2022, Vol. 57
重组人粒细胞集落刺激因子(recombinant human granulocyte colony stimulating factor, rhG-CSF) 是通过基因重组技术产生的由175个氨基酸组成的重组蛋白, 具有与天然G-CSF几乎相同的生物活性, 分子质量约为18.8 kDa[1-3]。在临床上, rhG-CSF已被批准用于下列适应症: 接受骨髓抑制化疗的癌症患者; 接受诱导或巩固化疗的急性髓性白血病患者; 接受骨髓移植(BMT) 的癌症患者; 接受外周血祖细胞收集和治疗的患者; 重度慢性中性粒细胞减少患者[4]。此外, rhG-CSF在缺血性脑梗死[5]、调节免疫[6]、鼻咽癌放射治疗引发的急性口腔黏膜炎[7]、受到大量辐射照射导致造血急性辐射综合征(ARS)[8]等方面也有不错的治疗效果。
目前, 主要有非糖基化rhG-CSF [如非格司亭(filgrastim)]、糖基化G-CSF [如来格司亭(lenograstim)] 和G-CSF的突变基因产物[如那托司亭(nartograstim)] 等短效rhG-CSF被应用于临床[9-11]。然而, 短效rhG-CSF存在体内稳定性差、半衰期短等问题, 需要反复多次给药, 容易使患者产生药物耐受性及免疫排斥等不良反应, 因此非常有必要开发长效化rhG-CSF来提高其临床药效[2, 12]。目前, 开发长效rhG-CSF的技术主要有化学修饰[如聚乙二醇(PEG) 修饰][13]、融合蛋白[14, 15]以及新剂型[16]等(图 1)。
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Figure 1 Strategies for the development of long-acting recombinant human granulocyte colony stimulating factor (rhG-CSF) |
PEG修饰具有延长药物体内半衰期、降低蛋白的免疫原性以及改善药物溶解性等优点, 是开发长效化蛋白类药物应用最成功的化学修饰技术[17-20]。目前, PEG修饰rhG-CSF的位点主要包括赖氨酸侧链的ε-氨基、N末端的α-氨基、半胱氨酸的巯基和苏氨酸的羟基, 其修饰方法如图 1B所示。
1.1 赖氨酸侧链ε-氨基修饰rhG-CSF上有4个赖氨酸侧链ε-氨基, 容易出现多修饰产物。而且, 对rhG-CSF的赖氨酸侧链ε-氨基的PEG修饰会干扰其与rhG-CSFR的结合, 从而影响到rhG-CSF的活性[4]。
1.2 N末端α-氨基修饰rhG-CSF的N末端远离其活性中心, 对其进行修饰不会影响其与rhG-CSFR结合。目前上市的PEG修饰rhG-CSF基本都是N末端修饰。其中, 培非格司亭(pegfilgrastim) 是Amgen公司在非格司亭(filgrastim, Neupogen®) 基础上开发的长效剂型, 是第一个上市的长效rhG-CSF药物(商品名Neulasta®)。由于rhG-CSF的N端α-氨基的pKa值约为7.6~8.0, 而赖氨酸侧链ε-氨基的pKa值约为10~10.2, 可以控制pH值在酸性条件下利用PEG醛基化衍生物对rhG-CSF的N端α-氨基进行位点特异性PEG修饰[21, 22]。与filgrastim (未修饰的rhG-CSF) 相比, pegfilgrastim的半衰期从3 h延长到33 h, 给药频率从一天一次延长到一个化疗周期给药一次, 减轻了患者的负担。在市场上, pegfilgrastim的销售额远高于filgrastim, 体现了长效化的优势。
硫培非格司亭(macapegfilgrastim) 是由江苏恒瑞公司开发的长效型rhG-CSF药物, 于2018年上市。Macapegfilgrastim是在rhG-CSF的N端引入一个巯基, 再通过迈克尔加成反应, 将一个19 kDa的PEG分子连接到rhG-CSF上[23]。Macapegfilgrastim给药后22 h达到最大血浆浓度, 半衰期可达到56 h[23], 在临床上主要用于预防非髓系恶性肿瘤接受骨髓抑制化疗的发热性中性粒细胞减少症。临床Ib期试验表明, 3组不同剂量的macapegfilgrastim (60、100和200 μg·kg-1·day-1) 与filgrastim (5 μg·kg-1·day-1) 对治疗非小细胞肺癌的安全性与耐受性进行比较, 最终确定了100 μg·kg-1 macapegfilgrastim最安全[24]。一项多中心、随机Ⅱ期临床试验表明, macapegfilgrastim的疗效在各方面均优于rhG-CSF, 安全性方面相当[25]。非小细胞肺癌[26]或乳腺癌[27]患者进行的两个多中心、随机临床Ⅲ期试验表明, 给药剂量6 mg的macapegfilgrastim与pegfilgrastim的疗效相当。
1.3 巯基修饰目前常用于蛋白多肽类药物半胱氨酸巯基PEG修饰的试剂有PEG-马来酰亚胺(PEG-MAL)、PEG-乙烯基砜(PEG-VS)、PEG-邻吡啶二硫醚(PEG-OPSS)[28]、聚乙二醇-硒(PEG-Se)[3]和聚乙二醇-多巴醌(PEG-DAQ)[29]等。
rhG-CSF存在一个游离半胱氨酸残基, 但存在于内部的疏水结构区域, 需要使用合适溶剂体系使半胱氨酸残基暴露出来与PEG修饰剂反应[30, 31]。Veronese等[30]使用盐酸胍或尿素溶液作为溶剂, 使rhG-CSF的半胱氨酸巯基暴露出来, 显著提高了PEG修饰效率, 而且rhG-CSF的二级结构也无明显变化。Peng等[31]使用DMSO作为溶剂, 使疏水端的半胱氨酸残基暴露出来, 同样显著提高了PEG修饰效率, 并且显著加快了反应速度。
另外, 还可以通过序列改造在rhG-CSF引入游离半胱氨酸巯基。如pegteograstim (Neulapeg) 是韩国Green Cross公司研制的一种新型的长效化rhG-CSF, 是在filgrastim的Gly136和Ala137之间插入一个半胱氨酸残基, 并将Cys18用Ser取代[32], 最后将20 kDa PEG-MAL与半胱氨酸巯基反应[33]。Ⅰ期临床研究表明, 与相同剂量的pegfilgrastim相比, pegteograstim对ANC和CD34+细胞的活性增强[34]。在人体pegteograstim单次给药30~100 μg·kg-1后的药代动力学反应与单剂量皮下注射neulasta 100 μg·kg-1的药代动力学反应相当[34]。Ⅱ/Ⅲ临床研究表明, pegteograstim在预防乳腺癌患者高危化疗后的严重中性粒细胞减少方面, 与pegfilgrastim一样有效[35]。
1.4 苏氨酸羟基修饰利培非格司亭(lipegfilgrastim) 是Teva公司研发的一种聚乙二醇化的糖基化长效型非格司亭, 于2013年经EMA批准上市, 商品名Lonquex®。Lipegfilgrastim是通过N-乙酰半乳糖氨基转移酶亚型2将N-乙酰氨基半乳糖(GalNAc) 结合到蛋白质肽链的Thr134的羟基上, 再用唾液酸转移酶将一种20 kDa的聚乙二醇-唾液酸衍生物连接到其O-糖链上[36]。
Lipegfilgrastim在给药30~36 h后血药浓度出现峰值, 半衰期为32~62 h, 比pegfilgrastim长7~10 h[37]。临床Ⅱ期试验确定lipegfilgrastim的给药剂量为6 mg[38]。对乳腺癌患者的临床Ⅲ期试验表明, lipegfilgrastim的安全性与药效都与pegfilgrastim相当[39]。单次注射6 mg lipegfilgrastim可有效地进行足够的干细胞动员、收集, 确保接受自体干细胞移植(ASCT) 的多发性骨髓瘤(MM) 患者在ASCT后以最小的毒性进行移植[40]。
2 rhG-CSF的融合蛋白技术融合蛋白是通过将药物分子跟其他蛋白融合在一起, 增加药物的分子质量, 降低其肾脏清除率, 从而延长半衰期[41, 42]。目前, 应用最为广泛的是Fc融合蛋白和人血清白蛋白(HSA) 融合蛋白技术(图 1C)。
2.1 Fc融合蛋白技术Fc融合蛋白技术是通过将蛋白或多肽药物与免疫球蛋白(IgG) 的Fc片段融合而获得的一种新型功能重组蛋白。Fc融合蛋白除通过增加分子质量提高药物半衰期外, 还可以与Fc受体结合, 避免融合蛋白被细胞中的溶酶体降解。治疗蛋白通常通过它们的羧基端连接到人IgG的Fc (铰链-CH2-CH3) 和CH (CH1-铰链-CH2-CH3) 结构域的氨基末端。当IgG融合蛋白在哺乳动物细胞中表达时, 由于位于IgG铰链区的半胱氨酸残基之间形成链间二硫键, IgG融合蛋白通常以二硫键连接的同型二聚体形式分泌。Cox等[43]构建了G-CSF融合人IgG1和IgG4的Fc和CH结构域N端的嵌合基因, 并在COS-1细胞中表达。结果发现, G-CSF/IgG-Fc的体外活性与G-CSF相当, 而G-CSF/IgG-CH的体外活性降至G-CSF的1/4~1/3。随后做了动物实验, 条件性位置厌恶反应(CPA) 治疗大鼠经一次性注射G-CSF/IgG1-Fc后, 其促进中性粒细胞恢复的作用与PEG-G-CSF效果相当[44]。以下主要介绍benefilgrastim、GX-G3和eflapegrastim这3种G-CSF-Fc融合蛋白候选药物。
Benefilgrastim是由亿帆医药研发的一种rhG-CSF二聚体(rhG-CSF/IgG2-Fc融合蛋白), 它在人体内的半衰期为43.9~62.8 h[45], 在美国和中国都完成了临床Ⅲ期试验(图 2A)。由于天然的IgG会有抗体依赖性细胞毒作用和补体依赖性细胞毒作用, 所以对Fc区的某些氨基酸进行了突变[46]。Benefilgrastim就是这种形式的突变体IgG2-Fc。乳腺癌化疗患者的临床Ⅱ期试验表明每个化疗周期单次注射240或320 μg·kg-1 benefilgrastim, 与pegfilgrastim的安全性相当[46]; 临床Ⅲ期试验结果表明, 给药剂量为20 mg的benefilgrastim与6 mg给药剂量pegfilgrastim疗效相当[46]。
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Figure 2 Structures of benefilgrastim, GX-G3, and eflapegrastim |
GX-G3是韩国Genexine公司研发的一种rhG-CSF-Fc融合蛋白药物[47], 其中使用的人Fc是IgD-Fc和IgG4-Fc的杂交形式(图 2B)。这种杂交Fc无抗体依赖性细胞毒作用(ADCC) 和补体依赖性细胞毒作用(CDC), 表现出较长的作用特性和较低的免疫原性[48]。SD大鼠实验表明, filgrastim-100 μg·kg-1和pegfilgrastiytim-100 μg·kg-1的半衰期分别为2.4 h和4.0 h, 而GX-G3-25 μg·kg-1和GX-G3-100 μg·kg-1的半衰期为13.1 h和9.1 h, 明显高于pegfilgrastim和filgrastim[49]。GX-G3对CPA治疗大鼠的作用也强于pegfilgrastim[49]。目前GX-G3已完成了Ⅰ期临床试验, 目前正在进行临床Ⅱ期试验。
Eflapegrastim是由Spectrum Pharmaceuticals, Inc.从韩美制药公司引进的, 通过柔性的PEG linker将IgG4来源的Fc结构域与rhG-CSF相连[50] (图 2C)。Eflapegrastim不同于上述提到的两款rhG-CSF融合蛋白候选药物在于它分子结构只含有一个rhG-CSF分子, 且采用非肽连接子PEG, 通过活化PEG两端的基团, 分别与rhG-CSF和IgG的Fc片段结合, 可以避免肽连接子可能在体内被蛋白酶水解的风险。药效学和药动学实验表明, eflapegrastim的体外活性和药动学(PK) 曲线与pegfilgrastim相似[51]。临床Ⅱ期试验表明, 患者注射eflapegrastim的适宜剂量为45~270 μg·kg-1 (相当于12.3~73.6 μg·kg-1 G-CSF)[52]。两次Ⅲ期临床试验(ClinicalTrials.gov Identifier: NCT02953340、NCT02643420)[53]表明给药剂量为13.2 mg的eflapegrastim (3.6 mg rhG-CSF) 与pegfilgrastim (6 mg rhG-CSF) 的效果相当; 体重对临床疗效无显著性影响; 每周给药一次剂量为13.2 mg (3.6 mg rhG-CSF) 的eflapegrastim对预防化疗诱导的中性粒细胞减少有效。
2.2 人血清白蛋白(HSA) 融合蛋白技术HSA占血浆蛋白质量的60%, 可以将营养物质运输到细胞内以及将代谢物质运输到肝脏进行清除, 平均半衰期为14天。而且肿瘤细胞对HSA的摄取量比正常细胞多[54]。与HSA融合的药物除通过增加分子质量延长半衰期外, HSA的结构域Ⅲ也可以与Fc受体结合, 防止溶酶体对蛋白的降解, 并实现靶向药物递送[15]。HSA融合蛋白可以将目标蛋白通过连接子结合到HSA的C端或N端, 这取决于目标蛋白的C端或N端是否是活性中心。另外, 连接子类型和长度也会影响到融合蛋白的活性、稳定性以及细胞内的表达[41]。下面主要介绍balugrastim和GW003这两种G-CSF-HSA融合蛋白候选药物。
Balugrastim是由Teva Pharmaceutical公司研发的一种由人血清白蛋白和rhG-CSF通过重组DNA技术在酿酒酵母中表达的重组蛋白, 它将rhG-CSF的N末端与白蛋白的C端结合得到一条由759个氨基酸组成的单肽链(分子质量为85 kDa), 半衰期可达36 h[55]。目前balugrastim已经完成了临床试验。临床Ⅰ/Ⅱa期试验表明, 给药450 μg·kg-1 (相当于30 mg) 的balugrastim, ANC恢复和安全性与6 mg pegfilgrastim相当[56]。临床Ⅱ期试验表明, 给药剂量为40或50 mg的balugrastim对接受多柔比星和多西他赛化疗的乳腺癌患者的安全有效性, 与6 mg pegfilgrastim的效果相当[57]。临床Ⅲ期试验结果表明, 每周注射一次40 mg balugrastim在治疗第一期严重的中性粒细胞减少症患者的持续作用时间与pegfilgrastim相当[58]。
GW003是由江苏泰康生物医药公司研发的一种由人血清白蛋白和突变的人G-CSF通过重组DNA技术在酵母中产生的重组蛋白, 其也是通过N端基因连接于重组HSA的C端, GW003的分子质量约为85 kDa[59]。与balugrastim不同的是, GW003对rhG-CSF的T1A、L3T、G4Y、P5R、K34H、L35I、K40H、L41I的位点进行了突变, 半衰期更长且活性更高[60]。GW003临床前实验表明, 食蟹猴内GW003的清除速率得到明显的下降, 半衰期是rhG-CSF的5倍(15.7 h), 但是随着GW003剂量的增加, 清除速率也在增加[59]。目前GW003正在进行临床Ⅰ期试验, 进一步研究其安全有效性。
另外, 天津溥瀛生物技术有限公司于2009年向国家药监局提交了“注射用重组人血清蛋白/粒细胞刺激因子融合蛋白”原创新药的注册申请, 2011年被批准开展Ⅰ~Ⅱ期临床试验。该rHSA/G-CSF创新药的半衰期(~38.6 h) 比rhG-CSF的半衰期(~2.54 h) 延长约10倍以上[61]。Ⅰ~Ⅱ期临床试验(Clinical rials.gov identifiers: NCT02465801、NCT03246009和NCT03251768)[61]结果表明, 给药剂量为1.5 mg的rHSA/G-CSF对接受含蒽环类药物化疗的乳腺癌患者的持续作用时间与吉赛欣®相当, 剂量范围为1.8~2.4 mg的rHSA/G-CSF的安全性被评估为可接受, 而重复给药2.4 mg HSA/G-CSF与吉赛欣®相比未能满足非劣效性。
2.3 其他融合蛋白技术除了G-CSF/IgG Fc和G-CSF/HSA融合蛋白之外, Huang等[62]合成了一种高亲水性人工明胶样蛋白(GLK), 将其跟G-CSF融合, 得到水动力半径大、稳定性好和生物活性保持高的GLK/G-CSF融合蛋白。Mickiene等[63]将干细胞因子(SCF) 通过一个柔韧性的α螺旋连接子与G-CSF融合(SCF-Lα-G-CSF)。由于SCF和G-CSF都是调节造血的重要造血生长因子, 融合蛋白能协同增强SCF和G-CSF的作用。连接子Lα的α-螺旋构象有效地分离了双功能融合蛋白的结构域, 保证了单体之间的距离, 从而使SCF-Lα-G-CSF保持高的G-CSF生物活性[64]。利用这两种方法开发的G-CSF融合蛋白均能保持G-CSF良好的生物活性, 目前处在临床前研究阶段。
3 新剂型新剂型是通过改变药物的传递系统来延长药物半衰期的手段(图 1D), 如微球、埋植剂、口服、鼻腔、肺部等给药途径和纳米技术[65]。早期, 研究者开发了鼻腔[66, 67]、肺[68, 69]和口服[70, 71]给药等rhG-CSF新剂型, 虽然一定程度上克服rhG-CSF半衰期短的问题以及提高患者依从性, 但这些非注射给药都存在药物吸收差、稳定性差、对肝脏的首过作用以及药物对作用部位的靶向性等方面的明显不足[65]。为了解决这些问题, 研究者使用一些药物载体材料对其进行包裹。Maeda等[72]在单层微丸外侧再包覆一层高密度胶原得到一种双层微丸, 延长了rhG-CSF的释放期, 血液中的rhG-CSF浓度可维持约一周, 并持续增加白细胞计数。另外, 聚乳酸-羟基乙酸共聚物(PLGA) 具有良好的生物相容性、成囊和成膜特性, 并且无毒, 已通过美国FDA认证, 并被正式作为药用辅料收录进美国药典(USP)[73]。但PLGA微粒在释放期间, PLGA会降解产生酸性单体和低聚物, 导致包裹的蛋白质在更大程度上变性和聚集[74]。而且PLGA单体及其低聚物的降解产物在酸性条件下与包裹的多肽和蛋白质的游离氨基化学偶联[75]。Choi等[76]设计了一种rhG-CSF PLGA纳米粒。由于PLGA纳米粒子的尺寸大小, 人PLGA纳米粒子比PLGA微米粒子在更大程度上减少酸性微环境问题, 并且酸性降解产物更容易从PLGA纳米粒子中扩散到外水介质中。该PLGA纳米粒中约90%的rhG-CSF在1周内从PLGA纳米粒中以持续的方式释放, 大大提高了rhG-CSF的释放率, 不足之处在于PLGA纳米粒对rhG-CSF的包覆率仅为37.8%。Liu等[77]使用S/O/O/W制备PLGA微球, 将G-CSF-葡聚糖纳米粒子微胶囊化成PLGA微球, 提高了PLGA对G-CSF的包覆率。葡聚糖纳米粒在释药过程中还能吸收水, 体积增大, 易于相互接触, 在PLGA微球内形成更多的扩散通道, 这些扩散通道使PLGA降解产生的酸从微球中扩散出来, 降低了微球内的酸含量。这种方法得到的G-CSF-PLGA微球能够有效地缓释G-CSF, 可持续两周, 并保持90%以上的生物活性。
目前, G-CSF的新剂型的研究虽然取得了一些不错的进展, 但均处于临床前阶段。
4 总结与展望目前, 利用PEG修饰、融合蛋白以及新剂型技术开发长效化rhG-CSF中, PEG修饰是最成功的。然而, PEG修饰经过了40多年的发展, 逐渐暴露出一些问题: ① PEG对蛋白药物的空间屏蔽作用会减弱蛋白药物与受体的相互作用, 降低其生物活性, 且分子质量越大活性降低越多[78]; ②高分子质量的PEG在体内不易被代谢, 容易聚集在肝脏等部位而引起大分子综合征, 长期使用会对肝脏造成损伤[79]; ③偶联在蛋白药物上的PEG可能会诱导产生抗PEG抗体, 降低药效和安全性[80, 81]。虽然近年来有抗PEG单克隆抗体用于开发PEG化治疗或药物释放的分析工具的报道, 但相关的研究仍处于临床前阶段[81]。
对于Fc融合蛋白延长药物半衰期, 已经应用到很多药物了, 如Enbrel、Orencia和Nplate等, 但Fc融合蛋白技术也仍有一些难题需要解决: ① Fc片段往往会形成二聚体, 如果单体的蛋白分子是生物活性的首选形式, 这样可能导致药物的异质性, 而影响药效。② Fc分别与FcγRs和C1q结合, 会导致ADCC和CDC, 如果Fc融合蛋白不需要抗体相关的细胞毒性, 所以需要仔细选择和修饰IgG亚型的Fc区域。HSA融合蛋白虽然可以有效地提高药物的半衰期, 但在融合蛋白的设计以及药物进行长期的检测和评估方面仍需考虑以下问题: ①与HSA融合会导致目标蛋白生物活性下降; ② HSA融合蛋白的给药剂量较高, 连续的高剂量给药可能会出现免疫原性或不良反应; ③ HSA融合蛋白由至少两个具有不同理化性质的组分组成, 可能引起纯化、配方和稳定性的问题[15]。
目前, 蛋白质药物新剂型研究中最常用的方法是微粒控制释放。而在微粒制备和应用中仍需考虑对蛋白质稳定性的影响、微粒的载药量和包封率低以及突释效应等问题[82]。蛋白药物新剂型的开发与药物载体新材料的开发密不可分, 但单一高分子材料仍存在一些问题。如聚乳酸、聚己内酯和PLGA都是疏水性可降解载体材料。这些材料在蛋白的包封过程中必须使用有机溶剂(如DMSO), 而这些聚合物会降解产生酸性物质, 可能会影响蛋白质药物的活性[73]。
此外, 脂肪酸链修饰也是一种发展前景不错的长效化技术, 具有如下优势[83, 84]: 脂肪酸是HSA的天然配体, 在体内能与大分子质量的HSA可逆性地结合, 延长药物体内半衰期; 某些情况下, 脂肪酸还能诱导药物自组装形成多聚体, 进一步延长药物体内半衰期; 脂肪酸是构成细胞膜磷脂及人体脂肪与类脂的重要成分, 几乎无免疫原性; 脂肪酸有助于提高亲水性药物的脂溶性, 增大肠道黏膜透过性, 因此脂肪酸链修饰蛋白多肽类药物有望制成口服制剂(如索马鲁肽[85])。此外, 脂肪酸链修饰还可以减少药物与组织相容性复合物Ⅱ分子结合, 干扰T细胞的活化, 从而降低免疫原性[86, 87]。目前, 上市的脂肪酸链修饰药物有由诺和诺德公司研发的地特胰岛素[88]、德谷胰岛素[89]、利拉鲁肽[90]和索马鲁肽[85]。目前, 本课题组正在开展利用脂肪酸链修饰开发长效rhG-CSF的研究。
总体而言, 目前已有好几款长效化rhG-CSF药物上市, 多款候选药物正在临床试验中。如2001年美国FDA批准上市了第一个长效化rhG-CSF, 即安进公司开发的PEG修饰rhG-CSF药物, 商品名Neulasta®。目前, Neulasta的累积销售额为554亿美元, 远超1991年获批的filgrastim。随着Neulasta的专利到期以及lipegfilgrastim的上市, Neulasta生物类似药也层出不穷[91]。截至目前, FDA已批准4款Neulasta生物类似药(Fulphila®、Udenyca®、Ziextenzo®及Nyvepria® [92]) 上市。这虽然导致Neulasta近年来的销售额逐步下降, 但在国外长效rhG-CSF市场占比仍然很高。目前国内市场上, 短效rhG-CSF药物占比很高, 有近20个。长效rhG-CSF目前有4个, 分别为津优力® [93]、新瑞白® [94]、药艾多®和申力达®。另外还有多个处于临床研究阶段, 如亿帆医药自主研发的benefilgrastim[45]、杭州九源基因工程有限公司的Neulasta生物仿制药(ClinicalTrials.gov Identifier: NCT01918241和NCT01637493)、北京双鹭药业股份有限公司的Neulasta生物仿制药(Trial registration: http://www.chinadrugtrials.org.cn/:CTR20170164)[95]、厦门特宝的Y型PEG化rhG-CSF[96]和天津溥瀛生物技术有限公司的Ⅰ类新药rHSA/G-CSF[61]等。在这种趋势下, 长效化rhG-CSF有望在未来几年内替代短效rhG-CSF在国内市场中占据主导地位。
作者贡献: 王旭东负责组织文章的框架、文章的撰写及修改; 刘家辉负责资料的收集和文章初稿撰写; 陈康楠负责资料的收集和整理; 梅建凤和易喻完善了文章思路并修改文章; 应国清负责文章的思路指导和审阅。
利益冲突: 所有作者声明不存在利益冲突。
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