CN113018317A - 间充质干细胞联合玻璃酸钠在治疗关节炎中的应用 - Google Patents
间充质干细胞联合玻璃酸钠在治疗关节炎中的应用 Download PDFInfo
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Abstract
本发明属于医学领域,具体涉及一种间充质干细胞联合玻璃酸钠在治疗关节炎中的应用。首先公开了间充质干细胞和玻璃酸钠的组合物在制备治疗类风湿性关节炎的药物中的应用。还公开了一种构建类风湿性关节炎模型的方法,包括:S1:制备胶原乳剂;S2:在动物皮内注射胶原乳剂;7‑8天后用减半剂量的胶原乳剂在相同部位加强免疫一次;S3:在动物身上注射间充质干细胞和玻璃酸钠的组合物,进行类风湿性关节炎治疗;S4、模型的评估。本发明首次发现了脐带间充质干细胞和玻璃酸钠混合液可用于治疗类风湿性关节炎,具有潜在的临床应用价值,给类风湿性关节炎患者带来了福音。
Description
技术领域
本发明属于医学领域,具体涉及一种间充质干细胞联合玻璃酸钠在治疗关节炎中的应用。
技术背景
类风湿关节炎(rheumatoid arthritis,RA)是一种自身免疫性疾病,表现为对称小关节的慢性关节滑膜炎症,而且以炎症的持续反复发作为主要特征。其病程主要表现为关节滑膜增生和血管翳形成,继而造成关节软骨的破坏,最终导致骨、关节坏死从而致残。
我国RA的患病率为0.32%~0.36%,略低于0.5%~1%的世界平均水平,是造成人类劳动力丧失和致残的主要因素之一。该疾病确切病因及发病机制尚未完全明确,目前尚无有效的治疗方法。有科学家发现,间充质干细胞对治疗类风湿性关节炎具有一定的临床疗效,而玻璃酸钠对受损的软骨细胞具有一定的保护和修复作用。然而两者的联合使用能否用于类风湿性关节炎的治疗还未见报道。
发明内容
本研究联合脐带间充质干细胞和玻璃酸钠治疗类风湿性关节炎,在大鼠模型中获得了较理想的治疗效果。
具体的,本发明的技术方案如下:
本发明第一个方面公开了一种间充质干细胞和玻璃酸钠的组合物在制备治疗类风湿性关节炎的药物中的应用。
优选的,所述间充质干细胞为脐带间充质干细胞。
本发明第二个方面公开了一种构建类风湿性关节炎模型的方法,包括:
S1:将牛Ⅱ型胶原溶液与IFA按照1:1的比例混匀后充分乳化,得到胶原乳剂;
S2:在动物皮内注射胶原乳剂;7-8天后用减半剂量的胶原乳剂在相同部位加强免疫一次;
S3:在动物身上注射间充质干细胞和玻璃酸钠的组合物,进行类风湿性关节炎治疗;
S4、模型的评估:如果检测到S3建立的模型中关节腔炎症细胞浸润较少,关节腔较规整,周围结缔组织中炎症细胞较少,则该模型建立成功。
优选的,在步骤S3中,在选自动物的裸关节、膝关节、裸关节或脚趾中至少一处注射间充质干细胞和玻璃酸钠的组合物。
本发明第三个方面公开了上述的方法制备得到的类风湿性关节炎模型。
本发明第四个方面公开了一种用于治疗类风湿性关节炎的药物组合物,所述药物组合物包括:有效量的间充质干细胞和玻璃酸钠。
优选的,还包括药学上可接受的载体。
优选的,所述药物组合物为静脉注射试剂,和/或关节腔内注射试剂。
优选的,所述间充质干细胞为脐带间充质干细胞。
本发明第五个方面公开了一种治疗类风湿性关节炎的方法,包括:采用脐带间充质干细胞联合玻璃酸钠治疗的方法,在患者的风湿性关节炎部位,注射脐带间充质干细胞和玻璃酸钠混合液。
与现有技术相比,本发明至少具有以下区别技术特征:
本发明首次发现了脐带间充质干细胞和玻璃酸钠混合液可用于治疗类风湿性关节炎,具有潜在的临床应用价值,给类风湿性关节炎患者带来了福音。
附图说明
图1为本发明实施例中脐带间充质干细胞的流式检测示意图一;
图2为本发明实施例中脐带间充质干细胞的流式检测示意图二;
图3为本发明实施例中成软骨细胞分化染色示意图;
图4为本发明实施例中成骨细胞分化示意图;
图5为本发明实施例中向脂肪细胞分化染色示意图;
图6为本发明实施例中大鼠体内造模成功示意图;
图7为本发明实施例中不同组别疗效分析示意图。
具体实施方式
下面通过详细的实施例对本申请进行进一步的阐述,应该理解,下述实施例仅是为了用于说明本申请,并不对发明内容进行限定。
实施例中所用仪器、设备、试剂均可通过各种渠道获得,例如购买得到,或可以制备而得。
实施例1
本实施例公开了一种脐带间充质干细胞的原代培养方法,包括:
(1)将脐带浸泡于含2%SP的DPBS中,在冰上运输。
(2)将脐带剪成2cm左右的小段,用含2%SP的DPBS洗至无血液存在。
(3)剔除脐带中的血管。
(4)将剔除血管后的脐带用眼科剪剪碎成大约1mm3细小组织块;加入同等体积0.1%或0.2%(1mg/mL或2mg/mL)胶原酶Ⅰ消化液,转移至15mL或50mL离心管中,再加入透明质酸酶至终浓度为0.2mg/mL,37℃恒温消化0.5~1h。
(5)加入与酶液等体积的间充质干细胞培养液,终止消化。
(6)尽量将组织吹散,过400目细胞筛,室温1000r/min离心10min。
(7)用间充质干细胞培养液重悬细胞,按每3.5cm培养皿5×105个细胞的密度接种标记为原代细胞(P0)。
(8)置于37℃、5%CO2、饱和湿度的培养箱中静置培养。
(9)24h后更换新鲜的间充质干细胞培养液,去除未贴壁细胞,以后每2~3天更换1次培养液。
(10)待细胞长至80%~90%融合时传代,得到脐带间充质干细胞
实施例2
本实施例将实施例1得到的脐带间充质干细胞进行表型鉴定,包括以下步骤:
(1)37℃预热MSCs培养液和0.05%Trypsin。
(2)弃掉旧培养液,加入室温放置的DPBs洗一遍,尽可能将残留的培养液洗净,吸去DPBS。
(3)加入适量(盖住皿底即可)0.05%Trypsin,37℃培养箱内孵育消化2~3min。
(4)显微镜下观察细胞变圆、漂起时,可加等体积MSCs培养液(为了节约成本,可以
用细胞基础培养液代替)终止消化。
(5)轻柔吹打细胞,收集细胞悬液到15mL离心管中,室温1000r/min离心3min,弃上清。
(6)加入2mL DPBS后将细胞重悬,室温1000r/min离心3min,弃上清。
(7)再加入2mL DPBS后将细胞重悬(DPBS的体积根据后面需要鉴定的管数和每管的体积决定),过40μm细胞筛。
(8)根据需要鉴定的CD分子数,将细胞均匀分到数个管中,要包括空白细胞对照管(不加抗体)、PE同型对照管和/或FITC同型对照管。一般1个100mm培养皿细胞长满可鉴定10个CD分子(根据不同的流式细胞仪和抗体决定)。
(9)向不同管中加入适当体积的抗体(不同抗体的稀释比例不同),总体积为200μL即可(为节约使用抗体,体积可适当减小)。
(10)室温避光孵育30min。
(11)1200r/min室温离心3min。
(12)加2mL DPBS重悬,1200r/min室温离心3min。
(13)加DPBS重悬(不同的流式细胞仪要求的最小体积不同),一般500μL即可,用流式细胞仪分析。
(14)实验结果
经过流式细胞术鉴定,MSCs的CD分子表达情况如下图1和图2所示。CD44与CD105阳性率大于95%;CD73阳性率大于95%;CD90阳性率大于95%;CD45阴性,阳性率不高于5%。
实施例3
本实施例鉴定实施例1制备得到的脐带间充质干细胞的分化能力,包括以下步骤:
一、软骨细胞分化:
1.分化培养液:
无血清高糖DMEM、10μg/L TGFβ1、10-7M Dex、50mg/L Vit C、100mg/L丙酮酸钠及50g/L ITS+Premix)
2.鉴定:
1)甲苯胺蓝母液:甲苯胺蓝Toluidine Blue O(SIGMA,T3260-5g)1.0g,70%酒精alcohol 100.0ml混合溶解,保存期6个月。工作液:甲苯胺蓝Toluidine blue,Stock5.0ml,1%氯化钠Sodium chloride 45.0ml新鲜配制,用后丢弃。
2)步骤:
a)脱蜡至蒸馏水。
b)甲苯胺蓝工作液1-2分钟。
c)蒸馏水洗涤三次。
d)快速95%和无水乙醇脱水。
e)二甲苯透明和封固。
3)结果:细胞紫红色,背景蓝色。如图3所示
二、成骨细胞分化:
1.分化培养液:
DMEM supplemented with 10%FBS,10mMβ-glycerol phosphate(Sigma,50020-100G,1241RMB),50μM ascorbate-2-phosphate(Sigma,)and 100ng/ml recombinanthuman bone morphogenic protein-2(Pepro Tech Inc.,Rocky Hill,NJ),10-7Mdexamethasone(BMSC)
2.鉴定:茜素红染色法(sigma,A5533-25G)
1)作用原理利用沉积的钙于茜素红发生显色反应,得到红色的染色效果。先配制好0.05M的Tris-HCl,调pH至4.2,4度保存.再在80ml的上述溶液中,加入100mg茜素红,使之充分溶解,然后定容到100ml。这就是0.1%的茜素红,现配现用。
2)步骤
a)培养皿用PBS冲洗2次,95%乙醇固定10min,双蒸水冲洗3次。
b)0.1%茜素红-Tris-Hcl(PH 4.2)37℃,30min。
c)蒸馏水冲洗,干燥,封片。
3)结果:染料品种的不同,矿化结节呈现不同的红色,如图4所示。
三、脂肪细胞分化:
1.分化培养液:
DMEM supplemented with 10%FBS,10-7M dexamethasone(Sigma),10μg/mLinsulin(Invitrogen),0.1mM 3-isobutyl-1-methyl-xanthine(Sigma),100μMindomethacin(Sigma)
2.鉴定:oil-red-o染色
1)染色:oil-red-o(sigma,O0625-25G)
称取预先研磨粉碎的0.5g油红干粉,溶于少量异丙醇中,然后加异丙醇至100ml,棕色瓶密封(或锡箔纸包裹避光)4℃保存,为储存液,可长期保存。用时取6ml加三蒸水4ml混匀,定性滤纸过滤,稀释后数小时内用完。
2)步骤:
a)4%多聚甲醛固定细胞10分钟,PBS清洗2次。
b)油红染色30min-1h。
c)异丙醇清洗1-2次,镜下观察。
3.结果:脂肪细胞被染成红色,如图5所示。
实施例4
本实施例构建一种大鼠风湿性关节炎模型的构建,包括以下步骤:
(1)牛II型胶原(CII)(2mg/mL溶于0.05M冰醋酸中)。
(2)CⅡ和IFA(弗氏不完全佐剂)等比例体积充分混匀,充分乳化得到胶原乳剂。
(3)准备wistar大鼠,雄性,大鼠体重160~300g,在其左后足底、尾根部、背部分3点皮内分别注射胶原乳剂0.2ml(D0)。
(4)7天后用减半剂量免疫原在相同部位加强免疫一次。
(5)给药
1)模型对照组:尾静脉注射生理盐水1毫升。
2)干细胞治疗组:在踝关节腔部位注射100微升脐带间充质干细胞与玻璃酸钠的混合液(其中包括:干细胞2×106/ml,50微升,玻璃酸钠溶液10毫克/毫升,50微升;利用注射器将两者混匀);膝关节30-50微升,踝关节和脚趾50微升左右。
3)地塞米松治疗组:肌肉注射给药200微升地塞米松,给药后第3天开始按照体重进行腹腔给药1mg/kg,3d(地塞米松药物浓度为5mg/ml)。
(4)第29天,进行踝关节的HE染色,观察治疗效果。
(5)结果分析
结果如图6和7所示,相对于模型对照组,干细胞治疗组关节腔炎症细胞浸润较少,关节腔较规整,周围结缔组织中炎症细胞较少。表面干细胞治疗确实改善了大鼠关节炎症状。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种间充质干细胞和玻璃酸钠的组合物在制备治疗类风湿性关节炎的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述间充质干细胞为脐带间充质干细胞。
3.一种构建类风湿性关节炎模型的方法,其特征在于,包括:
S1:将牛Ⅱ型胶原溶液与IFA按照1:1的比例混匀后充分乳化,得到胶原乳剂;
S2:在动物皮内注射胶原乳剂;7-8天后用减半剂量的胶原乳剂在相同部位加强免疫一次;
S3:在动物身上注射间充质干细胞和玻璃酸钠的组合物,进行类风湿性关节炎治疗;
S4、模型的评估:如果检测到S3建立的模型中关节腔炎症细胞浸润较少,关节腔较规整,周围结缔组织中炎症细胞较少,则该模型建立成功。
4.根据权利要求3所述的方法,其特征在于,在步骤S3中,在选自动物的裸关节、膝关节、裸关节或脚趾中至少一处注射间充质干细胞和玻璃酸钠的组合物。
5.根据要求3-4所述的方法制备得到的类风湿性关节炎模型。
6.一种用于治疗类风湿性关节炎的药物组合物,其特征在于,所述药物组合物包括:有效量的间充质干细胞和玻璃酸钠。
7.根据权利要求6所述的药物组合物,其特征在于,还包括药学上可接受的载体。
8.根据权利要求6所述的药物组合物,其特征在于,所述药物组合物为静脉注射试剂,和/或关节腔内注射试剂。
9.根据权利要求6所述的药物组合物,其特征在于,所述间充质干细胞为脐带间充质干细胞。
10.一种治疗类风湿性关节炎的方法,其特征在于,采用脐带间充质干细胞联合玻璃酸钠治疗的方法,在患者的风湿性关节炎部位,注射脐带间充质干细胞和玻璃酸钠混合液。
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CN112261944A (zh) * | 2018-06-05 | 2021-01-22 | Medipost株式会社 | 用于治疗软骨损伤相关疾病的包含透明质酸和干细胞的药物组合物 |
CN113424799A (zh) * | 2021-06-28 | 2021-09-24 | 中国人民解放军陆军特色医学中心 | 一种基于成骨龛微环境修饰的pdx模型的构建方法及应用 |
CN113424799B (zh) * | 2021-06-28 | 2022-09-02 | 中国人民解放军陆军特色医学中心 | 一种基于成骨龛微环境修饰的pdx模型的构建方法及应用 |
CN113975468A (zh) * | 2021-11-11 | 2022-01-28 | 成都中科娥皇健康咨询有限公司 | 一种骨膜组织老化修复剂及其制备工艺 |
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