CN117801091A - 一种子宫内膜干细胞在子宫内膜修复中的应用 - Google Patents
一种子宫内膜干细胞在子宫内膜修复中的应用 Download PDFInfo
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- CN117801091A CN117801091A CN202311836669.XA CN202311836669A CN117801091A CN 117801091 A CN117801091 A CN 117801091A CN 202311836669 A CN202311836669 A CN 202311836669A CN 117801091 A CN117801091 A CN 117801091A
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Abstract
本发明涉及一种子宫内膜干细胞在子宫内膜修复中的应用。具体的，将来自子宫内膜干细胞的外泌体与本发明多肽一起使用后能够有效的用于子宫内膜损伤的修复，将其制备成为药物组合物后，应用前景广阔。
Description
技术领域
本申请涉及生物治疗领域，具体的涉及一种子宫内膜干细胞在子宫内膜修复中的应用。
背景技术
子宫内膜存在着具有高度增殖、自我更新以及分化潜能的干细胞，命名为子宫内膜干细胞或者宫内膜干细胞EMSC。EMSC具有一般的干细胞生长特性。体外培养原代EMSC能够在2-24h内贴附在培养皿或者培养瓶的底面,并逐渐形成多个单层放射状的成纤维样细胞集落。细胞状态良好的EMSC在倒置显微镜呈梭形或者纺锤形,初始呈放射性生长,在密度达到一定程度时,呈现旋涡状生长。细胞核较大且不规则,核仁明显。研究表明,EMSC可分化成脂肪源性干细胞、心肌源性干细胞、成骨细胞、软骨细胞等谱系,与骨髓间充质干细胞的分化效率相当。EMSC也符合间充质干细胞的分化标准,但并不局限于向中胚层谱系细胞分化。在一定条件诱导下,甚至能够跨胚层分化成神经细胞、心肌细胞、胰岛细胞等。通过对在纤维蛋白凝胶中对EMSC进行三维培养,使其分化成Schwann细胞,再利用FGF2/FSK/HRG/RA等诱导介质来研究分化的Schwann细胞标记的表达；并利用细胞免疫组化技术,发现了分化的细胞表达S100和P75标记。这些结果表明,人类EMSC在二维和三维培养中,首次可以分化为Schwann细胞。但也有学者提出了不同的观点。据报道,EMSC与骨髓、脂肪干细胞相比成脂分化潜能较低,且分化率较低,在第5周时只有不到30％。
子宫内膜是一个高度再生的组织,主要由腺上皮细胞、基质细胞和血管组成。在结构上分为功能层和基底层,基底层与子宫肌层相连,在月经期间不会发生脱落。功能层靠近宫腔,受到卵巢激素的影响从而发生周期性变化。子宫内膜异位症的发病机制有多种解释,普遍认为是经血逆流所致,但不能解释发生在腹腔外的病灶。EMSC可能与子宫内膜异位症的发生和发展有关。研究证明,异位的子宫内膜细胞经过体外培养后也具有和间充质干细胞类似的集落形成能力和多分化潜能,并在小鼠模型中具有侵袭性,这说明异位的子宫内膜中存在干细胞类细胞。另外也有研究证明,从异位的子宫内膜中获取的EMSC和正常人的EMSC相比,在形态和生物学特性上有着显著的区别。研究发现,在整个月经周期中,子宫内膜异位症患者的子宫内膜所表达的Musashi-1要明显高于正常人,这说明,Musashi-1与子宫内膜异位症的发生密切相关。
目前，针对子宫内膜干细胞在子宫内膜修复中的应用研究还比较少，需要相应的研究来提供可选的子宫内膜修复新的治疗方法。
发明内容
本发明一方面，通过提供子宫内膜干细胞用于子宫内膜损伤修复。
具体的，本发明提供一种用于子宫内膜损伤修复的药物组合物，所述的药物组合物中含有子宫内皮干细胞外泌体以及其他辅助治疗药物。
另外一方面，本发明通过建库筛选，设计了一系列具有潜在修复作用的多肽，并通过实验研究筛选出了一种能够促进子宫内膜损伤修复的多肽。
所述筛选是基于子宫出血模型后，大鼠子宫组织VEGF的含量明显降低而MMP-1的含量明显增加，说明VEGF和MMP-1与子宫内膜异常出血密切相关。说明影响这两个因素的多肽会对对子宫内膜有明显的修复作用，因此，通过计算机模拟以及肽库筛选获得了能够上调VEGF并且下调MMP-1的多肽用于子宫内膜损伤修复。
具体的，所述的多肽序列如SEQ ID NO：1所示。
进一步的，本发明所述的多肽还可以被取代修饰或者保守性替换。
提供功能相似的氨基酸的保守替换表为本领域技术人员所公知。例如，氨基酸侧链的特性为疏水氨基酸(A、I、L、M、F、P、W、Y、V)、亲水氨基酸(R、D、N、C、E、Q、G、H、K、S、T)，以及具有如下共同的官能团或特性的侧链：脂肪族侧链(G、A、V、L、I、P)；含有侧链(S、T、Y)的羟基；含有侧链(C、M)的硫原子；含有侧链(D、N、E、Q)的羧酸和氨基化合物；含有侧链(R、K、H)的碱基；以及含有侧链(H、F、Y、W)的芳香烃。
更进一步的，本发明提供一种用于子宫内膜损伤修复的药物组合物，所述的药物组合物中含有子宫内皮干细胞外泌体以及多肽，所述的多肽序列如SEQ ID NO：1所示。
所述的药物组合物还要有药学上可接受的载体。
本发明还包含用于药品的额外常规载体或赋形剂。所述载体的实例包括但不限于崩解剂、粘合剂、润滑剂、助流剂、稳定剂和填充剂、稀释剂、着色剂、调味剂和防腐剂。本领域普通技术人员可关于剂型的特定所需特性通过常规实验并且无任何不当负荷来选择一种或多种上述载体。所用的各载体的量可在本领域中的常规范围内变化。以下均以引用的方式并入本文的参考文献公开用于配制口服剂型的技术和赋形剂。
药学上可接受的崩解剂的实例包括但不限于淀粉；粘土；纤维素；海藻酸盐；胶；交联聚合物，例如交联聚乙烯吡咯烷酮或交联聚维酮，例如来自International SpecialtyProducts(Wayne,NJ)的POLYPLASDONE XL；交联羧甲基纤维素钠(cross-linked sodiumcarboxymethylcellulose)或交联羧甲基纤维素钠(croscarmellose sodium)，例如来自FMC的AC-DI-SOL；和交联羧甲基纤维素钙；大豆多糖；和瓜尔胶。崩解剂可以所述组合物的约0重量％至约10重量％的量存在。在一个实施方案中，崩解剂是以组合物的约0.1重量％至约5重量％的量存在。
药学上可接受的粘合剂的实例包括但不限于淀粉；纤维素和其衍生物，例如微晶纤维素，例如来自FMC(Philadelphia,PA)的AVICEL PH、来自Dow Chemical Corp.(Midland,MI)的羟基丙基纤维素、羟基乙基纤维素和羟基丙基甲基纤维素METHOCEL；蔗糖；右旋糖；玉米糖浆；多糖；和明胶。粘合剂可以所述组合物的约0重量％至约50重量％，例如2-20重量％的量存在。药学上可接受的润滑剂和药学上可接受的助流剂的实例包括但不限于胶状二氧化硅、三硅酸镁、淀粉、滑石、磷酸三钙、硬脂酸镁、硬脂酸铝、硬脂酸钙、碳酸镁、氧化镁、聚乙二醇、粉末状纤维素和微晶纤维素。润滑剂可以所述组合物的约0重量％至约10重量％的量存在。在一个实施方案中，润滑剂可以组合物的约0.1重量％至约1.5重量％的量存在。助流剂可以约0.1重量％至约10重量％的量存在。药学上可接受的填充剂和药学上可接受的稀释剂的实例包括但不限于糖粉、可压缩糖、葡萄糖结合剂(dextrate)、糊精、右旋糖、乳糖、甘露醇、微晶纤维素、粉末状纤维素、山梨醇、蔗糖和滑石。填充剂和/或稀释剂例如可以所述组合物的约0重量％至约80重量％的量存在。
本发明可包含水溶性添加剂，目的是例如使释放速度最优化或使药物稳定。在体内环境中(即在中性pH和37℃下)，在此使用的水溶性添加剂在室温下为固体，且其1g可溶解在小于100mL的水中，优选小于10mL。只要是医学上/药学上可接受的，在此使用的水溶性添加剂就没有限制，并且包括，例如糖类、盐类、氨基酸类和胆汁盐。具体地，在此使用的糖类包括，例如葡萄糖、甘露醇、乳糖、海藻糖、蔗糖、赤藓糖醇、山梨糖醇、木糖醇；且优选葡萄糖、甘露醇和乳糖。在此使用的盐类包括，例如氯化钠、氯化钾和氯化钙；且优选氯化钠。在此使用的氨基酸类包括天然存在的20种不同的α-氨基酸，如甘氨酸、丙氨酸、脯氨酸、丝氨酸、精氨酸和谷氨酸；且优选甘氨酸。在此使用的胆汁盐包括，例如一级胆酸盐，如胆酸钠和鹅脱氧胆酸钠；二级胆酸盐，如脱氧胆酸钠和石胆酸钠；和共轭胆酸盐，如甘氨胆酸钠和牛磺胆酸钠；且优选胆酸钠、脱氧胆酸钠和甘氨胆酸钠。更优选地，水溶性添加剂为氯化钠和/或脱氧胆酸钠。本发明的固体制剂可包含一种或几种不同类型的上述水溶性添加剂。
根据本发明，治疗有效量的本发明的组合的各组合搭配物可同时或依序并且以任何顺序施用，且所述组分可分开地或作为固定组合施用。例如，根据本发明的治疗增生性疾病的方法可包括(i)施用呈游离或药学上可接受的盐形式的第一试剂(a)和(ii)施用呈游离或药学上可接受的盐形式的试剂(b)，其同时或以任何顺序依序，以联合地治疗有效量，优选地以协同有效量，例如以对应于本文所述量的每天或间接剂量施用。本发明的组合的各组合搭配物可在治疗过程期间的不同时间分开地或以分次或单一组合形式并行地施用。此外，术语“施用”还涵盖使用组合搭配物的前药，所述前药体内原样转化为所述组合搭配物。本发明因此应了解为包含同时或交替治疗的所有所述方案并且术语“施用”应相应地加以解释。
具体的，本发明的外泌体的给药剂量可以是1-100μg/kg/d。
具体的，本发明的多肽的给药剂量可以是1-100μg/kg/d。
进一步的，包含含量为按制剂全部重量计的40重量％以下，优选30重量％以下，更优选20重量％以下，且最优选10重量％以下的水溶性药物。
有益效果
本发明提供了宫内膜干细胞在子宫内膜修复中的应用，具体的，将来自子宫内膜干细胞的外泌体与本发明多肽一起使用后能够有效的用于子宫内膜损伤的修复，将其制备成为药物组合物后，应用前景广阔。
附图说明
图1多肽对子宫内膜间质细胞ESC的增殖活性的影响结果图
图2治疗各组对子宫的内膜厚度的影响结果图
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1多肽对子宫内膜间质细胞ESC的影响
提取大鼠增生期子宫内膜组织，所有组织在冰上使用平衡盐容易漂洗。剪碎，在DMEM/F12中加入0.25％胶原酶溶液中，在37℃、5％CO2培养箱内消化，再使用过滤器过滤，HBSS继续漂洗3遍后，细胞在培养皿内使用DMEM/F12培养基添加10％FBS、100U/ml青霉素和100pg/ml链霉素重悬并培养在37℃、5％CO2培养箱内，使用当细胞达到80％培养皿底部后进行1:2传代，用于本研究的细胞为P4代。使用免疫荧光鉴定发现ESCs均表达Vimentin，基本不表达cytokeratin，说明ESCs的纯度较高。
将ESCs细胞在DMEM/F12培养基添加10％FBS、100U/ml青霉素和100pg/ml链霉素重悬并培养在37℃、5％CO2培养箱内进行培养，其中培养基中分别添加0、10、50、100、200μg/ml终浓度的多肽，培养24h后，使用大鼠VEGF酶联免疫分析试剂盒和大鼠MMP-1酶联免疫分析试剂盒进行VEGF和MMP-1表达量测定，分析的细胞总量相同，以不加多肽的细胞蛋白表达量为基础，计算各添加多肽后的细胞中的蛋白相对表达量，结果如表1所示。
表1多肽对ESCs中VEGF和MMP-1相对表达量的影响
| 组别 | VEGF的相对不表达量 | MMP-1的相对表达量 |
| 10μg/ml多肽组 | 1.13±0.12 | 0.95±0.07 |
| 50μg/ml多肽组 | 1.45±0.10 | 0.84±0.04 |
| 100μg/ml多肽组 | 1.64±0.08 | 0.78±0.08 |
| 200μg/ml多肽组 | 1.72±0.09 | 0.73±0.05 |
从表1可以看出，随着多肽浓度的增加，其能够显著的增加VEGF的相对表达量，同时降低MMP-1的表达量。
另外，将ESCs细胞在DMEM/F12培养基添加10％FBS、100U/ml青霉素和100pg/ml链霉素重悬并培养在37℃、5％CO2培养箱内进行培养(24孔板接种量为1*105cells/well)，其中培养基中分别添加0、10、50、100、200μg/ml终浓度的多肽，24h换液，培养96h，使用CCK8试剂盒检测每孔吸光度。结果如图1所示。
从图1可以看出，该多肽能够剂量依赖性的促进细胞的增殖，具有促进子宫损伤细胞修复的功效，在200μg/ml的浓度下，吸光度达到了(3.75±0.10)，比对照组的(2.51±0.09)吸光度值显著的提高，差异显著(P<0.05)。
实施例2子宫内膜干细胞外泌体的制备
将大鼠断颈处死后，提起下腹部皮肤，剪开皮肤、腹壁肌肉、腹膜并撕开，暴露出腹腔脏器。找到并分离子宫，磷酸盐缓冲液(PBS)洗涤后除去系膜脂肪及其他结缔组织。剪开子宫，呈现米白色透明云朵状的子宫内膜层。撕下表面的内膜层，放入PBS内，加入胰蛋白酶+0.04％乙二胺四乙酸(EDTA)，提前预热，浓度0.25％，容量为1ml，重悬皿内子宫内膜组织块。在微型磁力搅拌器上进行消化，然后转移至15ml离心管中，加入等量SAIOS原代间充质干细胞培养基终止。
2000rpm/min离心5min，沉淀加入2ml SAIOS原代间充质干细胞培养基重悬并进行培养。采用流式细胞仪检测及分选大鼠子宫内膜干细胞，激发光波长为350nm，前射光及侧射光由100mW氩离子激光器于488nm波长激发，采集波长为450nm和675nm。分选后的得到的干细胞分别加入SAIOS原代间充质干细胞培养基于37℃、5％CO2的培养箱中培养备用，经鉴定过其CD105免疫荧光染色为阳性。
培养P3代的子宫内膜干细胞，当细胞融合至80％时，PBS冲洗细胞并更换SAIOS原代间充质干细胞培养基，培养48h后，收集细胞培养上清，ExoQuick-TCTM试剂盒提取exosome，根据外泌体提取试剂盒操作步骤进行。(1)将收集的细胞培养上清液3000×g高速离心15min，弃沉淀，去除细胞及细胞碎片，收集上清。(2)将收集上清液转移至无菌离心管中，按2：1(培养上清：提取试剂)加入exosome提取试剂，反复颠倒混匀后置于4℃冰箱过夜(至少12h)。(3)过夜后于4℃，1500×g，高速离心30min，管底可见黄白色沉淀，弃去上清。(4)重悬后于4℃，1500×g，高速离心5min，弃去上清液，取沉淀物，PBS 500μl重悬，置于-80℃保存。通过电镜检测，外泌体为圆形或者椭圆形的小囊泡，直径约为50-156nm，富含峰值粒径为121±10nm。使用BCA蛋白定量法检测提取的外泌体稀释10倍后，浓度为3.247μg/μl。
实施例3干细胞和多肽在模型中的效果验证
大鼠子宫损伤模型：SD大鼠，雌性，220g左右，经适应性饲养10周后进行动物实验。大鼠使用4％水合氯醛麻醉(10ml/kg)，麻醉成功后，纵行切开下腹部约3cm，暴露子宫并牵出子宫至腹壁外，距宫颈0.5cm处纵行切开子宫约3cm长将子宫翻开暴露内膜，使用T10手术刀片刮除内膜，直至出现明显的出血以及沙砾感，结束刮宫，PBS冲洗2遍，以6-0可吸收线缝合子宫，以3-0不可吸收线缝合腹壁。
按照如下各组方式给药：
A组：取exosome，PBS重悬调整蛋白含量为400μg/ml。通过微量进样针来吸取外泌体悬液，容量为50μl，进针的位置选择皮下注射，常规进行饲养。
B组：取多肽浓度为200μg/ml。通过微量进样针来吸取多肽，容量为50μl，进针的位置选择皮下注射，常规进行饲养。
C组：将A组中等量的外泌体和B组中等量的多肽组合使用，按照相同的方法，间隔10min给药，常规进行饲养。
D组：只给PBS，不给药物的模型组，方法同上。
E组：阳性对照组，黄体酮，用量为200μg/ml，用法同B组。
以上各组，每4d给药1次，给药10次后，再培养10d。分别处死大鼠分离子宫进行HE染色下我们观察了子宫的内膜厚度并做统计学分析。
如图2所示。统计学分析显示，各治疗组相比较模型组的内膜厚度均得到有效的提高，差异具有统计学意义(P<0.05)。多肽和外泌体单独均可以有效的提高内膜厚度，均比阳性组的内膜厚度要高，特别是外泌体和多肽组合使用后，能够显著的协同提高内膜厚度，达到了(590.4±29.7)μm。
此外还验证各组对大鼠生育功能重建性能的影响。将SD大鼠子宫内膜损伤模型如上所述，术后给药同前。术后60天，各组大鼠与公鼠合笼，观察阴道栓形成，在阴道栓形成后20天，大鼠二氧化碳处死，游离大鼠子宫，观察并计数妊娠子宫数量及各组的直径超过1cm的孕囊数量和孕囊总数。结果显示，正常大鼠的子宫的妊娠率为96％，模型组的妊娠率为0；多肽组和外泌体组的妊娠率分别为52％和47％，而多肽和外泌体组的妊娠率为79％。这个结果说明多肽和外泌体能够通过提高子宫内膜损伤修复来部分恢复子宫内膜损伤大鼠子宫的生育力。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种能够促进子宫内膜修复的多肽，其特征在于所述的多肽序列如SEQ ID NO：1所示。
2.一种用于子宫内膜修复的药物组合物，其特征在于由子宫内皮干细胞外泌体和多肽组成，所述的多肽序列如SEQ ID NO：1所示，所述的子宫内皮干细胞为非人的子宫内皮干细胞。
3.如权利要求2所述的药物组合物，其特征在于还含有药学上可接受的载体。
4.子宫内皮干细胞外泌体和多肽在制备用于治疗子宫内膜修复的药物组合物中的用途，其中，所述的多肽序列如SEQ ID NO：1所示，所述的子宫内皮干细胞为非人的子宫内皮干细胞。
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