CN113730557B - 重组i型人源化胶原蛋白在盆底修复中的用途 - Google Patents
重组i型人源化胶原蛋白在盆底修复中的用途 Download PDFInfo
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- CN113730557B CN113730557B CN202111031518.8A CN202111031518A CN113730557B CN 113730557 B CN113730557 B CN 113730557B CN 202111031518 A CN202111031518 A CN 202111031518A CN 113730557 B CN113730557 B CN 113730557B
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Abstract
本发明属于生物医药领域,涉及重组I型人源化胶原蛋白在盆底修复中的用途,具体而言,本发明拟提供了该种重组I型人源化胶原蛋白在制备用于预防和/或治疗盆底疾病(尤其是女性盆底功能障碍性疾病)的药物中的用途,所述重组I型人源化胶原蛋白包含以SEQ ID No.1所示的序列的n个重复,n为大于等于1的整数,其中当n为大于等于2的整数时,各重复序列之间是直接连接的。本发明在实验中证明重组I型人源化胶原蛋白可以增强膀胱储尿功能,改善腹部漏尿点压,增强阴道抗张能力,增厚阴道固有层和肌层,增加阴道固有层结缔组织的血管数量和胶原纤维,促进细胞外基质合成,促进骶韧带成纤维细胞早期黏附。
Description
技术领域
本发明属于生物医药领域,涉及重组I型人源化胶原蛋白在盆底修复中的用途。
背景技术
女性盆底功能障碍性疾病(female pelvic floor dysfunction,FPFD)是由盆腔支持结构损伤、退化或功能缺陷而引起的一组难治性妇科疾病。随着我国人口老龄化程度加深,FPFD的发病率将急剧增加,预计2050年,FPFD发病率将会超过50%。目前盆底重建手术是主要治疗方法,但复发率20%-40%。既往临床尝试使用动物源性胶原及盆底合成网片治疗FPFD,治疗有效率均在60%以上,但存在局部慢性糜烂、持续的疼痛、免疫反应等问题,目前已逐渐趋于淘汰。FPFD发生的主要组织学病理特征有:成纤维细胞合成I/III胶原蛋白合成减少、降解增多,盆底肌肉、韧带、筋膜等结缔组织力学抗性减退,细胞外基质代谢异常已被认为是FPFD发病过程中的重要病理基础。
目前已知成纤维细胞是盆底合成细胞外基质的主要效应细胞。动物源性I型胶原蛋白可以增强促进皮肤伤口、牙龈成纤维细胞粘附,促进细胞外基质合成能力,调节细胞外基质重塑,局部注射重组人I、III型胶原蛋白凝胶可以改善心肌细胞外基质重塑,减轻晚期心肌梗塞及心肌纤维化程度。发明人团队前期研究发现,局部应用重组人III型胶原蛋白可以促进阴道上皮增生及增强阴道上皮细胞外基质重塑。申请号为202110968550.2、发明名称为“一种重组I型人源化胶原蛋白多肽及其制备方法和用途”的专利记载了本发明的发明人在之前研究并生产的重组I型人源化胶原蛋白,但并未直接披露其在医药领域中的具体用途。
发明内容
发明要解决的问题
本发明拟提供利用申请号为202110968550.2的专利申请中的重组I型人源化胶原蛋白来恢复尿道动力学性能和阴道生物力学性能,改善局部组织结构,促进细胞外基质合成,进而对盆底疾病(特别是女性盆底功能障碍性疾病)的预防和/或治疗起到有效作用。
用于解决问题的方案
第一方面,本发明提供了重组I型人源化胶原蛋白在制备用于预防和/或治疗盆底疾病的药物中的用途,其中:所述重组I型人源化胶原蛋白包含以SEQ ID No.1所示的序列的n个重复,n为大于等于1的整数,其中当n为大于等于2的整数时,各重复序列之间是直接连接的;任选地,在所述重组I型人源化胶原蛋白多肽的N末端包含能够通过TEV蛋白酶切除的氨基酸序列。
进一步地,在上述用途中,所述能够通过TEV蛋白酶切除的氨基酸序列包含以SEQID No.2所示的序列;优选地,以SEQ ID No.2所示的序列与以SEQ ID No.1所示的序列直接相连。
优选地,在上述用途中,所述n为4。
进一步地,在上述用途中,所述重组I型人源化胶原蛋白包含以SEQ ID No.3所示的氨基酸序列。
进一步地,在上述用途中,所述盆底疾病为盆底功能障碍性疾病。
优选地,在上述用途中,所述盆底功能障碍性疾病为女性盆底功能障碍性疾病。
进一步地,在上述用途中,所述女性盆底功能障碍性疾病具有下列症状中的至少一种:尿道动力学性能下降、阴道生物力学性能下降、局部组织结构损伤、细胞外基质成分表达降低、骶韧带功能异常。
进一步地,在上述用途中,所述尿道动力学性能下降表现为下列特征中的至少一种:膀胱储尿功能下降、腹部漏尿点压降低。
进一步地,在上述用途中,所述局部组织结构损伤表现为下列特征中的至少一种:阴道和尿道周围组织上皮层变薄、固有层的结缔组织中的血管数量和胶原纤维减少、肌层萎缩断裂。
进一步地,在上述用途中,所述细胞外基质成分包含I型胶原蛋白和III型胶原蛋白中的至少一种。
进一步地,在上述用途中,所述骶韧带功能异常表现为下列特征中的至少一种:骶韧带成纤维细胞早期黏附能力下降、骶韧带成纤维细胞早期黏附过程细胞铺展面积减小。
发明的效果
本发明研究结果表明,重组I型人源化胶原蛋白可以明显增强膀胱储尿功能,改善腹部漏尿点压,增强阴道抗张能力,增厚阴道固有层和肌层,增多固有层的结缔组织中的血管和胶原纤维数量,促进细胞外基质成分的表达,促进骶韧带成纤维细胞早期黏附,从而为预防和/或治疗女性盆底功能障碍性疾病的药物制备奠定基础,可以用于预防和/或治疗女性盆底功能障碍性疾病。
附图说明
图1为各组大鼠膀胱尿动力学测试结果。
图2为各组大鼠阴道生物力学测试结果。
图3为各组大鼠阴道和尿道局部组织结构染色结果。
图4为各组大鼠阴道组织细胞外基质合成情况测试结果。
图5为大鼠盆底多点注射重组I型人源化胶原蛋白后骶韧带成纤维细胞表型变化情况的分析结果。
具体实施方式
以下将结合具体的实施例来进一步阐述本发明的技术方案。本发明所述重组I型人源化胶原蛋白为申请号为202110968550.2、发明名称为“一种重组I型人源化胶原蛋白多肽及其制备方法和用途”的专利记载。
本说明书中,使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。本说明书中,“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生,并且该描述包括该事件发生的情况和该事件不发生的情况。
在一项实施方案中,本发明的重组I型人源化胶原蛋白包含以SEQ ID No.1所示的序列(由60个氨基酸组成,从N端至C端分别为GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS)的n个重复,其中n为大于等于1的整数,当n为大于等于2的整数时,各重复序列之间是直接连接的;任选地,在所述重组I型人源化胶原蛋白的N末端包含能够通过TEV蛋白酶切除的氨基酸序列。
在一项优选的实施方案中,本发明的重组I型人源化胶原蛋白包含以SEQ ID No.1所示的序列的n个重复,其中n为大于等于1的整数,当n为大于等于2的整数时,各重复序列之间是直接连接的,并且在所述重组I型人源化胶原蛋白的N末端包含能够通过TEV蛋白酶切除的氨基酸序列。
在一项更优选的实施方案中,本发明的重组I型人源化胶原蛋白包含以SEQ IDNo.1所示的序列的n个重复,其中n为大于等于1的整数,当n为大于等于2的整数时,各重复序列之间是直接连接的,并且在所述重组I型人源化胶原蛋白的N末端包含以SEQ ID No.2所示的序列(由6个氨基酸组成,从N端至C端分别为ENLYFQ)。
在一项更优选的实施方案中,本发明的重组I型人源化胶原蛋白包含以SEQ IDNo.1所示的序列的n个重复,其中n为4,各重复序列之间是直接连接的,并且在所述重组I型人源化胶原蛋白的N末端包含以SEQ ID No.2所示的序列,以SEQ ID No.1所示的序列和以SEQ ID No.2所示的序列之间是直接连接的。
在另一项优选的实施方案中,本发明的重组I型人源化胶原蛋白包含以SEQ IDNo.1所示的序列的n个重复,其中n为大于等于1的整数,当n为大于等于2的整数时,各重复序列之间是直接连接的。
在另一项更优选的实施方案中,本发明的重组I型人源化胶原蛋白包含以SEQ IDNo.1所示的序列的n个重复,其中n为4,各重复序列之间是直接连接的,即包含以SEQ IDNo.3所示的序列。
GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS(SEQ ID No.3)
除非另有说明,本发明中使用的仪器、试剂、材料、实验动物等均可通过常规商业手段获得。
为更清楚地表述本发明的技术方案,下面结合具体实施例进一步说明,但不能用于限制本发明,此仅是本发明的部分实施例。
实施例1:重组I型人源化胶原蛋白对盆底功能障碍大鼠的治疗作用研究
动物模型建立:
所有的动物研究都得到了重庆医科大学伦理委员会的批准。39只雌性Sprague-Dawley大鼠(2月龄)由重庆医科大学实验动物中心提供,平均体重为220-260克。大鼠在温度为22±2℃、湿度为50-60%、光照/黑暗周期为12小时的环境下被喂食和自来水。大鼠分为假手术组(Control),模型组(FPFD),治疗组(FPFD+hrCOLI),每组13只。造模方法:腹腔注射乌拉坦(1.2g/kg)麻醉大鼠,切除双侧卵巢,然后用导尿法排空膀胱,将去掉尖端的F12导管插入大鼠阴道2-3cm,在球囊中注入3ml生理盐水后,将大鼠仰卧在桌子边缘,让导尿管悬空,在其自由悬空的一端连接0.15kg重物。4小时后,拔出导管,随后单次肌肉注射80000u青霉素钠预防感染。为了模拟临床多发性产伤,2周后对大鼠再次进行阴道球囊扩张术,4周后通过尿动力学试验和生物力学试验确认模型的成功建立。
给药方式:
对照组接受假手术。建立FPFD大鼠模型后,准备PBS(0.01mol/L)和hrCOLI(10mg/ml,例如使用以SEQ ID No.3所示的序列的蛋白)各200μl,在直视下分别于四点(三点、六点、九点、十二点方向)注射到整个阴道粘膜层至宫颈。注射2周后,进行后续实验。
1.重组I型人源化胶原蛋白可以促进盆底功能障碍大鼠尿道动力学恢复
(1)尿动力学测试过程
在测试前2天,将PE-50导管插入FPFD大鼠的膀胱中。大鼠腹腔注射尿烷(1.2g/kg)麻醉,膀胱排空后,测压导管与压力传感器(YPJ01,中国成都仪器厂)相连,灌注管与微量注射泵相连,连续向膀胱内注射生理盐水(注射速度12mL/h),在此过程中,记录波形图,根据灌注速度和波峰间隔,计算大鼠最大膀胱体积(MBV)。当膀胱容积达到一半时,用一根手指轻轻按压大鼠的腹部,增加膀胱压力,直到尿道口出现第一滴生理盐水。在这一点上,迅速移开手指并记录最大腹部漏尿压力(LPP)。膀胱被排空并重新填充,该过程在每只大鼠身上重复三次。
(2)实验结果
实验结果如图1所示,造模以后,和假手术组相比,模型组大鼠的最大膀胱容量和腹部漏尿点压力明显下降,说明造模的成功,经过hrCOLI治疗后,最大膀胱容量和腹部漏尿点压力明显增加,说明hrCOLI对盆底功能障碍大鼠的尿动力学功能恢复有治疗作用。
2.重组I型人源化胶原蛋白可以促进盆底功能障碍大鼠阴道生物力学抗性恢复
(1)生物力学实验方法
大鼠处死后,仔细分离阴道软组织,用0.9%氯化钠持续湿润,并在冰浴上操作,以防止组织破裂。沿纵轴将阴道和宫颈的远端5mm分别置于组织夹内,首先使用单轴力学拉伸仪ElectroForce 5500(德国Bose公司)给阴道组织预载至0.1N,在0-2mm之间以10mm/min的拉伸速率进行10个循环的预处理,然后在相同参数下,将阴道组织单轴拉伸至断裂,记录最大载荷(N)。
(2)实验结果
实验结果如图2所示,造模以后,和假手术组相比,模型组大鼠的阴道抗拉伸能力下降,这符合盆底功能障碍的特点,经过hrCOLI治疗以后阴道抗拉伸能力有所恢复,说明hrCOLI可以促进阴道力学性能恢复。
3.重组I型人源化胶原蛋白可以促进阴道和尿道局部组织结构恢复
(1)实验方法
测量MBV和LPP后,分别从注射PBS、hrCOLI溶液的大鼠中取出整个阴道。迅速将组织固定在4%多聚甲醛中,然后用石蜡包埋,沿横向切成0.5μm厚的切片,进行组织学观察,进行苏木精-伊红(HE)和masson(MT)染色。
(2)实验结果
实验结果如图3所示,假手术组大鼠的阴道上皮组织被复层鳞状上皮细胞覆盖,固有层的结缔组织富含血管和胶原纤维,肌层丰富。相反,模型组大鼠阴道上皮层较薄,血管数量较少,肌层萎缩断裂。重组I型人源化胶原蛋白处理后,阴道固有层和肌层的增厚,血管数量增多,胶原纤维增多。在尿道周围组织也观察到了同样的结果。
4.重组I型人源化胶原蛋白可以促进细胞外基质合成
(1)免疫组化实验方法
测量MBV和LPP后,分别从注射PBS、hrCOLI溶液的大鼠中取出整个阴道。迅速将组织固定在4%多聚甲醛中,然后用石蜡包埋,沿横向切成0.5μm厚的切片。为了进行免疫组化,石蜡切片在60℃下干燥2小时,然后用二甲苯进一步去石蜡化。用梯度酒精脱水后,将切片放在沸水中15min以修复抗原,然后用30%的H2O2在37℃孵化10分钟,切片用5%的山羊血清在37℃再阻断15分钟。然后用兔一抗进行孵育(Collagen I(1:250,14695-1-AP,Proteintech,中国)、Collagen III(1:250,22734-1-AP,Proteintech,中国)),并在4℃下孵育过夜,复温后用羊抗兔二抗在37℃下染色15分钟。最后,进行3,3'-二氨基联苯胺(DAB)染色,每个切片至少选择3个随机视野进行拍照,用尼康采集图像,用Image J软件进行结果分析。
(2)定量PCR实验方法
大鼠处死后,仔细分离阴道软组织,并迅速放入RNA保存液中保存,使用高速组织匀浆机破碎组织,使用Trizol制剂(Ambion,美国)分离阴道组织的总RNA,并按照制造商的说明用PrimeScript RT Master Mix(Takara,日本)进行逆转录,1ng RNA的逆转录酶用于反应以生成互补DNA(cDNA)。荧光定量聚合酶链反应用SYBR Premix Ex Taq II(Takara)进行。PCR的反应条件为95℃30秒,95℃5秒,60℃30秒,重复40个循环,实验重复三次。GAPDH作为内参基因。计算相对定量2-△△CT值。定量PCR分析的引物如下表所示:
正向引物 | 反向引物 | |
I型胶原蛋白 | GCTCCTCTTAGGGGCCACT | CCACGTCTCACCATTGGGG |
III型胶原蛋白 | CTGTAACATGGAAACTGGGGAAA | CCATAGCTGAACTGAAAACCACC |
GAPDH | AGGTCGGTGTGAACGGATTTG | TGTAGACCATGTAGTTGAGGTCA |
(3)实验结果
结果如图4所示,I型胶原蛋白(Collagen I)和III型胶原蛋白(Collagen III)在盆底功能障碍模型组中的表达是降低的,经过hrCOLI治疗以后,阴道组织中I型胶原蛋白和III型胶原蛋白的表达明显增加,说明hrCOLI有促进细胞外基质的主要成分(I型胶原蛋白、III型胶原蛋白)的合成的功能。
5.对骶韧带成纤维细胞表型影响检测
(1)细胞的培养
取大鼠骶韧带,清理干净,剪成1mm大小碎块,剪切过程中用DMEM保湿,将组织小块置于培养瓶中,并摆匀,每瓶25块左右,间隔0.5cm。放好后,将培养瓶翻转,让平底朝上,向瓶内加入2mL的培养液,盖好,放置于培养箱内(5%CO2,37%饱和湿度)。干贴壁3-4小时后,将培养瓶放平,继续培养。约25天长满瓶底(2-3天换液一次,用含20%胎牛血清的DMEM培养)。
待细胞长满瓶底,抛弃瓶内旧的培养液,加入0.25%胰蛋白酶,进行消化,用吸管吸掉大部分消化液,少量继续消化,待细胞大部分收缩变圆,加入含15%胎牛血清的DMEM,用吸管充分吹打瓶壁细胞,制成细胞混悬液,离心(1000r/min,5min),抛弃上清培养液,加入新的培养液,继续培养。
细胞纯化:
由于组织中可能含有少量的上皮细胞,因此取出先脱壁的细胞(往往是成纤维细胞),而后脱壁的细胞往往是上皮细胞。
(2)细胞黏附力测定
采用贝博(bestbio)细胞黏附检测试剂盒,将96孔板每孔加入100μL包被液,培养于2-8℃过夜,移除包被液,洗涤液洗涤。
将4.(1)处理好的细胞,用胰酶消化,PBS洗涤,然后用培养基重悬,制成细胞悬液。之后按照5×104细胞/孔接种96孔,放37℃培养箱孵育0.5-1小时。同时设立对照组(孵育后不弃上清液组)。取出培养板,吸弃培养基。再用相应的培养基洗涤2-3次,每孔加入100μL新鲜培养基。
黏附力测定:
96孔板每孔加入10μL细胞染色液,37℃孵育2小时之后测定孔OD值,选择450nm,以对照组(control组)OD为1.0,对比各实验组的OD值。
(3)细胞铺展面积
将5.(1)的细胞在培养液中培养8小时,并用电镜观察。计算其面积。
(4)实验结果:
细胞黏附性是维持组织结构稳定的基本条件,发挥生物学功能的调节因素。实验结果如图5所示,黏附实验显示:重组I型人源化胶原蛋白促进骶韧带成纤维细胞早期黏附,同时在早期黏附过程中细胞铺展面积明显增加。
综上所述,盆底功能障碍疾病目前发病率高,治疗方式有限,急需更加有效的治疗手段。重组人I型胶原蛋白具有高水溶性,高生物活性的特点,可能是一种有效的早期预防和治疗方法。现有实验证实,重组人I型胶原蛋白可以促进盆底功能障碍模型鼠尿道动力学和阴道力学性能恢复,并且可以改善局部组织结构,促进细胞外基质的合成。这将为临床早期预防和治疗盆底功能障碍的材料和药物开发提供了一定的理论基础。
序列表
<110> 山西锦波生物医药股份有限公司
重庆医科大学附属第二医院
<120> 重组I型人源化胶原蛋白在盆底修复中的用途
<130> 6C39-2163550I
<160> 3
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<213> 人工序列(Artificial Sequence)
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Claims (7)
1.重组I型人源化胶原蛋白在制备用于治疗盆底疾病的药物中的用途,
其中:
所述重组I型人源化胶原蛋白的氨基酸序列如SEQ ID No.3所示;
所述盆底疾病为盆底功能障碍性疾病。
2.根据权利要求1所述的用途,其特征在于,
所述盆底疾病为女性盆底功能障碍性疾病。
3.根据权利要求2所述的用途,其特征在于,
所述女性盆底功能障碍性疾病具有下列症状中的至少一种:尿道动力学性能下降、阴道生物力学性能下降、局部组织结构损伤、细胞外基质成分表达降低、骶韧带功能异常。
4.根据权利要求3所述的用途,其特征在于,
所述尿道动力学性能下降表现为下列特征中的至少一种:膀胱储尿功能下降、腹部漏尿点压降低。
5.根据权利要求3所述的用途,其特征在于,
所述局部组织结构损伤表现为下列特征中的至少一种:阴道和尿道周围组织上皮层变薄、固有层的结缔组织中的血管数量和胶原纤维减少、肌层萎缩断裂。
6.根据权利要求3所述的用途,其特征在于,
所述细胞外基质成分包含I型胶原蛋白和III型胶原蛋白中的至少一种。
7.根据权利要求3所述的用途,其特征在于,
所述骶韧带功能异常表现为下列特征中的至少一种:骶韧带成纤维细胞早期黏附能力下降、骶韧带成纤维细胞早期黏附过程细胞铺展面积减小。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012158169A1 (en) * | 2011-05-18 | 2012-11-22 | Affinergy, Inc. | Methods and compositions for tissue repair |
WO2013093921A1 (en) * | 2011-12-20 | 2013-06-27 | Collplant Ltd. | Collagen coated synthetic polymer fibers |
CN106215250A (zh) * | 2016-08-26 | 2016-12-14 | 易金阳 | 一种用于阴道松弛的重组人源i型胶原蛋白凝胶支架及其制备方法 |
CN109293783A (zh) * | 2018-10-25 | 2019-02-01 | 山西锦波生物医药股份有限公司 | 多肽、其生产方法和用途 |
CN109575126A (zh) * | 2018-07-06 | 2019-04-05 | 山西锦波生物医药股份有限公司 | 多肽、其生产方法和用途 |
CN109593126A (zh) * | 2018-11-28 | 2019-04-09 | 山西锦波生物医药股份有限公司 | 多肽、其生产方法和用途 |
CN112156173A (zh) * | 2020-09-10 | 2021-01-01 | 西安臻迈医疗科技有限公司 | 一种盆底肌功能障碍治疗用凝胶及其制备方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040146894A1 (en) * | 2002-10-14 | 2004-07-29 | Warrington Janet A. | Methods of diagnosing and treating stress urinary incontinence |
US20070027511A1 (en) * | 2005-08-01 | 2007-02-01 | Ty Fairneny | Collagen injection combined with focused ultrasound for the treatment of incontinence |
-
2021
- 2021-09-03 CN CN202111031518.8A patent/CN113730557B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012158169A1 (en) * | 2011-05-18 | 2012-11-22 | Affinergy, Inc. | Methods and compositions for tissue repair |
WO2013093921A1 (en) * | 2011-12-20 | 2013-06-27 | Collplant Ltd. | Collagen coated synthetic polymer fibers |
CN106215250A (zh) * | 2016-08-26 | 2016-12-14 | 易金阳 | 一种用于阴道松弛的重组人源i型胶原蛋白凝胶支架及其制备方法 |
CN109575126A (zh) * | 2018-07-06 | 2019-04-05 | 山西锦波生物医药股份有限公司 | 多肽、其生产方法和用途 |
CN109293783A (zh) * | 2018-10-25 | 2019-02-01 | 山西锦波生物医药股份有限公司 | 多肽、其生产方法和用途 |
CN109593126A (zh) * | 2018-11-28 | 2019-04-09 | 山西锦波生物医药股份有限公司 | 多肽、其生产方法和用途 |
CN112156173A (zh) * | 2020-09-10 | 2021-01-01 | 西安臻迈医疗科技有限公司 | 一种盆底肌功能障碍治疗用凝胶及其制备方法 |
Non-Patent Citations (4)
Title |
---|
Injectable recombinant human collagen-derived material with high cell adhesion activity limits adverse remodelling and improves pelvic floor function in pelvic floor dysfunction rats;Hu Li等;Biomaterials Advances;第134卷;第112715页 * |
Persistence of delayed hypersensitivity following transurethral collagen injection for recurrent urinary stress incontinence;K T Echols等;INTERNATIONAL UROGYNECOLOGY JOURNAL;第13卷(第1期);第52-54页 * |
胶原蛋白与盆底功能障碍性疾病的研究进展;严斌等;国际妇产科学杂志;第35卷(第06期);第422-425页 * |
胶原蛋白对去势SD大鼠萎缩阴道上皮作用探讨;游爽等;第三军医大学学报;第42卷(第06期);第632-639页 * |
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