Preliminary Exploration of Simple BH3 Profiling in Screening Drug Susceptibility of Blood Tumors
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摘要:目的
建立一种血液肿瘤BCL-2抗凋亡蛋白抑制剂药敏筛选的简易BH3分析法。
方法建立基于显微镜和JC-1染色的BH3分析方法，通过BH3-only模拟肽描绘K562、NB4细胞系BCL-2家族抗凋亡蛋白依赖谱，应用聚合酶链式反应（polymerase chain reaction，PCR）加以验证；回溯临床治疗疗效，检验该方法对临床样本BH3药敏分析结果的符合情况。
结果BH3分析结果显示NB4对抗凋亡蛋白的依赖程度依次为BCL2>MC1-1>BCL-XL；K562的依赖程度依次为BCL-XL>MC1-1>BCL2。PCR分析BCL-2家族基因表达量显示，K562抗凋亡蛋白MCL-1、BCL2、BCL-XL相对表达程度较高，BH3-only促凋亡蛋白中BIM、PUMA、NOXA高表达而HRK低表达，细胞抗凋亡能力更依赖于BCL-XL；NB4抗凋亡蛋白BCL-XL表达明显低于MCL-1与BCL-2，BH3-only促凋亡蛋白NOXA低表达，细胞抗凋亡更依赖于MCL-1与BCL-2而非BCL-XL，与BH3分析结果相符。对连续6例血液肿瘤患者骨髓进行BH3分析，结果显示6例患者均对HRK（BCL-XL抑制物）、VEN（BCL-2抑制物）不敏感；4例患者（例1~3，例5）对NOXA（MCL-1抑制物）表现为敏感，2例（例4、例6）的敏感度较低（<50%）。药敏预测结果显示6例患者均对BCL-2抑制剂耐药，例1~3、例5患者对MCL1抑制剂敏感，例4和例6对MCL1抑制剂可能耐药。回溯临床用药效果（4~6个疗程）显示例1~5患者对BCL-2抑制剂相关方案治疗无效，考虑与BCL-2抑制剂不敏感有关，例5更换西达苯胺治疗有效，例4和例6患者西达苯胺相关方案疗效不佳，考虑与MCL-1抑制物不敏感有关，上述临床结局与BH3预测结果一致。
结论基于荧光显微镜和JC-1染色的BH3分析是简便可行的，可用于检测血液肿瘤细胞对不同抗凋亡蛋白的依赖性，从而为BCL-2家族相关抑制剂的选择提供证据。
Abstract:ObjectiveTo establish a simple BH3 profiling for drug-sensitive screening of anti-apoptotic protein inhibitors of the BCL-2 family in blood tumors.
MethodTo establish the BH3 profiling method based on the microscope and JC-1 staining, describe the dependence of K562 and NB4 cell lines on anti-apoptotic proteins of the BCL-2 family by BH3-only simulated peptide, and verify it by polymerase chain reaction (PCR) method. Clinical drug efficacy was retrospectively reviewed to check the conformity of BH3 drug sensitivity analysis results.
ResultsBH3 profiling results showed that the anti-apoptotic protein dependence of the NB4 cell line was BCL2 > MC1-1 > BCL-XL; the K562 cell line was BCL-XL > MC1-1 > BCL2. PCR analysis of BCL-2 family expression showed that anti-apoptotic proteins MCL-1, BCL2, and BCL-XL were highly expressed in K562, while BIM, NOXA, and PUMA were highly expressed in BH3-only protein and HRK was low. The anti-apoptotic ability of cells depended more on BCL-XL. In NB4, the anti-apoptotic protein BCL-XL was significantly lower than MCL-1 and BCL-2, while NOXA in BH3-only protein was lower. NB4 cells were more dependent on MCL-1 and BCL-2 than BCL-XL; Which was consistent with BH3 profiling results. Bone marrow BH3 profiling results of 6 patients with hematologic tumors showed that HRK (BCL-XL inhibitor) and VEN (BCL-2 inhibitor) were not sensitive. NOXA (MCL-1 inhibitor) was sensitive to examples 1, 2, 3 and 5, while the sensitivity of examples 4 and 6 was low (< 50%). It was speculated that all 6 cases were resistant to BCL-2 inhibitor; Cases 1, 2, 3 and 5 were sensitive to MCL1 inhibitor, and cases 4 and 6 might be resistant to MCL1 inhibitor. Retrospective clinical efficacy (4~6 sessions): Cases 1~5 were not sensitive to the treatment regimen containing BCL-2 inhibitor, which was considered to be related to insensitivity to BCL-2 inhibitor. Chidamide was effective in case 5, but not effective in cases 4 and 6, which were considered to be related to MCL-1 inhibitor insensitivity. The above clinical outcomes were consistent with the predicted results of BH3 profiling.
ConclusionsBH3 profiling based on fluorescence microscopy and JC-1 staining is simple and feasible and can be used to detect the dependence of blood tumor cells on different anti-apoptotic proteins, thus providing evidence for the selection of inhibitors related to the Bcl-2 family.
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Keywords:
- BH3 profiling /
- BCL-2 /
- fluorescence microscopy /
- JC-1
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BH3分析（BH3 profiling）技术是近年兴起的一种检测细胞凋亡阈值的技术，最早由Letai教授提出[1]。该技术根据不同细胞BCL-2家族蛋白表达模式不一样，应用体外合成的BH3-only模拟肽（BH3 peptides）或小分子抗凋亡蛋白抑制剂作用于目的细胞，检测细胞是否发生凋亡启动从而判断肿瘤细胞凋亡倾向性。BCL-2家族蛋白成员接近20种，成员间相互作用关系繁杂而重要，单纯获知蛋白成员的表达水平无法确定成员间的相互作用的最终结果。BH3分析基于BCL-2家族蛋白促凋亡与抗凋亡的功能分类，不以BCL-2家族成员单一对应关系为研究对象，而是以BCL-2家族蛋白成员促/抗凋亡力量抗衡的最终结果为研究目标的功能性试验，其通过检测不同的BH3-only模拟肽作用后是否引起线粒体膜电位改变（△Ψm）/细胞色素C释放来间接评估线粒体启动状态,即线粒体外膜通透（mitochondrial outer membrane permeabilization，MOMP），或者说细胞为凋亡所做准备的程度，从而预测细胞拮抗凋亡主要作用的蛋白。但临床上往往发现并非所有患者对BCL-2抑制剂都有良好反应，因而寻找合适有效的检测“靶点”成为亟待解决的问题。而BH3分析技术可检测细胞对抗凋亡蛋白的依赖性，有望可为BCL-2相关抑制剂选择提供证据，从而实现精准治疗。常规BH3分析方法对使用荧光微孔板仪或流式细胞仪进行检测，对设备和实验要求较高。综合既往报道的BH3分析方法，我们改良并建立一种基于荧光显微镜和JC-1染色的BH3分析方法用于检测血液肿瘤对抗凋亡蛋白依赖性，该方法具有结果直观（可观测每个细胞凋亡状态）、操作简便快捷、支持少样本分析、成本低廉的优势。
1. 材料及方法
1.1 研究样本
研究共纳入2株细胞系及6例血液肿瘤患者骨髓样本。（1）2株细胞系NB4及K562，由本实验室购自ATCC，以细胞冻存液（STEM CELL）冻存于液氮中，解冻复苏后用新鲜介质洗涤重悬于含10% FBS（GIBCO）的RPMI 1640培养基（GIBCO），于含5% CO2、37 ℃加湿培养箱中培养。（2）6例白血病患者骨髓样本来自在广东省人民医院确诊的白血病患者，所有患者均经形态学、细胞遗传学、分子学、免疫学检测并严格按照WHO 2016（The 2016 revision of the World Health Organization classification）标准诊断。
1.2 主要材料
（1）药物及试剂：洋地黄皂苷（digitonin）（MCE，HY-N4000）；Hoechst 33342（碧云天，C1025）；JC-1（MCE，HY-15534）；CCCP（MCE，HY-100941）；ABT199（Active Biochem）；HEPES（GIBCO，15630106）；BSA（SIGMA，SRE0096），海藻糖（Macklin，D807342），EGTA（源叶，S30018），EDTA（Macklin，E809068），琥珀酸（源叶，S30140）。（2）BH3模拟肽（BH3 peptides）：包括hBIM、hBID-Y、mBAD、PUMA、mNOXA、MS-1、W-HRK，由上海吉尔生化合成，序列见表1[2]。（3）实验仪器：徕卡DM4000B荧光显微镜。
表 1 BH3模拟肽序列Table 1. BH3 mimics sequencesBH3
peptidesSequences hBIM Ac-MRPEIWIAQELRRIGDEFNA-NH2 hBID-Y Ac-EDIIRNIARHLAQVGDSMDRY-NH2 mBAD Ac-LWAAQRYGRELRRMSDEFEGSFKGL-NH2 mNoxaA Ac-AELPPEFAAQLRKIGDKVYC-NH2 MS-1 Ac-RPEIWMTQGLRRLGDEINAYYAR-NH2 Puma Ac-EQWAREIGAQLRRMADDLNA-NH2 W-Hrk Ac-WSSAAQLTAARLKALGDELHQ-NH2 1.3 研究方法
1.3.1 聚合酶链式反应（polymerase chain reaction，PCR）检测
收集2~4 mL样本，TRIzol法提取样本总RNA，按说明书合成cDNA和构建RCR反应体系，检测BIM、BID、BAD、PUMA、NOXA、HRK、BAX、BAK、BCL2、BCL-XL、MCL1、BCL-W等基因（引物序列见表2），以2−ΔΔCt计算相对表达量。
表 2 RT-qPCR引物序列Table 2. Primer sequences for RT-qPCRGene Sequences BIM-F AGCCCAGCACCCATGAGTTGTGAC BIM-R CTCTGGGCGCATATCTGCAGG BID-F ATGGACCGTAGCATCCCTCC BID-R GTAGGTGCGTAGGTTCTGGT BAD-F GTTTGAGCCGAGTGAGCAGG BAD-R ATAGCGCTGTGCTGCCCAGA NOXA-F ACCAAGCCGGATTTGCGATT NOXA-R ACTTGCACTTGTTCCTCGTGG HRK-F CAGGCGGAACTTGTAGGAAC HRK-R GCTGGATTTCCAAAGGGCTT PUMA-F GACCTCAACGCACAGTACGAG PUMA-R AGGAGTCCCATGATGAGATTGT BAX-F TCTGACGGCAACTTCAACTG BAX-R GGAGGAAGTCCAATGTCCAG BAK-F GTTTTCCGCAGCTACGTTTTT BAK-R GCAGAGGTAAGGTGACCATCTC BCL-2-F ATGTGTGTGGAGAGCGTCAA BCL-2-R GCCGGTTCAGGTACTCAGTC BCL-xL-F GGAGGCAGGCGACGAGTTTGAA BCL-xL-R AAGGGGGTGGGAGGGTAGAGTGG BCL-W-F GCGGAGTTCACAGCTCTATAC BCL-W-R AAAAGGCCCCTACAGTTACCA MCL-1-F CGGTAATCGGACTCAACCTC MCL-1-R CCTCCTTCTCCGTAGCCAA GAPDH-F AGAAGGCTGGGGCTCATTTG GAPDH-R AGGGGCCATCCACAGTCTTC 1.3.2 BH3分析
BH3分析体系部件：（1）配制DTEB缓冲液：10 mM HEPES pH 7.5，135 mM海藻糖，50 mM KCl，0.02 mM EGTA，0.02 mM EDTA，0.1% BSA，5 mM琥珀酸。（2）洋地黄皂苷（digitonin）：溶解于DMSO中制成5%储存液（50 mg/mL）。（3）JC-1：溶解于DMSO中准备5 mM储存液，并储存在−80 ℃；使用前稀释为200 μM工作液。（4）药物：ABT-199（BCL-2抑制剂）溶解于DMSO制作成1 mM溶液备用。（5）样本处理液：每1 mL的DTEB中含0.008%/0.004%洋地黄皂苷、20 mM β-巯基乙醇（洋地黄皂苷终浓度0.002%/0.001%)。（6）模拟肽：合成的BH3模拟肽溶解于DMSO中，浓度为1 mM；使用时根据工作浓度用DTEB稀释。
BH3谱系检测步骤：（1）含培养基的待测样本加入等体积Hoechst 33342染色10 min，经PBS洗涤后重悬于DTEB中，浓度为5×109/L，与等体积样本处理液混合。（2）同时取96孔板分别加入50 μL DTEB（含DMSO）、CCCP（50 uM）、hBIM（20 uM）、hBID-Y（20 uM）、mBAD（20 uM）、PUMA（20 uM）、mNOXA（20 uM）、MS-1（20 uM）、W-HRK（20 uM）、ABT-199（20 uM）、hBIM（1 uM）、hBID-Y（1 uM）、mBAD（1 uM）、PUMA（1 uM）、mNOXA（1 uM）、MS-1（1 uM）、W-HRK（1 uM）、ABT-199（1 uM）共18孔。（3）每孔加入步骤（1）中细胞混合液50 μL，混匀后37 ℃孵育30~60 min。（4）每孔加入1 μL JC-1（200 μM）37 ℃孵育30 min。（5）经离心洗涤后重悬于DTEB，取细胞悬液滴入玻片中待自然沉降后使用荧光显微镜观察，计数线粒体膜电位（△Ψm）降低（去极化）的细胞比率，根据公式修正去极化率，并绘制BH3谱系色阶图。（图1）色阶图中以肽空白孔（DMSO）为阴性对照（凋亡通常低于10%），CCCP孔为阳性对照（通常100%凋亡），各孔凋亡程度（去极化率）从低到高显示从绿到黄到红的不同颜色。去极化率计算公式如下：
$$ {{去极化率}}\text{%}{\boldsymbol{=}}\left[{1-}\frac{(肽处理孔-\text{CCCP})}{({\mathrm{DMSO}}{-{\mathrm{CCCP}}}}\right]\times \text{100\%} $$ 1.3.3 临床验证
6例血液肿瘤患者骨髓样本，按1.3.2步骤进行BH3分析，结果与临床实际药物疗效比对。
2. 结 果
2.1 细胞系（K562、NB4）BH3分析
根据BCL2家族抗凋亡蛋白和BH3-only蛋白相互作用模式，见表3，结果显示不同样本对BCL2家族蛋白有不用依赖性，有不同的BH3谱系结果。从谱系结果可推断抗凋亡蛋白的依赖性及依赖程度。
表 3 BCL2家族抗凋亡蛋白和BH3-only蛋白相互作用模式Table 3. Interaction model of BCL2 family anti-apoptotic protein and BH3-only proteinAnti-apoptotic protein BH3-only protein BH3 inhibitor BIM BID BAD PUMA NOXA MS1 HRK ABT199(VEN) S63845 WEHI539 BCL-2 BCL-XL BCL-W MCL-1 注：*红色代表强抑制，绿色代表弱抑制或者无抑制。
Note：* Red represented strong inhibition, and green represented weak or no inhibition.为充分体现细胞对不同BH3-only蛋白的反应程度，避免在某一浓度下结果接近无法分辨，实验中分别使用高低两种浓度的BH3模拟肽作用于细胞，计数△Ψm减低的细胞比例（见表4，图2），从△Ψm在不同浓度降低的比例可以得出抗凋亡蛋白是否依赖及依赖程度。结果显示K562细胞反应HRK>NOXA/MS1>VEN，即依赖程度BCL-XL>MC1-1>BCL2，而NB4细胞反应VEN>NOXA/MS1>HRK，即依赖程度BCL2>MC1-1>BCL-XL。
表 4 细胞系BH3分析结果(色阶图)Table 4. Cell line BH3 profiling results (level diagram)DMSO CCCP BIM BID NOXA MS1(syn） HRK BAD PUMA VEN NB4 1 μM 7.80% 100.00% 97.94% 90.56% 27.11% 67.90% 24.08% 100.00% 91.11% 94.90% 20 μM 7.80% 100.00% 100.00% 100.00% 100.00% 97.51% 83.73% 90.89% 100.00% 100.00% K562 1 μM 0.00% 100.00% 90.00% 0.00% 1.70% 4.10% 10.60% 87.30% 40.40% 1.30% 20 μM 14.60% 100.00% 67.80% 24.59% 40.40% 39.34% 43.44% 81.26% 71.43% 11.94% 注:阳性对照（CCCP）显示100%凋亡，空白对照低凋亡。连续色阶图显示对BH3模拟肽的敏感程度，从红到黄到绿代表敏感强度从高到低，去极化率%=1−[(肽处理孔−阳性对照）/(空白−阳性对照）]。
Note: Positive control (CCCP) showed 100% apoptosis, while blank control showed low apoptosis. The continuous color level diagram showed the sensitivity to BH3 mimic peptide, and the diagram from red to yellow to green represented the sensitivity intensity from high to low. %Depolarization=1−[(Sample−CCCP)/(Blank−CCCP)].![]()
图 2 （左）模拟肽作用于NB4，经JC-1染色后镜检；（右）模拟肽作用于K562，经 JC-1染色后镜检（200×）注：△Ψm降低细胞表现为JC-1红色荧光减弱或消失。Figure 2. (Left) BH3 peptide as a treatment for NB4, observed by microscope after JC-1 staining; (Right) BH3 peptide as a treatment for K562, viewed by microscope after JC-1 staining (200×)Note: Cells with reduced Ψm showed decreased or disappeared JC-1 red fluorescence.2.2 PCR检测
运用PCR检测BCL2家族相关基因基础表达量。BCL-2家族相互作用复杂，最终凋亡敏感性由各成员综合作用结果。分析BCL-2家族基因表达谱，K562抗凋亡蛋白中MCL-1、BCL-2、BCL-XL相对表达程度较高，BCL-W低表达；BH3-only中BIM、PUMA、NOXA表达程度较高，可能抵消MCL-1、BCL-2的抗凋亡作用，而与BCL-XL相互作用的HRK表达较低，从而可见在外源性引入HRK时可能更容易打破BCL-2家族的平衡导致细胞凋亡；而NB4抗凋亡蛋白中BCL-XL表达明显低于MCL-1与BCL-2，BH3-only中与MCL-1相互作用的NOXA表达则较低，显然NB4细胞更依赖于MCL-1与BCL-2而非BCL-XL，见图3。
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图 3 BCL-2家族基因表达量，（左）K562、（右）NB4Figure 3. Gene expression of BCL-2 family, (left) K562, (right) NB42.3 临床样本检测
2021年3月25日至2022年4月30日，对6例血液肿瘤患者骨髓细胞进行BH3分析并追踪临床疗效，分析结果及BCL-2抑制剂疗效预测如表示（表5、表6）。不同患者个体骨髓细胞BH3谱系结果不一：6例患者均表现为对HRK（BCL-XL）、VEN（BCL-2）不敏感，而对NOXA（MCL-1）敏感度存在异质性。如表5所示，例1~3、例5患者对NOXA敏感，而例4和例6的敏感度较低（<50%）；6例患者骨髓细胞对VEN均不敏感。上述结果提示6例患者抗调亡均不依赖于BCL-XL和BCL-2，而对MCL-1依赖性则有所不同。根据BH3分析原理，细胞对BH3-only模拟肽敏感度越高，则对相互对应的抗凋亡蛋白（表3）依赖程度较高，反之依赖程度低，因而推测6例均对BCL-2抑制剂（维奈克拉）、BCL-XL抑制剂（WEHI-539）耐药，例1~3、例5骨髓细胞对MCL1抑制剂（Servier63845、西达苯胺[3]、高三尖杉酯碱[4]）敏感，例4和例6对MCL1抑制剂敏感度可能较低或出现耐药。另外，例1对VEN、BAD、HRK敏感度均低，而BAD与BCL-2、BCL-W、BCL-XL均可相互作用，故综上例1抗凋亡也不依赖于BCL-W。回溯6例患者临床4~6个疗程治疗情况（表6）所示：例1~5患者对BCL-2抑制剂相关方案治疗无效，考虑与BCL-2抑制剂不敏感有关，例2患者西达苯胺有效，例4和例6患者西达苯胺相关方案疗效不佳，考虑与MCL-1抑制物不敏感有关，与BH3预测结果基本一致。
表 5 临床样本BH3分析结果Table 5. BH3 profiling results of clinical samplesCase DMSO CCCP BIM BID NOXA HRK BAD PUMA VEN 1 8.75% 100.00% 91.01% 86.74% 25.48% 15.40% 91.34% 5.75% 2 11.40% 100.00% 90.86% 41.08% 77.88% 14.00% 64.90% 98.19% 23.36% 3 20.00% 100.00% 93.38% 69.88% 73.50% 33.63% 85.63% 89.00% 15.13% 4 22.90% 100.00% 100.00% 82.88% 45.91% 0.52% 52.14% 93.77% 15.95% 5 11.30% 100.00% 92.78% 59.98% 77.11% 34.61% 96.96% 82.64% 27.40% 6 23.20% 100.00% 61.20% 72.53% 49.35% 16.41% 79.95% 51.69% 29.04% 注:阳性对照（CCCP）显示100%凋亡，空白对照低凋亡。去极化率%=1−[(肽处理孔−阳性对照）/(空白−阳性对照）]。
Note: The apoptosis of positive control (CCCP) was 100%, the apoptosis of blank control was low.%Depolarization=1−[(Sample−CCCP)/(Blank−CCCP)].表 6 药敏预测及临床样本疗效Table 6. Drug sensitivity prediction and clinical sample drug efficacyCase Predict sensitive targets Possible drug-resistant targets Clinical effects (4~6 sessions) 1 MCL-1 BCL-2, BCL-XL, BCL-W Regimens containing BCL-2 inhibitor was ineffective 2 MCL-1 BCL-2, BCL-XL Regimens containing BCL-2 inhibitor was ineffective 3 MCL-1 BCL-2, BCL-XL Regimens containing BCL-2 inhibitor was ineffective 4 BCL-2, BCL-XL, MCL-1 Regimens containing BCL-2 inhibitor was ineffective,
the addition of chidamide was ineffective5 MCL-1 BCL-2, BCL-XL Regimens containing BCL-2 inhibitor was ineffective,
remission was maintained with chidamide6 BCL-2, BCL-XL, MCL-1 Regimens containing chidamide was ineffective 3. 讨 论
WHO下属国际癌症研究机构发布的《2020年全球癌症负担报告》显示白血病无论在全球范围或中国均为死亡病例前十的恶性肿瘤[5]。BCL-2抑制剂（Venetoclax）作为新药已应用于急性髓性白血病（acute myeloid leukemia，AML）及慢性淋巴细胞性白血病（chronic lymphocytic leukemia，CLL）等血液肿瘤的治疗，其联合去甲基化药物（hypomethylating agent，HMA）可提高老年AML及复发难治白血病的疗效已被国外研究所报道[6-9]。目前临床尚无合适有效的检测“靶点”预测BCL-2抑制剂药敏。BCL-2家族蛋白成员众多，相互作用繁杂，孤立分析某个BCL-2家族相关基因或蛋白无法获得细胞总体的凋亡启动状态及对BCL-2家族蛋白的依赖程度。BH3分析作为一种活细胞功能性检查，最早便是为解决血液肿瘤化疗敏感性问题而提出，随着技术的发展及深入，应用越来越广泛；除检测化疗敏感性以外，还可用于判断预后、筛选药物[10-11]等。BH3分析技术将线粒体途径作为一个整体，不对内部相互作用过程分析而直接通过促凋亡/抗凋亡的最终效应来判读凋亡启动状态的结果，结果通俗易懂，且可反映细胞实际的线粒体凋亡相关蛋白依赖关系，将有望用于指导BCL-2、MCL-1、BCL-XL、BCL-W等抑制剂的应用。同时，肿瘤细胞的凋亡，往往与BCL-2家族蛋白失衡密切相关，而抗肿瘤药物如细胞毒药物与新型抗肿瘤药物大多通过线粒体通路诱导肿瘤细胞凋亡[12]，该项技术将为广泛抗肿瘤药物及方案筛选决策提供支持。
BH3分析方法常用的指示剂包括JC-1及细胞色素C抗体，检查手段包括有流式细胞术、酶标仪、荧光显微镜等[13-15]。我们发现相较于细胞色素C抗体，JC-1染料成本低廉，信号清晰易辩，结果更为直观。在检测手段上，因细胞经洋地黄皂苷透性处理后容易粘连，使用流式细胞术容易导致堵管风险，操作不便，而滤器过滤耗损细胞，影响检测结果准确性；荧光酶标仪亦非多大临床实验室所具备，而荧光显微镜则是大多血液实验室具备的常规实验设备，但至今尚未见报道在BH3检测终点时使用JC-1染色后荧光镜检。我们结合前述的方法组合优化出一种简便快捷、结果直观、适合小样本、且成本低廉的基于荧光显微镜和JC-1染色的BH3分析方法。
结果显示，基于荧光显微镜和JC-1染色的BH3分析技术可检测出肿瘤细胞的BH3依赖谱系，PCR分析BCL-2家族基因表达量显示与BH3分析结果一致，且K562的BH3分析结果[16]与文献报道相符（NB4尚未见有相关文献报道）。进一步应用临床样本分析发现，BH3分析结果提示VEN、BAD反应不佳的患者临床中容易发生BCL-2抑制剂耐药或反应不佳。同时，不同患者通常在NOXA或HRK上有不同的表现，提示不同患者对MCL1和BCL-XL起调节作用的药物敏感度不一，为临床单独或联合用药提供依据。本研究对BH3分析在血液肿瘤BCL-2家族蛋白抑制剂药敏筛选上的应用进行了初步探索，初步验证了BH3分析可预测BCL-2家族蛋白抑制剂的敏感性。本研究中分析的6例血液肿瘤患者均为维奈克拉治疗无效，没有维奈克拉治疗缓解的患者作为对照，故尚需进一步增加样本量，验证该项技术对血液肿瘤BH3药敏分析的准确性。
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图 2 （左）模拟肽作用于NB4，经JC-1染色后镜检；（右）模拟肽作用于K562，经 JC-1染色后镜检（200×）
注：△Ψm降低细胞表现为JC-1红色荧光减弱或消失。
Figure 2. (Left) BH3 peptide as a treatment for NB4, observed by microscope after JC-1 staining; (Right) BH3 peptide as a treatment for K562, viewed by microscope after JC-1 staining (200×)
Note: Cells with reduced Ψm showed decreased or disappeared JC-1 red fluorescence.
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图 3 BCL-2家族基因表达量，（左）K562、（右）NB4
Figure 3. Gene expression of BCL-2 family, (left) K562, (right) NB4
表 1 BH3模拟肽序列
Table 1 BH3 mimics sequences
BH3
peptidesSequences hBIM Ac-MRPEIWIAQELRRIGDEFNA-NH2 hBID-Y Ac-EDIIRNIARHLAQVGDSMDRY-NH2 mBAD Ac-LWAAQRYGRELRRMSDEFEGSFKGL-NH2 mNoxaA Ac-AELPPEFAAQLRKIGDKVYC-NH2 MS-1 Ac-RPEIWMTQGLRRLGDEINAYYAR-NH2 Puma Ac-EQWAREIGAQLRRMADDLNA-NH2 W-Hrk Ac-WSSAAQLTAARLKALGDELHQ-NH2 
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表 2 RT-qPCR引物序列
Table 2 Primer sequences for RT-qPCR
Gene Sequences BIM-F AGCCCAGCACCCATGAGTTGTGAC BIM-R CTCTGGGCGCATATCTGCAGG BID-F ATGGACCGTAGCATCCCTCC BID-R GTAGGTGCGTAGGTTCTGGT BAD-F GTTTGAGCCGAGTGAGCAGG BAD-R ATAGCGCTGTGCTGCCCAGA NOXA-F ACCAAGCCGGATTTGCGATT NOXA-R ACTTGCACTTGTTCCTCGTGG HRK-F CAGGCGGAACTTGTAGGAAC HRK-R GCTGGATTTCCAAAGGGCTT PUMA-F GACCTCAACGCACAGTACGAG PUMA-R AGGAGTCCCATGATGAGATTGT BAX-F TCTGACGGCAACTTCAACTG BAX-R GGAGGAAGTCCAATGTCCAG BAK-F GTTTTCCGCAGCTACGTTTTT BAK-R GCAGAGGTAAGGTGACCATCTC BCL-2-F ATGTGTGTGGAGAGCGTCAA BCL-2-R GCCGGTTCAGGTACTCAGTC BCL-xL-F GGAGGCAGGCGACGAGTTTGAA BCL-xL-R AAGGGGGTGGGAGGGTAGAGTGG BCL-W-F GCGGAGTTCACAGCTCTATAC BCL-W-R AAAAGGCCCCTACAGTTACCA MCL-1-F CGGTAATCGGACTCAACCTC MCL-1-R CCTCCTTCTCCGTAGCCAA GAPDH-F AGAAGGCTGGGGCTCATTTG GAPDH-R AGGGGCCATCCACAGTCTTC
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表 3 BCL2家族抗凋亡蛋白和BH3-only蛋白相互作用模式
Table 3 Interaction model of BCL2 family anti-apoptotic protein and BH3-only protein
Anti-apoptotic protein BH3-only protein BH3 inhibitor BIM BID BAD PUMA NOXA MS1 HRK ABT199(VEN) S63845 WEHI539 BCL-2 BCL-XL BCL-W MCL-1 注：*红色代表强抑制，绿色代表弱抑制或者无抑制。
Note：* Red represented strong inhibition, and green represented weak or no inhibition.
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表 4 细胞系BH3分析结果(色阶图)
Table 4 Cell line BH3 profiling results (level diagram)
DMSO CCCP BIM BID NOXA MS1(syn） HRK BAD PUMA VEN NB4 1 μM 7.80% 100.00% 97.94% 90.56% 27.11% 67.90% 24.08% 100.00% 91.11% 94.90% 20 μM 7.80% 100.00% 100.00% 100.00% 100.00% 97.51% 83.73% 90.89% 100.00% 100.00% K562 1 μM 0.00% 100.00% 90.00% 0.00% 1.70% 4.10% 10.60% 87.30% 40.40% 1.30% 20 μM 14.60% 100.00% 67.80% 24.59% 40.40% 39.34% 43.44% 81.26% 71.43% 11.94% 注:阳性对照（CCCP）显示100%凋亡，空白对照低凋亡。连续色阶图显示对BH3模拟肽的敏感程度，从红到黄到绿代表敏感强度从高到低，去极化率%=1−[(肽处理孔−阳性对照）/(空白−阳性对照）]。
Note: Positive control (CCCP) showed 100% apoptosis, while blank control showed low apoptosis. The continuous color level diagram showed the sensitivity to BH3 mimic peptide, and the diagram from red to yellow to green represented the sensitivity intensity from high to low. %Depolarization=1−[(Sample−CCCP)/(Blank−CCCP)].
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表 5 临床样本BH3分析结果
Table 5 BH3 profiling results of clinical samples
Case DMSO CCCP BIM BID NOXA HRK BAD PUMA VEN 1 8.75% 100.00% 91.01% 86.74% 25.48% 15.40% 91.34% 5.75% 2 11.40% 100.00% 90.86% 41.08% 77.88% 14.00% 64.90% 98.19% 23.36% 3 20.00% 100.00% 93.38% 69.88% 73.50% 33.63% 85.63% 89.00% 15.13% 4 22.90% 100.00% 100.00% 82.88% 45.91% 0.52% 52.14% 93.77% 15.95% 5 11.30% 100.00% 92.78% 59.98% 77.11% 34.61% 96.96% 82.64% 27.40% 6 23.20% 100.00% 61.20% 72.53% 49.35% 16.41% 79.95% 51.69% 29.04% 注:阳性对照（CCCP）显示100%凋亡，空白对照低凋亡。去极化率%=1−[(肽处理孔−阳性对照）/(空白−阳性对照）]。
Note: The apoptosis of positive control (CCCP) was 100%, the apoptosis of blank control was low.%Depolarization=1−[(Sample−CCCP)/(Blank−CCCP)].
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表 6 药敏预测及临床样本疗效
Table 6 Drug sensitivity prediction and clinical sample drug efficacy
Case Predict sensitive targets Possible drug-resistant targets Clinical effects (4~6 sessions) 1 MCL-1 BCL-2, BCL-XL, BCL-W Regimens containing BCL-2 inhibitor was ineffective 2 MCL-1 BCL-2, BCL-XL Regimens containing BCL-2 inhibitor was ineffective 3 MCL-1 BCL-2, BCL-XL Regimens containing BCL-2 inhibitor was ineffective 4 BCL-2, BCL-XL, MCL-1 Regimens containing BCL-2 inhibitor was ineffective,
the addition of chidamide was ineffective5 MCL-1 BCL-2, BCL-XL Regimens containing BCL-2 inhibitor was ineffective,
remission was maintained with chidamide6 BCL-2, BCL-XL, MCL-1 Regimens containing chidamide was ineffective
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