CN117025506A - 含肾前体样细胞的生物制剂及其制备方法和应用 - Google Patents
含肾前体样细胞的生物制剂及其制备方法和应用 Download PDFInfo
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- CN117025506A CN117025506A CN202310522421.XA CN202310522421A CN117025506A CN 117025506 A CN117025506 A CN 117025506A CN 202310522421 A CN202310522421 A CN 202310522421A CN 117025506 A CN117025506 A CN 117025506A
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Abstract
本发明提供了一种含肾前体样细胞的生物制剂及其制备方法和应用。所述生物制剂包括阳性表达CD133、CD24和CD44的肾前体样细胞，具有强的连续传代能力，能够抑制肾脏组织成纤维化进程，促进肾组织再生或修复。
Description
技术领域
本发明涉及生物技术领域，尤其涉及含肾前体样细胞的生物制剂及其制备方法和应用。
背景技术
急慢性肾脏疾病导致的肾功能衰竭严重威胁人类健康，传统的药物治疗和透析治疗难以逆转肾脏结构的损伤和肾功能的持续下降，Ⅰ型糖尿病肾病、肾小球肾病、慢性肾炎等在疾病进展过程对肾脏造成损伤，造成肾小球滤过率持续下降，肾实质内瘢痕的堆积成肾纤维化，纤维基质沉淀扰乱器官结构，减少血液供应，纤维化会降低组织修复能力，最后不可避免的会进展为终末期肾病。肾前体细胞(Renal progenitors)分布在肾小管和肾小囊中，能够分化成为肾小管细胞和肾足细胞，恢复肾功能，逆转肾损伤，是肾组织修复最重要的细胞。体外构建肾前体细胞培养体系，研究肾前体细胞对肾脏疾病模型的治疗作用，对于研究慢性和急性肾脏疾病的意义重大。
利用胚肾分离得到肾前体样细胞的难度大，细胞连续传代能力有限。从尿液中分离筛选取得肾前体细胞的方法存在引入外源微生物污染的问题，且该种方法得到的前体细胞通过血液系统回输后无法实现定植。由胚胎干细胞或iPS细胞诱导分化形成的肾前体样细胞有发展成畸胎瘤的风险。
因此，有必要开发新型的含肾前体样细胞的生物制剂。
发明内容
本发明的目的在于提供新型的含肾前体样细胞的生物制剂及其制备方法和应用，以利于受损肾组织的再生或修复。
为实现上述目的，本发明的生物制剂包括阳性表达CD133、CD24和CD44的肾前体样细胞，具有强的连续传代能力，能够抑制肾脏组织成纤维化进程，促进肾组织再生或修复。
优选的，所述肾前体样细胞为上皮组织来源前体样细胞。具体的，所述肾前体样细胞具有上皮前体细胞特征。
所述的“上皮组织”为被覆上皮。具体的，所述上皮组织来源前体样细胞来源于肾皮质组织。
优选的，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种。
优选的，所述肾前体样细胞阳性表达CD133、CD24、CD44、CD73和SOX9。
优选的，所述肾前体样细胞还阴性表达至少一种MHC二类分子。具体的，所述肾前体样细胞还阴性表达HLA-DR、HLA-DP和HLA-DQ的至少一种。
优选的，所述肾前体样细胞还阴性表达CD34和CD45中的至少一种。
优选的，所述肾前体样细胞阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45。
所述生物制剂的制备方法包括：使用扩增转化培养基对肾原代细胞进行体外培养，得到阳性表达CD133、CD24和CD44的肾前体样细胞。
所述扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成分：
15-25纳克/毫升的表皮细胞生长因子EGF，40-60纳克/毫升的碱性成纤维细胞生长因子bFGF，体积百分比为0.5％-1.5％的N2添加剂，体积百分比为0.5％-1.5％的B27添加剂，5-15微摩尔/升的ROCK激酶抑制剂，2-4微摩尔/升的糖原合成酶激酶3β抑制剂，0.5-1.5微摩尔/升的TGF-β信号抑制剂，体积百分比为0.5％-1.5％的补充剂ITS，不超过1纳摩尔/升的地塞米松，5-15纳克/毫升的血小板源生长因子PDGF，体积百分比为1-10％的血清。其中，补充剂ITS为胰岛素-转铁蛋白。
优选的，所述基础培养基由体积比为3:1-1:1的DMEM/F12培养基和MCDB培养基组成。
优选的，MCDB培养基为MCDB-131培养基。
优选的，所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的任意一种。
优选的，所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的任意一种。
优选的，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种。
本发明还提供了所述生物制剂在肾病治疗药物制备方面的应用。
附图说明
图1为本发明实施例的肾前体细胞CD133、CD24、CD44、CD73和SOX9表达情况示意图；
图2为本发明实施例的肾前体细胞HLA-DR、HLA-DP、HLA-DQ、CD34和CD45的表达情况示意图；
图3为本发明实施例的肾前体样细胞的光学显微镜照片；
图4为本发明实施例的肾前体样细胞和经对照培养得到的细胞两者的增殖性能对比图；
图5为本发明实施例的实验组、生理盐水对照组以及空白对照组各左侧肾脏照片的对比图；
图6为本发明实施例的实验组、生理盐水对照组和空白对照组的Masson染色照片；
图7为本发明实施例的实验组、生理盐水对照组和空白对照组的天狼星红染色照片；
图8为本发明实施例的正常对照组、模型对照组、受试物组A和受试物组B的Urea水平对比图；
图9为本发明实施例的正常对照组、模型对照组、受试物组A和受试物组B的Cr水平对比图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另外定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。
本发明各实施例中，如无特别说明，细胞培养均在37摄氏度环境下且二氧化碳浓度为5％的细胞培养箱中进行。细胞培养使用的培养基以及处理细胞所使用的各类试剂，例如缓冲液使用前均经无菌化处理以及0.22微米滤器过滤以去除杂质。
本发明各实施例中涉及统计学分析的数据，每组实验至少重复3次，实验结果数据利用GraphPad Prism 8.0软件进行分析。
实施例1
本实施例提供了肾原代细胞的获取方法。
首先，获取正常无病变的肾组织，该肾组织来源于仁济医院提供的患者手术样本。患者经医学检查无传染性病毒感染，患者在术前6个月内未使用过类固醇激素药物。患者在术前对手术样本的获取目的充分知情，并签署了知情同意书。
去除前述肾组织的外部组织、脂肪和囊状物后，将皮质从髓质上切下，去除髓质。使用无菌PBS缓冲液对2g的得到的皮质组织进行清洗和灭菌处理后，再使用由Ⅰ型胶原酶、无菌PBS缓冲液和胰酶消化液组成的共3毫升细胞消化液在37摄氏度下对组织消化90分钟得到原代细胞悬液。其中，无菌PBS缓冲液和胰酶消化液具有相同的体积，Ⅰ型胶原酶占改良缓冲液的体积百分比为1％。
然后，使用70微米的无菌筛网对所述原代细胞悬液在无菌PBS缓冲液的辅助下进行筛网分选，收集滤液并去除粘液和未消化的组织，以完成筛网分选。将得到的滤液离心并去除上清后，向得到的沉淀物加入红细胞溶解平衡液进行重悬后再次离心并重复上述过程直至再次离心后在细胞沉淀中观察不到红细胞为止，以完成红细胞裂解去除。具体的，每次离心的速率为1000g，离心时间为3分钟。得到的细胞沉淀中包含肾原代细胞。
实施例2
本实施例提供了对实施例1得到的肾原代细胞进行扩增转化培养的方法。
本实施例进行所述扩增转化培养所使用的扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成分：
20纳克/毫升的EGF，50纳克/毫升的bFGF，体积百分比为1％的N2添加剂，体积百分比为1％的B27添加剂，10微摩尔/升的Y-27632，3微摩尔/升的CHIR99021，1微摩尔/升的A83-01，体积百分比为1％的ITS，1纳摩尔/升的地塞米松，10纳克/毫升的血小板源生长因子，体积百分比为5％的FBS。
本实施例所用的基础培养基由体积比为3:2的DMEM/F12培养基和MCDB131培养基组成。
本实施例使用的DMEM/F12培养基和MCDB131来源于上海源培生物科技有限公司；EGF和bFGF来源于近岸生物；Y-27632、CHIR99021和A83-01来源于陶术生物；N2添加剂和B27添加剂来源于Invitrogen，货号分别为17502001和17504044；ITS来源于Merck，货号为I1884-1VL；，地塞米松来源于Selleck，货号为S1322，血小板源生长因子来源于Merck，货号为GF142，FBS来源于Corning，货号为35081-CV。
对实施例1得到的肾原代细胞进行扩增转化培养得到肾前体细胞的方法具体为：将实施例1得到的细胞沉淀按104个/平方厘米的接种面积接种于6孔板中，每孔加2毫升的扩增转化培养基培养5-7天直至细胞融合度不低于80％。向每孔加入0.5毫升胰酶消化液进行1-5分钟的消化直至连接细胞及培养基的蛋白质消化完全，再向每孔加入2毫升的扩增转化培养基终止消化，收集细胞悬液并对得到的细胞悬液在不超过20摄氏度的室温以及200g离心力下离心5分钟以收集细胞沉淀。用扩增转化培养基继续对得到的细胞沉淀进行传代培养，具体传代培养如下：使用扩增转化培养基重悬前述离心得到的细胞沉淀，按照104个/平方厘米的接种面积接种于T75细胞培养瓶中继续进行培养4-5天后，向每个细胞培养瓶加入2.0毫升胰酶消化液进行1-5分钟的消化直至连接细胞及培养基的蛋白质消化完全，再向每孔加入5.0毫升的扩增转化培养基终止消化，收集细胞悬液，200g离心5分钟，收集细胞沉淀，并用扩增转化培养基重悬细胞沉淀，按照按104个/平方厘米的接种面积接种于合适数量的T75细胞培养瓶中完成一次传代培养。重复上述传代培养过程直至传代到第十代以完成传代培养。
传代培养结束后，提取每孔中的细胞团后用无菌PBS缓冲液进行润洗后，用胰酶消化液消化连接细胞蛋白质，然后对得到的包含细胞的消化产物离心收集沉淀。向部分沉淀中加入100微升染色缓冲液重悬细胞至流式管中，再向流式管中加入5微升待测流式抗体孵育20分钟，然后向每管加入400微升的染色缓冲液重悬后用于表面抗体流式检测。得到图1所示的肾前体细胞CD133、CD24、CD44、CD73和SOX9表达情况示意图以及图2所示的肾前体细胞HLA-DR、HLA-DP、HLA-DQ、CD34和CD45的表达情况示意图。从图1和图2中可以看到，本实施例经扩增转化培养得到的细胞阳性表达CD133、CD24、CD44、CD73和SOX9，表达率分别为79.5％，73.0％，85.2％，92.6％和99.9％，阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45，表达率都低于1％。
CD133和CD24是肾祖细胞特征标志物，两者的双阳性表达说明本实施例得到的肾前体样细胞具有肾祖细胞特征，CD44、CD73和SOX9是干细胞特征标志物，其中CD133是本领域公知的肾损伤修复过程中上皮细胞去分化的特异性标志物，CD34和CD45是造血干细胞和白细胞标志物，CD133的阳性表达以及CD34和CD45的阴性表达说明本实施例得到的肾前体样细胞为上皮来源。HLA-DR、HLA-DP、HLA-DQ的阴性表达说明本实施例得到的肾前体样细胞对于人或哺乳动物的异体移植而言就有非免疫原性。
本实施例还通过光学显微镜观察传代培养至第十代后得到的细胞团中细胞的形态特征，得到图3所示的光学显微镜照片。从图3可以看到，细胞呈多边形，聚集生长，表现出了上皮前体细胞特征。
本实施例还使用对照培养基对实施例1得到的肾原代细胞进行对照培养。具体的培养方法与本实施例1的对实施例1得到的肾原代细胞进行扩增转化培养的方法区别仅在于：使用体积比为3：2，并含FBS(体积终浓度5％)的DMEM/F12培养基和MCDB131培养基替代扩增转化培养基。对比培养得到的细胞团为对比细胞团。
本实施例分别对扩增转化培养以及对照培养过程中不同代次(P0-P10，P0为原代细胞)细胞进行计数，考察细胞增殖性能，得到图4所示的增殖性能对比图。从图4可以看到，对照培养得到的细胞不具备增殖性能，而经扩增培养得到的细胞，即使传到第十代，仍具有稳定的增殖性能。
实施例3
本实施例提供了实施例2得到的肾前体细胞在肾病治疗药物，具体为慢性肾病治疗药物制备方面的应用。
本实施例的单侧输尿管结扎肾纤维化模型的造模方法如下：选择周龄9-10周体重在250-300g的若干雄性SD大鼠，将各大鼠用异氟烷麻醉后，腹部剃毛，仰卧位固定于手术板上并持续麻醉。腹部消毒，沿腹中线作手术切口，依次切开皮肤和肌肉，游离肾脏和输尿管，用组织钳托起左侧输尿管中段部位，在两端用缝合线结扎后，于中间离断输尿管。造模成功后，将硅胶管经股动脉插管入肾动脉，并留置注射管路。然后缝合肌肉和皮肤，用碘伏擦拭消毒后，放置于加热垫复温，待其苏醒后放回笼盒饲养。
将实施例2的包含细胞的消化产物离心收集沉淀后得到的一部分沉淀加入到50mL无菌离心管后在200g离心力的作用下离心5分钟，弃上清，用生理盐水将细胞稀释至2×107个/mL。通过各大鼠左肾动脉按照6.6×106个/公斤的剂量给药，作为实验组。生理盐水对照组按同等剂量注射生理盐水。具体给药操作方法为本领域技术人员的常规技术手段。
给药结束后的第28天将各大鼠安乐死，解剖取左侧肾脏拍照，得到图5所示的实验组、生理盐水对照组以及空白对照组各左侧肾脏照片的对比图。从图中可以看到，生理盐水对照组的肾脏出现了明显的水肿增大现象，而实验组的肾脏基本维持正常的形态。
将各大鼠左肾沿冠状面切开后取一半固定于10％福尔马林溶液，并分别取部分进行Masson和天狼星红染色，得到图6所示的实验组、生理盐水对照组和空白对照组的Masson染色照片，和图7所示的实验组、生理盐水对照组和空白对照组的天狼星红染色照片。在Masson染色照片中可以看到，与生理盐水对照组相比，实验组的肾脏纤维化程度显著降低，说明肾前体样细胞对缓解肾纤维化具有积极作用。在天狼星红染色照片中，与生理盐水对照组相比，实验组的肾脏纤维化程度显著降低，说明肾前体样细胞注射后有缓解肾纤维化的作用。
实施例4
本实施例提供了实施例2得到的肾前体细胞在肾病治疗药物，具体为急性肾病治疗药物制备方面的应用。
本实施例还取健康的周龄9-10周，体重在250-300g的雄性SD大鼠肾脏的皮质组织进行扩增转化培养得到含鼠源肾前体样细胞。具体培养过程与实施例2的人源的肾前体样细胞培养过程的区别仅在于肾皮质组织的来源不同。
本实施例中，甘油诱导急性肾损伤模型的建模方法如下：选择健康的周龄9-10周，体重在250-300g的若干只雄性SD大鼠适应性饲养3天并禁水24h后称重，按10mL/kg肌肉注射50％的甘油。
将实施例2的包含人源的肾前体样细胞的消化产物离心收集沉淀后得到的一部分沉淀加入到50mL无菌离心管后在200g离心力的作用下离心5分钟，弃上清，用生理盐水将细胞稀释至2×107个/mL，对15只甘油诱导急性肾损伤模型大鼠左肾动脉按照6.6×106个/公斤的剂量在注射甘油后的第24小时给药作为受试物组A。
将本实施例得到包含鼠源的肾前体样细胞的消化产物离心收集沉淀后得到的一部分沉淀加入到50mL无菌离心管后在200g离心力的作用下离心5分钟，弃上清，用生理盐水将细胞稀释至2×107个/mL，对15只甘油诱导急性肾损伤模型大鼠左肾动脉按照6.6×106个/公斤的剂量在注射甘油后的第24小时开始给药作为受试物组B。
取15只健康的周龄9-10周，体重在250-300g的雄性SD大鼠作为正常对照组。
取15只甘油诱导急性肾损伤模型大鼠作为模型对照组。各组大鼠分别于注射甘油后6h和72h后取大鼠用异氟烷麻醉，通过眼眶静脉丛取血1mL置于促凝管中，3000rpm离心10分钟，取血清检测Urea、Cr，得到图8所示的正常对照组、模型对照组、受试物组A和受试物组B的Urea水平对比图(每个时间点所对应的若干柱形图中，从左至右依次为正常对照组、模型对照组、受试物组A和受试物组B)，以及图9所示的正常对照组、模型对照组、受试物组A和受试物组B的Cr水平对比图(每个时间点所对应的若干柱形图中，从左至右依次为正常对照组、模型对照组、受试物组A和受试物组B)。参图可知，模型对照组、受试物组A和受试物组B的各大鼠注射甘油后6h，血清中Urea和Cr相较于正常对照组都显著升高(P<0.01)。注射后的24h，受试物组A和受试物组B分别使用人源的肾前体样细胞和鼠源的肾前体样细胞进行了治疗干预，治疗后48h(即注射甘油后72h)，相较于模型对照组，无论是受试物组A还是受试物组B的血清Urea和Cr水平显著降低，说明本实施例和实施例2的鼠源以及人源的肾前体样细胞有修复急性肾损伤的作用。而且，受试物组A和受试物组B各自相较模型对照组而言表现出的Urea和Cr水平变化趋势相当，说明人源的肾前体样细胞表现出与鼠源的肾前体样细胞相当的治疗效果，且不会引起异种排斥。
虽然在上文中详细说明了本发明的实施方式，但是对于本领域的技术人员来说显而易见的是，能够对这些实施方式进行各种修改和变化。但是，应理解，这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且，在此说明的本发明可有其它的实施方式，并且可通过多种方式实施或实现。
Claims (11)
1.一种生物制剂，其特征在于，包括肾前体样细胞，所述肾前体样细胞阳性表达CD133、CD24和CD44。
2.根据权利要求1所述的生物制剂，其特征在于，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种。
3.根据权利要求1所述的生物制剂，其特征在于，所述肾前体样细胞还阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45中的至少一种。
4.根据权利要求1所述的生物制剂，其特征在于，所述肾前体样细胞为上皮组织来源前体样细胞。
5.根据权利要求4所述的生物制剂，其特征在于，所述上皮组织来源前体样细胞来源于肾皮质组织。
6.一种生物制剂的制备方法，其特征在于，包括：
使用扩增转化培养基对肾原代细胞进行体外培养，得到阳性表达CD133、CD24和CD44的肾前体样细胞；
所述扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成分：
15-25纳克/毫升的EGF，40-60纳克/毫升的bFGF，体积百分比为0.5％-1.5％的N2添加剂，体积百分比为0.5％-1.5％的B27添加剂，5-15微摩尔/升的ROCK激酶抑制剂，2-4微摩尔/升的糖原合成酶激酶3β抑制剂，0.5-1.5微摩尔/升的TGF-β信号抑制剂，体积百分比为0.5％-1.5％的补充剂ITS，不超过1纳摩尔/升的地塞米松，5-15纳克/毫升的血小板源生长因子，体积百分比为1-10％的血清。
7.根据权利要求6所述的生物制剂的制备方法，其特征在于，所述基础培养基由体积比为3:1-1:1的DMEM/F12培养基和MCDB培养基组成。
8.根据权利要求6所述的生物制剂的制备方法，其特征在于，所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的任意一种，所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的任意一种，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种。
9.根据权利要求6所述的生物制剂的制备方法，其特征在于，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种，阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45中的至少一种。
10.一种生物制剂在治疗肾病药物的制备方面的应用，其特征在于，所述生物制剂包含阳性表达CD133、CD24和CD44的肾前体样细胞。
11.根据权利要求10所述的生物制剂在治疗肾病药物的制备方面的应用，其特征在于，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种，阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45中的至少一种。
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| CN112126618B (zh) * | 2019-06-24 | 2023-09-12 | 上海拜羡生物科技有限公司 | 一种人胆囊干细胞的获取和长期体外培养的方法 |
| WO2021047495A1 (zh) * | 2019-09-12 | 2021-03-18 | 海门雨霖细胞科技有限责任公司 | 体内外化学诱导成纤维细胞直接重编程为肝细胞的化学小分子组合物及方法 |
| CN111440761A (zh) * | 2020-04-09 | 2020-07-24 | 上海赛尔维医疗科技有限公司 | 胰腺细胞的扩增和分化方法以及应用 |
| CN115322947B (zh) * | 2022-06-25 | 2024-02-09 | 同济大学 | 一种采用成体肾前体细胞经悬浮分化培养构建肾微器官的方法和应用 |
| CN115992090A (zh) * | 2022-11-09 | 2023-04-21 | 同济大学 | 一种肾前体细胞来源的分泌组及其制备方法和应用 |
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2023
- 2023-05-09 CN CN202310522470.3A patent/CN117025508A/zh active Pending
- 2023-05-09 WO PCT/CN2023/092956 patent/WO2023217123A1/zh unknown
- 2023-05-09 WO PCT/CN2023/092969 patent/WO2023217134A1/zh unknown
- 2023-05-09 WO PCT/CN2023/092967 patent/WO2023217132A1/zh unknown
- 2023-05-09 CN CN202310519697.2A patent/CN117025502A/zh active Pending
- 2023-05-09 CN CN202310519743.9A patent/CN117025504A/zh active Pending
- 2023-05-09 WO PCT/CN2023/092958 patent/WO2023217125A1/zh unknown
- 2023-05-09 CN CN202310522421.XA patent/CN117025506A/zh active Pending
- 2023-05-09 WO PCT/CN2023/092963 patent/WO2023217128A1/zh unknown
- 2023-05-09 WO PCT/CN2023/092965 patent/WO2023217130A1/zh unknown
- 2023-05-09 WO PCT/CN2023/092964 patent/WO2023217129A1/zh unknown
- 2023-05-09 CN CN202310519740.5A patent/CN117025503A/zh active Pending
- 2023-05-09 CN CN202310522452.5A patent/CN117018032A/zh active Pending
- 2023-05-09 WO PCT/CN2023/092954 patent/WO2023217121A1/zh unknown
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- 2023-05-09 CN CN202310522433.2A patent/CN117025507A/zh active Pending
- 2023-05-09 CN CN202310519744.3A patent/CN117025505A/zh active Pending
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| WO2023217128A1 (zh) | 2023-11-16 |
| WO2023217126A1 (zh) | 2023-11-16 |
| CN117025503A (zh) | 2023-11-10 |
| CN117025502A (zh) | 2023-11-10 |
| CN117025505A (zh) | 2023-11-10 |
| CN117018032A (zh) | 2023-11-10 |
| WO2023217130A1 (zh) | 2023-11-16 |
| WO2023217121A1 (zh) | 2023-11-16 |
| WO2023217129A1 (zh) | 2023-11-16 |
| CN117025507A (zh) | 2023-11-10 |
| CN117025508A (zh) | 2023-11-10 |
| WO2023217134A1 (zh) | 2023-11-16 |
| WO2023217132A1 (zh) | 2023-11-16 |
| CN117018033A (zh) | 2023-11-10 |
| WO2023217125A1 (zh) | 2023-11-16 |
| CN117025504A (zh) | 2023-11-10 |
| WO2023217123A1 (zh) | 2023-11-16 |
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