CN116804176A - 一株阴道卷曲乳酸杆菌及其在制备防治慢性子宫内膜炎和由ce引起的女性不孕症的药物中的应用 - Google Patents
一株阴道卷曲乳酸杆菌及其在制备防治慢性子宫内膜炎和由ce引起的女性不孕症的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一株阴道卷曲乳酸杆菌及其在制备防治慢性子宫内膜炎和由CE引起的女性不孕症的药物中的应用。阴道卷曲乳酸杆菌的保藏编号为CGMCC No.23396,保藏日期为2021年09月13日。上述阴道卷曲乳酸杆菌用于制备防治慢性子宫内膜炎以及由慢性子宫内膜炎引发的女性不孕症的药物。本发明阴道卷曲乳酸杆菌可通过调节阴道微生态,抑制病原菌的生长,治疗慢性子宫内膜炎,影响炎症因子及着床相关蛋白、细胞因子的表达,进而通过NF‑kB及Wnt/beta‑catenin通路,而影响受精卵的着床。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株阴道卷曲乳酸杆菌及其在制备防治慢性子宫内膜炎和由CE引起的女性不孕症的药物中的应用。
背景技术
近年来,越来越多的证据表明子宫内膜与各种妇女生殖疾病、受精和后代健康有关,并且证实子宫内膜内有细菌定植。在人体体表和外界相通的腔道中共生着近千种微生物,它们发挥着对宿主有益的重要作用。众所周知,女性阴道内寄生着几十种微生物,包括白假丝酵母菌、加德纳菌、大肠埃希菌、肠球菌、放线菌、变形杆菌、表皮葡萄球菌、支原体等条件致病菌,同时也存在着双歧杆菌、乳酸杆菌等益生菌,致病菌与益生菌之间处于动态平衡的状态。妇女正常的阴道微生态是其生殖健康的重要指标,由于各种原因,阴道微生物之间的平衡被打破,将会导致局部的炎症,并可向上蔓延至子宫腔内、输卵管、甚至盆腹部,导致内生殖系统的炎症,并可能造成不良妊娠结局及不孕,从而严重影响患者的工作与生活质量。临床上常见的许多妇科感染性疾患都存在明显的阴道微生态系统失衡。
慢性子宫内膜炎(chronicendometritis,CE)是慢性盆腔炎症的一种,分为特异性和非特异性,临床上一般指非特异性慢性子宫内膜炎,是子宫内膜间质受炎症细胞浸润的一种病理状况。CE在育龄期高发、绝经后罕见,是引起女性反复流产的原因之一,慢性子宫内膜炎占不明原因不孕的比例为40.7%-55.7%。CE通常无症状或仅有轻微异常阴道流血、慢性盆腔痛或白带异常,仅靠临床症状诊断困难,易被妇科专家和病理学家忽视。子宫内膜炎发病率为10-11%,其中经期延长、宫腔操作史、输卵管阻塞、子宫内膜异位症是其发病的危险因素,子宫内膜炎发病的关键是宫腔内定植的微生物(细菌)与子宫内膜免疫之间的持续相互作用,正常微生物群、宿主和环境之间始终处于动态平衡状态,形成一个相互依存、相互制约的系统,这个平衡一旦破坏,就可能致病。CE主要病因为病原微生物的感染,可导致子宫内膜局部免疫环境的改变,同时也可能对子宫内膜容受性产生负面影响,导致胚胎种植失败和复发性流产,不利于女性的妊娠结局。子宫内膜炎对女性妊娠的影响在过去几年受到生殖医学专家的广泛关注,有临床研究发现生殖道微生物区系紊乱引起的CE影响不孕症妇女治疗后的妊娠率。此外局部内膜炎性细胞的浸润和炎症介质的渗出会出现细胞毒作用,不利于精子成活和受精卵着床,同时引起炎症的病原体可激发机体的免疫反应,产生大量致敏的活性细胞、炎性细胞及多种细胞因子,杀灭和吞噬精子。
研究结果发现微生物区系不良的患者的临床妊娠率为6%,而良好的患者的临床妊娠率为41%,表明CE与不孕症之间可能存在相关性。针对CE引起的生殖道微生物区系紊乱的有效治疗,可能是改善反复妊娠丢失和胚胎种植失败女性妊娠结局的重要方法之一。当前生殖道炎症的治疗主要是全身或局部抗生素药物治疗。而抗生素疗法的缺点是容易产生耐药性,并且易复发。而临床的治疗理念已从以往杀灭微生物转变到增加益生菌、恢复阴道正常微生态环境,进而提高治愈率,减少复发和再感染率。
乳酸杆菌是健康妇女生殖道的主要微生物种属,而卷曲乳酸杆菌是女性阴道中乳酸杆菌的常见菌种,对维持生殖道菌群平衡以及健康起关键作用。它能抑制多种生殖道病原细菌和真菌,例如加德纳氏菌和白色念珠菌等,也能降低HIV感染和性传播疾病的风险。因此研究提出假设,卷曲乳酸杆菌可能通过改善阴道微生态环境,从而改善因CE导致的生殖道微生态紊乱。
发明内容
针对现有技术中的不足与难题,本发明旨在提供一株阴道卷曲乳酸杆菌及其在制备治疗慢性子宫内膜炎和由CE引起的女性不孕症的药物中的应用。本发明分离出一株来自阴道的卷曲乳酸杆菌,其可通过调节阴道微生态,抑制病原菌的生长,治疗慢性子宫内膜炎,影响炎症因子及着床相关蛋白、细胞因子的表达,进而通过NF-kB及Wnt/beta-catenin通路,而影响受精卵的着床。
本发明通过以下技术方案予以实现:
本发明一方面提供一株阴道卷曲乳酸杆菌,所述卷曲乳杆菌(Lactobacilluscrispatus)保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC No.23396,保藏日期为2021年09月13日。
本发明还提供上述阴道卷曲乳酸杆菌的应用,其用于制备防治慢性子宫内膜炎以及由慢性子宫内膜炎引发的女性不孕症的药物。
进一步地,所述药物包括包括卷曲乳酸杆菌CGMCC No.23396、和其他药学上可接受的载体和辅料;所述药物通过阴道给药,所述药物与抗生素联合作用。
所述药物防治慢性子宫内膜炎主要体现在:(1)减少子宫内膜中淋巴细胞及浆细胞的浸润,并促进内膜的修复。(2)通过抑制子宫内膜组织中NF-κB炎症信号通路上关键蛋白的表达,从而实现降低子宫内膜炎症反应。(3)将所述卷曲乳酸杆菌定植于子宫内膜,并抑制子宫内膜中致病菌的生长。
所述药物防治制备防治由慢性子宫内膜炎引起的女性不孕症主要体现在:(1)所述药物提高体外授精-胚胎移植术IVF的临床妊娠率。(2)通过BMP2促进Wnt4的表达,进而激活子宫内膜组织中经典Wnt/β-catenin信号通路,进而促进胚胎着床。
与现有技术相比,本发明有益效果包括:
1、本发明将卷曲乳杆菌可有效防治慢性子宫内膜炎CE和由CE引起的女性不孕症。
2、本发明将卷曲乳杆菌通过阴道给药,可调节阴道微生态进而维持正常的阴道菌群,增加阴道优势菌群,改善子宫腔微生物环境,进而治疗和改善慢性子宫内膜炎。
3、本发明对CE的患者给予抗生素治疗后,阴道给予卷曲乳杆菌,帮助纠正患者的微生物紊乱,提高IVF的临床妊娠率。
4、本发明卷曲乳酸杆菌可通过影响炎症因子及着床相关蛋白、细胞因子的表达,进而通过NF-kB及Wnt/beta-catenin通路,而影响受精卵的着床。
附图说明
图1为本发明阴道卷曲乳酸杆菌的体外益生性质分析图。
图2为实施例2中各实验组对子宫及内膜组织的影像图。
图3为实施例2中各实验组子宫内膜组织中炎症相关信号通路蛋白水平的表达。
图4为实施例2中各实验组q-PCR检测干预后子宫内膜组织中炎症因子转录水平的表达。
图5为实施例2中各实验组q-PCR检测干预后子宫内膜组织中微生物群的组成。
图6为实施例2中不同分组雌鼠胚胎着床及生殖结局的变化。
图7为实施例2中各实验组的雌鼠子宫内膜组织中细胞增殖、分化相关信号通路蛋白水平的表达。
具体实施方式
下面结合附图,对本发明作进一步地说明。
实施例1阴道卷曲乳酸杆菌CGMCC No.23396的体外益生性质
阴道卷曲乳酸杆菌的菌株来源:健康女性生殖道,保存于实验室-80℃冰箱。使用液体乳酸杆菌条件培养基(MRS),pH值调至5-5.5(冰乙酸),复苏该阴道卷曲乳酸杆菌,培养条件:37℃摇床过夜培养,之后活化2次,点板计数后保留菌株备用。
卷曲乳杆菌(Lactobacillus crispatus)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23396,保藏日期为2021年09月13日,其菌株序列如SEQ ID NO.1所示。
对阴道卷曲乳酸杆菌进行耐酸实验、抗氧化性实验评价、抑菌试验,培养VK2E6E7细胞(人阴道上皮细胞)进行阴道卷曲乳酸杆菌的细胞粘附实验定植抵抗实验。
1、阴道卷曲乳酸杆菌的耐酸实验
(1)将卷曲乳杆菌用相应液体培养基37℃恒温培养箱中培养36~72h,保留菌株备用;
(2)取出100μL用PBS缓冲液稀释101、103、105倍,4,000rpm离心3min弃掉上清;加入pH=3、4、5、7的PBS缓冲液,放置4h后,混匀取10μL涂板,在37℃恒温培养箱或者厌氧培养箱中培养36~72h,活菌计数;
2、阴道卷曲乳酸杆菌的抗氧化性实验评价
(1)DPPH自由基清除能力的测定:
2mL细菌培养液上清加2mL(0.2mmol/L)DPPH甲醇溶液,黑暗下室温反映30min,取上清517nm测OD,对照为2mL去离子水加DPPH甲醇溶液:
DPPH自由基清除率=[1-A517(样品)-A517(对照)/A517(空白)]×100%
(2)羟基自由基清除能力的测定:
取2mmol/L的FeSO4溶液1mL,6mmol/L H2O2 1mL,6mmol/L水杨酸1mL,加入细菌培养液上清1mL,室温条件下静置30min,以去离子水为空白对照,测定510nm处吸光度,计算羟自由基的清除率:
羟基自由基清除率=[1-A510(样品)/A510(空白)]×100%
(3)超氧自由基清除能力的测定
在0.5mL细菌培养液中依次加入150mmol/L pH=8.0的Tris-Hcl溶液2mL,1.2mmol/L邻苯三酚溶液1mL,室温反应30min,测定330nm处吸光度:
超氧自由基清除率=[1-(A11-A10)/(A01-A00)×100%
A00:不含样品和邻苯三酚; A01:不含样品含邻苯三酚
A10:含样品不含邻苯三酚; A11:含样品和邻苯三酚
(4)对Fe2+的螯合能力的测定:
0.5mL样品溶液中加入0.4%的硫酸亚铁溶液0.1mL,上下轻轻颠倒混合均匀后,再加入0.1mL 1%的VC和0.2mol/L NaOH溶液1mL,室温条件,下反应20min,然后用10%的三氯乙酸,4℃离心机6,000rpm,10min以去除蛋白,取0.4mL上述溶液并加入0.1%邻二氮菲4mL,室温条件下反应10min,测定536nm处吸光度:
Fe2+的螯合能力=[A空白-A样品/A空白]×100%
(5)总还原力的测定
取1mL的细菌培养液,加入磷酸缓冲液1mL(pH为6.6)和1%K3[Fe(CN)6]溶液1mL,上下颠倒充分混匀,在恒温箱中50℃保温2min,加入10%的三氯乙酸溶液1mL,振荡混匀,取混合液1mL,加入去离子水4mL和0.1%的FeCl3溶液0.4mL,静置10min。然后用去离子水为空白,测定700nm处吸光度,吸光度值越大,说明待测物的还原力越强。
3、阴道卷曲乳酸杆菌的抑菌试验
(1)将卷曲乳杆菌接种于相应的液体培养基中,37℃下在二氧化碳培养箱中培养36~72h,6,000g,离心5min;
(2)将白色念珠菌、金黄色葡萄球菌、大肠杆菌O157、乙型溶血性链球菌、粪肠球菌、加德纳菌涂布LB固体培养基中。将牛津杯轻轻放于平板上,吸取益生菌培养上清250μL于牛津杯中(每组做两个重复),37℃恒温培养,每隔2h观察抑菌圈大小,8h后测量抑菌圈直径,做好实验记录。
4、阴道卷曲乳酸杆菌的细胞粘附实验
(1)将卷曲乳杆菌接种于相应的液体培养基中,37℃下在二氧化碳培养箱中培养,使其OD值等于0.6后终止培养,保留菌株备用;
(2)将在盖玻片上覆盖率为30%的VK2E6E7细胞培养板用无菌PBS缓冲液清洗一次,取1mL上述细菌培养液,与1mL含有少量VK2E6E7细胞培养液混合后,加入六孔板中,37℃细胞培养箱培养;
(3)经过l~1.5小时培养,取出六孔细胞培养板,吸出培养液,用PBS缓冲液反复清洗5次,甲醇固定,革兰染色,油镜观察。
5、阴道卷曲乳酸杆菌的定植抵抗实验
(1)24孔板接种VK2E6E7细胞,待每孔细胞汇合率达90%左右,用1mL灭菌的PBS漂洗2次,加入1mL培养基;
(2)细胞板中每孔加OD=0.6的卷曲乳酸杆菌与107CFU致病菌株放到细胞培养箱中共同孵育2.5h;
(3)对照组不加卷曲乳酸杆菌处理,加等体积细胞培养基代替所加入的卷曲乳酸杆菌使其与等量致病菌共孵育;
(4)接着各处理组细胞用PBS漂洗3次,加0.5%TritonX-100 200μL,放在37℃细胞培养箱,孵育8min以使细胞充分裂解;
(5)加入双蒸水250μL,用5mL移液枪反复轻轻吹打细菌与细胞混合培养物,甩干残余液体,加入1mL细菌培养基,倍比稀释后涂布于相应的琼脂平板,置37℃恒温培养箱中培养18~24h,计算菌落数。
如图1所示,通过绘制生长曲线、耐酸实验、抗氧化性实验(DPPH自由基、羟基自由基、超氧自由基清除能力的测定,对Fe2+的螯合能力的测定以及总还原力的测定)、抑菌实验和细胞黏附实验等体外实验评价卷曲乳酸杆菌的体外益生性质,该卷曲乳酸杆菌具有良好的体外益生性质。
实施例2阴道卷曲乳酸杆菌CGMCC No.23396制备的药物用于防治慢性子宫内膜炎CE、以及治疗因CE引起女性不孕症的实验
1、药物制备
本实施例将阴道卷曲乳酸杆菌CGMCC No.23396加入辅料后制成冻干粉,辅料为5%脱脂乳+3%海藻糖(均为食品级)。研究所用益生菌经分子生物学、菌落、菌体形态鉴定,菌粉冻干工艺和辅料均按照国家规定的工艺进行,对患者无任何毒副作用。此外,微生物药物实验室对筛选自健康妇女阴道中的益生菌均进行分子生物学鉴定,确保菌株的准确性和安全性。现我们拟将卷曲乳杆菌冻干粉制成胶囊,使用于CE患者,进行阴道给药,维持正常的阴道菌群,改善子宫腔微生物环境。
2、实验对象和分组
以BALB/c雌性小鼠(SPF级别,6-8周大小)和BALB/c雄性小鼠(SPF级别,6-8周大小)作为动物实验对象。为建造慢性子宫内膜炎小鼠模型,小鼠适应性饲养1周,单次子宫内注射常见致病菌(大肠杆菌;金黄色葡萄球菌∶乙型溶血性链球菌=2∶1∶1)溶液,20μl 1*103CFU造慢性子宫内膜炎小鼠模型,连续观察小鼠成模情况(预计21天),每5天收集一次小鼠的阴道分泌物,观察小鼠生殖道微生物的变化情况。术后21天,实验组及对照组收集血液及阴道分泌物,各处死2只小鼠,对比观察子宫组织的改变,收集小鼠的子宫,进行病理HE染色及免疫组化,统计造模的成功率,观察子宫内膜厚度、腺体及子宫内膜上皮细胞的变化。
成模后(子宫充血、水肿、炎性样改变)开始分批进行阴道治疗(预计7天,隔天根据分组给予处理),将60只BALB/c雌性小鼠随机分为6组,空白对照组(A组):不注射致病菌,阴道置入PBS明胶海绵,连续1周;子宫内膜炎组(B组):注射致病菌混合液造模成功后,阴道置入PBS明胶海绵,连续1周;子宫内膜炎+抗生素组(C组):注射致病菌混合液造模成功后,阴道置入抗生素明胶海绵,连续1周;子宫内膜炎+卷曲乳酸杆菌组(D组):注射致病菌混合液造模成功后,阴道置入卷曲乳酸杆菌明胶海绵,连续1周;子宫内膜炎+抗生素+卷曲乳酸杆菌组(E组):注射致病菌混合液造模成功后,阴道置入抗生素明胶海绵3天后,停用抗生素12小时后再置入卷曲乳酸杆菌明胶海绵4天,共1周。
每组10只小鼠,标准饲料喂养,自由饮水,室温23±3℃左右,湿度55%左右,适应性饲养一周,期间收集各组小鼠阴道分泌物-80℃保存。C、D、E、F组予以混合抗生素(氨苄西林(1g/L)、万古霉素(0.5g/L)、新霉素(1g/L)、甲硝唑(1g/L))3天,收集各组小鼠阴道分泌物-80℃保存,除空白对照组(A组)外,其余各组单次子宫内注射常见致病菌(大肠杆菌;金黄色葡萄球菌∶乙型溶血性链球菌=2∶1∶1)溶液,20μl 1*103CFU造慢性子宫内膜炎小鼠模型,连续观察小鼠成模情况(预计14天),每5天收集一次小鼠的阴道分泌物,观察小鼠生殖道微生物的变化情况。
治疗结束后3天,收集小鼠血液及阴道分泌物,各实验分组每组处死2只,取子宫组织进行病理学检查,评价治疗效果(可将治疗周期延长至2周,分批观察)。治疗结束后按雌雄比1∶1的比例合笼,上午检查雌鼠阴道涂片,下午将雄鼠与处于发情前期的雌鼠合笼。于次日早上检查阴道栓,发现阴道栓为受孕0天。合笼期不超过两周。受孕后第5天(着床期),收集小鼠阴道分泌物,每组处死2只小鼠,收集血液,子宫组织,观察胚胎着床数量,同时对比各组胚胎着床的差异,取器官组织进行WB、病理染色、qPCR、活菌计数等指标检测,每组留下2只监测产子数及子代情况。
3、各组实验进行样本采集
HE染色:观察小鼠子宫组织学改变,子宫及内膜组织增生、水肿、渗出及炎性细胞浸润等指标。子宫内膜细胞结构、大小、核质比、异型性;
免疫组化:根据qPCR及WB结果选择差异蛋白进行免疫组化的检测,包括Bmp2、Wnt4(细胞黏附动力学过程的关键蛋白)、VEGF(血管内皮生长因子)。
取阴道分泌物、子宫及内膜组织做活菌计数治疗开始至结束(1周),隔天收集小鼠阴道分泌物,qPCR检测阴道分泌物中卷曲乳酸杆菌的生物量。每组处死2只小鼠,无菌操作下解剖,取子宫进行称重,测量子宫的直径,之后剖开子宫,收集子宫内膜置于生理盐水中匀浆,制成混悬液,分别10倍比稀释,取10ul点板于LB培养基上,37℃、5%CO2培养箱中培养48h,之后对平板上的菌落进行计数,计算每克子宫内膜中所含的活菌数量,qPCR检测转录水平上的细菌生物量。
小鼠受孕及子代的观察:每组剩余4只小鼠,上午检查雌鼠阴道涂片,下午将雄鼠与处于发情前期的雌鼠合笼。次日早上检查阴道栓,发现阴道栓为受孕0天,合笼期不超过两周。
阴道涂片检查::雌鼠于开始交配前1周每日进行阴道涂片检查,掌握每只雌鼠的性周期,以检查是否对交配或交配前时间有影响。
小鼠剖检:孕鼠于受孕5天时,1/2雌鼠剖检观察外表、内脏器官眼观病变。剖检时确认是否妊娠。称子宫重量并记录;切开子宫壁,计数活胎数、死胎数及吸收胎数,并合计为着床数,同时确认有无胎盘异常,保存子宫、卵巢、阴道和异常器官标本,必要时切取一定大小的病变组织,用4%多聚甲醛的固定,石蜡包埋后切片,HE染色,镜检。
子宫形态学影响:实验结束时称量剩余雌鼠体重,然后处死动物,通过HE染色观察子宫内膜形态改变,并记录腺体数量,测量子宫内膜厚度并在显微镜下观察其组织病理学变化。
检测干预后阴道及子宫组织中细菌的生物量,包括:
(1)检测小鼠造模/治疗后子宫内的细菌量:①金黄色葡萄球菌:上游引物:5’-AATTAA CGAAAT GGG CAG AAA CA-3’;下游引物:5’-TGC GCA ACACCC TGA ACT T-3’;②大肠杆菌:上游引物:5’-GCC TCG CCT GGA GAA TGA-3’;下游引物:5’-CCT GAG ACT GCG GTGGAA-3’;③乙型溶血性链球菌:上游引物5’-GAT TTG GGA TAA CTA AGCT-3’;下游引物:5’-TTA CAT CGT TAA CTT GAG CT-3’;
(2)干预后监测乳酸杆菌在阴道及子宫内的细菌量以评估其产生的作用及作用时间,卷曲乳酸杆菌:乳酸菌属引物序列为:
(LbLMA1-rev:5′-CTCAAAACTAAACAAAGTTTC-3′;R16-1:5′-CTTGTACACACCGCCCGTCA-3′)。
4、指标检测及结果分析
各组实验对子宫及内膜组织的影响如图2所示,结果表明阴道卷曲乳酸杆菌有效改善了CE导致的组织病理学改变,并与抗生素治疗有协同作用,可显著减少CE雌鼠的子宫内膜中淋巴细胞及浆细胞的浸润,并促进内膜的修复。
子宫内膜组织中炎症相关信号通路蛋白TLR4、MyD88、p-p65、p-IkB、IkB、β-actin水平的表达如图3所示,q-PCR检测干预后子宫内膜组织中炎症因子转录水平的表达图图4所示,结果说明卷曲乳酸杆菌可以通过抑制子宫内膜组织中NF-κB炎症信号通路上关键蛋白的表达,从而降低CE雌鼠子宫内膜炎症反应。NF-kB信号通路可调控多种参与炎症反应的细胞因子(如IL-1、IL-6、TNF-α)、黏附因子和蛋白酶类基因的转录过程,以应答多种胞外信号刺激,包括病毒侵染、细菌和真菌感染、肿瘤坏死因子、白细胞介素等细胞因子,甚至离子辐射,产生免疫、炎症和应激反应,并影响细胞增殖、分化及发育。
q-PCR检测干预后子宫内膜组织中微生物群的组成如图5所示,结果表明,阴道卷曲乳酸杆菌可定植于子宫内膜,并可抑制CE雌鼠子宫内膜中致病菌的生长。
不同分组雌鼠胚胎着床及生殖结局的变化如图6所示,结果说明,CE显著降低了雌鼠的受孕率,并阻碍了胚胎着床。卷曲乳酸杆菌可在降低CE炎症水平的同时促进胚胎着床,提高雌鼠受孕率,并且在CE治疗过程中与抗生素联合使用有显著的协同促进作用。
子宫内膜组织中细胞增殖、分化相关信号通路蛋白水平的表达如图7所示,卷曲乳酸杆菌通过BMP2促进Wnt4的表达,进而激活子宫内膜组织中经典Wnt/β-catenin信号通路,发挥促进胚胎着床的作用。
实施例3阴道益生菌干预对子宫内膜炎宫腔微生物环境及妊娠的影响
招募了130名志愿者,对患者进行资格评估,慢性子宫内膜炎阳性对照组:入组患者50例,慢性子宫内膜炎抗生素治疗组:40人,慢性子宫内膜炎卷曲乳酸杆菌治疗组:40人,对患者临床数据进行分析。CE组给予抗生素+阴道益生菌干预:诊断为CE的患者在抗生素治疗的基础上,在移植前12天给予阴道益生菌干预,比较阴道益生菌干预后的临床妊娠率,结果如表1所示。
阴道益生菌干预采用的是益生菌冻干粉,益生菌冻干粉为南昌大学转化医学研究院微生物药物实验室研发的益生菌成分,其仅含有阴道益生菌卷曲乳杆菌CGMCC No.23396(分离健康妇女阴道),每粒含益生菌活菌数不低于1×106CFU,辅料为5%脱脂乳+3%海藻糖(均为食品级)。研究所用益生菌经分子生物学(生工生物工程股份有限公司)、菌落、菌体形态鉴定,菌粉冻干工艺和辅料均按照国家规定的工艺进行,对患者无任何毒副作用。此外,微生物药物实验室对筛选自健康妇女阴道中的益生菌均进行分子生物学鉴定,确保菌株的准确性和安全性;体外对益生菌耐酸、耐胆盐、抗氧化、细胞粘附能力和抑制致病菌能力进行了评价,表明选用菌具有良好的耐酸、耐胆盐、广谱的抑菌能力和强的细胞结合能力;小鼠实验证明使用该菌粉(10倍剂量)三个月,小鼠无任何不良反应。
表1基线特征及妊娠结局统计
注:子宫内膜阳性对照组、抗生素治疗组与抗生素治疗+益生菌干预组采用平均值±标准差表示,组间计量资料采用t检验,计数资料采用x2检验。
由表1可以看出,入组患者基线特征无显著性差异,阴道益生菌卷曲乳酸杆菌干预可有效改善女性不孕症患者胚胎移植后的妊娠结局。
以上所述仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一株阴道卷曲乳酸杆菌,其特征在于:所述卷曲乳酸杆菌(Lactobacilluscrispatus)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo.23396,保藏日期为2021年09月13日。
2.权利要求1所述的阴道卷曲乳酸杆菌在制备防治慢性子宫内膜炎药物的应用。
3.根据权利要求2所述的应用,其特征在于:所述药物包括卷曲乳酸杆菌CGMCCNo.23396、和其他药学上可接受的载体和辅料;所述药物通过阴道给药,所述药物与抗生素联合作用。
4.根据权利要求3所述的应用,其特征在于:所述药物减少子宫内膜中淋巴细胞及浆细胞的浸润,并促进内膜的修复。
5.根据权利要求3所述的应用,其特征在于:所述药物通过抑制子宫内膜组织中NF-κB炎症信号通路上关键蛋白的表达,从而实现降低子宫内膜炎症反应。
6.根据权利要求3所述的应用,其特征在于:所述药物将所述卷曲乳酸杆菌定植于子宫内膜,并抑制子宫内膜中致病菌的生长。
7.权利要求1所述的阴道卷曲乳酸杆菌在制备防治由慢性子宫内膜炎引起的女性不孕症的药物的应用。
8.根据权利要求7所述的应用,其特征在于:所述药物包括卷曲乳酸杆菌CGMCCNo.23396、和其他药学上可接受的载体和辅料;所述药物通过阴道给药,所述药物与抗生素联合作用。
9.根据权利要求8所述的应用,其特征在于:所述药物提高体外授精-胚胎移植术IVF的临床妊娠率。
10.根据权利要求8或9所述的应用,其特征在于:所述药物通过BMP2促进Wnt4的表达,进而激活子宫内膜组织中经典Wnt/β-catenin信号通路,进而促进胚胎着床。
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WO2021176387A1 (en) * | 2020-03-05 | 2021-09-10 | Antonio La Marca | New use of probiotics |
CN115607578A (zh) * | 2022-10-25 | 2023-01-17 | 哈尔滨美华生物技术股份有限公司 | 一株卷曲乳杆菌在制备治疗宫腔粘连药物中的应用 |
CN116286484A (zh) * | 2023-02-08 | 2023-06-23 | 南昌大学 | 卷曲乳杆菌及其用途 |
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US20200164007A1 (en) * | 2017-07-07 | 2020-05-28 | Osel, Inc. | Use of vaginal lactobacilli for improving the success rate of in vitro fertilization |
WO2021176387A1 (en) * | 2020-03-05 | 2021-09-10 | Antonio La Marca | New use of probiotics |
CN115607578A (zh) * | 2022-10-25 | 2023-01-17 | 哈尔滨美华生物技术股份有限公司 | 一株卷曲乳杆菌在制备治疗宫腔粘连药物中的应用 |
CN116286484A (zh) * | 2023-02-08 | 2023-06-23 | 南昌大学 | 卷曲乳杆菌及其用途 |
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