CN108384752A - 成软骨培养基及其在微分裂技术中膝关节软骨分化中的应用 - Google Patents
成软骨培养基及其在微分裂技术中膝关节软骨分化中的应用 Download PDFInfo
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Abstract
本发明提供一种成软骨培养基及其在微分裂技术中膝关节软骨分化中的应用，包括脂肪干细胞成软骨诱导分化基础培养基、地塞米松、抗坏血酸、ITS、丙酮酸钠、脯氨酸、TGF‑β3。其经过低氧处理，低氧预处理组的膝关节损伤修复情况相对非低氧组更好，暗示低氧处理对脂肪干细胞分化而来的软骨细胞更具有软骨修复能力。
Description
技术领域
本发明涉及生物细胞学领域，尤其涉及成软骨培养基及其在微分裂技术中膝关节软骨分化中的应用。
背景技术
膝关节软骨损伤临床上非常多见，常伴随有疼痛、肿胀、影响正常的活动并导致骨关节炎。由于软骨组织没有血液供应，营养来源于周围关节滑液的滋润，损伤后很难再生，因此，最终的治疗目标是通过再生的缺陷的透明软骨来恢复正常的膝关节功能。在此目的下，人们开创了各种方法用于软骨损伤的外科治疗，包括关节镜下微骨折手术(MFX)、膝关节修复技术、自体骨软移植、镶嵌式成形术和冷冻异体骨软移植，将关节透明软骨移植到受损部位。其中MFX因其微创性和操作简单而成为一种最受欢迎的治疗手段。然而，修复组织绝大多数是纤维软骨，而不是透明软骨，并且该技术是基于多能干细胞分化技术，对老年患者来说效果比较差。尽管有研究已经证实早期和中期治疗结果良好，但由于再生纤维软骨的生物力学缺陷和疤痕的产生，时间久了会造成病情恶化。因此寻找具有优良分化潜能和安全性的干细胞种子资源至关重要。其中脂肪来源的间充质干细胞细胞(ADSCs)似乎成了人们选择的理想种子材料，原因如下：容易获得和分离，且对供体损伤小(抽脂分离即可)；自体移植无排异反应；体外增殖需要的环境条件相对简单；在传代过程中仍人能维持这其软骨分化的潜能。近年来，利用ADSCs治疗膝关节软骨损伤已越来越受到专家们的关注，且大多数专家认为利用ADSCs进行治疗可促进伤口愈合和关节软骨再生。如图1所示，传统上人们在膝关节软骨损伤的干细胞治疗方面是利用分离的自体干细胞体外诱导成软骨细胞后再行定点注射，但受体内环境影响，治疗效果往往不佳。
究其原因，在一个缺血缺氧的环境中，移植的细胞存活受到影响。为了增加的细胞耐受这种缺氧环境的能力。
发明内容
为了克服上述现有技术的不足，本发明一方面提供一种成软骨培养基，其特征在于，包括以下成分，脂肪干细胞成软骨诱导分化基础培养基、地塞米松、抗坏血酸、ITS、丙酮酸钠、脯氨酸、TGF-β3。
本发明第二方面提供了一种上述成软骨培养基在微分裂技术中膝关节软骨分化中的应用，采用所述的成软骨培养基，在正常条件下诱导21天，每三天换一次液，并每12小时进行两次低氧胁迫诱导，每次胁迫诱导半小时。
较佳地，所述的低氧成分为1％ O2，5％ CO2，94％ N2。
与现有技术相比，本发明的有益效果如下：
本发明发现无论低氧处理还是正常培养下，经过21天都能促进脂肪干细胞成软骨分化(Safranin O染色)，暗示低氧处理对脂肪干细胞分化无影响。经治疗后，取膝关节软骨组织进行masson染色发现，低氧预处理组的膝关节损伤修复情况相对非低氧组更好，暗示低氧处理对脂肪干细胞分化而来的软骨细胞更具有软骨修复能力。
附图说明：
图1为现有技术中的微分裂技术定点注射图。
图2为本发明实施例中脂肪干细胞分离及鉴定。
图3为本发明实施例中成软骨分化鉴定图。
图4为本发明实施例中masson染色检测膝关节软骨受损恢复情况图。
具体实施方式
下面结合具体的实施例对本发明作进一步地说明，以更好地理解本发明。1、脂肪干细胞分离：
首先取新西兰大白兔腹股沟处的脂肪组织，放入含有青链霉素的杜氏磷酸盐缓冲液(DPBS)中洗净血污。转入一个新的培养皿中，充分剪碎，加入消化液(10％胎牛血清，0.09％胶原酶I，其余为DMEM/F12)，移液器充分打匀，直到组织可以通畅的出入吸嘴，将脂肪组织转入离心管中，37℃消化1h，定时摇晃，使组织和消化液充分混匀。消化完后，反复吹打消化液，分散组织块，制成细胞悬液。1200rpm离心5min，将上层的脂肪组织反复吹打最少1min，再次离心。弃掉上层成熟的脂肪组织和中层的消化液，再用基础培养基重悬底层的细胞。先将细胞悬液用250μM的尼龙滤网过滤细胞悬液，再将所得滤液用孔径为80μM的尼龙过滤，除去大部分的成熟脂肪细胞，收集滤液，每次过滤后注意再用适量的基础培养基洗涤滤网，减少滤网上的细胞残留。将最后得到的滤液离心，吸弃漂浮的脂肪细胞，用基础培养基反复洗涤3遍。细胞培养，最后一次用完全培养基重悬，接种，置于培养箱中常规培养。首次换液，弃去培养基，PBS反复洗涤5-6次，加入新鲜的完全培养基继续培养。重复3次换液直到细胞面基本干净。消化并扩大培养鉴定。
利用常规免疫荧光方法及流式细胞仪技术检测ADSCs相对特异的分子CD29，CD44，CD90，CD105等。流式细胞仪技术检测细胞周期，计算处于G0/G1期的细胞比例。通过不同诱导液对体外培养的ADSCs进行诱导分化，使ADSCs向软骨细胞、脂肪细胞及内皮细胞方向分化，采用茜索红、ALP、油红、番红-O-染色及CD31等染色方法。
2、成软骨分化：利用软骨分化诱导培养基(配方表1)，正常条件下(5％CO2，37℃)诱导21天，每3天换一次液。
表1 成软骨培养基配方：
3、低氧预处理：即每12h进行两次低氧(1％O2，5％CO2，94％N2)胁迫诱导30min，直到注射前一天为止。
4、番红-O-染色(Safranin O)
样品经4％多聚甲醛溶液固定后，20％蔗糖脱水4-6h，30％蔗糖溶液脱水过夜，入O.T.C包埋剂浸泡2h，-20℃过夜，室温复温2h后再次入-20℃过夜，冰冻切片机切片8μm，37℃干燥过夜。0.02％ fast green染色液染色4min，自来水漂洗，1％乙酸分色数秒钟，加入0.1％番红-O-染色液染色3min，自来水漂洗，干燥后中性树胶封片，移至显微镜下观察
5、masson染色：4周后杀死兔子，取膝关节软骨组织进行组化检测。
步骤：脱水后石蜡包埋，每例连续切片6张，组织厚度4μm，切片置入70℃恒温箱中烘烤15min，脱蜡，PBS冲洗3遍，每遍2min。标本放于抗原修复盒中，浸于0.01mol枸椽酸溶液中，放入微波炉中加热15min，冷却至室温。PBS冲洗3遍，每遍2min。除去PBS液，切片加山羊血清封闭，室温避光孵育20min。除去多余血清，不洗，切片滴加50μl一抗。除去PBS液，切片滴加50μl二抗，置入37℃恒温箱中40min，PBS震荡洗涤4遍，每遍5min，masson3染色30min，负染，苏木精病理染色液染色40min，脱水透明处理，中性树胶封片，室温晾干，显微镜观察拍片。
6、兔膝关节软骨损伤模型及微分裂技术治疗：取新西兰大白兔6只，6g/L戊巴比妥耳缘经脉注射麻醉，通过在实验兔后退关节的股髁关节面部位用电动骨钻，在控制其直径4.5mm，深度3m，造成其膝关节软骨缺损，2周后进行微分裂技术，如图2所示，注射正常分裂的软骨细胞和经低氧预处理的软骨细胞1×106，4周后杀死兔子，取膝关节软骨组织进行massoon染色鉴定修复情况。
结果
1、脂肪干细胞的分离和鉴定：对ADSCs进行分离和鉴定结果如图2所示，对表面标记物进行免疫荧光检测发现ADSCs特异性标记蛋白CD29，CD44，CD90和CD105均有表达，而内皮细胞表面标记物vWF和CD34无表达，说明脂肪干细胞分离成功。
2、如图3所示，研究发现，研究发现无论低氧处理还是正常培养下，经过21天都能促进脂肪干细胞成软骨分化(Safranin O染色)，暗示低氧处理对脂肪干细胞分化无影响。
3、如图4所示，经4周治疗后，取膝关节软骨组织进行masson染色发现，低氧预处理组的膝关节损伤修复情况相对非低氧组更好，暗示低氧处理对脂肪干细胞分化而来的软骨细胞更具有软骨修复能力。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
Claims (3)
1.一种成软骨培养基，其特征在于，包括以下成分，脂肪干细胞成软骨诱导分化基础培养基、地塞米松、抗坏血酸、ITS、丙酮酸钠、脯氨酸、TGF-β3。
2.一种权利要求1所述的成软骨培养基在微分裂技术中膝关节软骨分化中的应用，其特征在于，采用所述的成软骨培养基，在正常条件下诱导21天，每三天换一次液，并每12小时进行两次低氧胁迫诱导，每次胁迫诱导半小时。
3.根据权利要求2所述的成软骨培养基在微分裂技术中膝关节软骨分化中的应用，其特征在于，所述的低氧成分为1％O2，5％CO2，94％N2。
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