CN115192611B - 一种具有修复输卵管功效的复合制剂 - Google Patents
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Abstract
本发明公开了一种具有修复输卵管功效的复合制剂,属于生物医药技术领域。所述复合制剂由以下组分组成:输卵管内膜干细胞悬液,1~3ml;脱细胞输卵管内膜,2~4g;PRP,0.5~1.2ml。本发明的具有修复输卵管功效的复合制剂,含有输卵管内膜干细胞,可以修复输卵管内膜;含有PRP,提供生长因子;含有脱细胞输卵管内膜作为内膜干细胞的支架;所有组分共同作用,可起到修复输卵管内膜的治疗效果。
Description
技术领域
本发明涉及一种具有修复输卵管功效的复合制剂,属于生物医药技术领域。
背景技术
众所周知,受孕是个很复杂的过程,它要求精卵结合形成受精卵,最后着床于宫腔。除了要有正常的精子、卵子和适当的子宫内环境外,使精子、卵子能够相遇并顺利运送到宫腔也是受孕过程中的重要环节,这个任务是由输卵管来完成的。输卵管不仅仅是连接卵巢和子宫的渠道,而且还具有排卵、贮卵、输精、提供精卵结合的场所、输送孕卵至宫腔以便及时到达宫腔内膜的功能。因输卵管有炎症,导致输卵管堵塞,精子不能通过并与卵子相遇造成的不孕,称为输卵管性不孕。因此如何修复输卵管是治疗输卵管性不孕的有效措施。
人类输卵管是一个管状器官,由粘膜和两个交织在一起的光滑肌层组成,覆盖着浆膜。黏膜上皮为单层柱状,由纤毛细胞和分泌细胞组成。纤毛细胞在漏斗部和壶腹部最多,峡部和子宫部则逐渐减少。纤毛向子宫方向的摆动有助于卵子的运送。夹在纤毛细胞之间的分泌细胞虽无纤毛,但有微绒毛,其分泌物构成输卵管液,含有氨基酸、果糖及少量乳酸等,该分泌物在纤毛表面形成粘稠的膜,这不仅对卵细胞有营养作用,而且还有助于卵子向子宫输送,并防止病菌从子宫经输卵管入腹腔。输卵管粘膜上皮在卵巢激素的影响下随月经周期而发生周期性变化。
与子宫内膜类似,输卵管粘膜中存在多能干细胞,输卵管粘膜是自体多能干细胞的新来源。可以通过活检获得的输卵管粘膜是一种可用于再生医学的新工具。2009年,Jazedje等首次证实外科手术废弃的输卵管组织中存在间充质干细胞(MSC)。2012年Jazedje等又报道将人输卵管间充质干细胞(FTMSC)移植到骨损伤大鼠体内,可增强其骨再生能力。2013年Indumathi等再次证实了输卵管中MSC的存在,并对输卵管间充质干细胞与骨髓MSC的生物学特性进行了比较,认为FTMSC可以取代骨髓MSC用于再生医学,作为一种新的干细胞来源。
富血小板血浆(Platelet-rich plasma,PRP)为自体周围静脉血经细胞分离提取得到的浓缩血小板血浆。PRP富含血小板源生长因子,血管内皮生长因子、转化生长因子、表皮生长因子、胰岛素生长因子、成纤维细胞生长因子等,可调节细胞间的迁移、扩散、分化,促进细胞外基质的积累。另外活化的PRP促进了人类子宫内膜干/祖细胞的迁移,如经血间充质干细胞、子宫内膜间充质干细胞和骨髓间充质干细胞等。促进间充质细胞和上皮细胞之间的转化。PRP在眼科、整形外科、骨科等领域应用广泛,其能刺激脂肪、皮肤和黏膜等软组织以及肌腱、骨骼等组织的再生。
中国发明专利申请CN 112535767 A公开了一种输卵管支架的制备方法,其中使用了生物降解材料进行熔融纺丝和编织,得到具有三维立体结构的输卵管支架。但是该组合物虽然是一种可以降解的生物材料,具有一定的力学性能,但是没有细胞成份和生长因子,难以获得满意的治疗效果。
发明内容
针对上述现有技术,本发明提供了一种具有修复输卵管功效的复合制剂。
本发明是通过以下技术方案实现的:
一种具有修复输卵管功效的复合制剂,由以下组分组成:输卵管内膜干细胞悬液,1~3ml;脱细胞输卵管内膜,2~4g;PRP,0.5~1.2ml。
所述输卵管内膜干细胞悬液是通过以下方法制备得到的:
洗去输卵管内膜组织表面残留血液;剪碎,置于胶原酶溶液中消化;离心收集组织块及细胞,过细胞滤网收集细胞;将细胞接种于间充质干细胞培养基中,置入培养箱内,37℃、5%CO2、饱和湿度培养,每3天换液一次;待原代收集后每3~4天使用胰蛋白酶消化传代培养,所得细胞悬液即为输卵管内膜干细胞悬液,调整细胞密度至0.5×107~3×107个/ml。
进一步地,所述间充质干细胞培养基是以BI基础培养基为基础,添加10%的无血清添加剂、10ng/ml的CD10、20ng/ml的CD14和50ng/ml的重组人Wnt3a蛋白而成。
所述脱细胞输卵管内膜是通过以下方法制备得到的:
(1)洗去输卵管组织表面的残留血液;剪碎得输卵管组织碎片;
(2)输卵管组织碎片在-80℃和37℃下分别进行三次冷冻/解冻循环;
(3)输卵管组织碎片在室温下、在1%的Triton X-100溶液、50rpm条件下孵化15小时;
(4)将输卵管组织碎片取出,置于浓度1%的SDS溶液中72小时;
(5)将输卵管组织碎片取出,冲洗,即得脱细胞输卵管内膜。
进一步地,所述PRP,最好是自体PRP,以避免排异反应、过敏反应等。
所述具有修复输卵管功效的复合制剂在制备具有修复输卵管内膜功效的制剂中的应用。
现有技术中使用的支架材料主要是凝胶或胶原材料,其生物特性无法与真正的输卵管内膜相比,而脱细胞的输卵管内膜含有丰富的细胞外基质,可以模拟输卵管的微环境,如三维结构及其细胞与细胞外基质的相互作用。本发明的脱细胞输卵管内膜,可以支持人输卵管内膜干细胞的体外培养,可以提高输卵管内膜干细胞的增殖率。
本发明的具有修复输卵管功效的复合制剂,含有输卵管内膜干细胞,可以修复输卵管内膜。含有PRP,提供生长因子。含有脱细胞输卵管内膜作为内膜干细胞的支架。所有组分共同作用,起到修复输卵管内膜的治疗效果。实际应用时,临床上由于输卵管积水等原因经常切除一段输卵管,为输卵管内膜脱细胞支架的制备创造了一定的条件。
附图说明
图1:内膜干细胞流式细胞鉴定结果示意图。
图2:HE染色结果示意图,其中,A:空白组;B:模型组;C:治疗组。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1输卵管内膜干细胞的培养及鉴定
1.输卵管内膜干细胞的培养:无菌采集输卵管组织,可保存于混有青霉素+链霉素+两性霉素B(碧云天,C0224)的DMEM培养液(青霉素、链霉素、两性霉素B的浓度分别为0.1kU/ml,0.1mg/ml,0.25μg/ml),保存时间不超过8h。
用PBS缓冲液洗去输卵管组织表面的残留血液;剪碎至1~2mm3的组织块,置于0.5%胶原酶(merck,C2674-100mg)溶液中消化1小时。2000r/min离心8min收集组织块及细胞,过100μm细胞滤网,收集细胞,PBS缓冲液(Gibco,C10010)洗涤2次。将细胞接种于间充质干细胞培养基(BI,05-200-1A,添加10%无血清添加剂,Life Sciences,15950)中,置入培养箱内,37℃、5%CO2、饱和湿度培养,每3天换液一次。待原代收集后每3~4天使用胰蛋白酶消化传代培养,直到细胞密度达到90%汇合时,收集细胞悬液用于后续实验。
所述间充质干细胞培养基中添加有10ng/ml的CD10(Acro)、20ng/ml的CD14(Acro)和50ng/ml的重组人Wnt3a蛋白(abcam)。
2.输卵管内膜干细胞的鉴定:
采用流式细胞技术检测细胞表面标志物,其中上皮细胞标志物为:CD9,CD326;周细胞标志物为:SUSD2,CD140;间充质干细胞标志物为:CD73,CD90,CD105,HLA-DR;造血干细胞标志物为:CD34,CD45,单核细胞标志物:CD14,B细胞标志物为CD19。
(1)将1ml细胞悬液转移至1.5ml离心管中。4℃,300g离心5分钟,仔细吸取上清。
(2)用适量PBS缓冲液清洗细胞,4℃,300g离心5分钟,仔细吸取上清。
(3)用预冷(4℃)的PBS缓冲液重悬细胞,调整细胞终浓度为1×107细胞/ml,轻轻吹打混匀。
(4)取100μl细胞悬液作为空白对照组,取100μl细胞悬液作为同型对照组加入同型对照抗体,取200μl作为实验组加入检测抗体(均购自BD公司),4℃孵育40分钟。
(5)加入适量PBS缓冲液清洗细胞,4℃,300g离心5分钟,仔细吸取上清。
(6)用500μl的PBS缓冲液重悬细胞,上机检测。
结果:如图1所示,低表达cd34,cd45和HLA-DR。具体为:
周细胞标志物:SUSD2+80.32%,CD140+1.00%;
上皮细胞标志物:CD9+98.69%,CD326+1.58%;
间充质干细胞的标志物:cd73+99.98%,cd90+99.26%,cd105+98.97%,HLA-DR0.78%;
造血干细胞标志物:cd34+0.04%,cd45+1.60%。
B细胞标志物:cd19+0.50%。
单核细胞标志物:cd14+1.18%。
结果分析:通过分析,培养的输卵管内膜细胞为间充质干细胞,高表达cd73,cd90,cd105;低表达cd34,cd45和HLA-DR,部分表达周细胞和上皮细胞的标志物,低表达B细胞和单核细胞的标志物。
实施例2PRP的制备
(1)提供健康志愿者的传染病检测报告信息(包括乙肝、丙肝、HIV、梅毒),确保传染病检测的真实性和准确性,传染病阳性的需除外。
(2)严格按照无菌要求采集外周血50ml,使用专用血袋(已包含抗凝剂)采集血液,采集过程中,边采集边缓慢上下摇晃血袋,以防止凝血。
(3)采集外周血后,采血袋外粘贴标签,标注姓名等相关信息。
(4)采集血液在24h内进行制备,轻离心,收集上层富含血小板的血浆。
(5)重离心,调整血小板浓度到合适水平,放置50min,加入凝血酶(蓬莱诺康药业,H20041419)及葡萄糖酸钙(上海浦津林州制药有限公司,H41022648)(PRP/激活剂9:1),3min之内形成凝胶。
实施例3脱细胞输卵管内膜的制备
(1)无菌采集输卵管组织,用PBS缓冲液洗去输卵管组织表面的残留血液;剪碎至1~2mm3的组织块,得输卵管组织碎片。
(2)输卵管组织碎片在-80℃和37℃下分别进行三次冷冻/解冻循环。
(3)输卵管组织碎片在室温下、在1%的Triton X-100中、在旋转器(50rpm)中孵化15小时。
(4)将输卵管组织碎片取出,置于浓度1%的SDS溶液中72小时。
(5)将输卵管组织碎片取出,在室温下、在旋转器(50rpm)中用PBS缓冲液冲洗48小时,即得脱细胞输卵管内膜。
实施例4复合制剂的制备
将实施例1的干细胞悬液调整到密度为1×107个/ml(加入生理盐水稀释)。取2ml,加入3g的脱细胞输卵管内膜和1ml的PRP(实施例2制备),即得复合制剂。
实验1大鼠输卵管损伤模型的建立
将SPF级雌性SD大鼠(鼠龄10周,体重270克)用10%水合氯醛溶液腹腔麻醉,向输卵管注入4×109CFU/ml的金黄色葡萄球菌菌液0.1ml,待大鼠伤口愈合(1周)后,行声光电、夹尾、倾斜鼠笼复合刺激,持续3周。即建立得到输卵管损伤的大鼠模型。
实验2复合制剂的动物临床实验
1.将输卵管损伤的大鼠分为两组:模型组和治疗组,每组4只;同时以健康的SPF级SD大鼠作为空白组(4只)。
治疗方式:治疗组注射0.1ml的复合制剂(实施例4制备)(用异氟醚麻醉,体温保持在37±0.5℃;然后打开腹壁,输卵管暴露出来,向输卵管一侧注射0.1ml的复合制剂),注射一次;14天后观察治疗效果。模型组与治疗组的不同之处在于:采用生理盐水代替复合制剂。空白组与治疗组的不同之处在于:采用生理盐水代替复合制剂。
2.HE染色输卵管组织形态观察:治疗第14天,每组随机取3只大鼠,开腹后肉眼观察输卵管形态、色泽、弹性、管壁厚度,有无积水脓肿,管壁有无血管肿胀及与周围组织有无黏连等,取双侧输卵管,进行HE染色观察其病理变化,结果如图2所示。
结果分析:空白组大鼠输卵管管壁颜色红润,质地光滑,粗细均匀,弹性良好,与周围组织无粘连,未见炎性细胞浸润,结构良好。模型组大鼠输卵管管壁颜色紫暗,增粗积水,弹性差,与周围组织粘连,可见输卵管周围大量炎细胞浸润(淋巴细胞为主),组织肿胀明显,黏膜上皮细胞脱落,输卵管组织结构排列紊乱。治疗组大鼠输卵管组织周围见少量炎性细胞,上皮细胞轻度脱落。这表明,复合制剂对输卵管损伤具有修复作用。
实验3体外共培养实验
使用移液枪向事先加入了输卵管内膜细胞(细胞浓度为1×105个/ml)的六孔板(每孔500μl)内分别加入复合制剂(制备方法见实施例4)、PRP凝胶(制备方法见实施例2),每孔500μl,轻轻摇动6孔板,使充分混匀,37℃静置20min,使其充分凝固。再在其上加入含10%胎牛血清的DMEM培养液3ml,放置在培养箱中37℃、饱和湿度培养,4天后用四甲基偶氮唑盐染色,倒置显微镜下计数直径超过0.5mm的细胞克隆,根据公式计算克隆形成率:克隆形成率=(细胞克隆数*100/接种的细胞数)。结果如表1所示。
表1细胞克隆形成率
组别 | 克隆形成率 | P值 |
复合制剂处理组 | 16.8±1.02 | |
PRP凝胶处理组 | 11.2±1.25 | 0.005 |
结果表明,经过四天的培养,输卵管内膜细胞逐渐形成细胞克隆,复合制剂处理形成的克隆明显多于PRP凝胶,具有统计学意义。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (4)
1.一种具有修复输卵管功效的复合制剂,其特征在于,由以下组分组成:输卵管内膜干细胞悬液,2 ml;脱细胞输卵管内膜,3 g;PRP,1 ml;其中,所述输卵管内膜干细胞悬液的细胞密度为1×107个/ml。
2.根据权利要求1所述的具有修复输卵管功效的复合制剂,其特征在于,所述输卵管内膜干细胞悬液是通过以下方法制备得到的:
洗去输卵管内膜组织表面残留血液;剪碎,置于胶原酶溶液中消化;离心收集组织块及细胞,过细胞滤网收集细胞;将细胞接种于间充质干细胞培养基中,置入培养箱内,37℃、5%CO2、饱和湿度培养,每3天换液一次;待原代收集后每3~4天使用胰蛋白酶消化传代培养,得输卵管内膜干细胞悬液,调整细胞密度至1×107个/ml。
3.根据权利要求2所述的具有修复输卵管功效的复合制剂,其特征在于:所述间充质干细胞培养基是以BI基础培养基为基础,添加10%的无血清添加剂、10 ng/ml的CD10、20 ng/ml的CD14和50 ng/ml的重组人Wnt3a蛋白而成。
4.根据权利要求1所述的具有修复输卵管功效的复合制剂,其特征在于,所述脱细胞输卵管内膜是通过以下方法制备得到的:
(1)洗去输卵管组织表面的残留血液;剪碎得输卵管组织碎片;
(2)输卵管组织碎片在-80℃和37℃下分别进行三次冷冻/解冻循环;
(3)输卵管组织碎片在室温下、在1%的Triton X-100溶液、50 rpm条件下孵化15小时;
(4)将输卵管组织碎片取出,置于浓度1%的SDS溶液中72小时;
(5)将输卵管组织碎片取出,冲洗,即得脱细胞输卵管内膜。
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CN112043727A (zh) * | 2020-09-17 | 2020-12-08 | 山西宾大干细胞生物科技有限公司 | 一种细胞复合型子宫内膜修复凝胶的制备方法及应用 |
CN113244272A (zh) * | 2021-06-18 | 2021-08-13 | 陕西中鸿科瑞再生医学研究院有限公司 | 用于改善卵巢早衰的组合物及其制备方法和应用 |
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CN105663168A (zh) * | 2016-01-27 | 2016-06-15 | 深圳爱生再生医学科技有限公司 | 用于修复卵巢功能的细胞制剂 |
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CN112043727A (zh) * | 2020-09-17 | 2020-12-08 | 山西宾大干细胞生物科技有限公司 | 一种细胞复合型子宫内膜修复凝胶的制备方法及应用 |
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