CN117925533A - 一种宫内膜干细胞在阴道结缔组织弹性黏膜修复中的应用 - Google Patents
一种宫内膜干细胞在阴道结缔组织弹性黏膜修复中的应用 Download PDFInfo
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Abstract
本发明涉及干细胞修复技术领域,提供了一种宫内膜干细胞在阴道结缔组织弹性黏膜修复中的应用。本发明提供了利用修复肽基因改造宫内膜干细胞,提高其阴道结缔组织弹性黏膜修复功能,同时,该修复肽也具备良好的抑菌活性。经改造后的宫内膜干细胞能够有效抑制阴道细菌的生长,改善阴道健康状况,同时兼具结缔组织弹性黏膜修复功能,能有效修复子宫内膜,恢复其正常厚度,有效提高了生育能力。
Description
技术领域
本发明涉及干细胞修复技术领域,特别涉及一种宫内膜干细胞在阴道结缔组织弹性黏膜修复中的应用。
背景技术
子宫是女性极其重要的生殖器官,同时,在受精卵着床、胚胎发育以及胎儿分娩过程中,子宫具有不可替代的作用。然而,子宫的这些重要功能主要通过子宫内膜得以实现。
子宫内膜是由胚胎发育过程中的中胚层组织(包括中肾管组织和苗勒管)完全融合而成,位于子宫腔与子宫肌层之间,是人体新陈代谢和自我更新最为活跃的组织之一。子宫内膜分为功能层和基底层2层。功能层,由基底层再生而来,受卵巢性激素的影响发生周期性变化;基底层靠近子宫肌层,不受卵巢性激素周期性变化的影响。研究表明,在女性的育龄期,子宫内膜需经历400多次的再生、分化和剥脱的循环过程。正常的月经周期中,当功能层剥脱后,基底层即开始增生,促进其修复和再生。因此,基底层一旦受到损伤,会影响功能层对性激素的应答,导致子宫内膜的再生能力下降。
女性阴道上皮柔软易弯曲,得益于雌激素、乳酸杆菌、阴道pH等维持精密的平衡机制。其可以保持正常水平的水合作用和润滑度。在进行清宫术、诊断性刮宫、子宫内膜消融术、子宫整形术等各种宫腔操作中,如果损伤子宫内膜基底层可导致子宫内膜修复障碍,是薄型子宫内膜形成的重要原因。其中较常见的情况是人工流产后宫腔粘连,即Asherman综合征。子宫内膜长期暴露于孕激素的作用下,可能导致孕激素拮抗雌激素对子宫内膜的增生作用,从而最终引起子宫内膜萎缩,引起细胞微环境改变,不利于子宫内膜的修复。
子宫内膜干细胞是作为修复子宫内膜的一种新型治疗方向,经血中包含的子宫内膜脱落细胞可在体外分离并培养出子宫内膜间充质干细胞,具有复制快、来源广、取材易、含量多、无创获得等优点,其大量制备子宫内膜干细胞提供支持。然而,现有的子宫内膜干细胞制剂无法兼具抑菌和修复功能,往往是需要与抑菌药物组合使用,势必会引起耐药性,且对子宫内膜产生持续再生的作用有限。
发明内容
针对现有技术所存在的技术问题,本发明提供了一种宫内膜干细胞在阴道结缔组织弹性黏膜修复中的应用。首次利用修复肽基因改造宫内膜干细胞,提高其阴道结缔组织弹性黏膜修复功能,同时,该修复肽也具备良好的抑菌活性。经改造后的宫内膜干细胞能够有效抑制阴道细菌的生长,改善阴道健康状况,同时兼具结缔组织弹性黏膜修复功能,能有效修复子宫内膜,恢复其正常厚度,有效提高了生育能力。
本发明的首要目的是提供了一种宫内膜干细胞制备方法,其特征在于,所述方法包括如下步骤:
1)宫内膜干细胞的分离;
2)宫内膜干细胞的培养;
5)宫内膜干细胞的鉴定;
6)利用修复肽改造宫内膜干细胞;
优选地,步骤1)包括:
取子宫内膜组织,剔除脂肪、系膜,并用1×PBS(pH=7.0)进行冲洗,并用手术剪将子宫内膜组织剪碎;采用0.25%胰蛋白酶,37℃震荡酶解消化子宫内膜组织碎片40-60min;并向消化液中加入等体积含10%FBS的DMEM完全培养基终止消化;收集消化液,1000rpm离心10-20min,弃掉上清液,将离心后所得沉淀加入DMEM完全培养基吹打均匀后,接种于25ml培养瓶中,在37℃、5%CO2培养箱内培养,每隔2天更换新鲜培养液。
优选地,步骤2)包括:待步骤1)中的细胞长至约80%融合时进行传代;在超净工作台内弃掉培养瓶中旧培养液,用1×PBS(pH=7.0)冲洗培养瓶3遍,并着重冲洗培养瓶底部后,加入胰蛋白酶-EDTA消化液(0.25%)10ml,于培养箱内消化5min,取出后于倒置显微镜下观察,若贴壁细胞数量较多,轻轻晃荡培养瓶,当大部分细胞漂浮时,加入等体积的DMEM完全培养基终止消化;最后,1000rpm离心10-20min,并将细胞以1:2进行传代,标记为P1;此后,每隔2天更换新鲜培养液,重复上述步骤进行P2、P3代稳定细胞。
优选地,步骤3)包括:取生长状态良好的P3细胞,胰蛋白酶-EDTA消化液(0.25%)消化细胞,加入DMEM培养基,重悬,制成1.0×106/ml的细胞悬液,利用流式细胞仪检测CD29,CD44,CD73,CD90,CD105,CD146、CD45、CD34和CD117标记;结果显示:P3代细胞高表达CD29,CD44,CD73,CD90,CD105,CD146(>95.3%),低表达CD34,CD45、CD117(<5.7%)。
优选地,步骤4)包括:将修复肽SEQ ID NO.1的编码序列SEQ ID NO.2与pEGFP-N1载体连接,通过电穿孔方式导入步骤3)宫内膜干细胞进行稳定表达。
本发明的另一目的在于提供了一种宫内膜干细胞水凝胶的制备方法,其特征在于,所述方法包括如下步骤:
1)将10-40mg海藻酸钠和1.0×106-109cells实施例1得到的宫内膜干细胞溶解于50-100mL生理盐水;
2)向步骤1)中缓慢滴加5-10mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置5-10min即可得到宫内膜干细胞水凝胶。
进一步优先地,所述宫内膜干细胞水凝胶的制备方法包括如下步骤:
1)将10mg海藻酸钠和1.0×106cells实施例1得到的宫内膜干细胞溶解于50mL生理盐水;
1)向步骤1)中缓慢滴加5mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置5min即可得到宫内膜干细胞水凝胶。
进一步优先地,所述宫内膜干细胞水凝胶的制备方法包括如下步骤:
1)将30mg海藻酸钠和1.0×108cells实施例1得到的宫内膜干细胞溶解于70mL生理盐水;
2)向步骤1)中缓慢滴加8mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置8min即可得到宫内膜干细胞水凝胶。
进一步优先地,所述宫内膜干细胞水凝胶的制备方法包括如下步骤:
1)将50mg海藻酸钠和1.0×109cells实施例1得到的宫内膜干细胞溶解于100mL生理盐水;
2)向步骤1)中缓慢滴加10mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置10min即可得到宫内膜干细胞水凝胶。
进一步地,本发明还提供了一种宫内膜干细胞水凝胶在制备阴道结缔组织弹性黏膜修复药物中的用途。
本发明的优点如下:本发明首次提供了利用修复肽基因改造宫内膜干细胞,提高其阴道结缔组织弹性黏膜修复功能,同时,该修复肽也具备良好的抑菌活性。经改造后的宫内膜干细胞能够有效抑制阴道细菌的生长,改善阴道健康状况,同时兼具结缔组织弹性黏膜修复功能,能有效修复子宫内膜,恢复其正常厚度,有效提高了生育能力。
附图说明
图1.不同处理组下的抑菌能力分析;
图2.不同处理下对子宫内膜细胞角蛋白,波形蛋白,血管内皮生长因子和雌激素受体α表达量的影响分析。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
以下各实施例,仅用于说明本发明,并不用来限制本发明的范围。基于本发明中的具体实施例,本领域普通技术人员在没有做出创造性劳动的情况下,所获得的其他所有实施例,都属于本发明的保护范围。
在本发明实施例中,若无特殊说明,所有原料组分均为本领域技术人员熟知的市售产品;在本发明实施例中,若未具体指明,所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1
一种宫内膜干细胞制备方法,其特征在于,所述方法包括如下步骤:
1)宫内膜干细胞的分离
取子宫内膜组织,剔除脂肪、系膜,并用1×PBS(pH=7.0)进行冲洗,并用手术剪将子宫内膜组织剪碎。采用0.25%胰蛋白酶,37℃震荡酶解消化子宫内膜组织碎片40-60min。并向消化液中加入等体积含10%FBS的DMEM完全培养基终止消化。收集消化液,1000rpm离心10-20min,弃掉上清液,将离心后所得沉淀加入DMEM完全培养基吹打均匀后,接种于25ml培养瓶中,在37℃、5%CO2培养箱内培养,每隔2天更换新鲜培养液。
2)宫内膜干细胞的培养
待步骤1)中的细胞长至约80%融合时进行传代。在超净工作台内弃掉培养瓶中旧培养液,用1×PBS(pH=7.0)冲洗培养瓶3遍,并着重冲洗培养瓶底部后,加入胰蛋白酶-EDTA消化液(0.25%)10ml,于培养箱内消化5min,取出后于倒置显微镜下观察,若贴壁细胞数量较多,轻轻晃荡培养瓶,当大部分细胞漂浮时,加入等体积的DMEM完全培养基终止消化。最后,1000rpm离心10-20min,并将细胞以1:2进行传代,标记为P1;此后,每隔2天更换新鲜培养液,重复上述步骤进行P2、P3代稳定细胞。
7)宫内膜干细胞的鉴定
取生长状态良好的P3细胞,胰蛋白酶-EDTA消化液(0.25%)消化细胞,加入DMEM培养基,重悬,制成1.0×106/ml的细胞悬液,利用流式细胞仪检测CD29,CD44,CD73,CD90,CD105,CD146、CD45、CD34和CD117标记。结果显示:P3代细胞高表达CD29,CD44,CD73,CD90,CD105,CD146(>95.3%),低表达CD34,CD45、CD117(<5.7%)。
8)宫内膜干细胞基因改造
将修复肽SEQ ID NO.1的编码序列SEQ ID NO.2与pEGFP-N1载体连接,通过电穿孔方式导入步骤3)宫内膜干细胞进行稳定表达。
实施例2
一种宫内膜干细胞水凝胶的制备,其特征在于,所述方法包括如下步骤:
2)将10mg海藻酸钠和1.0×106cells实施例1得到的宫内膜干细胞溶解于50mL生理盐水;
3)向步骤1)中缓慢滴加5mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置5-10min即可得到宫内膜干细胞水凝胶。
实施例3
一种宫内膜干细胞水凝胶的制备,其特征在于,所述方法包括如下步骤:
3)将30mg海藻酸钠和1.0×108cells实施例1得到的宫内膜干细胞溶解于70mL生理盐水;
4)向步骤1)中缓慢滴加8mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置8min即可得到宫内膜干细胞水凝胶。
实施例4
一种宫内膜干细胞水凝胶的制备,其特征在于,所述方法包括如下步骤:
3)将50mg海藻酸钠和1.0×109cells实施例1得到的宫内膜干细胞溶解于100mL生理盐水;
4)向步骤1)中缓慢滴加10mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置10min即可得到宫内膜干细胞水凝胶。
对比例1
方法步骤同实施例2,区别在于,宫内膜干细胞未经基因改造。
试验例1
抗菌性能分析:将阴道乳杆菌和阴道大肠埃希菌分别在LB液体培养基中培养12h,37℃,150rpm。然后将细菌在无菌的LB液体培养基中稀释至OD600=2.0,取实施例和对比例水凝胶200μL至1.5mL Ep管中,并取200μL稀释细菌加入所述Ep管,然后置摇床中37℃,150rpm。培养12h后测OD600,以各管中所取菌液的OD600为纵坐标作柱状图,以PBS溶液作为空白组,验证宫内膜干细胞水凝胶的抗菌活性。
试验例2
子宫内膜机械损伤模型构建:取动情期BALB/C裸雌性鼠,空腹注射1.5%戊巴比妥钠进行麻醉处理,剂量为80mg/kg;待裸鼠无肢体反应后,采用仰卧位,将其四肢固定在铺有无菌垫巾的操作台上。用75%酒精消毒术区,于下腹部正中线膀肤上方1.5cm处行纵行切口,用眼科镊提起皮层,眼科剪纵行逐层剪开,于膀肤后方找到子宫,缓慢挑出;于“丫”型子宫,子宫充盈。用直镊夹起子宫输卵管端,充分暴露子宫角,将右侧子宫角切开切口,用1ml注射器针头从子宫角处进入子宫腔三分之二处,进行旋转和来回搔刮4次机械损伤子宫内膜,复位子宫。
其中,空白组模型小鼠不经任何治疗直接进行复位子宫;实验组小鼠在机械损伤子宫内膜后,用微量注射器取25μl的实施例2所述的宫内膜干细胞水凝胶液,自右侧子宫角切口处进针,将细胞混悬液缓慢注射至右侧宫腔内,留针20s,复位子宫;对照组进行同样处理,不同在于其注射凝胶为对比例1。
子宫复位后,用青霉素钠盐溶液1-2ml滴洒于腹腔;在切口处滴洒青霉素钠盐溶液,逐层缝合肌肉及皮肤。将裸鼠移至37℃恒温台上,做好标记,待其自然苏醒后,移入SPF级饲养室饲养。饲养7d后脱颈处死,解剖暴露双侧子宫,做好标记,置于4%多聚甲醛中固定行冰冻切片,并对冰冻切片进行HE染色。采用IMS图像分析系统测量子宫内膜厚度,每张待测片选取3~4个区域测量子宫内膜厚度,取平均值作为被测子宫内膜的子宫内膜厚度。并对角蛋白,波形蛋白,血管内皮生长因子和雌激素受体α进行免疫组化鉴定。
表1子宫内膜厚度
组别 | 子宫内膜厚度(μm) |
实施例2 | 398.5±12.25 |
实施例3 | 400.65±13.69 |
实施例4 | 410.54±20.25 |
对比例1 | 152.87±10.58 |
空白组 | 101.3±25.36 |
结果显示:与空白组相比,实施例2-4的子宫内膜平均厚度得到明显改善,差异具有显著性(P<0.05),厚度也逐步恢复到正常水平。进一步证实,本发明实施例2-4所述的宫内膜干细胞水凝胶具备良好的修复功能,能有效促进阴道结缔组织弹性黏膜修复,提高子宫内膜整体上的厚度。
另外,如图2所示,免疫组织化学结果显示实施例2-4处理下,子宫内膜细胞角蛋白,波形蛋白,血管内皮生长因子和雌激素受体α表达量明显升高,与对比例1相比,差异显著(P<0.05)。该四种蛋白的表达上调,说明本发明实施例提供的宫内膜干细胞水凝胶对小鼠子宫内膜具有修复作用。
在此有必要指出的是,以上实施例仅限于对本发明的技术方案做进一步的阐述和说明,并不是对本发明的技术方案的进一步的限制,本发明的方法仅为较佳的实施方案,并非用于限定本发明的保护范围。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)
1.一种宫内膜干细胞制备方法,其特征在于,所述方法包括如下步骤:
1)宫内膜干细胞的分离;
2)宫内膜干细胞的培养;
3)宫内膜干细胞的鉴定;
4)利用修复肽改造宫内膜干细胞。
2.如权利要求1所述的方法,其特征在于,步骤1)包括:
取子宫内膜组织,剔除脂肪、系膜,并用1×PBS(pH=7.0)进行冲洗,并用手术剪将子宫内膜组织剪碎;采用0.25%胰蛋白酶,37℃震荡酶解消化子宫内膜组织碎片40-60min;并向消化液中加入等体积含10%FBS的DMEM完全培养基终止消化;收集消化液,1000rpm离心10-20min,弃掉上清液,将离心后所得沉淀加入DMEM完全培养基吹打均匀后,接种于25ml培养瓶中,在37℃、5%CO2培养箱内培养,每隔2天更换新鲜培养液。
3.如权利要求1所述的方法,其特征在于,步骤2)包括:待步骤1)中的细胞长至80%融合时进行传代;在超净工作台内弃掉培养瓶中旧培养液,用1×PBS(pH=7.0)冲洗培养瓶3遍,并着重冲洗培养瓶底部后,加入胰蛋白酶-EDTA消化液(0.25%)10ml,于培养箱内消化5min,取出后于倒置显微镜下观察,若贴壁细胞数量较多,晃荡培养瓶,当大部分细胞漂浮时,加入等体积的DMEM完全培养基终止消化;最后,1000rpm离心10-20min,并将细胞以1:2进行传代,标记为P1;此后,每隔2天更换新鲜培养液,重复上述步骤进行P2、P3代稳定细胞。
4.如权利要求1所述的方法,其特征在于,步骤3)包括:取生长状态良好的P3细胞,胰蛋白酶-EDTA消化液(0.25%)消化细胞,加入DMEM培养基,重悬,制成1.0×106/ml的细胞悬液,利用流式细胞仪检测CD29,CD44,CD73,CD90,CD105,CD146、CD45、CD34和CD117标记;结果显示:P3代细胞高表达CD29,CD44,CD73,CD90,CD105,CD146(>95.3%),低表达CD34,CD45、CD117(<5.7%)。
5.如权利要求1所述的方法,其特征在于,步骤4)包括:将修复肽SEQ ID NO.1的编码序列SEQ ID NO.2与pEGFP-N1载体连接,通过电穿孔方式导入步骤3)宫内膜干细胞进行稳定表达。
6.一种宫内膜干细胞水凝胶的制备方法,其特征在于,所述方法包括如下步骤:
1)将10-40mg海藻酸钠和1.0×106-109cells权利要求1-5任一项所述方法得到的宫内膜干细胞溶解于50-100mL生理盐水;
2)向步骤1)中缓慢滴加5-10mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置5-10min即可得到宫内膜干细胞水凝胶。
7.如权利要求6所述的方法,其特征在于,所述宫内膜干细胞水凝胶的制备方法包括如下步骤:
1)将10mg海藻酸钠和1.0×106cells实施例1得到的宫内膜干细胞溶解于50mL生理盐水;
2)向步骤1)中缓慢滴加5mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置5min即可得到宫内膜干细胞水凝胶。
8.如权利要求6所述的方法,其特征在于,所述宫内膜干细胞水凝胶的制备方法包括如下步骤:
1)将30mg海藻酸钠和1.0×108cells实施例1得到的宫内膜干细胞溶解于70mL生理盐水;
2)向步骤1)中缓慢滴加8mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置8min即可得到宫内膜干细胞水凝胶。
9.如权利要求6所述的方法,其特征在于,所述宫内膜干细胞水凝胶的制备方法包括如下步骤:
1)将50mg海藻酸钠和1.0×109cells实施例1得到的宫内膜干细胞溶解于100mL生理盐水;
2)向步骤1)中缓慢滴加10mM CaCl2水溶液,滴加过程同时进行涡旋混匀处理,至溶液透亮发白为止,静置10min即可得到宫内膜干细胞水凝胶。
10.一种如权利要求6-9任一项所述方法制备得到的宫内膜干细胞水凝胶在制备阴道结缔组织弹性黏膜修复药物中的用途。
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