CN110438069B - 一种连翘脂素用于体外促进人脂肪间充质干细胞成软骨分化的用途 - Google Patents
一种连翘脂素用于体外促进人脂肪间充质干细胞成软骨分化的用途 Download PDFInfo
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Abstract
本发明公开了一种连翘脂素用于体外促进人脂肪间充质干细胞成软骨分化的用途。糖胺聚糖和Ⅱ型胶原常用作判断间充质干细胞向软骨细胞分化的特征性标志，甲苯胺蓝染色用于检测糖胺聚糖，RT‑PCR测定Ⅱ型胶原的表达水平。本发明实施例说明，hADSCs经连翘脂素诱导后，糖胺聚糖的含量和Ⅱ型胶原表达水平显著升高，说明连翘脂素可以促进hADSCs成软骨分化，且不会影响其增殖，可以将连翘脂素添加到L‑DMEM培养基制成人脂肪间充质干细胞成软骨分化培养基，为再生医学领域提供软骨细胞。
Description
技术领域
本发明涉及干细胞诱导分化，具体涉及一种连翘脂素用于体外促进人脂肪间充质干细胞成软骨分化的用途。
背景技术
膝关节骨性关节炎为骨科的常见病和多发病，发病原因有高龄、肥胖、外伤、遗传等。多种细胞因子代谢紊乱，蛋白聚糖及Ⅰ、Ⅱ型胶原降解，透明质酸减少等原因共同导致关节内软骨细胞代谢紊乱，最终导致骨性关节炎的发生。目前尚缺乏有效的途径来对抗软骨细胞外基质的丢失和软骨细胞的凋亡。
间充质干细胞是一类多向分化的干细胞，具有分化为软骨细胞并治疗软骨病损的潜力。脂肪间充质干细胞(ADSCs)具有来源广、容易获得、低免疫原性等特点，已成为组织工程种子细胞的来源之一，是干细胞移植和再生医学领域重要的种子细胞。
文献公开了瑞香素联合IGF-1对大鼠脂肪间充质干细胞成软骨分化的影响，发现瑞香素联合IGF-1转染可以有效促进脂肪间充质干细胞成软骨分化(瑞香素联合IGF-1对大鼠脂肪间充质干细胞成软骨分化的影响，中国修复重建外科杂志，2019年06期)。
天然产物具有多种药理活性，为生物医药领域寻找活性化合物提供了宝贵的化合物库。连翘脂素是分离自连翘中的一种天然产物，具有多种药理活性，目前没有文献公开其具有体外促进人脂肪间充质干细胞成软骨分化的活性。
发明内容
本发明是为了促进人脂肪间充质干细胞成软骨分化，特提供一种连翘脂素用于体外促进人脂肪间充质干细胞成软骨分化的用途。
技术方案如下：
一种连翘脂素用于促进脂肪间充质干细胞成软骨分化的用途。
进一步地，所述脂肪间充质干细胞为人脂肪间充质干细胞。
进一步地，用于体外促进脂肪间充质干细胞成软骨分化。
一种连翘脂素用于制备体外促进人脂肪间充质干细胞成软骨分化的培养基的用途。
进一步地，所述培养基包括连翘脂素和L-DMEM培养基。
技术效果：
本发明发现：连翘脂素在体外可以有效促进人脂肪间充质干细胞成软骨分化，连翘脂素可以用于制备体外促进人脂肪间充质干细胞成软骨分化的培养基。
附图说明
图1为hADSCs的细胞形态观察结果；
图2为hADSCs的细胞表型鉴定结果；
图3为不同处理hADSCs的增殖曲线；
图4为甲苯胺蓝染色结果；
图5为琼脂糖凝胶电泳结果。
具体实施方式
一、实验材料
双抗溶液和PBS溶液自制，4℃保存。Ⅰ型胶原酶购自泽叶生物。连翘脂素购自源叶生物。
胎牛血清、高糖DMEM培养基、低糖DMEM培养基购自GIBCO。
荧光标记单克隆抗体购自美国Bioscience公司。
脂肪组织取自身体健康的腹部脂肪抽脂术者，签署知情同意书，仅用于科研用途。抽取后于4℃保存，12h内参照文献方法进行脂肪间充质干细胞培养和传代。
甲苯胺蓝染料试剂盒购于Solarbio公司。
二、实验方法
1、hADSCs的培养、传代、形态观察和表型鉴定
取脂肪组织用含有双抗的PBS溶液冲洗3次，用0.25％Ⅰ型胶原酶于37℃、搅拌条件下消化30min，加入等体积含10％胎牛血清的高糖DMEM培养基终止消化，200目滤网过滤，再用1200×g离心5min，沉淀用适量含10％胎牛血清的高糖DMEM培养基重悬，加入5倍体积红细胞裂解液，室温孵育5min，1200×g离心5min，沉淀用适量含15％胎牛血清的低糖DMEM培养基(完全培养基)重悬，计数，以1×106/皿的密度接种于细胞培养皿中，每3d换液1次，待细胞长至80％-90％时，用胰蛋白酶消化后以1:3的比例传代。
取第3代hADSCs进行细胞形态观察、表型鉴定和后续实验。
流式细胞仪进行表型鉴定：消化处于对数生长期的第3代hADSCs，制成单细胞悬液，取适量细胞悬液于流式管中，PBS洗涤2次，将荧光标记单克隆抗体CD73、CD90、CD105和CD34、CD45、HLA-DR加入到流式管中，鼠同型对照抗体作为参照，轻轻混匀，4℃避光孵育30min，流式细胞仪检测细胞表面抗原标记。
2、MTT法测定连翘脂素对hADSCs增殖的影响
消化处于对数生长期的第3代hADSCs，制成单细胞悬液，接种于24孔板中，1×104/孔，分为实验组和对照组，24h后，实验组更换为含有不同浓度连翘脂素(100nM、200nM)的完全培养基继续培养，对照组更换为新鲜完全培养基继续培养，继续培养24、48、72h后，吸弃上清，PBS洗涤3次，每孔加入20μL 5g/L的MTT溶液，继续培养4h，弃上清后加入150μL二甲基亚砜，振荡10min，酶标仪检测各孔吸光度值(λ＝490nm)。使用3块24孔板平行操作，每块24孔板上各组的不同时间点各设2个复孔。
根据吸光度值绘制hADSCs增殖曲线。
3、测定连翘脂素对hADSCs成软骨分化能力的影响
3.1甲苯胺蓝染色
消化处于对数生长期的第3代hADSCs，制成单细胞悬液，1×106/皿的密度接种于细胞培养皿中，细胞融合生长至85％左右时，给药组更换为含有100nM连翘脂素的完全培养基，对照组更换为新鲜完全培养基，诱导至第14天消化诱导的细胞，按甲苯胺蓝染色剂说明进行细胞染色。
3.2RT-PCR测定Ⅱ型胶原表达水平
消化处于对数生长期的第3代hADSCs，制成单细胞悬液，1×106/皿的密度接种于细胞培养皿中，细胞融合生长至85％左右时，给药组更换为含有100nM、200nM连翘脂素的完全培养基，对照组更换为新鲜完全培养基，诱导至第14天消化诱导的细胞，洗涤收集细胞，采用Trizol法抽提取细胞总RNA，于紫外分光光度计上检测总RNA在260或280nm波长处的光吸收值(OD值)。通过一步法将RNA逆转录为cDNA，最后进行琼脂糖凝胶电泳。
Ⅱ型胶原上游引物：TTTCCCAGGTCAAGATGGTC
Ⅱ型胶原下游引物：TCACCTGGTTTTCCACCTTC
GAPDH上游引物：ACCACAGTCCATGCCATCAC
GAPDH下游引物：TCCACCACCCTGTTGCTGTA
4、统计学分析
结果以均值±标准差表示，应用SPSS 17.0进行数据分析进行统计学比较。
三、实验结果
1、hADSCs形态观察和表型鉴定结果
倒置显微镜下观察显示细胞形态呈长梭形，呈漩涡状生长，排列有序，符合hADSCs的细胞形态学特点，如图1所示。
流式细胞仪鉴定结果显示，细胞阳性表达CD73、CD90和CD105，阴性表达CD34、CD45和HLA-DR，符合hADSCs的表型特点，流式图如图2所示。表达率如表1所示。
表1第3代hADSCs的细胞表型测定结果
| 单克隆抗体 | 表达率 |
| CD73 | 99.8％ |
| CD90 | 97.1％ |
| CD105 | 99.5％ |
| CD34 | 0.72％ |
| CD45 | 0.69％ |
| HLA-DR | 0.61％ |
2、连翘脂素对hADSCs增殖的影响
不同组hADSCs增殖曲线如图3所示，与对照组相比，hADSCs经100nM、200nM连翘脂素干预培养24、48、72h后增殖率无显著差异，说明连翘脂素基本不影响hADSCs增殖。
490nm吸光度值如表2所示。
表2 490nm吸光度值
| 组别 | 24h | 48h | 72h |
| 对照组 | 0.17±0.05 | 0.28±0.05 | 0.47±0.06 |
| 实验组(100nM) | 0.20±0.05 | 0.26±0.04 | 0.51±0.07 |
| 实验组(200nM) | 0.19±0.04 | 0.29±0.06 | 0.52±0.06 |
3、连翘脂素对hADSCs成软骨分化能力的影响
甲苯胺蓝染色结果如图4所示，对照组未见明显染色，给药组可见明显的染色，且可见细胞聚集。图4中，A为对照组，B为给药组。
RT-PCR测定结果如图5所示，对照组未见明显的Ⅱ型胶原表达，给药组可见明显的Ⅱ型胶原表达。图5中，A为对照组，B为给药组(100nM)，C为给药组(200nM)。
糖胺聚糖和Ⅱ型胶原常用作判断间充质干细胞向软骨细胞分化的特征性标志，甲苯胺蓝染色用于检测糖胺聚糖，RT-PCR测定Ⅱ型胶原的表达水平。上述实施例说明，hADSCs经连翘脂素诱导后，糖胺聚糖的含量和Ⅱ型胶原表达水平显著升高，说明连翘脂素可以促进hADSCs成软骨分化，且不会影响其增殖，可以将连翘脂素添加到L-DMEM培养基制成人脂肪间充质干细胞成软骨分化培养基，为再生医学领域提供软骨细胞。
Claims (1)
1.一种连翘脂素用于体外促进脂肪间充质干细胞成软骨分化的用途，所述脂肪间充质干细胞为人脂肪间充质干细胞。
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