CN114774545A - 一种结直肠癌化疗药物敏感性预测标志物探针及应用 - Google Patents
一种结直肠癌化疗药物敏感性预测标志物探针及应用 Download PDFInfo
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Abstract
本发明属于基因检测技术领域，具体公开了一种结直肠癌化疗药物敏感性预测标志物探针，包括用于检测PHLPP1和PHLPP2的PHLPP探针，用于检测PHLPP1的探针核苷酸序列为SEQ ID NO.1至SEQ ID NO.8中的一个或两个以上；用于检测PHLPP2的探针核苷酸序列为SEQ ID NO.9至SEQ IDNO.16中的一个或两个以上。本发明还公开了上述探针用于检测结直肠癌化疗药物敏感性预测标志物的检测方法，以及上述探针及其检测方法在制备检测试剂盒中的应用。本发明公开的探针能够通过检测外周血CTC中PHLPP mRNA水平以预测患者对奥沙利铂、5‑氟尿嘧啶、卡培他滨等化疗药物的敏感性，以及识别不同表型外周血CTC中PHLPP mRNA表达水平。
Description
技术领域
本发明属于基因检测技术领域，尤其涉及一种结直肠癌化疗药物敏感性预测标志物探针及应用。
背景技术
结直肠癌(Colorectal cancer,CRC)是威胁人类健康的主要恶性肿瘤之一，2020年全球癌症数据显示，CRC发病率占全球所有癌症的10.0％，死亡率占癌症死亡总数的9.4％；在中国，CRC的新发病人数仅次于肺癌，成为第二大癌症。流行病学数据显示，近年来，我国CRC的发病率和死亡率均呈上升趋势。CRC临床表现主要为排便习惯的改变，腹痛，贫血和肠出血。但大多数病人早期无明显症状，导致CRC病人初诊时多为中晚期。
目前针对中晚期无法手术切除和不适合使用免疫治疗的CRC患者的治疗方式仍然以化疗为主。最新NCCN(美国国立综合癌症网络)更新的2021第二版(V2)肠癌指南仍然推荐使用FOLFOX(奥沙利铂(Oxaliplatin)+亚叶酸钙+5-氟尿嘧啶(5-Fluorouracil，5-FU))或CAPEOX(奥沙利铂+卡培他滨)作为主要的辅助治疗手段。
近年来，以循环肿瘤细胞(circulating tumor cell，CTC)分型检测为代表的液体活检技术是肿瘤研究领域的一个新兴方向，在肿瘤患者早期诊断、分期、疗效监测、预测评估方面均显示出较好的应用价值。大部分中晚期CRC患者在接受化疗后会复发，5年生存率低于15％，而化疗耐药是导致患者预后差和复发的主要原因。
现有的技术手段主要通过组织活检或血液样本对某些与肿瘤药物相关的基因(例如TS基因、MTHFR基因等)进行检测来预测患者对化疗药物敏感性，然而组织活检取样难度大且不能反复多次取样，因而不利于动态评估耐药情况；而通过外周血浆或血清进行药物基因检测，主要检测的是肿瘤细胞坏死后释放的片段化DNA，不能真实地反应肿瘤的实际情况。
PHLPP磷酸酶家族有PHLPP1和PHLPP2两种亚型，属于丝氨酸/苏氨酸蛋白磷酸酶的PPM超家族。在最初发现PHLPP作为AKT的蛋白磷酸酶之后，许多研究证明PHLPP通过抑制多种致癌信号通路从而抑制结直肠癌的发生和发展。申请人之前的研究(Cell Death andDisease(2021)12:960；https://doi.org/10.1038/s41419-021-04251-0)发现：PHLPP下调是造成结直肠癌化疗耐药的重要原因，并探明了PHLPP引起结直肠癌耐药的机制，说明PHLPP可充当结直肠癌化疗药物敏感性预测的标志物，但目前临床上并没有可行的试剂盒和相应的检测手段检测PHLPP mRNA的表达。
发明内容
本发明的目的在于提供结直肠癌化疗药物敏感性预测标志物探针及应用，以解决上述技术问题。
本发明目的之一在于提供：一种结直肠癌化疗药物敏感性预测标志物探针，包括用于检测PHLPP1和PHLPP2的PHLPP探针，用于检测PHLPP1的探针核苷酸序列为SEQ ID NO.1至SEQ ID NO.8中的一个或两个以上；用于检测PHLPP2的探针核苷酸序列为SEQ ID NO.9至SEQ ID NO.16中的一个或两个以上。
优选地，还包括上皮型外周血CTC特异性捕获探针EpCAM、CK8、CK18、CK19，核苷酸序列分别如SEQ ID NO.17-SEQ ID NO.22、SEQ ID NO.23-SEQ ID NO.28、SEQ ID NO.29-SEQ ID NO.34、SEQ ID NO.35-SEQ ID NO.40所示；还包括间质型外周血CTC特异性捕获探针Vimentin和Twist，核苷酸序列分别如SEQ ID NO.41-SEQ ID NO.46、SEQ ID NO.47-SEQID NO.52所示；还包括白细胞表型特异性捕获探针CD45，核苷酸序列如EQ ID NO.53-SEQID NO.58所示。
本发明目的之二在于提供：上述探针在制备检测试剂盒中的应用。
本发明目的之三在于提供：上述探针用于检测结直肠癌化疗药物敏感性预测标志物的检测方法，包括以下步骤：
S1、采用滤膜截留外周血CTC；
S2、采用三种特异性捕获探针对截留的外周血CTC进行分型检测：
三种特异性捕获探针分别为：
①上皮型外周血CTC特异性捕获探针EpCAM、CK8、CK18、CK19；
②间质型外周血CTC特异性捕获探针Vimentin和Twist；
③白细胞表型特异性捕获探针CD45进行。
S2′、采用PHLPP探针在外周血CTC中检测PHLPP mRNA的表达情况。
本发明目的之四在于提供：上述检测方法在制备检测试剂盒中的应用。
本发明的原理和有益效果在于：
1、根据前期已公开发表的研究成果，PHLPP表达下调是造成结直肠癌化疗耐受的重要原因。
2、本发明通过设计的用于检测PHLPP1和PHLPP2的PHLPP探针，采用纳米膜过滤结合mRNA原位杂交技术检测外周血CTC PHLPP mRNA水平，从而预测患者对奥沙利铂、5-氟尿嘧啶、卡培他滨等化疗药物的敏感性，动态地对肿瘤细胞的耐药情况进行检测和评估。
3、采用滤膜截留外周血CTC后，需要探针对其特异性进行鉴定和分型，而在探针进行鉴定和分型过程中，本发明可以实现CTC分型检测和PHLPP mRNA检测同步进行，互不干扰，鉴定和分型检测效率高。
4、本发明采用mRNA原位杂交技术检测外周血CTC PHLPP mRNA水平的主要原因在于：
(1)CTC与组织样本一致性较高，样本可反复多次取样，能及时捕捉到患者将发生耐药的时间点。
(2)mRNA原位杂交技术特异性高、敏感性高、操作简单快捷且成本低。
附图说明
图1为荧光显微镜下的镜检图，图中红色点状荧光代表外周血CTC上皮标志物：EpCAM、CK8、CK18和CK19基因表达情况。绿色点状荧光代表外周血CTC间质型标志物Vimentin和Twist基因表达情况。紫色点状荧光代表外周血CTC PHLPP1和PHLPP2基因的表达情况。白色信号点代表白细胞标志物CD45基因表达情况。A图和C图是多个荧光通道合成图，B图和D图是单个荧光通道图(科研探针表达信号图)。
具体实施方式
下面通过具体实施方式进一步详细说明：
1.1实验设计
表1实验设计
1.2 PHLPP探针
结直肠癌化疗药物敏感性预测标志物探针的设计主要使用在线Primer5软件，该软件本身会通过序列比对，优选出特异性的探针序列。探针序列由Invitrogen公司合成，最终本发明通过高中低表达内参基因(B2M、TBP和TFRC)的表达情况的预实验，根据内参基因的表达情况验证探针序列的特异性，即为表2所列出的序列。
PCR实验时，为了去除不同标本在RNA的产量、质量和逆转录效率上的可能存在的差别而获得目标基因特异性表达的真正差异，通常会选择一定的内参基因(housekeepinggene)进行校准和标准化。本发明所采用的内参基因B2M、内参基因TBP和内参基因TFRC分别对应高表达、中表达和低表达。
表2 PHLPP探针
1.3实验方法
S1、采用滤膜截留外周血循环肿瘤细胞(外周血CTC)。
1.使用EDTA抗凝采血管采集患者外周血样本5ml，颠倒混匀，加入15ml红细胞裂解液(SurExam Inc.USA)混匀，室温静置30min裂解红细胞。
红细胞裂解液配方为：154mM NH4Cl、10mM KHCO3和0.1mM EDTA。
2.500×g离心5min，去除血液样本上清。
3.使用PBS缓冲液(武汉博士德生物工程公司，货号为AR0030)重悬进行细胞沉淀。
4.使用终浓度为4％甲醛固定剩余细胞沉淀8min。
5.将固定后的细胞转移至含滤膜(美国BD公司，8μM)的过滤管中，使用真空抽滤泵(天津奥特赛恩斯仪器有限公司，货号为AP-01P)将细胞过滤至滤膜上。
6.过滤后的滤膜样本，使用4％甲醛继续室温固定1h。
本实施例采用滤膜孔径为8μM，并根据滤膜滤法截留外周血CTC(ISET法)，有效去除白细胞并截留外周血CTC。
S2、采用三种特异性捕获探针对截留的外周血CTC进行分型检测。
1.将固定后的滤膜样本使用PBS缓冲液洗涤三次，置于24孔板中。
2.加入0.1mg/ml蛋白酶K(Sigma,St.Louis,USA，CAS号为：39450-01-6)进行处理，室温静置1h，增加细胞膜通透性。
3.采用PBS缓冲液洗涤三次，为了区分循环肿瘤细胞型别，加入三种特异性捕获探针进行杂交。
三种特异性捕获探针(具体序列见表3)分别为：
①上皮型外周血CTC特异性捕获探针EpCAM、CK8、CK18、CK19；②间质型外周血CTC特异性捕获探针Vimentin和Twist；③白细胞表型特异性捕获探针CD45。
在40℃杂交反应3h。未结合特异性捕获探针用1000μl洗脱液洗涤3次。洗脱液配方：0.1×盐水柠檬酸钠(SSC)(Thermo，货号为：AM9765)。
4.加入100μl预扩增液。
预扩增液配方：30％马血清，1.5％十二烷基硫酸钠(Sigma,St.Louis,USA，货号为：L5750-500G)、3mM Tris-HCl(pH 8.0)(Sigma,St.Louis,USA，货号为：T3038-1L)，0.5fmol预扩增探针(序列见表4)，置于40℃孵育30min，进行信号扩大探针反应。
5.将膜进行冷却：用1000μl洗脱液洗脱三次(0.1×SSC)，然后与100μl扩增溶液、1fmol预扩增探针(序列见表4)进行孵育40℃30min。
扩增溶液配方：30％马血清、1.5％十二烷基硫酸钠和3mM Tris-HCl(pH 8.0)。
6.加入三种标记荧光蛋白，分别为荧光染料Alexa Fluor 594(用于标记上皮型外周血CTC特异性捕获探针EpCAM、CK8/18/19)，Alexa Fluor 488(用于标记间质型外周血CTC特异性捕获探针Vimentin和Twist)以及Alexa Fluor 750(用于标记白细胞表型生物标记物CD45)，置于40℃孵育30min。
7.用0.1×SSC进行洗脱，然后使用DAPI(SIGMA，货号为：S26939)进行细胞核染色5min，在100倍油镜下使用自动化荧光扫描显微镜对样本进行观察。
实验结果如图1所示，红色点状荧光代表外周血CTC上皮标志物：EpCAM、CK8、CK18和CK19基因表达情况。绿色点状荧光代表外周血CTC间质型标志物Vimentin和Twist基因表达情况。白色信号点代表白细胞标志物CD45基因表达情况。这里的判读标准不需要分为高中低表达水平，如果荧光信号点的数量大于7个以上，则判定为阳性。
表3核酸探针序列
表4 bDNA信号放大探针的序列
备注：bDND探针和CTC分型检测技术平台关联。
本实验所采用的试剂盒、厂家及货号如下：
CD45检测试剂盒，益善生物技术股份有限公司，货号为22030301。EP-CAM检测试剂盒，益善生物技术股份有限公司，货号为22030302。细胞角蛋白8检测试剂盒，益善生物技术股份有限公司，货号为22030303。细胞角蛋白18检测试剂盒，益善生物技术股份有限公司，货号为22030304。细胞角蛋白19检测试剂盒，益善生物技术股份有限公司，货号为22030305。Vimentin/twist检测试剂盒，益善生物技术股份有限公司，货号为22030306。
S2′、采用PHLPP探针在外周血CTC中检测PHLPP mRNA的表达情况。
本步骤与S2的检测方法大致相同，具体实验步骤如下：
1.将固定后的滤膜样本使用PBS缓冲液洗涤三次，置于24孔板中。
2.加入0.1mg/ml蛋白酶K(Sigma,St.Louis,USA，CAS号为：39450-01-6)进行处理，室温静置1h，增加细胞膜通透性。
3.采用PBS缓冲液洗涤三次，加入PHLPP探针进行杂交，在40℃杂交反应3h。未结合PHLPP探针用1000μl洗脱液洗涤3次。洗脱液配方：0.1×SSC。
4.加入100μl预扩增液。
预扩增液配方：30％马血清，1.5％十二烷基硫酸钠、3mM Tris-HCl(pH 8.0)，0.5fmol预扩增探针(序列见表4)，置于40℃孵育30min，进行信号扩大探针反应。
5.将膜进行冷却：用1000μl洗脱液洗脱三次(0.1×SSC)，然后与100μl扩增溶液、1fmol预扩增探针(序列见表4)进行孵育40℃30min。
扩增溶液配方：30％马血清、1.5％十二烷基硫酸钠和3mM Tris-HCl(pH 8.0)。
6.加入荧光染料Alexa Fluor 647(标记为紫色)，置于40℃孵育30min。
7.用0.1×SSC进行洗脱，然后使用DAPI进行细胞核染色5min，在100倍油镜下使用自动化荧光扫描显微镜对样本进行观察。
紫色信号点代表PHLPP基因表达，根据信号点的数量进行PHLPP高中低表达水平划分：
表5 PHLPP高中低表达水平划分
实验结果如图1所示：本实施例的检测方法可以在治疗前，预测患者对物奥沙利铂、5-氟尿嘧啶、卡培他滨等化疗药物的敏感性，并且能够在治疗过程中，动态地对肿瘤细胞的耐药情况进行检测和评估，观察产生化疗耐药的时间点。
CTC是液体活检非常重要的检测项目之一，无论从形态、来源还是成分都与组织活检达到最大的一致，所以通过采集外周血液可以反复多次获取CTC，检测CTC中PHLPP mRNA水平可以动态地监测结肠癌患者化疗敏感性的变化。本发明通过设计的PHLPP探针，采用纳米膜过滤结合mRNA原位杂交技术检测完成了对结肠癌患者外周血CTC中PHLPP1和PHLPP2的mRNA水平的检测，发现检测到CTC中PHLPP1和PHLPP2mRNA水平高的，患者化疗敏感性好；CTC中PHLPP1和PHLPP2mRNA水平低，患者化疗效果不好。因此，采用本发明提供的PHLPP探针及其检测方法检测外周血CTC中PHLPP mRNA水平可以在治疗前很好的预测患者化疗的敏感性。
本发明采用mRNA原位杂交技术检测外周血CTC PHLPP mRNA水平的主要因为在于：
(1)CTC与组织样本一致性较高，样本可反复多次取样，能及时捕捉到患者将发生耐药的时间点。(2)mRNA原位杂交技术特异性高、敏感性高、操作简单快捷且成本低。
实施例2
本实施例提供了一种用于识别不同表型外周血CTC中PHLPP mRNA表达水平的检测试剂盒，试剂盒具体包括以下成分：
表6检测试剂盒成分
本试剂盒的检测方法参见S1和S2，本实施例不再赘述。
实施例3
本实施例提供了一种结直肠癌化疗药物敏感性检测试剂盒，能够在治疗前，预测患者对奥沙利铂、5-氟尿嘧啶、卡培他滨等化疗药物敏感性，以及在治疗过程中，动态地对肿瘤细胞的耐药情况进行检测和评估，观察产生化疗耐药的时间点。试剂盒具体成分如下表所示：
表7检测试剂盒成分
本试剂盒的检测方法参见S1和S2′。
5-氟尿嘧啶(5-FU)是结直肠癌、胃癌等肿瘤的一线化疗药物，通过抑制胸腺嘧啶核苷酸合成酶的活性而减缓DNA的生物合成，从而达到抑制肿瘤细胞增殖的目的。临床试验证明5-FU疗效较佳，但患者的个体间反应差异很大，甚至出现了不良反应。现有技术中通常检测患者叶酸代谢通路的关键酶-亚甲基四氢叶酸还原酶(MTHFR)活性，预测人体肿瘤细胞对5-FU的化疗敏感性。卡培他滨(Capecitabine)是一种可以在体内转变成5-FU的抗代谢氟嘧啶脱氧核苷氨基甲酸酯类药物。奥沙利铂是用于治疗结直肠癌患者的一线化疗药物，但大约有50％的结直肠癌患者对以奥沙利铂为基础的化疗会产生耐药性。
实施例3
本实施例提供了一种能够用于预测患者对奥沙利铂、5-氟尿嘧啶、卡培他滨等化疗药物敏感性，以及识别不同表型外周血CTC中PHLPP mRNA表达水平的检测试剂盒，检测试剂盒的成分如下表所示：
表8检测试剂盒成分
本试剂盒的检测方法参见S1、S2和S2′。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
序列表
<110>一种结直肠癌化疗药物敏感性预测标志物探针及应用
<120>重庆大学附属肿瘤医院
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<212>DNA
<213>artificial sequence
<400>33
aagtcatcag cagcaagacg 20
<210>34
<211>20
<212>DNA
<213>artificial sequence
<400>34
ctgcagtcgt gtgatattgg 20
<210>35
<211>20
<212>DNA
<213>artificial sequence
<400>35
ctgtaggaag tcatggcgag 20
<210>36
<211>20
<212>DNA
<213>artificial sequence
<400>36
aagtcatctg cagccagacg 20
<210>37
<211>20
<212>DNA
<213>artificial sequence
<400>37
ctgttccgtc tcaaacttgg 20
<210>38
<211>20
<212>DNA
<213>artificial sequence
<400>38
ttcttcttca ggtaggccag 20
<210>39
<211>20
<212>DNA
<213>artificial sequence
<400>39
ctcagcgtac tgatttcctc 20
<210>40
<211>20
<212>DNA
<213>artificial sequence
<400>40
gtgaaccagg cttcagcatc 20
<210>41
<211>20
<212>DNA
<213>artificial sequence
<400>41
gagcgagagt ggcagaggac 20
<210>42
<211>20
<212>DNA
<213>artificial sequence
<400>42
ctttgtcgtt ggttagctgg 20
<210>43
<211>20
<212>DNA
<213>artificial sequence
<400>43
catattgctg acgtacgtca 20
<210>44
<211>20
<212>DNA
<213>artificial sequence
<400>44
gagcgcccct aagtttttaa 20
<210>45
<211>20
<212>DNA
<213>artificial sequence
<400>45
aagattgcag ggtgttttcg 20
<210>46
<211>20
<212>DNA
<213>artificial sequence
<400>46
ggccaatagt gtcttggtag 20
<210>47
<211>20
<212>DNA
<213>artificial sequence
<400>47
acaatgacat ctaggtctcc 20
<210>48
<211>20
<212>DNA
<213>artificial sequence
<400>48
ctggtagagg aagtcgatgt 20
<210>49
<211>20
<212>DNA
<213>artificial sequence
<400>49
caactgttca gacttctatc 20
<210>50
<211>20
<212>DNA
<213>artificial sequence
<400>50
cctcttgaga atgcatgcat 20
<210>51
<211>20
<212>DNA
<213>artificial sequence
<400>51
tttcagtggc tgattggcac 20
<210>52
<211>20
<212>DNA
<213>artificial sequence
<400>52
ttaccatggg tcctcaataa 20
<210>53
<211>20
<212>DNA
<213>artificial sequence
<400>53
tcgcaattct tatgcgactc 20
<210>54
<211>20
<212>DNA
<213>artificial sequence
<400>54
tgtcatggag acagtcatgt 20
<210>55
<211>20
<212>DNA
<213>artificial sequence
<400>55
gtatttccag cttcaacttc 20
<210>56
<211>20
<212>DNA
<213>artificial sequence
<400>56
ccatcaatat agctggcatt 20
<210>57
<211>20
<212>DNA
<213>artificial sequence
<400>57
ttgtgcagca atgtatttcc 20
<210>58
<211>20
<212>DNA
<213>artificial sequence
<400>58
tacttgaacc atcaggcatc 20
<210>59
<211>18
<212>DNA
<213>artificial sequence
<400>59
ctacaaacaa acaatatt 18
<210>60
<211>13
<212>DNA
<213>artificial sequence
<400>60
cgcagcctca gcc 13
<210>61
<211>13
<212>DNA
<213>artificial sequence
<400>61
cccagaccct acc 13
<210>62
<211>18
<212>DNA
<213>artificial sequence
<400>62
cttctcaata actaacat 18
<210>63
<211>13
<212>DNA
<213>artificial sequence
<400>63
gacggtcggc gtt 13
<210>64
<211>13
<212>DNA
<213>artificial sequence
<400>64
gtcaccgctc cac 13
<210>65
<211>18
<212>DNA
<213>artificial sequence
<400>65
gtaaaaagaa aggtataa 18
<210>66
<211>13
<212>DNA
<213>artificial sequence
<400>66
aattatacat ctc 13
<210>67
<211>13
<212>DNA
<213>artificial sequence
<400>67
gaaatgaatg aat 13
Claims (5)
1.一种结直肠癌化疗药物敏感性预测标志物探针，其特征在于，包括用于检测PHLPP1和PHLPP2的PHLPP探针，用于检测PHLPP1的探针核苷酸序列为SEQ ID NO.1至SEQ ID NO.8中的一个或两个以上；用于检测PHLPP2的探针核苷酸序列为SEQ ID NO.9至SEQ ID NO.16中的一个或两个以上。
2.根据权利要求1所述的探针在制备检测试剂盒中的应用。
3.根据权利要求1或2所述的探针用于检测结直肠癌化疗药物敏感性预测标志物的检测方法，其特征在于，包括以下步骤：
S1、采用滤膜截留外周血CTC；
S2′、采用PHLPP探针在外周血CTC中检测PHLPP mRNA的表达情况。
4.根据权利要求3所述的检测方法在制备检测试剂盒中的应用。
5.一种结直肠癌化疗药物敏感性检测试剂盒，其特征在于，包括用于检测PHLPP1和PHLPP2的PHLPP探针。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559883A (zh) * | 2011-12-27 | 2012-07-11 | 芮屈生物技术（上海）有限公司 | 前列腺癌变前期PTEN和PHLPP1的mRNA水平原位杂交检测试剂盒及检测方法和应用 |
| US9200328B1 (en) * | 2012-03-14 | 2015-12-01 | New York University | Methods and kits for diagnosing the prognosis of cancer patients |
| CN105209636A (zh) * | 2013-03-15 | 2015-12-30 | 麦塔马克基因股份有限公司 | 用于癌症预后的组合物和方法 |
| CN110806479A (zh) * | 2019-11-15 | 2020-02-18 | 复旦大学附属肿瘤医院 | 一种乳腺癌相关的激酶变异的检测panel及其应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559883A (zh) * | 2011-12-27 | 2012-07-11 | 芮屈生物技术（上海）有限公司 | 前列腺癌变前期PTEN和PHLPP1的mRNA水平原位杂交检测试剂盒及检测方法和应用 |
| US9200328B1 (en) * | 2012-03-14 | 2015-12-01 | New York University | Methods and kits for diagnosing the prognosis of cancer patients |
| CN105209636A (zh) * | 2013-03-15 | 2015-12-30 | 麦塔马克基因股份有限公司 | 用于癌症预后的组合物和方法 |
| CN110806479A (zh) * | 2019-11-15 | 2020-02-18 | 复旦大学附属肿瘤医院 | 一种乳腺癌相关的激酶变异的检测panel及其应用 |
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