CN117503799A - 一种脐带间充质干细胞上清液的制备方法及其在眼腺体修复中的应用 - Google Patents
一种脐带间充质干细胞上清液的制备方法及其在眼腺体修复中的应用 Download PDFInfo
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Abstract
本发明采用分离制备的脐带间充质干细胞,通过对其进行无血清培养,对收集的培养基的上清中主要的细胞因子的含量进行了测定,其中在培养基中添加了KRN 7000可以明显提高培养基中细胞因子EGF、IGF‑1、KGF‑2以及IL‑10的含量。将收集的培养基上清液进一步制备成滴眼液,用于治疗干眼症模型的白兔,发现KRN 7000诱导的培养基上清制备的滴眼剂治疗的腺体中的凋亡细胞数低于未添加KRN 7000的培养基上清制备的滴眼剂,而Fas表达降低以及Bcl‑2蛋白的表达量明显增加。表明两种滴眼剂对于白兔干眼模型眼腺体均有一定的修复作用,且KRN 7000诱导的培养基上清制备的滴眼剂治疗的修复效果更优。
Description
技术领域
本发明涉及间充质干细胞培养领域,具体涉及一种脐带间充质干细胞上清液的制备方法及其在眼腺体修复中的应用。
背景技术
干眼综合征是因泪液的质或者量发生改变而导致泪膜相对稳定性下降及眼球表面功能的侵害的一类眼表疾病,临床表现为眼部的刺痛感、干涩感、畏光感、异物感、灼烧感、眼表损害、视觉功能紊乱等。它可引起不适、视力障碍和泪膜不稳定。国际干眼工作组于2007年定义干眼为:多种因素所致的一种泪液和眼表疾病,包括眼表不适症状,视力变化和泪膜不稳定并且有潜在眼表损害,伴随泪液渗透压升高和眼表炎症反应。常见的症状为:干涩感、异物感、烧灼感、痒感、畏光、眼红、视物模糊、视力波动等。严重干眼者可引起视力明显下降从而影响其正常的工作和生活,甚至导致失明。
引起干眼症的起始病因很多,这些因素均可造成眼表面的病理生理改变。虽然干眼症的临床表现不同,但其病理生理改变是相似的。炎症是干眼症发病机制中最关键的因素,而细胞凋亡、性激素等也共同参与了干眼症的发病过程。干眼症治疗的常规方法是提供润滑的眼药水或泪液替代品,而新的治疗方法则是针对干眼症潜在的病因而不是单纯的缓解症状。
随着对干眼症发病机制的不断深入探索,其治疗也在不断的完善,干眼症治疗的终极目标是保护患者的视功能,抑制眼表的炎性反应,恢复眼表面的正常结构及功能。根据不同的病情,有以下几种方法。药物治疗随着对干眼症病理生理学研究的不断深入,干眼症的治疗方法也从单纯的缓解症状转变为针对不同病因的个体化治疗,从而达到既治标又治本的目的。人工泪液能够及时地缓解症状,诸多抗炎药物可针对不同的病因进行治疗,中西医结合治疗也是治疗的有效方法。泪液替代疗法人工泪液仍然是治疗干眼症的主要方法。人工泪液主要由纤维素衍生物构成并决定其黏度、存留时间。理想的人工泪液的渗透压、pH离子成分等应与泪液相同,并含有模拟黏蛋白的成分,黏度接近泪液的黏度,所含的防腐剂对角膜和结膜无害。应用人工泪液治疗可相对改善眼表炎症,增加眼表润滑和眼表湿度,营养眼表和视神经,改善对比敏感度,甚至有助于提高视力。人工泪液常含有防腐剂,特别是苯扎氯胺,当点药频繁时,眼表面炎性反应较重、泪液动力学异常或脂质层异常患者常不能耐受。这些患者宜选用不含防腐剂的人工泪液,以减小防腐剂对眼表面上皮细胞的影响。
目前西医治疗干眼综合征主要是以缓解症状、改善体征为主,治疗方法主要包括物理治疗、药物治疗、自体血清及富血小板血浆替代治疗等。当物理治疗和药物治疗效果欠佳时,可考虑给予手术治疗。手术方式主要是显微探针疏通术、泪道塞法与腺体移植术。间质性干细胞移植也可对干眼综合征进行有效治疗。中医治疗干眼综合征能起到一些辅助作用。
间充质干细胞是一种具有自我更新功能、增殖和多向分化潜能的细胞,同时具有抗炎、抗免疫及修复受伤组织的功能,已经用于治疗多种疾病(系统性红斑狼疮、系统性硬化、1型糖尿病等)。理论上间充质性干细胞移植可以修复受损的泪腺组织,促进泪液的分泌。但间充质干细胞移植后对受损的泪腺、是否可定向分化为正常的泪腺、副泪腺,替代原本已经受损的组织来发挥作用,需要进一步研究证明。
间充质干细胞的培养上清液中包含有多种细胞因子,间充质干细胞的培养上清液已经被广泛应用于多个领域,经研究表明,MSCs具有自分泌和旁分泌功能,能够在局部分泌多种蛋白,引导细胞的迁移、分化,改善局部供血功能、消除局部炎症反应。MSCs分泌的活性蛋白主要涉及促细胞迁移类、受体类、促细胞分化类、调节类和血管生成等几类。这些不同种类的活性蛋白在改善修复靶器官组织功能、抗炎、抗凋亡、免疫调节等方面发挥了重要的作用,是其重要的治疗机制之一。
发明内容
本发明目的在于针对现有技术中干眼症治疗方案不足的问题,提供一种一种脐带间充质干细胞上清液的制备方法及其在眼腺体修复中的应用。
为解决上述技术问题,本发明采用如下技术方案:
一种脐带间充质干细胞上清液的制备方法,包括如下步骤
1)取新生儿脐带,清洗并去除外膜及血管,获得华通氏胶;
2)将华通氏胶剪碎后,分别用I型胶原酶和胰酶处理;
3)用200目细胞过滤器进行过滤,PBS重悬清洗;
4)接种细胞至含有10%胎牛血清的DMEM培养液中培养至2-3代;
5)收集细胞,进一步采用无血清DMEM培养液培养至少2-3代;
6)离心收集培养上清液;
优选的,步骤5)中采用的无血清DMEM培养液中添加KRN 7000。
优选的,所述KRN 7000的浓度为2-5μg/L。
优选的,在步骤3)之后还包括对获得的间充质干细胞进行表面抗原标记检测。
优选的,在步骤3)之后还包括对所述细胞采用无血清DMEM培养液进行清洗的步骤。
优选的,采用所述的任一所述的方法制备获得。
本发明公开了所述的脐带间充质干细胞上清液在制备治疗干眼症的试剂中的应用。
本发明公开了所述的脐带间充质干细胞上清液在制备修复眼睛腺体的试剂中的应用。
本发明公开了所述的脐带间充质干细胞上清液在制备抑制眼睛腺体FAS蛋白表达的试剂中的应用。
本发明公开了所述的脐带间充质干细胞上清液在制备促进眼睛腺体Bcl-2蛋白表达的试剂中的应用。
本发明公开了一种滴眼剂,其包含所述的脐带间充质干细胞上清液。
本发明采用分离制备的脐带间充质干细胞,通过对其进行无血清培养,对收集的培养基的上清中主要的细胞因子的含量进行了测定,其中在培养基中添加了KRN 7000可以明显提高培养基中细胞因子EGF、IGF-1、KGF-2以及IL-10的含量。将收集的培养基上清液进一步制备成滴眼液,用于治疗干眼症模型的白兔,发现KRN 7000诱导的培养基上清制备的滴眼剂治疗的腺体中的凋亡细胞数低于未添加KRN 7000的培养基上清制备的滴眼剂,而Fas表达降低以及Bcl-2蛋白的表达量明显增加。表明,两种滴眼剂对于白兔干眼模型的腺体均有一定的修复作用,且KRN 7000诱导的培养基上清制备的滴眼剂治疗的修复效果更优。
附图说明
图1.分离培养的脐带间充质干细胞的显微照片。
图2.间充质干细胞细胞表面抗原标记的检测图。
图3.脐带间充质干细胞上清液细胞因子的测定结果,其中图3A为EGF含量检测结果(pg/mL),图3B为IGF-1检测结果(pg/mL),图3C为KGF-2含量检测结果(pg/mL),图3D为IL-10含量检测结果(pg/mL);a为原始基础培养基,b为未添加KRN 7000的培养基上清液,c为添加KRN 7000的培养基上清液。
图4.干眼模型兔经滴眼剂治疗后的眼腺体的凋亡细胞染色图。其中图4a为采用的滴眼剂类型为培养基上清液(未添加KRN 7000),图4b为滴眼剂类型为培养基上清液(KRN7000诱导)治疗效果。
图5.干眼模型兔经滴眼剂治疗后的眼腺体的Bcl-2的表达,其中,图5a为未添加KRN 7000的培养基上清制备的滴眼剂治疗的腺体中的Bcl-2的表达,图5b为KRN 7000诱导的培养基上清制备的滴眼剂治疗的腺体中的Bcl-2的表达。
图6.干眼模型兔经滴眼剂治疗后的眼腺体的Fas的表达,其中,图6a为未添加KRN7000的培养基上清制备的滴眼剂治疗的腺体中的Fas的表达,图6b为KRN 7000诱导的培养基上清制备的滴眼剂治疗的腺体中的Fas的表达。
具体实施方式
以下结合附图对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
实施例1脐带间充质干细胞的分离培养
取新生儿脐带标本(经孕妇知情同意),迅速转移至实验室洁净工作台内处理。
首先用含20U/mL肝素钠、80U/mL青霉素、80U/mL链霉素的PBS浸泡;用无菌剪刀将其剪成长约0.5-1cm的小段,用PBS多次冲洗脐带,洗去残留的血凝块,将脐带外膜及脐动脉、脐静脉仔细剥除,以获得华通氏胶(WJ)。尽可能剪碎WJ,取适量剪碎的WJ置于离心管中,加入适量I I型胶原酶,浸没组织块。将其放入37℃、5%CO2、90%湿度的培养箱中消化16-18h,待WJ完全消化后,充分吹打混匀,进行离心,转速2500rpm离心20min,倒掉上清液,加入适量0.25%胰酶,吹打混匀后置于培养箱中消化30min。加入相同体积0.5%胰酶抑制剂终止消化。
充分混匀后用200目细胞过滤器进行过滤,收集滤液,进行离心,转速1200rpm离心20min;加适量PBS重悬细胞沉淀,再次离心,转速1000rpm离心5min,重复2次后,用适量DMEM培养液(含10%FBS)重悬细胞沉淀,用细胞计数板进行细胞计数后,接种细胞至细胞培养皿中,细胞浓度为1.0xl05/mL。置于37℃,5%CO2,85%湿度的细胞培养箱中培养。24小时后镜下观察贴壁情况,视贴壁情况决定是否更换培养基。每天常规察看细胞形态、生长状况并拍照记录。隔天更换为新鲜培养基继续培养。待观察到细胞生长至85%融合时,进行传代培养并保存。细胞的显微拍摄照片参见图1。
实施例2间充质干细胞细胞表面抗原标记的检测
选择传代培养生长状态良好,处于对数增长期的脐带间充质干细胞,经0.2%胰酶消化制成单细胞悬液进行流式细胞术检测其表面抗原的表达情况。
1)培养的细胞在汇合至80%左右融合时,除去培养液和悬浮细胞,以0.01M PBS连续漂洗2-3次,弃洗液;
2)加入0.2%胰蛋白酶溶液,边消化边在倒置显徼镜下观察细胞形态的变化;
3)见细胞间隙变大,胞质回缩,少量细胞脱离瓶底漂起后,立即加入含10%FBS的用DMEM培养基终止消化,用吸管反复轻柔吹打,使贴壁细胞脱落下来;
4)将消化的细胞悬液转移到10mL离心管中,室温,转速1000rpm,离心5min,去掉上清,重悬细胞;
5)计数、离心,重新吹打成单细胞悬液,分装于EP管中;
6)以流式标本缓冲液(含2%FBS,0.1%NaN3的PBS液)在冰上孵有30min;
7)分别与小鼠抗单克隆抗体CD44-PE,CD105-PE,CD19-PE,HLA-ABC-PE,CD31-FITC和HLA-DR-FITC反应,以FITC或PE标记的大鼠抗小鼠IgG染色的细胞作为阴性对照。
8)0.0 1M PBS重复洗涤细胞2次,加入0.01M PBS重悬细胞。
9)细胞悬液用流式细胞仪(BECTON DICKISON)检测,每管样本至少检测10,000个细胞。检测结果参见图2。
其中,流式细胞仪检测所制备的间充质干细胞的表面表达抗原CD44、CD105以及I类抗原HLA-ABC,但是不表达CD19、CD31以及II类抗原HLA-DR,证明制备的间充质干细胞正常。
实施例3脐带间充质干细胞的培养以及上清液的分离
将保存的传代的第3代细胞接种于含10%FBS的DMEM培养基中培养,根据细胞融合情况,培养传代2-3代。
离心收集培养的细胞,进一步采用无血清DMEM培养基(加2μg/L KRN 7000)中置于37℃,5%CO2,90%湿度的细胞培养箱中培养。24小时后镜下观察贴壁情况,视贴壁情况决定是否更换培养基。每天常规察看细胞形态、生长状况并拍照记录。若更换培养基,则继续采用无血清DMEM培养基(加2μg/L KRN 7000)培养。约培养至第6-7天后,并观察到细胞生长至90%融合时,12000g离心收集培养基上清液,保存备用。
其中无血清培养时,以不添加KRN 7000的培养基上清作为培养对照,并离心收集上清液,保存备用。
实施例4脐带间充质干细胞上清液细胞因子的测定
1)标准品制备:取一个标准品的冻干粉,准备7个无菌的EP管,依次标记S0、S1、S2、S3、S4、S5、S6,每个EP管中加入250uL的样本稀释液,从标准品中吸取250uL加入到EP管(S6),动作轻柔地吹打均匀后,更换枪头,从EP管(S6)吸取250uL加
2)入到EP管(S5),按照以上步骤,将标准品进行梯度稀释,EP管(S0)是样品稀释液;
2)将200uL各个梯度的标准品样本稀释液加入到对应的孔板中,然后用移液枪向各组每孔加入待测样品液,然后标准品溶液为对照组溶液。用封板膜封住孔板后,进行2小时的室温孵育;
3)用洗涤液对孔板进行洗涤3次,然后再向每孔中加入洗涤液300uL,洗涤最后一遍后,倒扣在吸干纸上,用力拍干孔板,保证孔内干结,无洗涤液残留;
4)将200uL的对应的抗体(抗IGF-1、EGF、KGF-2、IL-10)加入到每孔中,封膜封住板孔,继续室温孵育2小时;
5)用洗涤液对孔板进行洗涤3次,倒扣在吸干纸上,用力拍干孔板,保证孔内干结,无洗涤液残留;
6)每孔加入底物显色溶液200uL,避光,室温孵育20min;
7)在孔板的每个孔中加入50uL终止反应液,观察颜色变化,然后在酶标仪560nm波长下进行吸光度检测即:OD值。
8)根据建立的标准曲线以及样本测定的OD值计算获得两种脐带间充质干细胞上清液细胞因子的。
检测结果参见图3,其中图3A为EGF含量检测结果(pg/mL),图3B为IGF-1检测结果(pg/mL),图3C为KGF-2含量检测结果(pg/mL),图3D为IL-10含量检测结果(pg/mL)。从测定结果来看,通过添加KRN 7000的培养基上清,其中的细胞因子igf-1、EGF、KGF-2、IL-10的表达均大幅度提高。
实施例5兔干眼症模型的建立及治疗
建立兔维生素A缺乏干眼症模型并初步进行Schirmer试验完成对眼表损害的诊断。
一、兔维生素A缺乏干眼症模型
1).取健康1月龄新西兰白兔9只,体重0.5士0.1kg,雌雄兼用,其中,对照组3只兔。
2).以干酪素为主的VA完全缺乏饲料喂养实验兔6个月,饲料配方参照下表1,其中对照组喂以正常饲料,含VA1200μg/kg。
表1:维生素A完全缺乏饲料配方
二、Schirmer试验操作步骤
1).取对照组白兔以及维生素A缺乏饲料喂养后不同时间的白兔;
2).取5mmX40mm号滤纸条,折出5mm置于下穹窿结膜囊内的外1/3处,其余部分突出眼外,5min后测量滤纸湿润长度,并取测定的平均值记录,测量结果参见下表2。
表2:VA缺乏模型兔Schirmer试验试纸长度测定结果(mm)
类型 | 喂养后2月 | 喂养后3月 | 喂养后4月 | 喂养后5月 | 喂养后6月 |
对照白兔 | 24.3±3.8 | 25.1±3.6 | 25.5±4.0 | 24.8±3.2 | 25.8±3.4 |
VA缺乏白兔 | 23.2±4.1 | 21.2±3.4 | 16.8±2.9 | 8.6±2.1 | 7.8±2.5 |
从测定结果可以看出,对照组试纸的浸润长度基本没有变化,而维生素A缺乏组从第4个月开始试纸浸湿长度明显缩短,初步表明,通过维生素A缺乏饲料的喂养,成功建立的兔干眼症的模型。
实施例6干眼模型兔的滴眼剂治疗以及Schirmer试验试纸长度测定
1).取实施例3中收集的培养基上清液,以1:99的体积比和0.01%氯化钠溶液混合后,作为滴眼剂。
2).对干眼模型兔采用两种滴眼剂(KRN 7000诱导和未添加KRN 7000),每种滴眼剂治疗3只模型兔,每天早晚各一次进行等量滴眼治疗,治疗期间干眼症模型喂养的饲料组分不改变。
3).连续治疗3周后,对两种滴眼剂治疗的白兔进行Schirmer试验试纸长度测定,测定结果如下表3.
表3:滴眼剂治疗的白兔Schirmer试验试纸长度测定结果(mm)
滴眼剂类型 | 治疗3周后 |
培养基上清液(KRN 7000诱导) | 14.6±2.9 |
培养基上清液(未添加KRN 7000) | 9.2±3.5 |
从测定结果可以看出,采用制备的滴眼剂进行一段时间的治疗后,白兔的干眼症症状均有所缓解,其中,添加KRN 7000诱导的培养基上清制备的滴眼剂,恢复效果明显由于未添加KRN 7000的培养基上清制备的滴眼剂。
实施例7干眼模型兔的眼腺体的组织病理观察
对于采用两种滴眼剂治疗的白兔,在治疗3周后正常处死,并对眼睛的泪腺体组织制备石蜡切片后进行病理染色观察,具体操作如下。
原位细胞凋亡检测,采用凋亡检测试剂盒进行操作,主要步骤如下:
切片脱蜡至水,放入新配制的3%过氧化氢中,水溶液室温灭活内源性过氧化物酶,0.01MPBS洗3次。PBS稀释蛋白酶K储备液,切片入湿房箱置37℃恒温箱10-20min,双蒸水洗5次以上。切片上加标记缓冲液,室温10min后甩掉,勿洗。将TdT及Biotin-11-dUTP经离心机离心,再于混匀器上混匀后加入标记缓冲液中,再在混匀器上混匀。37℃恒温箱标记15min,MPBs洗3次。将20xSSC液稀释10倍,然后将切片浸泡于2xSSC中室温15min,0.01MPBS洗3次,切片加封闭液,室温30min后甩掉,勿洗。以封闭液按1:50配制Avidin-HRP为工作液,37℃恒温箱反应30min。0.01MPBS洗3次。DAB显色:取1ml蒸馏水,加试剂盒中A、B、C试剂各1滴,混匀后加至切片,室温显色,镜下控制反应时间,双蒸水洗。苏木素轻度复染,脱水,透明,封片。显微镜下观察。检测结果如图4。其中图4a为采用的滴眼剂类型为培养基上清液(未添加KRN 7000),图4b为滴眼剂类型为培养基上清液(KRN 7000诱导)治疗效果。其中图4b的凋亡细胞的数目明显少于图4a中的细胞。
免疫组织化学染色,采用bcl-2以及Bax免疫组织化学检测试剂盒进行,主要操作步骤如下:
切片脱蜡至水放入新配制的3%过氧化氢水溶液室温10min灭活内源性过氧化物酶,蒸馏水洗3次。切片入湿房箱。Fas及Bcl-2染色切片浸入0.01M磷酸盐缓冲液,微波炉加热至沸腾,蒸馏水洗3次。所有切片滴加正常山羊血清封闭液,室温10min,甩掉多余液体,勿洗。分别滴加鼠抗Fas、Bcl-2多克隆抗体,37℃恒温箱反应60min。0.01MPBS洗2次,滴加生物素化山羊抗鼠IgG,37℃20min,PBS洗3次。加试剂盒中A、B、C试剂各1滴,混匀后加至切片,室温显色,镜下控制反应时间,蒸馏水洗。苏木素轻度复染,脱水,透明,封片。显微镜下观察。
检测结果参见图5和图6。其中,图5a为未添加KRN 7000的培养基上清制备的滴眼剂治疗的腺体中的Bcl-2的表达,图5b为KRN 7000诱导的培养基上清制备的滴眼剂治疗的腺体中的Bcl-2的表达,可见,采用KRN 7000诱导的培养基上清制备的滴眼剂更加促进了腺体中Bcl-2的表达。图6a为未添加KRN 7000的培养基上清制备的滴眼剂治疗的腺体中的Fas的表达,图6b为KRN 7000诱导的培养基上清制备的滴眼剂治疗的腺体中的Fas的表达,采用KRN 7000诱导的培养基上清制备的滴眼剂使得腺体中Fas的表达降低。
从检测结果来看,VA缺乏模型兔经滴眼剂治疗3周后,对于两种不同的治疗剂,KRN7000诱导的培养基上清制备的滴眼剂治疗的腺体中的凋亡细胞数低于未添加KRN 7000的培养基上清制备的滴眼剂,而Fas表达降低以及Bcl-2蛋白的表达量明显增加。表明,两种滴眼剂对于白兔干眼模型的腺体均有一定的修复作用,且KRN 7000诱导的培养基上清制备的滴眼剂治疗的修复效果更优,基于目前的证据来看,可能是KRN 7000诱导的培养基上清中的细胞因子表达更加丰富的原因。
本发明通过上述实施例来说明本发明的工艺方法,但本发明并不局限于上述工艺步骤,即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (10)
1.一种脐带间充质干细胞上清液的制备方法,其特征在于,包括如下步骤:
1)取新生儿脐带,清洗并去除外膜及血管,获得华通氏胶;
2)将华通氏胶剪碎后,分别用I型胶原酶和胰酶处理;
3)用200目细胞过滤器进行过滤,PBS重悬清洗;
4)接种细胞至含有10%胎牛血清的DMEM培养液中培养至2-3代;
5)收集细胞,进一步采用无血清DMEM培养液培养至少2-3代;
6)离心收集培养上清液;
其中,步骤5)中采用的无血清DMEM培养液中添加KRN 7000。
2.根据权利要求1所述的制备方法,其特征在于,所述KRN 7000的浓度为2-5μg/L。
3.根据权利要求1所述的制备方法,其特征在于,在步骤3)之后还包括对获得的间充质干细胞进行表面抗原标记检测。
4.根据权利要求1所述的制备方法,其特征在于,在步骤3)之后还包括对所述细胞采用无血清DMEM培养液进行清洗的步骤。
5.一种脐带间充质干细胞上清液,其特征在于,采用权利要求1-4任一所述的方法制备获得。
6.权利要求5所述的脐带间充质干细胞上清液在制备治疗干眼症的试剂中的应用。
7.权利要求5所述的脐带间充质干细胞上清液在制备修复眼睛腺体的试剂中的应用。
8.权利要求5所述的脐带间充质干细胞上清液在制备抑制眼睛腺体FAS蛋白表达的试剂中的应用。
9.权利要求5所述的脐带间充质干细胞上清液在制备促进眼睛腺体Bcl-2蛋白表达的试剂中的应用。
10.一种滴眼剂,其特征在于,包含有权利要求5所述的脐带间充质干细胞上清液。
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