CN108524601A - 一种含有多效因子的阴道凝胶及其制备方法 - Google Patents
一种含有多效因子的阴道凝胶及其制备方法 Download PDFInfo
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- CN108524601A CN108524601A CN201810495512.8A CN201810495512A CN108524601A CN 108524601 A CN108524601 A CN 108524601A CN 201810495512 A CN201810495512 A CN 201810495512A CN 108524601 A CN108524601 A CN 108524601A
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Abstract
本发明涉及阴道炎药物制备技术领域，具体涉及一种含有多效因子的阴道凝胶及其制备方法，解决了目前的阴道凝胶产品抑菌、润滑、保湿效果不够理想的技术效果。所述阴道凝胶含有间充质干细胞的条件培养基提取液和中药提取物，可有效的抑制阴道炎相关细菌的滋生和提高阴道湿润度，用于治疗阴道炎症、缓解阴道干燥症状或作为人体润滑剂，同时具有促进阴道黏膜鳞状上皮细胞层修复的作用，可改善阴道黏膜层变薄和修复阴道内小的损伤，使阴道黏膜恢复弹性和柔软，适用于阴道炎和阴道干燥症人群。
Description
技术领域
本发明涉及阴道炎药物制备技术领域，具体涉及一种含有复方中药提取物及间充质干细胞的多效因子的阴道凝胶制备方法。
背景技术
阴道炎是妇女的常见病和多发病，包括细菌性阴道炎、霉菌性阴道炎和滴虫性阴道炎。阴道炎的临床症状主要表现为阴道分泌物增多且伴有异味、外阴瘙痒，并引起宫颈、子宫体感染及盆腔炎症性疾病。阴道干燥症或萎缩性阴道炎的主要人群是绝经后妇女，据调查统计半数左右的绝经后妇女存在阴道干涩问题。阴道黏膜由含丰富糖原的非角化复层鳞状上皮和含丰富纤维的基底膜组成。生育期时复层鳞状上皮富含糖原，阴道黏膜滋润有弹性。在绝经后由于雌激素减少，上皮萎缩，鳞状细胞层消失，只保留了营养不良且绒毛易掉的基底层和副基底层细胞，这种情况下阴道黏膜易受细菌感染和有毒代谢物渗透，而且容易形成小裂伤造成感染。在正常情况下阴道上皮具有平衡机制能够使阴道内足够的水化和润滑，保证阴道组织柔软而又弹性。在女性绝经后由于阴道分泌物减少，阴道润滑不足，不能形成生理保护层，上皮细胞易于损伤或凋亡，而引起阴道干涩等症状。
现阴道炎或阴道干燥症的治疗产品分为三类：1.西药，如甲硝唑、洗必泰、咪康唑和酮康唑等实现杀菌抗菌，西药见效快但容易产生耐药性且有副作用；2.中药，如蛇床子、苦参、黄柏、白矾、甘草、冰片和地肤子等具有清热解毒、杀虫止痒功效成分，单纯使用中药见效慢不能及时缓解症状；3.器械类，分为两种，一是用臭氧或红光等理疗设备治疗，需要病人定期去医院做治疗或自行购买治疗设备成本较高，二是壳聚糖、银离子等阴道抑菌凝胶，采用生物抑菌原料，即时、方便、快捷、经济的治疗阴道炎，凝胶的使用和疗效得到广泛认可。而目前的阴道凝胶产品抑菌、润滑、保湿效果不够理想。
发明内容
本发明的目的是为了解决上述技术的不足而提供一种能安全、有效地治疗阴道炎和缓解女性阴道干燥萎缩症状的阴道凝胶及其制备方法。
为了达到上述目的，本发明采用的技术方案是:一种含有多效因子的阴道凝胶制备方法，其特征在于，包括以下步骤：
步骤一：间充质干细胞的制备
分离培养间充质干细胞条件培养基提取液，保存待用；
步骤二：制备复方中药提取物
取苦参、蛇床子、金缕梅、积雪草和薄荷五味中药，加水煎煮两次，每次加水20倍量，使溶液按照体积比为1:20的比例浓缩后过滤，合并两次滤液，备用；
步骤三：制备基础凝胶
将壳聚糖、水及步骤一中制得的条件培养基提取液按照体积比为1：10：0.01的比例混合均匀，溶胀至呈凝胶；
步骤四：制备阴道凝胶
a)将氧化银、乙二胺和水混合，制备成银离子溶液，备用；
b)冰片加乙醇溶解，备用；
c)将水与卡波姆混合加热，并以120r/min的速度搅拌，温度升至85℃时，加入NaOH溶液中和至PH为6.2-6.5，其后冷却；待混合溶液降温至35℃时，加入步骤a)、步骤b)和步骤二中的备用液混匀；待溶液降温至30℃时，加入步骤三中的基础凝胶混合均匀，灌装到无菌西林瓶中，保存待用，得到阴道凝胶。
所述间充质干细胞为脐带间充质干细胞、脂肪间充质干细胞或骨髓间充质干细胞。
上述的制备方法制得的含有多效因子的阴道凝胶。
相对于现有技术，本发明具有以下优点：
1、选取临床对于治疗外阴、阴道干燥症具有较好的清热消毒和杀虫止痒功效及可有效的改善阴道内环境的中药处方；
2、将间充质干细胞条件培养基制备成凝胶剂型，使得细胞分泌的条件培养基得到了保存和利用，而且存活的时间可以更长，可达到长时间促进表皮细胞增长，可以缩短创口的愈合时间，并起到消除炎症，促进经久不愈的溃疡愈合等作用。
3、本发明的含有复方中药提取物及间充质干细胞的多效因子的阴道凝胶，有抗炎活性，可有效抑制了阴道炎相关细菌的滋生和提高阴道湿润度，用于治疗阴道炎症、缓解阴道干燥症状或作为人体润滑剂，同时具有促进阴道黏膜鳞状上皮细胞层修复的作用，可改善阴道黏膜层变薄和修复阴道内小的损伤，使阴道黏膜恢复弹性和柔软，适用于阴道炎和阴道干燥症人群。
具体实施方式
下面对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明的药物配伍合理,其组份作用是：
苦参，味苦，性寒，具有抗菌、抗炎作用。
蛇床子，味辛、苦，性温；有小毒。有抗真菌、抗霉菌、解毒杀虫、燥湿、祛风之功能。
金缕梅，味甘、平。有益气，主劳伤乏力作用。
积雪草，味苦，性寒，具有清热利湿，消肿解毒的功效。
薄荷，味辛、凉。有散热，辟秽，解毒之功效
间充质干细胞(MSC)是最重要的多能成体干细胞之一，具有自我更新和自我分化潜能，以及分泌多种因子参与损伤的组织与器官的修复与再生而被广泛应用于多种疾病的治疗。MSCs在体内或体外特定的诱导条件下，可分化为胰岛、神经、血管内皮、骨、软骨、肌肉、肝脏、心肌等多种组织细胞。越来越多的研究证实间充质干细胞(MSCs)通过分泌的细胞因子可发挥免疫调节与降低炎症的作用。目前的研究认为，这些生物活性物质使间充质干细胞具有免疫调节、降低炎症的功能，所有的MSC分泌物中的生物活性因子和细胞因子可以在条件培养基中收集。
本发明的含有多效因子的阴道凝胶的制备方法，包括以下步骤：
步骤一：间充质干细胞的制备
分离培养间充质干细胞条件培养基提取液，保存待用；
步骤二：制备复方中药提取物
取苦参、蛇床子、金缕梅、积雪草和薄荷五味中药，加水煎煮两次，每次加水20倍量，使溶液按照体积比为1:20的比例浓缩后过滤，合并两次滤液，备用；
步骤三：制备基础凝胶
将壳聚糖、水及步骤一中制得的条件培养基提取液按照体积比为1：10：0.01的比例混合均匀，溶胀至呈凝胶；
步骤四：制备阴道凝胶
a)将氧化银、乙二胺和水混合，制备成银离子溶液，备用；
b)冰片加乙醇溶解，备用；
c)将水与卡波姆混合加热，并以120r/min的速度搅拌，温度升至85℃时，加入NaOH溶液中和至PH为6.2-6.5，其后冷却；待混合溶液降温至35℃时，加入步骤a)、步骤b)和步骤二中的备用液混匀；待溶液降温至30℃时，加入步骤三中的基础凝胶混合均匀，灌装到无菌西林瓶中，保存待用，得到阴道凝胶。
所述间充质干细胞为脐带间充质干细胞、脂肪间充质干细胞或骨髓间充质干细胞。
间充质干细胞的制备。
一、脐带间充质干细胞的制备
在超净工作台中进行操作，取新鲜足月健康胎儿脐带，用含2倍100U/mL青霉素和链霉素的生理盐水冲洗去除血渍，将华尔通氏胶剥离，剪成1立方毫米以下块状，均匀涂布，缓慢加入10mL无血清培养基，置37℃、体积分数为5％的二氧化碳、饱和湿度培养箱中培养。将原代细胞培养7天后，更换无血清培养液，培养至细胞达到70％的融合，移去无血清培养液，添加浓度分别为0.25％和0.1％的胰蛋白酶-乙二胺四乙酸钠(EDTA)消化1min，脐带MSC收缩脱离瓶壁，此时加入先前移去的培养上清液，中和胰蛋白酶-EDTA溶液，并使用移液管轻轻吹打，将脐带MSC悬液移入50mL离心管。1000rpm离心8min，去除上清液；重新加入无血清完全培养基将MSC重悬，并计数。按照1传1.5接种10cm培养皿。MSC培养融合70％左右时参照上述方法传代培养。
选择第三代培养的MSC，在对数生长期，且细胞融合程度不超过80％时按照传代的要求消化、中和、洗涤、收集细胞，采用细胞冻存液(90％胎牛血清和10％二甲亚砜(DMSO))重悬MSC获得条件培养基，充分混匀，每管1mL分装于冻存管密封。将冻存管置于装有异丙醇的冻存盒中，于-80℃冰箱过夜，次日移入液氮罐保存。
MSC复苏的步骤为：取出MSC冻存管，立即浸入37℃温水中溶解；取适量无血清培养基混悬细胞并离心，弃去上清液；细胞重悬于无血清培养基中，接种1×105个细胞，置于37℃、体积分数为5％的二氧化碳、饱和湿度培养箱中培养72h，72h后收集上清液。
二、骨髓间充质干细胞的制备
采用常规方法于髂前上棘穿刺点，用骨穿针抽取50mL骨髓液，注入50mL的无菌离心管中；用等量含20u/mL肝素的PBS液稀释，并沿管壁加入等体积的比重为1.073g/ml的淋巴细胞分离液；2000rmp离心30min，吸取单个核细胞层；无菌生理盐水离心洗涤2次，每次1000rpm离心5min，弃去上清液，取沉淀；将上述得到的骨髓MSC，用无血清培养基混悬，计数细胞，按5×106个/mL接种于的培养瓶中，置37℃体积分数为5％的二氧化碳、饱和湿度的培养箱中培养。培养3天后，更换无血清培养液，弃去未贴壁细胞，以后每3天1次更换培养液；7～9天后，骨髓MSC融合达50％～60％时，进行第一次传代。移去无血清培养液，采用浓度分别为0.25％和0.1％的胰蛋白酶-EDTA 37℃消化1min，骨髓MSC收缩脱离瓶壁，加入先前移去的培养上清液，中和胰蛋白酶-EDTA，并使用移液管轻轻吹打，将骨髓间充质细胞悬液移入50mL无菌离心管中。1000rpm离心8min，弃去上清液；重新加入无血清完全培养基重悬，计数，按照1传1-1.5接种10cm培养皿。骨髓MSC培养融合70％左右时如上述方法传代培养。
三、脂肪间充质干细胞的制备
按照常规方法分离腹部白色脂肪组织，剔除可见微血管及肌肉组织，无菌PBS洗涤三遍，然后用无菌剪刀将脂肪组织剪碎至1立方毫米以下的糊状，添加无菌消化液(含有0.1％胶原酶Ⅰ型和0.05％胰蛋白酶的无血清DMEM培养基)，37℃水浴中匀速搅拌45-50min，至组织块消化干净；200目筛网过滤收集滤液，然后使用含有10％胎牛血清(FBS)的低糖DMEM培养基等量中和，1500rpm离心10min，弃去上清液；以1×106个/mL的细胞密度，用无血清培养基重悬脂肪MSC，并接种于培养瓶中，37℃、体积分数为5％二氧化碳、饱和湿度培养箱中培养，当脂肪MSC生长至接近80％融合时，使用0.25％胰蛋白酶溶液消化MSC细胞，以1：3的接种比例接种到新的培养瓶中进行传代培养。脂肪MSC融合至不超过80％，1000rpm离心8min，弃去上清液，重新加入无血清完全培养基重悬MSC，计数，按照1传1-1.5接种10cm培养皿。脂肪MSC培养融合70％左右时如上述方法传代培养。
含有多效因子的阴道凝胶的抗炎作用评估：
根据本发明阴道凝胶针对的细菌性阴道炎、霉菌性阴道炎和滴虫性阴道炎，选择白色念珠菌、大肠杆菌和金黄色葡萄球菌为实验菌株。
将多效因子凝胶和水按照1:1稀释配成溶液使用；对照组为不含多效因子的水凝胶。
方法：
制备两种培养基：沙氏葡萄糖琼脂培养基、胰酪大豆琼脂培养基、甘露糖高盐琼脂培养基各25ml，在层流罩下分别导入直径15cm的培养皿，待凝固。分别取0.4ml白色念珠菌和大肠杆菌混悬液，白色念珠菌接种于沙氏葡萄糖琼脂培养基，大肠杆菌接种于胰酪大豆琼脂培养基，金黄色葡萄球菌接种于甘露糖高盐琼脂培养基。涂布菌液待干燥后，在层流罩下用灭菌刀片将平皿分成大小相等的2部分，用无菌器具分装入不同培养皿。将高压灭菌过的滤纸小片浸入多效因子水凝胶样品和阴道水凝胶样品，后用灭菌镊子分别夹起放入分好平皿中，盖上平皿杆子，37℃下培养40h，然后分别测定抑菌圈大小。
结果：本发明阴道凝胶对白色念珠菌、大肠杆菌和金黄色葡萄球菌抑菌效果见表1
表1.阴道凝胶的抑菌效果
| 菌株 | 多效因子水凝胶1:1稀释样品液 | 水凝胶1:1稀释样品液 |
| 白色念珠菌 | 48.5 | 40 |
| 大肠杆菌 | 41.2 | 35 |
| 金黄色葡萄球菌 | 55.6 | 48 |
表1
结论：由表1所示，多效因子凝胶对白色念珠菌、大肠杆菌和金黄色葡萄球菌有明显抑菌效果。
含有多效因子的阴道凝胶对阴道炎治愈能力的评估：
为了探索多效因子水凝胶在阴道炎症中的效果，我们选取了74名志愿者，进行了临床试验观察。
入选标准：1、生育期女性，有正常性生活，月经正常，半年内曾患过滴虫性阴道炎、细菌性阴道病、念珠性外阴阴道病或需氧菌性阴道炎至少一次。2、3天内有阴道炎症状，但未治疗，经阴道分泌物检查，可确诊为滴虫性阴道炎、细菌性阴道病、念珠性外阴阴道病或需氧菌性阴道炎。
排除标准：1、入组前两周内接受了全身性或阴道抗真菌/抗生素治疗；2、怀孕；3、除阴道炎外无其他疾病。4、淋病、梅毒、尖锐湿疣、非特异性泌尿生殖道炎等疾给药方法。
观察组：应用多效因子活性水凝胶每晚一支，连续使用7天；对照组1应用正常水凝胶每晚1支，连续使用7天；对照组2应用多效因子活性水凝胶每晚1支，连续使用4天；对照组3应用正常水凝胶每晚1支，连续使用4天。
检测方法：对滴虫、念珠菌、细菌性阴道病进行检测。
疗效痊愈判定标准:停药一周后复诊，症状及体征消失，阴道分泌物正常，清洁度≤II度，镜检病原体阴性。
结果：用药过程中，对照组2有1人放弃治疗。本发明阴道凝胶对阴道炎治愈率见表2
表2.给药后治愈率情况
表2
结论：由表2所示，用药后，观察组治愈率高于对照组1、对照组2和对照组3，P<0.05，差异显著。多效因子凝胶显著提高阴道炎治愈率。
经试验，本产品对于细菌性阴道炎、滴虫性阴道炎和念珠菌性阴道炎等阴道疾病具有良好的效果，如患者在使用前存在白带增多或有臭气、外阴有瘙痒、灼热，阴道松弛、性交痛等症状均在使用3—7天后得到较大的改善。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。
Claims (3)
1.一种含有多效因子的阴道凝胶的制备方法，其特征在于，包括以下步骤：
步骤一：间充质干细胞的制备
分离培养间充质干细胞条件培养基提取液，保存待用；
步骤二：制备复方中药提取物
取苦参、蛇床子、金缕梅、积雪草和薄荷五味中药，加水煎煮两次，每次加水20倍量，使溶液按照体积比为1:20的比例浓缩后过滤，合并两次滤液，备用；
步骤三：制备基础凝胶
将壳聚糖、水及步骤一中制得的条件培养基提取液按照体积比为1：10：0.01的比例混合均匀，溶胀至呈凝胶；
步骤四：制备阴道凝胶
a)将氧化银、乙二胺和水混合，制备成银离子溶液，备用；
b)冰片加乙醇溶解，备用；
c)将水与卡波姆混合加热，并以120r/min的速度搅拌，温度升至85℃时，加入NaOH溶液中和至PH为6.2-6.5，其后冷却；待混合溶液降温至35℃时，加入步骤a)、步骤b)和步骤二中的备用液混匀；待溶液降温至30℃时，加入步骤三中的基础凝胶混合均匀，灌装到无菌西林瓶中，保存待用，得到阴道凝胶。
2.根据权利要求1所述的含有多效因子的阴道凝胶的制备方法，其特征在于，所述间充质干细胞为脐带间充质干细胞、脂肪间充质干细胞或骨髓间充质干细胞。
3.如权利要求1-2任一项所述的制备方法制得的含有多效因子的阴道凝胶。
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