CN112877364B - 软骨下骨细胞向关节软骨细胞直接转化的重编程诱导方案 - Google Patents
软骨下骨细胞向关节软骨细胞直接转化的重编程诱导方案 Download PDFInfo
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Abstract
本发明属于生物技术领域，公开了一种经重编程软骨下骨成骨细胞向功能性关节软骨细胞直接转化的诱导方案，适用于关节软骨重度缺损的再生修复治疗。本发明的诱导方案包括：将Sox9、Sox5与Plagl1或与Sox8及Zcchc5基因导入软骨下骨细胞，获得软骨样细胞。
Description
技术领域
本发明属于生物技术领域，涉及细胞与分子生物学、真核细胞重编程、细胞转化与分化、组织再生修复，也涉及利用细胞重编程技术方案在关节软骨损伤治疗上的应用。
背景技术
关节软骨组织(articular cartilage)具有精细的分层结构，含有不同功能的软骨细胞(chondrocyte)。中深层结构中缺乏血管、神经和淋巴管，软骨细胞数量稀少。虽然基质中主要包含胶原蛋白(collagen)、蛋白多糖(proteoglycan)和非胶原蛋白等成分，发挥缓冲和润滑作用，但缺乏自我更新能力(Bhosale and Richardson2008)。若关节活动造成高强度冲击和过度磨损，软骨发生严重损伤，软骨组织无法自我修复。
随着我国人口老龄化的快速进程，关节软骨损伤的发病率逐年攀升；而全民健身运动的迅猛发展，也使得软骨病患人群呈现年轻化趋势。作为高发关节疾病的骨关节炎(osteoarthritis)，我国的患者数量目前已达总人口10％以上，其中出现症状者多已发生关节软骨缺损等病理学改变(Bhosale and Richardson 2008)。由于临床干预有限，病程迁延不愈，创面及周边进一步磨损，逐步发展成为全层软骨缺损(full-thickness defect)，造成软骨下方骨板裸露(Frobell,Wirth et al.2010)，使关节运动受限加重，最终致残。
软骨重度缺损患者只有通过全关节置换术才能改善生活质量、避免残疾(Juni,Reichenbach et al.2006)，但同时也需要经历可称之为最艰难的术后康复过程，还要面对假体维护及不可忽视的并发症等一系列问题，而对于广大老年患者来说，无疑需要承受更严峻的挑战。因此，开发风险低、安全性高的微创治疗方法具有十分重要的意义。
目前，针对软骨缺损的再生修复治疗手段主要包括内源细胞招募和外源细胞移植两种方式。微骨折术(microfracture)通过骨面钻孔技术促进含有间充质干细胞的骨髓血渗出，填补缺损，修复损伤软骨。但该治疗存在术后易形成纤维软骨、有别于透明软骨的问题，削弱了关节功能修复作用(Oussedik,Tsitskaris et al.2015)。虽然采用非负重部位软骨组织的自体软骨细胞移植(autologous chondrocyte implantation,ACI or matrix-induced autologous chondrocyte implantation,MACI)具有较好的透明软骨修复效果，但也存在无法避免的问题，如需要手术取材，自体软骨来源有限，体外培养中去分化现象以及供区术后并发症。由于关节软骨的分层结构特点，单纯移植分化成熟的软骨细胞，仍较难达到正常生理状态的修复水平。以间充质干细胞作为种子细胞近年来备受关注，成为基础和临床的研究热点(Harrell,Markovic et al.2019)。然而实际应用中，还面临着细胞获取、培养和保存等体外操作及精确调控细胞定向分化避免骨化和钙化等诸多问题，技术瓶颈有待突破。
就软骨全层缺损的再生修复而言，软骨下骨细胞(subchondral bone cell)因具有以下独特优势，可能是潜在的种子细胞：(1)软骨细胞与软骨下骨细胞位置毗邻，暴露的软骨下骨细胞成为创面(lesion)；(2)软骨下骨板承载软骨下骨细胞，形成天然的细胞支架复合体(cell-scaffold complex)；(3)两者皆为中胚层来源，属于同一谱系(lineage)；软骨细胞经软骨内骨化(endochondral ossification)途径向成骨细胞转分化(chondrocyte-to-osteoblast transdifferentiation)(Yang,Tsang et al.2014)；(4)软骨下骨细胞经骨骼干细胞终末分化，来源充足。由此，将软骨下骨细胞转变为具有修复功能的软骨细胞成为实现原位修复软骨全层缺损的首要关键。
经外源过表达转录因子组合Oct4、Sox2、KLF4和c-Myc可诱导成纤维细胞重编程(cell reprogramming)转变为诱导型多潜能干细胞(induced pluripotent stemcells,iPSC)(Takahashi and Yamanaka 2006)，而基于此项技术，还可将其他成体细胞重编程转化为多潜能干细胞，其中包括由骨关节炎患者自体来源的软骨细胞、滑膜细胞向iPSC的直接诱导转化(Kim,Son et al.2011,Wei,Zeng et al.2012)，进而再分化为软骨细胞。由iPSC分化而来的软骨细胞可能比间充质干细胞显示更好的软骨基质形成能力(Ko,Kim etal.2014)。鉴于iPSC自身潜在的致瘤性风险使其在临床应用方面受到的制约，通过导入若干关键转录因子来诱导一类成体细胞直接转化(direct conversion)为另一类成体细胞，可越过细胞去分化再定向分化的过程，避免经过iPSC阶段所带来的安全隐患。
早在1987年曾经报道MyoD转录因子诱导成纤维细胞直接转换为肌细胞(Davis,Weintraub et al.1987)。近些年发现成纤维细胞经Gata4、Mef2c和Tbx5三因子诱导可直接转化为心肌样细胞(Ieda,Fu et al.2010)，软骨细胞也可由成纤维细胞、胎盘细胞、肌腱细胞直接重编程(direct reprogramming)转化形成(Hiramatsu,Sasagawa et al.2011,Ishii,Kami et al.2012,Takimoto,Oro et al.2012)。由此可见，直接重编程技术可更直接利用成体细胞进行再生修复。
针对软骨全层缺损的修复，创面底部暴露的软骨下骨细胞可直接用于向软骨细胞定向诱导转化的种子细胞。如果进行原位诱导，则只需要关节注射重编程因子即可实现，相当于极微创操作，患者近乎无痛，不但可弥补ACI/MACI疗法的不足，也开启利用天然支架移植技术的新模式。
发明内容
本发明的目的在于提供一种软骨下骨细胞向关节软骨细胞的直接转化路径和细胞重编程诱导方案。
本发明所指的来源于软骨下骨板的特定骨组织细胞，包括成骨细胞、骨母细胞、骨细胞或破骨细胞，分布于关节软骨下骨板，通常由于位于其上方的关节软骨组织损伤或磨损而导致其裸露于关节表面。
本发明所指的含有软骨下骨板的骨关节，包括膝关节、髋关节、椎关节、骶髂关节、肋椎关节、胸锁关节、肩锁关节、肩关节、肘关节、腕关节、足关节、手指关节。
本发明所指的由软骨下骨细胞向关节软骨细胞的直接转化，包括由单一或多个转录因子作用于细胞内特定靶基因启动子区域，导致来源于软骨下骨板的特定骨组织细胞发生细胞直接重编程而失去原有特定类型细胞标志性功能与表型特征，同时定向转变成为另一特定类型并具有关节软骨细胞标志性功能与表型特征的关节软骨样细胞。
本发明所指的关节软骨细胞，具有标志性功能与表型特征，包括表达高水平II型胶原蛋白、蛋白多糖，适量表达水平的IX和XI型胶原蛋白，呈现多边形铺路石状形态，不表达或低表达I型胶原蛋白，碱性磷酸酶，骨钙蛋白。
本发明所述的由软骨下骨细胞向软骨细胞的直接转化需要导入至少一个重编程因子，具有瞬时或稳定的表达水平，能直接或间接诱导至少一个细胞内源基因的表达，促进形成稳定的、确定的、属于关节软骨细胞的标志性功能与表型特征。
所述的重编程因子可通过参与染色质表观修饰，促进至少一个重编程因子直接或间接诱导至少一个促进关节软骨细胞形成的细胞内源基因的表达。
所述的重编程因子中，至少有一个因子可阻止原有骨组织细胞标志性功能与表型特征的维持或促进丧失原有骨组织细胞标志性功能与表型特征的形成。
所述的重编程因子不应促进向非骨组织细胞或软骨细胞的第三类细胞转化，包括纤维细胞、肌细胞、神经细胞、上皮细胞、内皮细胞、造血细胞、淋巴细胞。
本发明所述的重编程因子包括Sox9与Sox5、Sox6、Sox8其中之一或多个因子形成组合物，与Foxa3、Id1、Id3、Ndn、Peg3、Plagl1、Zcchc5、Zfp354c以及Zfp385b其中之一或多个因子组合诱导及促进软骨下骨细胞定向转化成为软骨样细胞，还可与AEBP1、ATF5、CREB3L2、Eid1、Foxa2、Foxc1、Foxo1、Foxp2、Hoxa10、Hoxa9、Hoxc10、KLF2、Nr4a1、Nfix、Nrep、Pitx1、Pou3f3、Prickle1、RARG、Runx1、Sfr1、Shox2、Snai2、Sp1以及Trps1其中之一或多个因子组合提高诱导或促进软骨下骨细胞的向软骨细胞的直接转化。
本发明所述的重编程因子导入软骨下骨细胞可通过病毒载体、质粒转染、核酸形式、蛋白质介导、转座子、纳米粒的方法运送，以核酸、蛋白、多肽、小分子化合物的形式作用于特定靶基因启动子区域。
一些组合中的全部或部分组分的基因片段构建到同一个重组表达载体上，转入细胞内进行共表达。
所述重编程因子组合物发挥诱导时间为1–8周，表达至少一种稳定的、确定的、属于关节软骨细胞的标志性功能与表型特征为至少1–8周。
所述直接重编程诱导的软骨样细胞可在体外三维培养形成软骨微球，在体内形成软骨组织。
本发明提供的方法可把软骨下骨细胞作为原位种子细胞，进行有效的直接重编程转化成为软骨样细胞，满足各种伴随重度软骨损伤的关节疾病治疗需求，包括骨关节炎、类风湿性关节炎、软骨损伤、外伤性破裂或脱落、软骨发育不全以及强直性脊柱炎。
本发明优势在于：
软骨下骨细胞是软骨缺损创面的一部分，就地取材，来源充足，是原位再生修复的天然种子细胞，而且同谱系逆向诱导有助于诱导效率和转化可靠性的提高。
软骨下骨细胞与骨板形成天然的细胞支架复合体，有利于大面积缺损的修复，降低对人工支架的依赖。
附图说明
图1是转染Sox9、Sox5与Plagl1因子组合后，免疫荧光检测软骨细胞特异性标志II、IX和XI型胶原蛋白及蛋白聚糖的表达。
图2是转染Sox9、Sox5、Sox8与Zcchc5因子组合后，免疫荧光检测软骨细胞特异性标志II、IX和XI型胶原蛋白及蛋白聚糖的表达。
图3是转染Sox9、Sox6与Zcchc5因子组合后，免疫荧光检测软骨细胞特异性标志II、IX和XI型胶原蛋白及蛋白聚糖的表达。
具体实施方式
以下结合附图对本发明的实施例分别进行阐述，下面描述的所有特定组合物、物质和方法，其全部或部分地在本发明的范围之内。这些特定的组合物、物质和方法并非意在限制发明，而仅仅是举例说明在本发明范围之内的特定实施方式。可以理解，对本发明描述的方案可以进行多种改变，但仍然在本发明的范围之内。
实施例1
一、实验材料
小鼠膝关节软骨下骨成骨细胞、293T细胞
慢病毒包装质粒psPAX2,包膜质粒pMD2.G和穿梭质粒pSIN4-CMV
二、实验方法
1.a.构建携带重编程因子基因片段的慢病毒载体
将从cDNA中扩增出的Sox9、Sox5和Plagl1重编程因子基因构建至pSIN4慢病毒载体骨架上，将所得质粒纯化，通过第二代慢病毒包装系统获得滴度在1–3×108TU/ml的慢病毒颗粒。
表1引物序列
1.b.基于小鼠膝关节软骨下骨成骨细胞的重编程因子诱导
软骨下骨成骨细胞以5000/cm2接种24小时后，重编程因子慢病毒以Sox9、Sox5、Sox6、Sox8、Zcchc5或Plagl1单因子及Sox9、Sox5和Plagl1组合转导的方式在感染复数等于100的条件下加入至含有8μg/ml聚凝胺的培养液中，孵育16h后去除含病毒的培养基，DPBS清洗2次，更换为软骨细胞完全培养基。
1.c.免疫荧光法检测软骨特异性蛋白表达
在诱导14天后，将所获得的诱导细胞用4％多聚甲醛固定15min,DPBS清洗5min,重复2次。0.2％TritonX-100通透细胞膜10min,DPBS清洗3min,重复3次。3％BSA室温封闭1h,加入一抗COL2A1(1:500)、ACAN(1:750)、COL9A1(1:1000)或COL11A1(1:1000),4℃过夜孵育。吸除一抗，DPBS清洗3min,重复4次，加入AF568标记的山羊抗小鼠IgG2b二抗(1:2000)和AF647标记的驴抗兔二抗(1:750)，室温孵育1h，加入DAPI(1:1000)继续孵育5min,DPBS清洗5min,重复4次。高内涵成像系统观察、记录细胞荧光表达。
三、实验结果
免疫荧光染色结果及荧光表达强度统计结果如图1和表2所示。未经转录因子组合诱导的成骨细胞、空载体对照及Sox9、Sox5、Sox6、Sox8、Zcchc5或Plagl1单因子转导组无II、IX和XI型胶原蛋白及蛋白聚糖的表达；Sox9、Sox5和Plagl1组合诱导细胞表达软骨细胞标志性功能蛋白II、IX和XI型胶原蛋白及蛋白聚糖,且形态由梭形锯齿状转化为多边形铺路石状。
上述结果表明，Sox9、Sox5、Sox6、Sox8、Zcchc5或Plagl1单因子转导不足以诱导软骨下骨细胞向软骨细胞转化，Sox9、Sox5和Plagl1因子组合才能够有效重编程软骨下骨细胞，使其直接转化为表达构建软骨组织所需关键功能蛋白的软骨样细胞。
表2诱导后细胞蛋白表达平均荧光强度
实施例2
一、实验材料
小鼠膝关节软骨下骨成骨细胞、293T细胞
慢病毒包装质粒psPAX2,包膜质粒pMD2.G和穿梭质粒pSIN4-CMV
二、实验方法
2.a.构建携带重编程因子基因片段的慢病毒载体
将从cDNA中扩增出的Sox9、Sox5、Sox8和Zcchc5重编程因子基因构建至pSIN4慢病毒载体骨架上，将所得质粒纯化，通过第二代慢病毒包装系统获得滴度在1–3×108TU/ml的慢病毒颗粒。
表3引物序列
2.b.基于小鼠膝关节软骨下骨成骨细胞的重编程因子诱导
软骨下骨成骨细胞以5000/cm2接种24小时后，重编程因子慢病毒以Sox9、Sox5、Sox8和Zcchc5组合在感染复数等于100的条件下加入至含有8μg/ml聚凝胺的培养液中，孵育16h后去除含病毒的培养基，DPBS清洗2次，更换为软骨细胞完全培养基。
2.c.免疫荧光法检测软骨特异性蛋白表达
在诱导14天后，将所获得的诱导细胞用4％多聚甲醛固定15min,DPBS清洗5min,重复2次。0.2％TritonX-100通透细胞膜10min,DPBS清洗3min,重复3次。3％BSA室温封闭1h,加入一抗COL2A1(1:500)、ACAN(1:750)、COL9A1(1:1000)或COL11A1(1:1000),4℃过夜孵育。吸除一抗，DPBS清洗3min,重复4次，加入AF568标记的山羊抗小鼠IgG2b二抗(1:2000)和AF647标记的驴抗兔二抗(1:750)，室温孵育1h，加入DAPI(1:1000)继续孵育5min,DPBS清洗5min,重复4次。高内涵成像系统观察、记录细胞荧光表达。
三、实验结果
免疫荧光染色结果及荧光表达强度统计结果如图2和表4所示。未经转录因子组合诱导的成骨细胞、空载体对照组无II、IX和XI型胶原蛋白及蛋白聚糖的表达；Sox9、Sox5、Sox8和Zcchc5组合诱导细胞表达软骨细胞标志性功能蛋白II、IX和XI型胶原蛋白及蛋白聚糖,且形态由梭形锯齿状转化为多边形铺路石状。
上述结果表明，Sox9、Sox5、Sox8和Zcchc5因子组合具有有效重编程软骨下骨细胞，使其直接转化为表达构建软骨组织所需关键功能蛋白的软骨样细胞的作用。
表4诱导后细胞蛋白表达平均荧光强度
实施例3
一、实验材料
小鼠膝关节软骨下骨成骨细胞、293T细胞
慢病毒包装质粒psPAX2,包膜质粒pMD2.G和穿梭质粒pSIN4-CMV
二、实验方法
3.a.构建携带重编程因子基因片段的慢病毒载体
将从cDNA中扩增出的Sox9、Sox6和Zcchc5重编程因子基因构建至pSIN4慢病毒载体骨架上，将所得质粒纯化，通过第二代慢病毒包装系统获得滴度在1–3×108TU/ml的慢病毒颗粒。
表5引物序列
3.b.基于小鼠膝关节软骨下骨成骨细胞的重编程因子诱导
软骨下骨成骨细胞以5000/cm2接种24小时后，重编程因子慢病毒以Sox9、Sox6和Zcchc5组合在感染复数等于100的条件下加入至含有8μg/ml聚凝胺的培养液中，孵育16h后去除含病毒的培养基，DPBS清洗2次，更换为软骨细胞完全培养基。
3.c.免疫荧光法检测软骨特异性蛋白表达
在诱导14天后，将所获得的诱导细胞用4％多聚甲醛固定15min,DPBS清洗5min,重复2次。0.2％TritonX-100通透细胞膜10min,DPBS清洗3min,重复3次。3％BSA室温封闭1h,加入一抗COL2A1(1:500)、ACAN(1:750)、COL9A1(1:1000)或COL11A1(1:1000),4℃过夜孵育。吸除一抗，DPBS清洗3min,重复4次，加入AF568标记的山羊抗小鼠IgG2b二抗(1:2000)和AF647标记的驴抗兔二抗(1:750)，室温孵育1h，加入DAPI(1:1000)继续孵育5min,DPBS清洗5min,重复4次。高内涵成像系统观察、记录细胞荧光表达。
三、实验结果
免疫荧光染色及荧光表达强度统计结果如图3和表6所示。未经转录因子组合诱导的成骨细胞、空载体对照组无II、IX和XI型胶原蛋白及蛋白聚糖的表达；Sox9、Sox6和Zcchc5组合诱导细胞表达软骨细胞标志性功能蛋白II、IX和XI型胶原蛋白及蛋白聚糖,且形态由梭形锯齿状转化为多边形铺路石状。
上述结果表明，Sox9、Sox6和Zcchc5能够重编程软骨下骨细胞，使其直接转化为表达构建软骨组织所需关键功能蛋白的软骨样细胞。
表6重编程后细胞蛋白表达平均荧光强度
Claims (7)
1.一种由骨组织细胞向关节软骨细胞定向转化的重编程诱导方法，其特征在于，来源于软骨下骨板的特定骨组织细胞发生细胞直接重编程，失去原有特定类型细胞标志性功能与表型特征，同时定向转变成为另一特定类型并具有关节软骨细胞标志性功能与特征的关节软骨样细胞；
所述软骨下骨板的特定骨组织细胞发生细胞直接重编程，需将重编程因子组合物转入软骨下骨板的骨组织细胞内，作用于细胞内特定靶基因启动子区域，诱导骨组织细胞向关节软骨样细胞的转化；所述重编程因子组合物选自Sox9、Sox5与Plagl1，或Sox9、Sox5、Sox8与Zcchc5，或Sox9、Sox6与Zcchc5。
2.根据权利要求1所述的重编程诱导方法，其特征在于，重编程因子组合物在骨组织细胞内表达，诱导细胞内源基因及其产物的表达，形成稳定的、确定的、属于关节软骨细胞但应不足以维持原有骨组织细胞或诱导及促进第三类细胞的标志性功能与表型特征。
3.根据权利要求2所述重编程诱导方法，包括在体内或体外将目的蛋白编码基因接触导入体细胞的步骤。
4.根据权利要求3所述重编程诱导方法，包括如下步骤：将所述重编程因子组合物的蛋白编码基因插入表达载体，得到重组载体。
5.根据权利要求3所述重编程诱导方法，其特征在于，所述重编程因子组合物发挥诱导时间为1–8周。
6.一种由骨组织细胞向关节软骨细胞定向转化的重编程诱导用组合物，其特征在于，所述组合物为Sox9、Sox5与Plagl1，或Sox9、Sox5、Sox8与Zcchc5，或Sox9、Sox6与Zcchc5，诱导及促进软骨下骨细胞定向转化成为软骨样细胞。
7.权利要求6所述的重编程诱导用组合物在制备治疗重度软骨缺损药物中的应用。
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