth factor血小板原性生长因子) 家族包括PDGF-A、PDGF-B、PDGF-C及PDGF-D,这些异构体能够以二硫键聚合为同源二聚体或异源二聚体(AA,AB,BB,CC以及DD)。这些二聚体经过适当的剪切修饰后,它们就能够与细胞膜表面的酪氨酸激酶受体PDGFR-α或PDGFR-β组成的二聚体(αα,αβ,ββ)结合进而发挥自己的生物学功能。在正常情况下,PDGF信号通路对胚胎发育及维持人体内环境稳态起着重要的作用,但PDGF通路的过度表达也与多种疾病相关,已知PDGFR (Platelet derivedgrowth factor receptor,血小板原性生长因子受体)信号通路过度表达与肝癌、前列腺癌、小细胞肺癌以及结直肠癌等多种肿瘤疾病相关,与此同时,在非肿瘤性疾病如各种纤维化疾病(肝、肺、肾、心脏及系统性硬化)中也发现了PDGFR信号通路的过度表达3,4。已有研究显示使用PDGFR抑制剂能够减轻系统性硬化(SSc)动物模型的纤维化损伤5。
其中,最为经典的纤维化通路是TGF-β信号通路,然而,TGF-β1/Smad通路却与放射性肠病关系不大。Haydont,V.等人的研究发现在放射性肠病患者受照射肠道组织中,TGF-β1下游因子CTGF高表达,而TGF-β1无明显增高6。他们通过筛查放射性肠病患者受照射肠道组织及正常肠道组织的基因表达情况,发现Rho/ROCK通路可能与放射性肠病肠壁纤维化及CTGF持续高表达有关,他们利用Rho激酶抑制剂处理离体培养的受照射肠道平滑肌细胞(radiation enteropathy derived smooth muscle cell,RE-SMC)以及离体的受照射肠道组织块,发现Rho激酶抑制剂能够下调RE-SMC及离体组织块中CTGF以及I型胶原纤维等纤维标志物的表达7,8,9。他们随后建立了放射性肠道损伤大鼠模型,发现给大鼠服用Rho激酶抑制剂Pravastatin能够减轻慢性期肠道纤维化,但对于急性期损伤,如肠粘膜细胞坏死、凋亡等无明显改善作用8,10。
可见,虽然放射性肠病是以纤维化损伤为重要病理表现,但经典的TGF-β纤维化信号通路却与放射性肠病无关,由此可见放射性肠病的复杂机制,以及由此给相关研究人员所带来的挑战。
发明内容
本发明目的在于提供一种预防或减轻放疗引起的消化道并发症的药物/方法。
本发明的另一个目的在于提供一种PDGFR信号通路抑制剂用于制备放射性肠病的药物的应用。
本发明的目的通过以下技术手段实现:
本发明提供了PDGFR信号通路抑制剂在制备治疗放射性肠病的药物中的应用。
PDGF属于血管内皮生长因子家族,其受体PDGFR是一种酪氨酸激酶受体,具有蛋白质酪氨酸激酶活性,与配体PDGF结合后,通过特异的酪氨酸残基去磷酸化作用启动并放大信号,促使肌动蛋白重排和发挥促有丝分裂、趋化等生理作用。发明人经研究发现, PDGFR信号通路的多个配体及受体在放射性肠病患者切除的肠道组织中高表达,进一步推断,PDGFR信号通路在受照射肠道组织中高表达与放射性肠道损伤存在一定的相关性,抑制PDGFR信号通路可能缓解放疗引起的肠道损伤。经实验证实,无论是通过敲除PDGF基因抑制配体,还是采用小分子抑制剂抑制PDGFR,均可以到达治疗放疗引起的肠道损伤。
其中,所述的PDGFR信号通路抑制剂包括PDGFR受体抑制剂和/或配体PDGF抑制剂。
根据一个优选的实施方案,所述抑制剂与受体PDGFR和/或配体PDGF结合。在另一个实施方案中,PDGFR/PDGF相互作用可以被抑制。
根据一个优选的实施方案,所述的PDGFR信号通路抑制剂为核酸效应分子。
所述核酸效应分子可以为DNA、RNA、PNA或DNA-RNA-杂合体。所述核酸效应分子可以是单链的或双链的。可以使用衍生自逆转录病毒、腺病毒、疱疹病毒或痘苗病毒或者衍生自各种细菌质粒的表达载体来将核苷酸序列递送至所靶器官、组织或细胞群。可以使用这样的构建体来将不可翻译的正义或反义序列引入到细胞中。甚至在不存在整合到DNA中的情况下,此类载体也可以继续转录RNA分子直至它们由于内源核酸酶而丧失能力。用非复制型载体,瞬时表达可以持续一个月或更久。
所述核酸效应分子可以特别地优选自能够抑制PDGF和/或PDGFR基因的表达小的抑制核酸分子,例如短干扰RNA(siRNA),双链RNA(dsRNA),microRNA(miRNA),核酶,以及小发夹RNA(shRNA),这些都能减弱或消除PDGF和/或PDGFR蛋白的表达。
这些小的抑制核酸分子可能包括第一、第二链,二者杂交彼此形成一个或多个双链区,每条链大约18~28个核苷酸的长度,大约18~23个核苷酸的长度,或者18,19,20,21,22个核苷酸的长度。另外,单链也可能包含能够相互杂交形成双链的区域,例如在shRNA 分子中。
这些小的抑制核酸分子在保持这种减弱或消除PDGF和/或PDGFR的表达的能力时,可能包括修饰性核苷酸。修饰性核苷酸可用于改善体外或体内特性,如稳定性、活性和/或生物利用度。举个例子,这些修饰性核苷酸可能含有脱氧核苷酸、2’-甲基核苷酸、2’-脱氧-2’-氟核苷酸、4’-三核苷酸、锁核酸(LNA)核苷酸和/或2’-O-甲氧乙基核苷酸等。小的抑制核酸分子,如短干扰RNA(siRNA),也可能含有5’-和/或3’-帽结构,以此来防止核酸外切酶对其降解。
在一些实施例中,小抑制核酸分子组成的双链核酸含有两端钝、或悬垂的核苷酸。其他核苷酸可能包括会导致错位、凸起、循环、或摆动碱基对的核苷酸。小抑制核酸分子可以设计配方以便施用,例如,通过脂质体包裹,或掺入其他载体(如可生物降解聚合物水凝胶,或环糊精)。
在一些实施例中,所述的PDGFR信号通路抑制剂包括PDGFR和/或PDGF基因表达抑制工具,包括但不限于RNA干扰(RNAi)microRNA以及基因编辑或敲除等工具手段。
根据一个另一个优选的实施方案,所述的PDGFR信号通路抑制剂为抗体或其功能片段。所述的抗体或其功能性片段特异性地与PDGFR和/或PDGF结合,从而封闭受体或者配体,进而阻断信号通路。
所述抗体可以为例如单克隆抗体、多克隆抗体、重组抗体、人源化抗体、人抗体、嵌合抗体、多特异性抗体或其抗体片段(例如,Fab片段、Fab'片段、F(ab')2片段、Fv片段、双抗体或单链抗体分子)。所述抗体可以是IgG1、IgG2、IgG3或IgG4类型的。
所述抗体可以以具有或没有修饰的形式进行使用,并且可以共价地或非共价地进行标记,用例如报道基团或效应基团。
根据本发明的“抗体片段”呈现与相应的抗体基本上相同的表位结合位点,和/或具有与相应的抗体实质上相同的PDGFR和/或PDGF抑制活性。“抗体片段”包含全长抗体的一部分,一般为其抗原结合区或可变区。抗体片段的实例包括,例如:Fab、Fab’、F(ab’)2和Fc片段;双抗体;CDR(互不决定区);微抗体;线性抗体;抗-独特性(抗-ID)抗体;Fab表达文库制备的片段;以免疫特异性方式结合抗原或抗原的任意的表位-结合片段;单-链抗体分子;由抗体片段形成的多特异性抗体。
本发明中的抗体可以选自现有技术中的抗体,现有技术中,已经公开多种PDGF抗体或 PDGFR抗体,如anti-PDGFC抗体,anti-PDGFRα抗体,和anti-PDGFRβ抗体,等等中的一种或几种。作为更优选的实施方式,所述的抗体选自AF1560(anti-PDGFC)、AF-307-NA(anti-PDGFRα)和AF385(anti-PDGFRβ)中的一种或几种。作为还更优选的实施方式,所述的AF1560、AF-307-NA和AF385选自R&D公司,上述的这些抗体均可用于本发明中,用于治疗放射性肠病。当然,也可以根据PDGFR或PDGF的序列,自行设计和合成特异性抗体。
用于产生本发明的抗体的方法是本领域技术人员已知的。
根据另一个优选的实施方案,所述的PDGFR信号通路抑制剂为PDGFR小分子抑制剂。
更进一步地,所述的PDGFR小分子抑制剂为ATP竞争性抑制剂或PDGF拮抗剂。
ATP竞争抑制剂靶向于PDGFR激酶的ATP结合位点,阻断磷酸化过程。
PDGF拮抗剂结构上与PDGF亚型相似,这类化学物能连接到PDGFR蛋白表面,抑制PDGF与PDGFR的结合。
或者,所述的PDGFR小分子抑制剂为苯胺衍生物,如伊马替尼和SU101;或者为吲哚酮衍生物,如苏尼替尼和SU6668;或者为喹喔啉衍生物,如7d-6等;或者为喹唑啉衍生物,如CT52923。
在本发明的优选的实施例中,所述的PDGFR小分子抑制剂为crenolanib、imatinib(伊马替尼),axitinib(阿西替尼),sorafenib(索拉非尼),CP-673451中的一种或几种。这些化合物可以但不限于通过商品化的方式购买获得。
当然,所述小分子抑制物也可以是上述的各种化合物的具有PDGFR信号通路抑制特性的衍生物之一,其盐、溶剂化物、互变异构体、或同分异构体。
所述的crenolanib具有如下的结构式:
根据另一个优选的实施方案,所述的PDGFR信号通路抑制剂为PDGF小分子抑制剂。
PDGF是由A、B、C、D 4条多肽链(即PDGF-A、PDGF-B、PDGF-C及PDGF-D)通过二硫键形成的同源或异源二聚体分子(AA,AB,BB,CC以及DD)。
进一步地,所述的PDGF抑制剂为PDGF-A、PDGF-B、PDGF-C及PDGF-D中的一个或几个。所述的PDGF抑制剂可以在基因水平,也可以在蛋白水平抑制PDGF-A、PDGF- B、PDGF-C及PDGF-D中的一种或几个。
作为本发明优选地实施方式,所述的PDGF抑制剂用于抑制PDGF-C。
本发明PDGFR信号通路的抑制剂可以以药物组合物的形式来进行提供。该组合物可以包含药学上可接受的承载体、稀释剂和/或助剂等。
另一方面,本发明还提供了一种个性化的放射性肠病用药系统,该系统包括PDGFR信号通路检测试剂或检测系统,以及上述的PDGFR信号通路抑制剂。
其中,所述的PDGFR信号通路检测试剂或检测系统为检测受体PDGFR和/或配体PDGF表达的检测试剂或系统;所述的放射性肠病为急性放射性肠病或者慢性放射性肠病。
本发明经实验发现,放射性肠病的肠组织和患者血浆中,PDGFR信号通路的多个配体及受体的表达均高于正常对照组的肠组织和血浆。
在本发明中所使用的药物组合物可以通过许多途径来进行施用,所述途径包括但不限于:口服、静脉内、肌内、动脉内、髓内、鞘内、心室内、透皮、皮下、腹膜内、鼻内、肠、局部、舌下或直肠方式。
适合于在本发明中使用的药物组合物包括其中以对于达到预期目的来说有效的量包含活性成分的组合物。有效剂量的确定完全在本领域技术人员的能力之内。治疗有效
剂量是指足以治疗特定病状的活性成分(例如,本发明的核酸或蛋白质,或者抗体)的量。准确的用药量将会由医师按照与需要治疗的受试者相关的因素来确定。
本发明的PDGFR信号通路抑制剂用于治疗放射性肠病(RE)。放射性肠病的治疗效果通常较差,临床中较少有治愈成功的案例。目前,放射性肠病的治疗主要以缓解临床症状为目的,包括药物、物理及手术治疗。
本发明的有益效果:
1.目前暂未有针对特定分子靶点的RE治疗方案,也没有能够有效根治或控制RE患者症状的药物,本发明首次发现PDGFR信号通路中,多个配体和受体高表达,并通过PDGFR抑制剂,有效治疗了放射性肠病,尤其是慢性反射性肠病。目前对于慢性放射性肠病无有效的治疗方案或者药品,临床上仅能做到缓解该疾病的症状。本发明的技术方案通过特定的靶点,特异性治疗放射性肠病,对于放疗患者的健康改善具有突破性的意义。
2.本发明无论是抑制PDGF,还是抑制其受体PDGFR,均可以抑制PDGFR信号通路,为放射性肠病的治疗提供了新的思路。
附图说明
图1在放射性肠病(RE)小鼠模型中,盆腔放射引起显著的远端结肠壁纤维化;
A.小鼠照射示意图;
B.对照组小鼠与25Gy照射组小鼠受照射肠段代表性HE染色与Masson染色病理切片(20 倍镜下观察);
C.对照组小鼠与25Gy照射组小鼠肠道组织病理切片放射损伤评分(Radiationinjury score,每组小鼠n=6,p<0.0001,Mann-Whitney test);
D.对照组小鼠与25Gy照射组小鼠肠道纤维化标志物fibronectin,Type1collagen,Type 3 collagen的WesternBlot(WB)结果示意图。
图2PDGFC基因敲除后阻止了放射性肠病(RE)小鼠模型中辐射诱导的直肠纤维化;
A.对照组野生型小鼠、对照组PDGFC KO鼠、25Gy照射组野生型小鼠、25Gy照射组PDGFC KO鼠照射8周后受照射肠段代表性HE染色、Masson染色以及纤维化标志物fibronectin、Type 1collagen,Type 3collagen的IHC染色病理切片(20倍镜下观察);
B.25Gy照射组野生型小鼠与25Gy照射组PDGFC KO鼠肠道组织病理切片放射损伤评分 (Radiation injury score,每组小鼠n=4,p=0.0286,Mann-Whitney test);
C.25Gy照射组野生型小鼠与25Gy照射组PDGFC KO鼠在照射后第8周照射野部位皮肤代表性图片;
D.对照组野生型小鼠、对照组PDGFC KO鼠、25Gy照射组野生型小鼠、25Gy照射组PDGFC KO鼠照射8周后受照射肠段粘膜下层厚度占肠壁全层厚度比例柱状图(每组小鼠 n=4,p<0.05,Kruskal-Wallis test);
E.对照组野生型小鼠、对照组PDGFC KO鼠、25Gy照射组野生型小鼠、25Gy照射组PDGFC KO鼠照射8周后受照射肠段粘膜下层厚度占肠壁全层厚度比例矩形图。
图3施用PDGF抑制剂crenolanib减轻了放射性肠病(RE)小鼠模型的直肠纤维化并提高了RE小鼠模型的存活率;
A.给药组小鼠及对照组小鼠照射及给药时间示意图;
B.Crenolaninb给药组小鼠及Vehicle对照组小鼠照射后每周体重/照射前一天初始体重变化折线图;
C.Crenolaninb给药组小鼠及Vehicle对照组小鼠受照射直肠组织HE,Masson及纤维标志物fironectin/Type 1collagen/Type 2collagen代表性免疫组化染色病理切片;
D.Crenolanib处理组小鼠与Vehicle对照组小鼠肠道组织病理切片放射损伤评分(Radiation injury score,Crenolanib处理组小鼠n=5,Vehicle对照组小鼠n=5,p<0.0001,Mann-Whitney test)。
图4人结肠成纤维细胞(CCD-Co18)在照射后高表达PDGF-CC及Fibronectin;
A.人结肠成纤维细胞(CCD-Co18)对照组、10Gy单次照射组24h后细胞培养液中PDGF- CC含量柱状图(p>0.05,unpair t test);
B.人结肠成纤维细胞(CCD-Co18)对照组、10Gy单次照射组6h、24h后细胞PDGF-CC及fibronectin的WB结果示意图。
图5PDGF抑制剂crenolanib能够下调在CCD-Co18中PDGF-CC诱导的纤维化标志物的表达;
A:Crenolanib处理CCD-Co18与空白对照处理CCD-Co18,纤维标志物fibronectin,Type 1 collagen,Type 3collagen的WB结果示意图;
B:对照组CCD-Co18、PDGF-CC共培养组CCD-Co18,PDGF-CC+PDGFRα抗体组和 PDGF-CC+PDGFRβ抗体组,CCD-Co18中fibronectin(FN1)的qPCR结果示意图。
图6在放射性肠病(RE)小鼠模型和CRP患者中PDGFCC上调;
A.PDGFC mRNA在对照组及25Gy照射组小鼠受照射肠段中的qPCR结果示意图(每组小鼠数量n=6,p=0.0373,unpaired t test);
B.PDGFC mRNA在放射性肠病(Radiation enteropathy,RE)患者手术标本组织及结直肠癌患者近端正常肠道组织中的qPCR结果示意图(RE组n=14,正常对照组n=8, p=0.0004,unpaired t test);
C.PDGFCC蛋白在对照组及25Gy照射组小鼠受照射肠段中的WB结果示意图(每组小鼠数量n=6,p=0.0015,unpaired t test);
D.PDGFCC蛋白在放射性肠病(Radiation enteropathy,RE)患者手术标本组织及结直肠癌患者近端正常肠道组织中的WB结果示意图(每组数量n=6,p=0.013,unpaired ttest);
E RE患者及作为对照组的I期CRC患者血浆PDGFCC含量示意图(RE组n=8,I期肠癌患者对照组n=30,p<0.001,Mann-Whitney test)。
图7crenolanib的化学结构式。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案,具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
本发明中,所述“PDGFR信号通路抑制剂”可以是至少部分地干扰或阻断PDGFR信号通路的任何物质,化学类物质或生物类物质。本发明的抑制剂可以在蛋白质水平和/或核酸水平起作用。在蛋白质水平起作用的抑制剂可以选自抗体、蛋白质和/或小分子。在核酸水平起作用的抑制剂例如反义分子,RNAi分子和/或核酶。
本发明中,在某些时候,PDGFC和PDGFCC可以理解为表示同一种物质。本领域公知,一般蛋白和刺激因子用PDGFCC,基因和mRNA用PDGFC,蛋白用两种都可以。
一、实验材料
1、实验动物
健康雌性SPF级C57BL/6J小鼠,体重16-18g,购于中山大学动物实验中心。PDGFC基因敲除小鼠,体重16g-20g由中山大学眼科中心李旭日教授提供。
2、实验细胞
人结肠成纤维细胞(CCD-Co18),购于ATCC。
3、实验用药及主要试剂
细胞培养相关试剂
DMEM培养基,胎牛血清,PBS粉末,胰蛋白酶,青、链霉素双抗,HEPES, NaHCO3,EDTA
HE&MASSON染色
Harris苏木素,伊红染液,苏木精,无水氯化铁,酸性品红,磷钼酸,冰醋酸,苯胺蓝浓盐酸
IHC
抗原修复液,DAB中杉金桥
WB
一抗稀释液,PDGFC抗体,Fibronectin抗体,T 1collagen抗体,T 3collagen抗体,Gapdh抗体,β-actin抗体,抗山羊二抗,抗兔二抗,抗鼠二抗,ECL发光试剂盒,BCA蛋白定量试剂盒,PVDF膜,脱脂奶粉,蛋白分子量Marker,SDS,甘氨酸,Tris base
QPCR
引物,Trizol,SYBR Green染料,PCR逆转录试剂盒
3、主要实验仪器
PCR仪,实时荧光定量PCR仪,RS2000小动物辐照仪,显微镜,Mini-PROTEAN3电泳系统,Mini Trans-Blot电转系统,-80℃超低温冰箱,冷冻高速离心机,电子分析天平,培养板/瓶/皿
4、主要试剂配制
4.1动物实验主要试剂配制
5%水合氯醛:用10ml双蒸水溶解0.5g水合氯醛警晶体,常温放置备用。
Crenolanib-DMSO-matrigel混合液:将Crenolanib粉剂溶于DMSO中,调节浓度至6mg/ml,使用当天将Crenolanib-DMSO溶液加入4℃液态matrigel中,调节浓度至1mg/ml, 4℃保存。
4.2细胞实验主要试剂配制
10%胎牛血清DMEM:吸取100ml胎牛血清至1000mlDMEM培养液中混匀,4℃保存。
PBS缓冲液:用2000ml三蒸水溶解一包PBS粉末,调p H值至7.2~7.4,分装后高压蒸汽灭菌,于4℃保存。
Crenolanib处理液:称取适量Crenolanib粉末溶解于30%PEG400+0.5%吐温80+5%丙二醇水溶液中,调节浓度至5mM/ml,4℃保存。
PDGFCC处理液:取适量PDGFCC(peprotech,100-00CC-20)粉末溶解于PBS缓冲液中,-80℃保存。
4.3MASSON染色主要试剂配制
Weigent氏苏木素:A液:将1g苏木素加入100ml无水乙醇中微热溶解,室温保存;B液:将4ml 30%无水氯化铁溶液,1ml浓盐酸以及100ml蒸馏水混合,室温保存。使用时将 A液B液等量混合即可,一般现混现用。
Masson丽春红酸性复红液:将丽春红0.7g,酸性复红0.3g,蒸馏水99ml,冰醋酸1ml混合溶解,室温保存。
1%冰醋酸水溶液:将冰醋酸1ml加入100ml水中,室温保存。
1%磷钼酸水溶液:将磷钼酸1g溶解于100ml水中,室温保存。
苯胺蓝水溶液:将苯胺蓝2g溶于98ml蒸馏水及2ml冰醋酸中,室温保存。
1%盐酸酒精:将1ml浓盐酸加到1000ml无水乙醇中,室温保存。
4.4免疫组织化学染色主要试剂配制
柠檬酸修复液:直接取一包柠檬酸钠粉剂加入1L双蒸水中,配成10×柠檬酸钠修复液,使用时直接量取100ml 10×柠檬酸钠修复液,双蒸水定容至1L,现配现用。
4.5Western Blot主要试剂配制
10%过硫酸铵(AP):过硫酸铵10g溶于100ml双蒸水中,于4℃保存,有效使用期不超过30天。
1×电泳缓冲液(25mM Tris,0.25M甘氨酸,1%SDS):
Tris base(MW 121.14g/mol)2.27g,甘氨酸(MW 75.07g/mol)10.8g,SDS0.75g,加蒸馏水至750ml,室温溶解,现配现用。
1×电转液(48mM Tris,39mM甘氨酸,20%甲醇):
Tris base(MW 121.14g/mol)5.8g,甘氨酸(MW 75.07g/mol)2.9g,SDS0.37g,加蒸馏水至 800ml,室温溶解,于4℃冰箱中预冷,最后在临用前加入200ml甲醇搅拌均匀,现配现用。
10%十二烷基硫酸钠聚丙烯酰氨凝胶(SDS-PAGE):按照下表1配置
表1
加TEMED后,立即混匀灌胶。
1×TBST缓冲液(含0.01%Tween20):
Tris base(MW 121.14g/mol)2.42g,NaCl 8.01g,加蒸馏水800ml室温溶解,使用浓盐酸将pH值调到7.4~7.6后,加蒸馏水定容至1000ml,4℃保存。
5%脱脂牛奶封闭液:
1g脱脂奶粉加入到20ml 1×TBST缓冲液中,充分溶解,现配现用。
5、主要抗体
IHC:
Fibronectin抗体 稀释度1:400
T 1collagen抗体 稀释度1:50
T 3collagen抗体 稀释度1:100
WB:
实施例1动物实验
1.1建立放射性肠病动物模型
根据已有文献报道,使用6-8周龄雌性C57BL6/J小鼠作为建模对象。本实验程序及动物处理方式均遵从中山大学实验动物伦理与福利委员会的动物实验指导方针与法则。
(1)从中山大学动物实验中心购买8周龄左右雌性C57BL6/J小鼠,体重约18g-20g,将其置于中山大学北校区动物实验中心适应性饲养一周后照射。
(2)照射前一天对小鼠进行打耳标编号,并采用Excel表生成随机数法将小鼠分入照射组及对照组,同时对两组小鼠进行称重并做记录。
(3)照射当天,照射前半小时用5%水合氯醛对小鼠进行腹腔注射麻醉,麻醉剂量为 250mg/kg。
(4)将麻醉的小鼠俯卧位平放于3.5×6cm铅盒内,铅盒上方为可前后滑动的铅盖,将铅盖下缘移动至距小鼠肛门所在平面约1cm左右的位置,使小鼠肛门往上1cm肠段暴露在X线照射范围内(如图1A所示),将铅盒至于RS2000小动物辐照仪内,调节辐射源电压为160KV,照射时间1430s(总剂量25Gy,剂量率为1.05Gy/min),关闭辐照仪箱门后按“start”开始照射。
(5)照射结束后,待小鼠自然苏醒后带回中山大学北校区动物实验中心继续饲养,照射后每天观察动物状态及可能出现的症状表现,每七天称量动物体重,观察终点设为照射后第八周,若动物在观察终点前死亡,则记录死亡小鼠编号、死亡时间及照射剂量,并按动物中心规定对动物尸体进行无害化处理。
(6)到达观察终点后,将所有小鼠进行颈椎脱臼处死,在超净台内,以75%的酒精浸泡胸部及腹部皮肤后,用眼科剪从小鼠耻骨上联合部位剪开腹壁至剑突,用止血钳分开腹壁暴露腹腔内脏器,用眼科剪剪断耻骨联合,游离结肠至肛缘,齐回肠末端、肛缘离断结肠上下两端,取出整个小鼠结肠。以钢尺为参照,从结肠末端往上剪下约1cm肠段,用眼科剪剖开剪下的1cm肠段,再用手术刀片将肠段纵向分为3等份,一份用于提取RNA,加入装有500微升RNA later液的1.5ml EP管中4℃过夜,第二天吸去RNA later液至于-80℃冰箱保存;一份用于提取蛋白,直接放入1.5ml EP管至于-80℃冰箱保存;一份用于制作病理切片,至于装有500微升10%中性福尔马林的1.5ml EP管中常温保存。
1.2使用PDGFC基因敲除小鼠构建放射性肠病动物模型
(1)本实验所使用的PDGFC基因敲除小鼠均由中山大学眼科中心李旭日教授提供(具体方法参考Ding H,et al.(2004)A specific requirement for PDGF-C in palateformation and PDGFR-alpha signaling.Nat Genet 36:1111–1116.
(2)PDGFC基因敲除小鼠的饲养、麻醉、照射、处死及取材方法与1.1所述方法相同。作为对照组的PDGFC基因敲除小鼠在照射时不放入RS2000小动物照射仪进行照射,其余操作与照射组相同。
1.3使用Crenolanib处理放射性肠病动物模型
在上述第3步麻醉小鼠后,使用两套小鼠12号灌胃针及1ml针筒分别吸取事先准备好的4℃保存的Crenolanib-DMSO-matrigel混合液及作为Vehicle对照的DMSO-matrigel混合液,将灌胃针经肛门插入小鼠结直肠约2cm,然后缓慢退出肛门,同时注入Crenolanib-DMSO-matrigel混合液或对照DMSO-matrigel混合液约0.1ml进入小鼠结直肠,推注时保持用力均匀,尽量使药液均匀分布在小鼠肛门往上2cm肠段内表面上。每只小鼠Crenolanib给药剂量约为5mg/kg体重。在照射后第2天、第4天再按照上述方法给药两次。
结果
1、单次盆腔外照射能够引起小鼠受照射肠段纤维化
我们对C57BL/6J小鼠进行25Gy盆腔外照射(示意图1A),照射后8周取受照射部位肠道组织制作蜡块并切片染色,发现与空白对照组小鼠相比,受照射小鼠受照射部位皮肤溃烂,毛发脱落、发白,肠道组织病理切片粘膜下层纤维化增厚明显(图1B),RIS评分明显增高 (图1C),纤维化标志物fibronectin、T 1collagen及T 3collagen在蛋白水平表达增高(图 1D)。
2、敲除PDGFC基因能够减轻盆腔外照射后小鼠肠道纤维化损伤
我们使用PDGFC基因敲除鼠进行25Gy盆腔外照射,照射后8周发现PDGFC基因敲除鼠肠道组织病理切片纤维化标志物IHC染色较野生型小鼠显著减弱(图2A),RIS评分显著降低(图2B),受照射部位皮肤溃烂较轻(图2C),粘膜下层厚度占肠壁全层百分比也显著下降(图2D、图2E)。
3、PDGFR抑制剂crenolanib对放射性直肠损伤动物模型的影响
3.1PDGFR抑制剂对放射性直肠损伤动物模型体重的影响
在照射前1天以及照射后每七天我们都会称量每只存活小鼠的体重并做记录(图3A)。我们将每只小鼠在测量当天的体重除以照射前1天的体重所得的百分数称为每只小鼠相对于初始体重的百分比,将所有小鼠相对于初始体重的百分比按组别及时间作折线图即得到图 3B。使用2way ANOVA法比较两组小鼠体重变化曲线发现两组间无统计学差异(F=1.159, P=0.1776),由此可知PDGFR抑制剂相对空白对照组并不会影响小鼠体重。
3.2PDGFR抑制剂对电离辐射损伤肠道组织纤维标志物fibronectin、Type1collagen、Type 2collagen蛋白表达的影响
Fibronectin、T 1collagen、T 3collagen是三种广泛表达的纤维蛋白,是常用的纤维化标志物。在图3D中我们可以看到,在照射后8周,Vehicle对照组小鼠相对于Crenolanib处理组小鼠的直肠病理切片RIS评分显著增高。且Fibronectin(纤连蛋白)、T1collagen(I型胶原纤维)、T 3collagen(III型胶原纤维)三种纤维化标志物在前者的肠道组织中表达明显增高(图3C)。这说明PDGFR抑制剂能显著减轻小鼠直肠电离辐射诱导的肠壁纤维化。
实施例2细胞培养及处理
(1)CCD-18Co(人结肠成纤维细胞)细胞培养
将液氮保存的CCD-18Co细胞置于37℃水浴锅内快速复温3-5min,然后重悬于含有10%胎牛血清的DMEM培养液中,于37%,5%CO2的条件下培养,待细胞生长至90%单层时按1:3~1:5传代。
(2)Crenolanib处理CCD-18Co细胞
待细胞生长至80%~90%单层时,吸去待处理细胞培养皿中的含10%胎牛血清培养液,加入适量无血清DMEM培养液饥饿过夜,然后将配置好的Crenolainib(CP-868596)溶液 (5mM/ml)加入到含10%胎牛血清的DMEM培养液中,调节处理浓度至1μM/ml,继续培养48h后收取细胞蛋白。
(3)抗PDGFRα或抗PDGFRβ抗体处理CCD-18Co细胞
待细胞生长至80%~90%单层时,吸去待处理细胞培养皿中的含10%胎牛血清培养液,加入适量无血清DMEM培养液饥饿过夜,然后将配置好的抗PDGFRα抗体(R&D,AF-307-NA)或抗PDGFRβ抗体(R&D,AF-385)加入到含10%胎牛血清的DMEM培养液中,调节处理浓度至1μg/ml,继续培养24h后收取细胞蛋白。
(4)PDGFCC处理CCD-18Co细胞
待细胞生长至80%~90%单层时,吸去待处理细胞培养皿中的含10%胎牛血清培养液,加入适量无血清DMEM培养液饥饿过夜,然后吸去无血清培养液,将配置好的 PDGFCC(peprotech,100-00CC-20)溶液加入到含10%胎牛血清的DMEM培养液中,调节处理浓度至50ng/ml,继续培养48h后收取细胞蛋白。
(5)X线照射CCD-18Co细胞
待细胞生长至80%~90%单层时,用封口胶密封盖口与培养皿缝隙,放入保温盒内带至中山大学附属第六医院放射科进行X线外照射,照射剂量为10Gy,照射前对放置培养皿的平面进行喷洒75%酒精消毒,照射完毕后放入保温盒内带回细胞房培养箱内继续培养,在规定的时间点收取细胞培养液及细胞蛋白。
结果
1、人结肠成纤维细胞(CCD-Co18)在照射后高表达PDGFCC及Fibronectin
我们使用10Gy X线照射CCD-Co18细胞,照射后6h、24h分别收取细胞培养液、细胞蛋白进行ELISA及WB检测。ELISA在细胞培养液中检测到照射后PDGFCCC高表达(图 4A,p>0.05,unpair t test)。PDGFCC在细胞照射后的6h及24h表达增高;Fibronectin也被检测到在蛋白水平高表达(图4B)。
2、PDGFR小分子抑制剂crenolanib能抑制PDGF-CC诱导的人结肠成纤维细胞(CCD-Co18)纤维标志物fibronectin、Type 1collagen、Type 3collagen的表达,拮抗PDGFRα受体也能达到上述效果。
PDGF-C是PDGFs家族中的一员(PDGF-A/PDGF-B/PDGF-C/PDGF-D),已有报道证实PDGF-C的活性二聚体PDGF-CC能与PDGFRαα结合激活PDGFR下游通路。使用 crenolanib抑制PDGFR受体,能显著降低PDGF-CC刺激CCD-Co18细胞引起的纤维化标志物fibronectin、T1collagen及T 3collagen的表达(图5A)。同时我们使用抗PDGFRα抗体代替crenolanib特异性抑制PDGFRα,发现抗PDGFRα抗体能够实现与crenolanib相近的抑制纤维标志物表达的效果(图5B,QPCR,FN1:fibronectin)。
实施例3动物组织的HE、MASSON、免疫组化染色及RIS评分
(1)HE染色
用于HE(伊红染色法hematoxylin-eosin staining)染色的小鼠肠道组织至于10%中性福尔马林固定液中24h,经流水冲洗、脱水、透明、石蜡包埋,制备4μm厚的连续石蜡切片。石蜡切片经常规脱蜡至水,苏木素室温染10min,自来水冲洗30-60s;1%盐酸酒精分化 1s,自来水冲洗1min;伊红室温染5-10min;梯度酒精脱水;二甲苯透明;中性树胶封片。
(2)MASSON染色
用于MASSON染色的小鼠肠道组织至于10%中性福尔马林固定液中24h,经流水冲洗、脱水、透明、石蜡包埋,制备4μm厚的连续石蜡切片。石蜡切片经常规脱蜡至水, Weigent氏苏木素室温染5min,自来水冲洗3min;1%盐酸酒精分化1s,自来水冲洗1min,蒸馏水浸洗30s;酸性品红室温染5min,蒸馏水浸洗30s;滴加1%磷钼酸,显微镜下观察至肌纤维显红色,粘膜下层显淡粉红色,不水洗直接苯胺蓝室温染3min,1%冰醋酸浸洗 30-60s;梯度酒精脱水;二甲苯透明;中性树胶封片。
(3)纤维标志物免疫组织化学染色
用于纤维标志物免疫组织化学染色的小鼠肠道组织至于10%中性福尔马林固定液中 24h,经流水冲洗、脱水、透明、石蜡包埋,制备4μm厚的连续石蜡切片。石蜡切片经常规脱蜡至水,浸没于枸橼酸钠抗原修复液(10mM,pH6.0)高压复性,待上汽3min后缓慢冷却,冷却后PBS浸洗2次,5min/次;兔血清封闭液室温封闭15min;分别直接滴加 fibronectin、T1collagen、T 3collagen一抗4℃12h,PBS浸洗2次,5min/次;滴加生物素化二抗,室温40min,PBS浸洗2次,5min/次;滴加三抗,室温40min,PBS浸洗2次,5min/ 次;DAB显色,镜下观察,适时终止,自来水冲洗5min;苏木素复染,室温30s,1%盐酸酒精分化1s,自来水冲洗5min;梯度酒精脱水;二甲苯透明;中性树胶封片。
(4)RIS评分
表2半定量组织病理学评分系统
注:该评分由一名病理科医生采用单盲方法独立评分,每项评分范围为0-2或0-3,每张
病理切片各组织分层单项评分的总和为该切片的RIS评分。
实施例4动物组织、人体组织及细胞样品的Real-time RT-PCR检测
(1)动物及人体组织RNA的提取
1)将前期准备好的小鼠肠道组织或人体肠道组织置于2ml EP管中,并加入1mlTRIzol;
2)向EP管中加入高压灭菌的直径2mm钢珠2个,直径5mm钢珠1个,封口胶密封管口,置于震荡仪中60Hz震荡至组织完全碎裂;
3)取出钢珠,室温放置5min;
4)每管EP管内加入0.2ml氯仿,,盖紧管盖,在振荡器上震荡10s;
5)室温静置至管内液体分层,4℃12000rpm离心15min;
6)小心取出EP管,此时样品分为三层:上层为含有RNA的无色水相层,中层为 DNA、脂类和蛋白质等,下层为红色的苯酚-氯仿层。取上清透明溶液至新的EP管中,能吸取的水相层体积大约为400-500μl;
7)再向新的EP管中每管加入0.5ml异丙醇,振荡器震荡10s,室温放置20min后4℃12000rpm离心10min,小心吸去上清,留下白色沉淀;
8)向沉淀中加入1ml新鲜配制的75%酒精,颠倒5次混匀后12000rpm 4℃离心5min;
9)小心吸去上清,将管子倒扣在吸水纸上吸去管口溶液,管口敞开在通风橱内风干 10min;
10)用20μl DEPC水室温5min溶解RNA后测浓度。
(2)细胞RNA的提取
1)倒掉处理后的CCD-Co18细胞培养皿中的培养基,用预冷的PBS轻柔洗涤细胞2-3次,最后一次吸干残留的PBS;
2)向培养皿中加入1ml TRIzol,至于摇床上轻柔摇晃5min;
3)用1ml移液枪吸取培养皿中的TRIzol,并轻柔吹打培养皿底部,尽量将贴附在培养皿底部的细胞完全吹打下来,然后将溶解了细胞的TRIzol转移至1.5ml EP管中;
4)以下步骤同(1)中步骤4)至10)。
(2)RNA定量
使用Nanodrp 2000微量定量仪检测定量。打开计算机中“Nanodrop”软件,选择“nuclear Acid”“RNA”进行机器自检。打开盖子并在定量处滴入1μl DEPC水,按“blank”扣去容积的影响。然后用擦镜纸擦掉DEPC水,再滴入1μl待测RNA样品,执行“measure”操作,即可得到RNA的绝对浓度。一般所有样品的RNA浓度都会用DEPC水调制1000μg/ml。
(4)逆转录合成cDNA
1)参考TOYOBO公司RT-PCR试剂盒说明书,第一步按照如下反应体系进行加样:
表3
反应条件为37℃5min。
2)第二步反应体系加样:
表4
反应条件为37℃15min,98℃5min,4℃保存。
(5)Real-Time PCR扩增
1)引物设计:
使用Primer premier 5.0软件设计相应的引物序列,采用序列相似性查询系统(BLAST)分析引物的同源性,并仔细选择引物使其跨越外显子,并采用Oligo 6软件分析引物热动力学特征。所有引物均由广州艾基生物公司合成,实验所需具体引物序列如下:
表5
2)参照SYBR Green Master Mix试剂说明书,按照以下反应体系进行加样:
表6
3)PCR扩增反应
瞬时离心混匀,然后将反应管放入PCR仪中,按照以下参数设置进行Real-TimePCR 反应:95℃,7min;95℃,10s;60℃30s,循环45次;60℃30s;溶解曲线60℃-98℃; 20℃10s。每组样品设置3个复孔,以保证实验结果的有效性和重复性。
结果
PDGFC在RE小鼠模型肠道组织中表达增高,同时也在RE患者肠道组织及血浆中表达增高
(1)PDGFC mRNA在RE动物模型受照射肠道组织中表达增高(图6A);
(2)我们收集了14例未接受新辅助放疗的I期结直肠癌患者肠道手术标本近端正常组织及 8例RE患者手术肠道标本组织进行qPCR检测,发现相较正常对照组,PDGFC在RE患者中表达增高(图6B)。
实施例5动物组织、人体组织及细胞样品的Western-blot检测
(1)动物组织及人体组织总蛋白的提取
1)将前期准备好的小鼠肠道组织或人体肠道组织置于2ml EP管中,每管加入200ul蛋白裂解液(已按1:50比例加入蛋白酶抑制剂及磷酸酶抑制剂);
2)向EP管中加入高压灭菌的直径2mm钢珠2个,直径5mm钢珠1个,封口胶密封管口,置于震荡仪中60Hz震荡至组织完全碎裂;
3)取出钢珠,EP管冰上静置30min;
4)将EP管置于离心机,4℃12000rpm离心10min;
5)将离心后的上清吸取至另一新的1.5ml EP管中,立即定量或冻存与-80℃以用于下一步实验。
(2)细胞总蛋白的提取
1)倒掉处理后的CCD-Co18细胞培养皿中的培养基,用预冷的PBS轻柔洗涤细胞2-3次,最后一次吸干残留的PBS;
2)在每个培养皿中加入预冷的蛋白裂解液100ul(已按1:50比例加入蛋白酶抑制剂及磷酸酶抑制剂),冰上裂解5-8min后,用细胞刮刮干净皿上的细胞,并将含有细胞的裂解液吸至1.5ml EP管中,冰上静置30min;
3)将超声探头置于含有细胞裂解液的液面下,65Hz超声裂解3次,10s/次;
4)将EP管置于离心机,4℃12000rpm离心10min;
5)将离心后的上清吸取至另一新的1.5ml EP管中,立即定量或冻存与-80℃以用于下一步实验。
(3)总蛋白含量的测定
1)按照50:1(A:B)的比例配置MIX(A+B)溶液。取1mg/ml的BSA标准溶液,根据下表配置一系列浓度梯度的BSA溶液:
表7
将上述梯度浓度溶液加入96孔板中,37℃孵育30min,采用酶标仪测定562mm波长的 OD值,其中以浓度为横坐标,OD值为纵坐标,绘制标准曲线。
2)根据标准曲线公式计算各样品蛋白浓度,若样品间蛋白浓度相差较大,则用裂解液将各EP管蛋白样品浓度补齐,蛋白样品浓度补齐后一般为2μg/ml。
(4)Western-blot检测操作步骤
1)蛋白样品前处理:各蛋白样品上样量均为10-15μl,以4:1的体积比将蛋白原液与 5×Loading Buffer混匀后,于95℃水浴锅中煮5min,冷却至室温后放-20℃保存待用。
2)制胶:配制10%的分离胶,1h以后待胶凝固后配制5%的浓缩胶,插梳。
3)SDS-PAGE电泳:将准备好的样品蛋白加入各电泳泳道中,浓缩胶电压80V,电泳39-40min;待溴酚蓝前沿进入分离胶后,将电压提高到110-120V,电泳60-70min,继续电泳至溴酚蓝到达分离胶底部,剪取PVDF膜,用甲醇浸泡5min后,浸于电转液中。其他电转部件浸泡于回收的电转液内。
4)转膜:电泳完成后,取出玻璃板,厚板朝下,小心撬开薄板,将膜贴上,按照膜的大小切割凝胶,将胶和膜共置于新鲜电转液中浸泡10min以上。转膜按照Western经典三明治方法,负极朝下依次放置海绵、三层滤纸、凝胶、PVDF膜、绿植及海绵,并将“三明治”固定好插入电转槽中,然后加入电转液,保证凝胶在负极,PVDF膜在正极方向。电专条件为4℃,60mA恒流,12h。
5)封闭:转膜完成后,用TBST洗膜3次,每次5min,然后将PVDF膜置于5%脱脂牛奶TBST中,室温下轻摇封闭60min。
6)孵育一抗:参照抗体说明书将抗体用一抗稀释液稀释至实验用浓度;均匀覆盖PCDF膜表面,4℃冰箱孵育过夜。孵育完成后,在摇床上用TBST洗三次,每次10min。
7)孵育二抗:将相应种属的二抗用含有5%脱脂牛奶的TBST稀释至1:10000,并均匀与膜接触,室温下孵育1h后,用TBST在室温下TBST漂洗三次,每次10min。
8)显影:配置发光液,按照一张膜1-2ml发光也的量,将溶液A和溶液B等体积均匀混合。在暗室中,将膜置于压片盒里,使附有蛋白的膜面朝上,均匀滴加发光液,使发光液完全覆盖膜表面,盖上塑料透明膜,红光下观察膜上蛋白的发光强度,估计压片时间;剪取一定数量的胶片压在塑料透明膜上,设定好时间后盖上压片盒开始压片,到时间后将膜放入洗片机内进行显影和定影,最后挑取符合要求的胶片进行扫描并存储分析图像。
结果
PDGFCC在RE小鼠模型肠道组织中表达增高,同时也在RE患者肠道组织及血浆中表达增高
(1)PDGFCC蛋白在RE动物模型受照射肠道组织中表达增高(图6C);
(2)我们挑选了6对适合做WB的患者组织,发现在RE患者组中,PDGFCC在蛋白水平上表达增高(图6D)。
实施例6人体血液标本及细胞培养液的ELISA检测
(1)制备乏血小板血浆
使用EDTA抗凝管收集患者血液,在取血后将血液至于4℃离心机内1000×g离心15分钟,然后吸取上层血浆至EP管中,继续4℃10000×g离心10分钟,立即用于后续ELISA检测或 -80℃保存。
(2)测定人体乏血小板血浆及细胞培养液中的PDGFCC含量
1)将PDGF-CC标准蛋白粉末溶于1ml双蒸水中,得到浓度为40000pg/ml的标准蛋白溶液;取100μl 40000pg/mlPDGF-CC标准蛋白溶液,按照下表8所示配置用于血浆或细胞上清液检测所用的不同浓度PDGF-CC标准蛋白溶液:
表8
2)向ELISA板中每孔加100μl RD1-63液;
3)再按50μl/孔的量向ELISA板中加梯度浓度PDGF-CC标准蛋白液及样品,室温放置2小时;
4)甩去板孔中的液体,用试剂盒自带的wash buffer清洗板孔4次,每次都要甩干孔内液体;
5)按200μl/孔的量向ELISA板中加PDGF-CC conjugate液,室温放置2小时;
6)重复步骤4);
7)按200μl/孔的量向ELISA板中加底物溶液,避光室温静置30分钟;
8)按50μl/孔的量向ELISA板中加反应停止液,充分混匀孔内溶液,孔内溶液颜色应由蓝变黄,在30分钟内采用酶标仪测定450nm波长及540nm波长OD值,以450nm波长与540nm 波长OD值差值为横轴,标准蛋白浓度为纵轴绘制标准蛋白曲线,进而计算样品浓度。
结果
PDGFCC在RE患者肠道组织及血浆中表达增高
我们收集了30例未接受新辅助放疗的I期结直肠癌患者血浆标本及8例放射性肠炎患者血浆标本进行ELISA检测,发现相较对照组,PDGFCC在RE患者血浆中表达增高(图6E)。
实施例7统计学分析
实验数据均以均数±标准差表示,采用SPSS 17.0统计分析软件进行统计分析,运用Mann-Whitney检验比较两组样本RIS评分及患者组与对照组血浆PDGFCC含量差异,运用Kruskal-Wallis法对照组野生型小鼠、对照组PDGFC KO鼠、25Gy照射组野生型小鼠、25Gy照射组PDGFC KO鼠肠道粘膜下层厚度占肠壁全层厚度百分比差异,运用2 wayANOVA法比较Crenolaninb给药组小鼠及Vehicle对照组小鼠照射后体重变化差异,运用Log-Rank检验比较Crenolaninb给药组小鼠及Vehicle对照组小鼠间生存曲线差异,运用非配对t检验比较两组样本均数(qPCR结果、WB灰度值)差异。实验整理时采用 GraphPadPrism 5.0软件(GraphPad software Inc.Jolla,CA,USA)进行作图,当P<0.05认为差异具有统计学意义。
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序列表
<110> 中山大学附属第六医院
中山大学
<120> PDGF抑制剂用于制备治疗肠道炎症疾病的药物方面的用途
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aggcggaatc caacctgagt a 21
<210> 2
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cttgtactcc gttctgttcc ttgt 24
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gatgccgatc agaagtttgg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggttgtgcag atctcctcgt 20
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctgggctaca ctgagcacc 19
<210> 6
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagtggtcgt tgagggcaat g 21
<210> 7
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatggaagtg ttttaggacg ct 22
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tggtttctgt gacttgtggc a 21
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aggtcggtgt gaacggattt g 21
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggggtcgttg atggcaaca 19
Claims (24)
1.配体PDGF-C抑制剂在制备治疗放射性肠病的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述的配体PDGF-C抑制剂与配体PDGF-C结合。
3.根据权利要求1所述的应用,其特征在于,所述的配体PDGF-C抑制剂为核酸效应分子。
4.根据权利要求3所述的应用,其特征在于,所述的核酸效应分子为DNA、RNA、PNA 或DNA-RNA- 杂合体。
5.根据权利要求3所述的应用,其特征在于,所述的核酸效应分子抑制PDGF-C基因的表达。
6.据权利要求3所述的应用,其特征在于,所述的核酸效应分子选自siRNA, dsRNA,miRNA,核酶,以及shRNA中的至少一种。
7.根据权利要求1所述的应用,其特征在于,所述的配体PDGF-C抑制剂为抗体或其功能性片段。
8.根据权利要求7所述的应用,其特征在于,所述的抗体或其功能性片段特异地与配体PDGF-C结合。
9.根据权利要求7所述的应用,其特征在于,所述的抗体选自anti-PDGFC抗体。
10.根据权利要求7所述的应用,其特征在于,所述的抗体选自AF1560。
11.根据权利要求1所述的应用,其特征在于,所述的配体PDGF-C抑制剂为PDGF-C小分子抑制剂。
12.受体PDGFR抑制剂在制备治疗放射性肠病的药物中的应用,其特征在于,
所述受体PDGFR抑制剂为吲哚酮衍生物、喹喔啉衍生物、喹唑啉衍生物,或
crenolanib,axitinib,sorafenib,CP-673451或其具有PDGFR信号通路抑制特性的衍生物之一,或其药学上可接受的盐,或
核酸效应分子,或
抗体或其功能性片段。
13.根据权利要求12所述的应用,其特征在于,所述的PDGFR抑制剂与受体PDGFR结合。
14.根据权利要求12所述的应用,其特征在于,所述的核酸效应分子为DNA、RNA、PNA 或DNA-RNA- 杂合体。
15.根据权利要求12所述的应用,其特征在于,所述的核酸效应分子抑制PDGFR基因的表达。
16.根据权利要求12所述的应用,其特征在于,所述的核酸效应分子选自siRNA,dsRNA, miRNA,核酶,以及shRNA中的至少一种。
17.根据权利要求12所述的应用,其特征在于,所述的抗体或其功能性片段特异地与受体PDGFR结合。
18.根据权利要求12所述的应用,其特征在于,所述的抗体选自anti-PDGFRα抗体和anti-PDGFRβ抗体中的一种或两种。
19.根据权利要求12所述的应用,其特征在于,所述的抗体选自AF-307-NA和AF385中的一种或两种。
20.根据权利要求1-19任一所述的应用,其特征在于,所述的放射性肠病为急性放射性肠病或者慢性放射性肠病。
21.根据权利要求1-19任一所述的应用,其特征在于,所述的放射性肠病为放射性直肠炎。
22.配体PDGF-C表达的检测试剂在制备放射性肠病的检测试剂中的应用。
23.根据权利要求22所述的应用,其特征在于,所述的放射性肠病为急性放射性肠病或者慢性放射性肠病。
24.根据权利要求22所述的应用,其特征在于,所述的放射性肠病为放射性直肠炎。
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