CN117660530A - 一种脐带间充质干细胞球的制备方法及其所得产品、应用 - Google Patents
一种脐带间充质干细胞球的制备方法及其所得产品、应用 Download PDFInfo
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Abstract
本发明涉及再生医学领域，尤其涉及一种脐带间充质干细胞球制备方法及其所得产品、应用。本发明通过注射FIH‑1基因敲低表达的间充质干细胞球，相对于注射离散的MSC而言，延长了MSC在膝关节腔和软骨组织损伤部位的存留时间与活力，显著增强MSC对多种有害刺激的抵御能力、低氧和炎症微环境的耐受性，从而有望提高MSC的移植效率和对软骨组织损伤的疗效。此外，低氧培养条件下间充质干细胞球能有效维持其细胞干性和多向分化潜能，进而发挥治疗效果。本发明为MSC在临床应用中提供了一种新的使用途径和研究方法。
Description
技术领域
本发明涉及再生医学领域，尤其涉及一种脐带间充质干细胞球制备方法及其所得产品、应用。
背景技术
由于软骨没有血管、淋巴结和神经的组织学特性，损伤的关节软骨的自我再生能力非常有限，在再生医学中仍是一个巨大的挑战。目前临床主要通过关节置换术、软骨移植和髌骨重排列手术等治疗膝关节软骨损伤。但上述治疗方法手术费用高，手术并发症多、风险大且维持时间有限。
间充质干细胞（MSC）具有良好的软骨修复能力，并且具有低免疫原性和高抗炎潜力等优势，是治疗膝关节软骨损伤修复的理想种子细胞。但离散的MSC在关节腔内对多种有害刺激的抵御能力和对低氧、炎症微环境的耐受性较差，影响其治疗效果。与传统的贴壁培养方式相比,间充质干细胞成球培养可显著提高细胞的抗炎,抗凋亡和旁分泌等功能。这些特性使其在膝关节软骨损伤的治疗中具有潜在的应用价值。
间充质干细胞的增殖和分化均是在复杂的环境条件下进行的。因此，包括氧气体积分数、温度、生长因子和pH值等均能影响间充质干细胞的存活、增殖、分化等生物学特性。其中，氧气体积分数是非常重要的影响因素。目前，临床试验常规使用的氧气体积分数为21%，而人体组织和组织间隙中的氧气体积分数为1%~3%。现有技术中，尚未有将脐带间充质干细胞球应用于治疗膝关节损伤中的相关记载。
发明内容
针对现有技术中存在的问题，本发明提供了一种脐带间充质干细胞球的制备方法。
本发明还提供了一种利用上述制备方法制备得到的脐带间充质干细胞球。
本发明的另一目的为提供了上述脐带间充质干细胞球在作为治疗膝关节损伤的药物中的应用。
为了实现上述目的，本发明的技术方案为：
本发明提供了一种脐带间充质干细胞球的制备方法，包括以下步骤：将FIH-1基因敲低表达的脐带间充质干细胞接种到装有MSC完全培养基的旋转式生物反应器中，低氧条件下成球培养。
本发明的目的通过以下技术手段得以实现：利用shRNA去干扰FIH-1，进而调控HIF-1α 信号通路，增强间充质干细胞球适应低氧和炎症等复杂微环境的能力。
本发明所述敲低FIH-1基因表达的shRNA的序列为：
正链（如SEQ ID NO.1所示）：
GATCCGCTAGCAAACACTGAGCTTTGCTCGAGCAAAGCTCAGTGTTTGCTAGCTTTTTG。
反链（如SEQ ID NO.2所示）：
AATTCAAAAAGCTAGCAAACACTGAGCTTTGCTCGAGCAAAGCTCAGTGTTTGCTAGCG。
本发明提供的制备方法中，所述接种的细胞接种密度为1×106~3×106cells/mL。
本发明培养脐带间充质干细胞球所使用的MSC完全培养基的组成为：MSC基础培养基（MSCBM）、5~10%血清替代物、10~20 ng/ml 表皮生长因子（EGF）、15~20 ng/ml成纤维细胞生长因子（FGF）和5~10 ng/ml胰岛素样生长因子（IGF）。
优选的，所述MSC完全培养基的组成为：MSC基础培养基（MSCBM）、5%血清替代物、15ng/ml 表皮生长因子（EGF）、15 ng/ml成纤维细胞生长因子（FGF）和5 ng/ml胰岛素样生长因子（IGF）。
上述血清替代物为UltroGRO-advanced。
进一步的，所述旋转式生物反应器的参数为：24h内以30rpm 5min，0rpm 20min，间歇搅拌；24h后以30rpm恒速搅拌。
进一步的，所述成球培养的培养条件为：培养温度为36~37℃，CO2体积浓度为5~6％，O2体积浓度为1%~3%；培养时间为2~3天；优选的，所述成球培养的条件为：培养箱中温度为37℃，CO2体积浓度为5％，O2体积浓度为2%；培养时间为2天。
本发明还提供了一种利用上述制备方法制备得到的脐带间充质干细胞球，其特征在于，所述脐带间充质干细胞球的直径为100~300μm。
本发明的另一目的为提供了一种上述脐带间充质干细胞球在作为治疗膝关节损伤的药物中的应用。
进一步的，所述脐带间充质干细胞球采用膝关节腔内注射法进行应用时，所用脐带间充质干细胞球的量为2×107cells~6×107cells。
本发明将FIH-1基因敲低表达的脐带间充质干细胞球注射进入关节腔内作为一种新的间充质干细胞（MSC）治疗软骨损伤的方案。注射间充质干细胞球，相对于注射离散的MSC而言，延长了MSC在膝关节腔和软骨组织损伤部位的存留时间与活力，显著增强MSC对多种有害刺激的抵御能力和对低氧、炎症微环境的耐受性，从而有望提高MSC的移植效率和对软骨组织损伤的疗效。此外，低氧培养条件下间充质干细胞球能有效维持其细胞干性和多向分化潜能，进而发挥治疗效果。
本发明的有益效果为：
（1）本发明首次提出将FIH-1基因敲低表达的脐带间充质干细胞球注射作为一种膝关节软骨损伤的治疗方案。
（2）相对于向膝关节腔内注射离散的MSC（MSCdiss）而言，本发明提供的MSC细胞球（MSCsp）延长了MSC在膝关节腔和软骨损伤部位的存留时间与活力，从而提高治疗效果。
（3）本发明提供的MSC细胞球（MSCsp），尤其意外的发现是，接受MSC细胞球注射后的宿主（猪）并无不良反应，更无致残现象。因此，本发明提供的技术方案为MSC在临床的应用提供了一种新的使用途径和研究方法。
附图说明
图1为本发明实施例1的MSC单层培养（MSC2D）和MSC成球培养（MSCCQ）第2天细胞形态；
图2为本发明实施例1的MSC单层培养（MSC2D）和MSC成球培养（MSCCQ）2天后干性基因和增殖基因mRNA水平的表达结果；
图3为本发明实施例1的MSC单层培养（MSC2D）和MSC成球培养（MSCCQ）2天后成软骨标志基因的mRNA水平的表达结果；
图4为本发明实施例1的MSC单层培养（MSC2D）和MSC成球培养（MSCCQ）2天后收取的培养基上清中炎症因子的含量检测结果；
图5为本发明实施例2的离散的MSC（MSCdiss）和成球的MSC（MSCsp）治疗16周后软骨损伤修复情况、整合度、外观及整体评价；
图6为本发明实施例2的离散的MSC（MSCdiss）和成球的MSC（MSCsp）治疗16周后软骨组织番红O-固绿染色染色结果；
图7为本发明实施例2的离散的MSC（MSCdiss）和成球的MSC（MSCsp）治疗16周后软骨组织苏木精-伊红染色结果；
图8为膝关节软骨损伤干细胞/球治疗模式图。
具体实施方式
为下面详细叙述本发明的实施例，需要说明的是，本实施例是叙述性的，不是限定性的，不能以此限定本发明的保护范围。
本发明中所使用的原料，如无特殊说明，均为常规的市售产品；本发明中所使用的方法，如无特殊说明，均为本领域的常规方法。
实施例1
待P3人脐带间充质干细胞汇合度达到80%左右，进行消化；弃去培养瓶中的培养基，使用室温下的生理盐水洗去培养基（8 mL/T175培养瓶）；添加TryplE Select， 4 mL/T175培养瓶，室温孵育2~5min，镜下观察细胞呈椭圆状脱落时，轻拍培养瓶至细胞全部脱落后，每瓶加入2倍体积MSCBM，终止消化，将细胞悬液倒至离心管中；向培养瓶中加入MSC基础培养基，8 mL/T175培养瓶，拧紧瓶盖，轻晃培养瓶，清洗瓶内的各个平面，将细胞悬液倒至离心管中，600xg，离心5 min。对收集后的细胞悬液，600xg，离心5min。离心后弃上清，沉淀使用MSC完全培养基重悬，5 mL/T175培养瓶，进行细胞计数。
FIH-1基因敲低的MSC成球培养组（MSCCQ）：委托质粒公司商业合成一条FIH-1shRNA干扰质粒序列（pGreenPruo-FIH-1）。将MSC以6000cells/cm2的密度接种到6孔板中，待细胞汇合度达到70%-80%时，用Lipofectamine 2000 将干扰质粒转染至MSC细胞，48h后，加入1.2μg/ml嘌呤霉素筛选相应的干扰FIH-1的稳转细胞株。将筛选出的稳转细胞株作为FIH-1 shRNA组，加入 RIPA 蛋白裂解液裂解细胞，Western blot 检测对照组和FIH-1shRNA组中FIH-1蛋白表达，检测敲低效果，确认获得FIH-1低表达的MSC细胞株。取P7代稳定转染的细胞（3*107cells）接种到装有30ml MSC完全培养基的旋转式生物反应器中，24h内以30rpm 5min，0rpm 20min，间歇搅拌；24h后以30rpm恒速搅拌，使MSC聚集成球，最终以3×107cells/瓶的密度，低氧（2%氧浓度）培养2天。
MSC单层培养组（MSC2D）：将P7代未转染的MSC（3*107cells）接种到装有30ml MSC完全培养基的普通T175瓶中低氧（2%氧浓度）培养2天。
细胞形态观察：细胞接种第二天显微镜下观察MSC细胞形态，如图1所示，大量MSC细胞球悬浮在旋转式生物反应器中（图1）。
基因表达检测结果：用RT-qPCR法定量测定了细胞增殖和干性相关基因的表达情况，用β-actin作为内参。与MSC单层培养组相比，MSC成球培养组中增殖相关基因（PCNA）和干性相关基因（OCT4）表达显著上调（图2）。而且MSC成球培养组的成软骨相关标志基因（Sox-9和Col-Ⅱ）的表达水平也明显增强（图3）。因此，成球培养MSC有利于维持人脐带间充质干细胞的增殖能力和干性，同时有利于增强其成软骨分化的潜能。
炎症因子分泌量检测结果：收获MSC单层培养组和成球培养组的培养基上清。用ELISA法测定不同组别培养基上清中炎症因子（促炎因子：IL6、IL8；抗炎因子IL10）的含量。与MSC单层培养组相比，MSC成球培养组培养基上清中IL6和IL8的含量显著下降，同时IL10的含量显著上升（图4）。因此，说明MSC成球培养较单层培养具有高抗炎能力。
实施例2
巴马小型猪称质量后，用3%戊巴比妥钠静脉注射麻醉，侧卧固定在手术台
上。通过髌骨内侧入路，切开皮肤、皮下组织后，暴露膝关节，使膝关节置于最大屈曲位，找出股骨内侧髁，使用专业手术器械在猪双膝股骨内侧髁的软骨上各制造一处直径5 mm，深 2 mm 的关节软骨损伤，不伤及软骨下骨组织，然后复位髌骨，缝合关节囊、皮下组织、皮肤。创面形成四周后，膝关节内注射人脐带间充质干细胞/球，具体模式图如图8所示。右侧膝关节软骨损伤注射人脐带间充质干细胞球，使细胞球完全填充缺损处，作为实验组；左侧注射离散的人脐带间充质干细胞，作为对照组，完成手术。术后猪两条腿用石膏固定制动一两天，之后正常活动、豢养。软骨修复时间为8~16周。治疗后 8，12，16周每个时间点分别处死 3，4，3 头猪，观察软骨损伤部位修复情况。获取软骨及骨组织进行石蜡切片，番红O-固绿染色、苏木精-伊红染色鉴定。
表1动物信息以及分组情况
软骨损伤修复情况、整合度、外观及整体评价结果：根据 ICRS 评分系统评价软骨修复情况，从软骨修复程度、整合度、外观及整体评价 4 个方面进行评分及统计，结果发现：软骨修复程度（P=0.007，n=10）、整合度 (P=0.003， n=10)外观 (P=0.0002，n=10) 及整体修复情况 (P=0.004，n=10) 有显著差异（图5）。说明，与离散的人脐带间充质干细胞相比，成球培养后的MSC对软骨损伤具有更强的修复作用。
治疗后软骨组织番红O-固绿染色染色结果：对治疗16周后的软骨组织进行番红O-固绿染色，可见蛋白聚糖被番红O染为红色。对照组治疗效果不明显，治疗组可见大量的成熟软骨细胞分布在软骨损伤部位，向上逐渐过渡为幼稚的软骨细胞，表层仍可见残存的肉芽组织。（图6）。
治疗后软骨组织苏木精-伊红染色结果：对治疗16周后的软骨组织进行苏木精-伊红染色。如图7可见，与对照组相比，实验组表面出现较多纤维肉芽，平行交错排列胶原纤维束呈嗜酸性，被染成红色。中层位置：对照组虽然也有少量纤维肉芽组织逐渐变为透明软骨，但软骨数量较少，治疗效果较差，而实验组中出现大量透明软骨，并且，细胞从外至内由少变多，由小变大，说明软骨细胞由幼稚转变为成熟。最深层位置：对照组含有少量纤维的类骨质、软骨囊和透明软骨；治疗组软骨囊呈强嗜碱性，大量软骨囊被染成蓝色。
综上所述，FIH-1基因敲低的间充质干细胞球膝关节腔注射可能作为一个新的MSC给药方案，延长了MSC在膝关节腔和软骨组织损伤部位的存留时间与活力，显著增强MSC对多种有害刺激的抵御能力和对低氧、炎症微环境的耐受性，从而有望提高MSC的移植效率和对软骨组织损伤的疗效。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种脐带间充质干细胞球的制备方法，其特征在于，包括以下步骤：将FIH-1基因敲低表达的脐带间充质干细胞接种到装有MSC完全培养基的旋转式生物反应器中，低氧条件下成球培养。
2.根据权利要求1所述的制备方法，其特征在于，敲低FIH-1基因表达的物质为特异性干扰FIH-1基因表达的shRNA；所述敲低FIH-1基因表达的shRNA序列：
正链：
GATCCGCTAGCAAACACTGAGCTTTGCTCGAGCAAAGCTCAGTGTTTGCTAGCTTTTTG。
反链：
AATTCAAAAAGCTAGCAAACACTGAGCTTTGCTCGAGCAAAGCTCAGTGTTTGCTAGCG。
3.根据权利要求1所述的制备方法，其特征在于，所述接种的细胞接种密度为1×106~3×106 cells/mL。
4.根据权利要求1或3所述的制备方法，其特征在于，所述MSC完全培养基的组成为：MSC基础培养基（MSCBM）、5~10%血清替代物、10~20 ng/ml 表皮生长因子（EGF）、15~20 ng/ml成纤维细胞生长因子（FGF）和5~10 ng/ml胰岛素样生长因子（IGF）。
5.根据权利要求4所述的制备方法，其特征在于，所述MSC完全培养基的组成为：MSC基础培养基（MSCBM）、5%血清替代物、15 ng/ml 表皮生长因子（EGF）、15 ng/ml成纤维细胞生长因子（FGF）和5 ng/ml胰岛素样生长因子（IGF）。
6.根据权利要求4或5所述的制备方法，其特征在于，所述血清替代物为UltroGRO-advanced。
7.根据权利要求1-6任一项所述的制备方法，其特征在于，所述旋转式生物反应器的参数为：24h内以30rpm 5min，0rpm 20min，间歇搅拌；24h后以30rpm恒速搅拌。
8.根据权利要求1-7任一项所述的制备方法，其特征在于，所述成球培养的培养条件为：培养温度为36~37℃，CO2体积浓度为5~6％，O2体积浓度为1%~3%；培养时间为2~3天；优选的，所述成球培养的条件为：培养箱中温度为37℃，CO2体积浓度为5％，O2体积浓度为2%；培养时间为2天。
9.一种利用权利要求1-8任一项所述的制备方法制备得到的脐带间充质干细胞球，其特征在于，所述脐带间充质干细胞球的直径为100~300μm。
10.一种如权利要,9所述的脐带间充质干细胞球在作为治疗膝关节损伤的药物中的应用，其特征在于，所述脐带间充质干细胞球采用膝关节腔内注射法进行应用时，所用脐带间充质干细胞球的量为2×107 cells~6×107cells。
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