CN116836276B - 一种用于修复膝关节损伤的干细胞制剂及其制备工艺 - Google Patents
一种用于修复膝关节损伤的干细胞制剂及其制备工艺 Download PDFInfo
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Abstract
本发明涉及一种用于修复膝关节损伤的干细胞制剂及其制备工艺。本发明提供了一种特异性针对NGF的单克隆抗体，所述单克隆抗体为鼠单克隆抗体。该抗体单独或者联合脐带间充质干细胞可以有效的抑制骨关节炎中炎性因子的产生，能够有效的促进骨关节炎的愈合，具有较好的制药用途。
Description
技术领域
本申请涉及生物治疗领域，具体的涉及一种用于修复膝关节损伤的干细胞制剂及其制备工艺。
背景技术
膝关节是全身最大的屈戍关节，同时由于它的外形也决定了它不是一个十分稳定的关节，因此膝关节的韧带结构在保持膝关节的正常功能和稳定性起很大的作用。膝关节虽然是一个屈戍关节，但在屈膝时也能作轻度的磨动和旋转。膝关节的主要功能为负重、传递载荷、参加运动为小腿活动提供力偶。膝关节不如髋关节灵活，主要为屈伸运动，但因其位于下肢的中部，位于身体两个最大的杠杆臂之间，承受较大的力，易引起扭伤和骨折。尤其在体育活动中，韧带和半月板的损伤最为常见。膝关节骨性关节炎是一种以退行性病理改变为基础的疾患。其症状多表现为膝盖红肿痛，如不及时治疗，可以引起关节畸形。在膝关节部位还常患有膝关节滑膜炎、韧带损伤、半月板损伤等关节疾病。膝关节骨性关节炎的发生一般由膝关节退行性病变、外伤、过度劳累等因素引起。另外，不正确的走路姿势、膝关节的受凉、受寒也是导致膝关节骨性关节炎的原因。
临床上对膝关节骨关节炎的病情进展仍无有效的控制措施。虽然针对早期膝关节骨关节炎的治疗方法种类繁多，包括口服药物治疗、关节腔药物注射、佩戴支具、理疗和改变生活方式等，但疗效均差强人意。非手术治疗失败后可以采取关节镜下软骨修复或移植，或进行截骨矫形、单髁及全膝关节置换等手术治疗。目前，随着干细胞研究的深入以及生物工程技术的普遍应用，越来越多的研究者在探索这些技术方法在骨关节炎治疗领域中的应用可行性。
研究发现MSCs可以源源不断分化为软骨细胞，可以将其用来治疗骨关节炎。MSCs能够进行培养扩增，且不会失去多向分化潜能；不论在体还是离体培养，MSCs都有软骨分化潜能。MSCs分布广泛，例如骨髓、骨膜、骨小梁、脂肪垫组织、滑膜、骨骼肌以及乳牙等组织都可以分离出MSCs。无论这些细胞是何种组织来源，都具有向多种细胞系分化的能力，包括***细胞，如骨、脂肪、软骨和肌肉等。在此认识的基础上，人们开创了越来越多的细胞技术治疗膝关节骨关节炎的方法。
MSCs相对容易分离，具有良好的体外扩增能力，扩增过程中可保留其干细胞特性，促进软骨细胞增生，因此MSCs是可用于关节软骨细胞来源的另一选择。最初人们从骨髓中分离出MSCs并认识到它的特性，随后不断被从其他***中分离出来，包括骨膜、关节滑膜以及脂肪组织等部位。动物实验研究发现扩增培养后的MSCs能够修复软骨和软骨下骨，并能控制继发性骨关节炎进展。用兔作为实验对象，将悬浮有骨髓或骨膜MSCs的胶原蛋白凝胶注入全层损伤的股骨外髁内侧，观察到关节内很快形成了玻璃样修复组织，而注入不含MSCs的胶原蛋白则形成了纤维样组织。虽然随时间推移玻璃样修复组织逐渐变薄、退化，生物力学特性较原始软骨组织差，且软骨面更加粗糙不平，但仍然优于纤维样组织。近年来，不断有MSCs治疗骨关节炎的相关研究进入临床试验阶段。临床试验显示，将自体MSCs进行体外扩增培养后注入体内，结果在损伤区域出现再生软骨，并因此认为这一疗法安全可行。局部应用MSCs有许多优点，不仅能够强化关节修复，还可减缓骨关节炎导致的退变，同时也是治疗骨关节炎最简单易行的方法。将从成纤维细胞生长因子受体阳性大鼠获取并培养的MSCs移植到成纤维细胞骨关节炎小鼠的股骨间隙，发现这一方法具有治疗效果，移植的细胞能够分化成软骨细胞。研究发现向骨关节炎公山羊移植人骨髓MSCs也能观察到治疗效果，因此认为局部细胞治疗能够刺激半月板组织再生、减轻损伤区域的进一步破坏。通过24周的MRI跟踪监测发现，植入自体MSCs能够刺激软骨生长，减轻退变关节的疼痛症状。从这些研究可以发现，MSCs具有一定的治疗骨病的效果，但是目前研究的方法还不够多，特别是单一的治疗的效果目前还不够好，开发组合的治疗药物组合物进而提高治疗效果是进一步的开发方向。
此外，神经生长因子(Nervegrowthfactor,NGF)属于与外周神经***和脑细胞发育相关的分泌蛋白。除在神经元生长、分化、成熟，以及损伤修复过程中发挥重要作用外，临床研究数据也表明，患有类风湿性关节炎和其他类型关节炎(包括KOA)患者滑液中检测到NGF含量显著升高。而在炎症或退化关节部位，NGF表达水平异常和慢性炎症反应被认为是导致患者疼痛的主要原因。动物模型及临床研究显示，抑制NGF可以减轻疼痛和痛觉过敏，NGF抗体L148M通过下调VEGFA和Ang-1蛋白水平抑制软骨下骨异常血管生成，缓解关节炎症疼痛反应；并通过抑制IL-1β、TNF-α和MMP表达，减轻软骨和软骨下骨病理损伤。开发特异性的NGF抗体用于骨关节炎的治疗也是重要的研究方向。
发明内容
本发明提供了一种脐带间充质干细胞联合NGF抗体一起用于治疗骨关节炎。
具体的，一方面，本发明提供了一种特异性针对NGF的单克隆抗体，所述单克隆抗体为鼠单克隆抗体。
进一步的，所述单克隆抗体命名为3N12，通过测序，鉴定获得其轻链可变区序列如SEQ ID NO：1所示，其重链可变区序列如SEQ ID NO：2所示。
在一些实施方案中，本发明的轻链可变区包含与选自SEQ ID NO:1的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(i)包含选自SEQ ID NO:1的氨基酸序列或由所述氨基酸序列组成：或者
(ii)包含与选自SEQ ID NO:1的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的重链可变区
i)包含与选自SEQ ID NO:2的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
ii)包含选自SEQ ID NO:2的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含与选自SEQ ID NO:2的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
本发明的抗体的片段和衍生物(如在本申请中所使用的，其由术语“抗体”所涵盖，除非另有说明或明显与上下文相矛盾)，优选为可以通过本技术领域已知的技术来产生。“免疫反应片段”包括完整抗体的一部分，通常是抗原结合部位或可变区。抗体片段的实例包括：Fab、Fab′、Fab′-SH、F(ab′)2、以及Fv片段；双功能抗体；任何抗体片段，其是具有一级结构的多肽，其中一级结构由邻近氨基酸残基的一个未中断序列(在本文中称作“单链抗体片段”或“单链多肽”)构成，包括但不限于(1)单链Fv(scFv)分子(2)仅包含一个轻链可变结构域的单链多肽，或其包含轻链可变结构域的三个CDR、而没有相联的重链部分的片段以及(3)仅包含一个重链可变区的单链多肽，或其包含重链可变区的三个CDR、而没有相联的轻链部分的片段；以及形成自抗体片段的多特异性抗体。例如，Fab或F(ab′)2片段可以按照常规技术通过蛋白酶消化分离的抗体来产生。应当明了，可以利用已知方法来修饰免疫反应片段。
进一步的，本发明还提供了NGF的单克隆抗体3N12在制备用于治疗骨关节炎的药物组合物中的用途。
更进一步的，本发明还提供了NGF的单克隆抗体3N12联合脐带间充质干细胞在制备用于治疗骨关节炎的药物组合物中的用途。
具体的，本发明的药物组合物中可以使用本领域普通技术人员公知的任何合适的载体，载体的类型将随着施用的方式不同而异。对于肠胃外施用(如皮下注射)，优选的载体含有水、盐水、醇、脂肪、蜡或者缓冲液。对于口服施用，可以使用上述的任何一种载体或固体载体，如甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、滑石、纤维素、葡萄糖、蔗糖和碳酸镁。可被生物降解的中心体(例如聚乳酸盐聚羟乙酸盐)也可以作为本发明药物组合物的载体。
可以在这些组合物中使用的药用可接受载体包括但不限于：离子交换剂，氧化铝，硬脂酸铝，卵磷脂，血清蛋白，如人清血白蛋白，缓冲物质如磷酸盐，甘氨酸，山梨酸，山梨酸钾，饱和植物脂肪酸的部分甘油酯混合物，水，盐或电解质，如硫酸鱼精蛋白，磷酸氢二钠，磷酸氢钾，氯化钠，锌盐，胶体二氧化硅，三硅酸镁，聚乙烯吡咯烷酮，纤维素基物质，聚乙二醇，羧甲基纤维素钠，聚丙烯酸酯，蜡，聚乙烯-聚氧化丙烯嵌段聚合物，聚乙二醇以及羊毛脂。
本发明的组合物可以用于增强患者对骨关节炎的修复的方法中。该方法包括用所述组合物与所述患者或生物样品接触的步骤。这样的方法将可用于诊断和治疗目的。为了与生物样品一起使用，可以通过与样品简单混合或直接施加于该样品来给予抗体组合物，其取决于样品的特性(液体或固体)。生物样品可以与在任何适宜装置(平板、囊、瓶等)中的抗体直接接触。为了与患者一起使用，该组合物必须加以配制以便为患者给药。本发明的组合物可以通过下述方式给药：口服、局部经过植入的贮存器。如在本文中所使用的，术语“肠胃外”包括皮下，病灶内注射或输注技术。
具体的，本发明的药物组合物中涉及的脐带间充质干细胞可以是商业购买的，也可以是从脐带中分离制备获得的。所述的分离制备方法是本领域公知的方法，其提的细胞浓度可以适当调整，这属于本领域常规技术。
有益效果
本发明提供了一种特异性针对NGF的单克隆抗体，所述单克隆抗体为鼠单克隆抗体。该抗体单独或者联合脐带间充质干细胞可以有效的抑制骨关节炎中炎性因子的产生，能够有效的促进骨关节炎的愈合，具有较好的制药用途。
附图说明
图1NGF单克隆抗体的特异性鉴定结果图
图2各治疗组的关节软骨评分结果图
具体实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
尽管本文使用了特定的术语，但它们仅用于一般性和描述性的意义，而不是为了限制的目的。除非另有定义，否则本文使用的所有技术和科学术语具有与本公开描述的主题所属领域的普通技术人员通常理解的相同的含义。
实施例1NGF单克隆抗体的制备
重组人NGF蛋白产品编号:519648纽普生物作为免疫原。采用如下方法进行免疫：
第1次免疫：选取3只18～22g体重的Balb/c雌性小鼠，将NGF蛋白蛋白与弗氏完全佐剂等体积混合乳化后，采用腹部皮下多点注射的方式进行首次免疫,剂量为80μg/只。第2次免疫：时间与第1次免疫间隔1个月，将NGF蛋白与弗氏不完全佐剂等体积混合乳化，转移至1mL注射器中，腹部皮下多点注射免疫，免疫剂量为70μg/只。二免1月后，采用同样的方法进行第3次免疫，免疫剂量为50μg/只。ELISA分别测定3次免疫后小鼠的多克隆抗体效价，选取效价最高的2号小鼠进行杂交瘤细胞融合，融合前3d对该鼠进行腹腔加强免疫,免疫剂量为50μg/只。
免疫小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0的细胞数目比为10:1，融合时间为2min，融合试剂为50％聚乙二醇；细胞融合前通过显微镜观察SP2/0的形态，融合5d后换半液继续培养，培养到第7d换全液，镜检观察换半液及换全液时细胞的生长状态，融合后7～10d左右，收取细胞板培养液上清，采用间接ELISA筛选阳性细胞板孔，采用有限稀释法对阳性的细胞进行4次亚克隆后，获得4株阳性单克隆，其中阳性反应最强的克隆命名为3N12，随后进行扩瓶放大培养。
取8周龄左右Balb/c小鼠，腹腔注射0.5mL液体石蜡，7d后腹腔注入5××105个3N12杂交瘤细胞，待小鼠腹部胀起后收集腹水。采用辛酸-硫酸铵法对腹水进行纯化，PBS透析后，采用BCA法进行蛋白浓度测定，调整蛋白浓度为1mg/mL置于-20℃保存。
实施例2NGF单克隆抗体的特异性鉴定
将NGF重组蛋白、BSA蛋白与上样缓冲液3:1混合，100℃煮沸3～5min变性处理之后进行SDS-PAGE电泳。恒压50V开始电泳至样品溶液进入分离胶后电压调至100V，至电泳结束。凝胶浸于TF缓冲液30min，用半干胶转移仪转移至硝酸纤维素膜，按0.8mA/cm2硝酸纤维素膜，恒电压18V转移30min。封闭液37℃封闭60min,TBST缓冲洗1次，弃去液体，加入单克隆抗体反应1h后，TBST缓冲洗1次，弃去液体，再加入碱性磷酸酶标记的抗鼠二抗，37℃摇动反应40min。TBST缓冲液洗5次，每次8min。加入混合好的BCIP/NBT显色液反应5min，观察结果，如图1所示。
从图1的单克隆抗体杂交分析结果可以看出，3N12单克隆抗体能够特异性的和NGF重组蛋白在泳道2约27KD处出现特异性条带，且单抗在对照蛋白泳道1内无条带，表现了出较好的特异性。
实施例3NGF单克隆抗体的亲和力及效价测定
采用生物薄膜干涉仪测定NGF单克隆抗体的亲和力，具体方法为：将NGF蛋白吸附于生物传感器[Anti-Penta-HIS(HIS1K)Biosensors]，投入纯化后的3N12单克隆抗体，在ForteBioBlitz生物分子相互作用分析***中进行亲和力常数测定。
采用间接ELISA方法测定单克隆抗体的效价，将1μg/mL NGF蛋白以，100μL/孔加入酶标板孔内，4℃条件下，过夜包被，PBST洗板后，5％脱脂牛奶封闭2h。弃去孔内液体，PBST洗板3次，取单抗用PBS梯度稀释后加入酶标板孔中反应45min，洗板后加入HRP酶标记的羊抗鼠IgG抗体(1:5000)孵育45min。弃去孔内液体，PBST洗板后，加入TMB显色液100μL/孔，避光显色7min后，加入2mol/L硫酸终止反应，置于酶标仪，450nm波长下测定其OD值。结果如表1所示。
表1 3N12单克隆抗体效价及亲和力鉴定结果
| 抗体名称 | 效价 | 亲和力 |
| 3N12单克隆抗体 | 1:2048000 | 1.437±0.054×10-9mol/L |
从表1可以看出，本发明的3N12单克隆抗体具有较好的效价及亲和力，活性较好。
实施例3脐带间充质干细胞的分离及制备
HUCMSC的分离和培养：取新鲜的足月剖宫产新生儿脐带，用含有青霉素/链霉素双抗的PBS缓冲液反复冲洗，去除残留血液。将脐带组织剪成0.5mm3左右大小的碎片后转移到培养皿中，用含10％去外泌体胎牛血清、1％双抗(100U/mL青霉素、100mg/mL链霉素)的高糖DMEM培养基培养，放置在37℃5％CO2培养箱中20h，加入5mL培养基，每3d换液1次，7d后细胞生长状态利用倒置显微镜观察，汇合度达到80％～90％时，用0.25％EDTA胰酶消化，1∶3传代。P3代细胞进行表明抗原检测，收集对数生长期细胞，标记荧光抗体CD29-PE，CD166-PE，CD73-PE，CD14-PE，CD144-PE，CD34-FITC，CD45-FITC，HLA-ABC，HLA-DR，CD44-FITC，CD105-FITC，流式细胞仪检测，结果显示，脐带间充质干细胞强表达CD29(99.5％)、HLA-ABC(98.2％)、CD166(95.3％)、CD105(90.5％)、CD73(98.9％)、CD44(98.2％)，几乎不表达CD34、CD14、HLA-DR、CD45、CD31、CD144。制备得到了脐带间充质干细胞。将干细胞调整到5×107浓度后备用。
实施例4NGF单克隆抗体和/或脐带间充质干细胞功能验证
将C57BL/6小鼠随机分为7组，每组10只：
阴性对照组，小鼠麻醉后右膝关节腔内注射50μL无菌生理盐水；
以下各组用1％戊巴比妥钠将小鼠麻醉，右膝手术部位常规碘伏消毒，使右膝屈膝90度，27G针垂直髌韧带穿刺至有落空感。根据不同分组，向膝关节腔内缓慢注射50μL无菌生理盐水或MIA溶液。拔针后用纱布压迫10s，再屈伸膝关节5次。结束后将小鼠置于温暖处，待小鼠苏醒再放回动物房，允许自由进食。
其中，模型对照组，小鼠麻醉后右膝关节腔内注射50μL 20mg/mL MIA溶液，手术后2周于腹腔注射无菌生理盐水(10mg/kg)；
阳性对照组，小鼠麻醉后右膝关节腔内注射50μL 20mg/mL MIA溶液，手术后2周于腹腔注射L148M(10mg/kg)；
低剂量3N12单抗组，小鼠麻醉后右膝关节腔内注射50μL 20mg/mL MIA溶液，手术后2周于腹腔注射本发明NGF单克隆抗体(1mg/kg)；
高剂量3N12单抗组，小鼠麻醉后右膝关节腔内注射50μL 20mg/mL MIA溶液，手术后2周于腹腔注射本发明NGF单克隆抗体(10mg/kg)；
干细胞治疗组，小鼠麻醉后右膝关节腔内注射50μL 20mg/mL MIA溶液，手术后2周于关节腔内注射实施例3制备的脐带间充质干细胞1×106；
干细胞联合3N12单抗治疗组，小鼠麻醉后右膝关节腔内注射50μL 20mg/mL MIA溶液，手术后2周于关节腔内注射实施例3制备的脐带间充质干细胞1×106，同时腹腔注射本发明NGF单克隆抗体(10mg/kg)；
在MIA造模后第4周(药物干预后第2周)，随机从各组各抽取5只小鼠，分别经腹膜注射水合氯醛麻醉，颈部脱臼法处死后，用咬骨钳和手术刀分离取出小鼠右膝关节，仔细清理膝关节周围软组织及肌肉。HE染色，OARSI关节软病理评分法进行关节软骨评分。结果如图2所示。
OARSI评分结果显示，模型组小鼠OARSI评分(8.27±0.43)显著高于阴性对照组(P＜0.05)，而高剂量单抗组小鼠OARSI评分(3.03±0.23)明显低于模型组(P＜0.05)，也比阳性对照组的效果要好。特别是干细胞联合单抗一起使用后，小鼠膝关节相较于对照组，软骨和软骨下骨结构更加完整，治疗效果更加显著。
将前述剩余各组小鼠的部分软骨部分用含1％PMSF的RIPA裂解缓冲液提取膝关节软骨细胞总蛋白，软骨组织置于在冰上超声破碎，12000×g4℃离心30min。使用BCA蛋白测定试剂盒检测蛋白浓度。将50μg总蛋白上样至10％SDS-PAGE凝胶，然后在300mA恒定电流下转移到PVDF膜。将膜在含0.1％Tween-20和5％脱脂奶粉的Tris缓冲液中室温孵育2h，再用TBS-T洗涤3次。将膜与抗INOS(1：1000)、COX-2(1：1000)、NGF(1：1000)一抗4℃孵育过夜。再用TBS-T清洗膜3次。然后在室温下与二抗孵育2h。用TBS-T洗涤后，使用增强化学发光试剂盒进行检测，并通过QuantityONE定量分析，以GAPDH蛋白的表达量作为相对表达基础。结果如表2所示。
表2各组中相关蛋白表达量分析结果
*表示与模型组相比，各治疗组差异极其显著(P<0.01)。
Westernblotting检测结果显示，与对照组比较，模型组小鼠INOS和COX-2、NGF蛋白表达水平均升高(P<0.05)；与模型组比较，阳性对照组、单抗之劳组以及单抗联合干细胞治疗组小鼠NGF、INOS和COX-2表达水平均降低(P<0.01)。本研究实验结果显示，3N12单抗能够显著抑制小鼠软骨细胞INOS和COX-2以及NGF蛋白表达水平，COX-2是关节炎疼痛和炎症反应的关键效应因子，通过抑制NGF的表达进而抑制COX-2和INOS的表达可以有效保护关节软骨结构可以减少疼痛抑制炎症。并且，单克隆抗体与干细胞一起联用后，具有比单抗单独治疗更好的效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种NGF单克隆抗体，其特征在于该抗体的轻链可变区序列如SEQ ID NO：1所示，其重链可变区序列如SEQ ID NO：2所示。
2.一种用于修复膝关节损伤的药物组合物，其特征在于由脐带间充质干细胞和权利要求1所述的NGF单克隆抗体组成，所述的膝关节损伤对应的是膝关节骨关节炎。
3.脐带间充质干细胞和NGF单克隆抗体在制备用于治疗膝关节骨关节炎的药物组合物中的用途，其中NGF单克隆抗体的轻链可变区序列如SEQ ID NO：1所示，其重链可变区序列如SEQ ID NO：2所示。
4.如权利要求3所述的用途，其特征在于所述的药物组合物中还含有赋形剂。
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