CN113262295A - 人羊膜间充质干细胞外分泌蛋白postn的鉴定和应用 - Google Patents
人羊膜间充质干细胞外分泌蛋白postn的鉴定和应用 Download PDFInfo
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Abstract
本发明涉及人羊膜间充质干细胞外分泌蛋白POSTN在制备促进精原干细胞增殖制品中的应用及分泌蛋白POSTN的鉴定方法，鉴定方法的步骤为人羊膜间充质干细胞条件培养基的收取；质谱分析条件培养基中羊膜间充质干细胞外分泌蛋白，筛选出高表达蛋白POSTN。本发明鉴定了羊膜间充质干细胞外分泌蛋白组，并对结果进行了分析整合，筛选出精原干细胞增殖积极因子POSTN，对后续羊膜干细胞旁分泌作用的研究提供了详实的基础和新的思路。POSTN为精原干细胞体外增殖提供了新的细胞因子，为更好的挖掘羊膜干细胞外分泌蛋白的特点提供了方向。
Description
技术领域
本发明涉及生物技术领域，具体涉及人羊膜间充质干细胞外分泌蛋白-骨膜蛋白(periostin，POSTN)的鉴定和应用。
背景技术
间充质干细胞（Mesenchymal stem cells，MSCs）已被证明能够分泌各种自分泌/旁分泌因子，包括生长因子和细胞因子，这些因子在细胞治疗过程中起很大作用。人羊膜在分娩后常被当作生物废物丢弃，成为羊膜间充质干细胞（hAMSCs）的重要来源。此外，它还有许多其他优点，如抗炎、抗菌、抗血管生成特性和低免疫原性。HAMSC分泌因子用于急性脑损伤的无细胞治疗。我们在hAMSCs条件培养基（hAMSCs-CM）中发现了许多与细胞增殖有关的蛋白质。
骨膜蛋白(periostin, POSTN)是一种由836个氨基酸组成、大小约90kD的细胞外基质蛋白。POSTN在体内广泛表达，在主动脉、胃、下消化道、胎盘、子宫、甲状腺组织和乳腺中水平最高。它主要在个体发育过程中和成人结缔组织暴露于机械负荷时产生，如心脏瓣膜、皮肤、牙周韧带、肌腱和骨骼。此外，POSTN在受伤后的愈合过程中也起着关键作用。POSTN有两种作用途径，一种是通过与细胞外基质成分相互作用来驱动胶原纤维形成和重塑，另外一种是通过细胞表面整合素受体传递信号来促进细胞粘附、迁移和增殖。目前并没有在人羊膜间充质干细胞外分泌蛋白中鉴定出POSTN，也未见相关的鉴定方法报道，同时对POSTN的应用研究尚未深入，对于人羊膜间充质干细胞外分泌蛋白POSTN与精原干细胞增殖作用的关系及在临床上男性不育的治疗及男性生育力保存作用方面的研究未见报道。
发明内容
发明目的：本发明提出一种人羊膜间充质干细胞外分泌蛋白POSTN的鉴定和应用，其目的在于提出一种从羊膜间充质干细胞外分泌蛋白中鉴定出POSTN的方法，并验证该蛋白有促进精原干细胞增殖的作用。
技术方案：
人羊膜间充质干细胞外分泌蛋白POSTN在制备促进精原干细胞增殖制品中的应用。
所述制品为药品、保健品和食品。
一种人羊膜间充质干细胞外分泌蛋白POSTN的鉴定方法，步骤为：
1）人羊膜间充质干细胞条件培养基的收取：在产妇的羊膜组织中提取人羊膜间充质干细胞，对提取的羊膜间充质干细胞进行流式细胞检测；满足阳性率条件后，人羊膜间充质干细胞进行成骨、成脂分化诱导：获得诱导后的人羊膜间充质干细胞；对诱导后的羊膜间充质干细胞采用条件培养基培养，收集条件培养基；
2）质谱分析条件培养基中羊膜间充质干细胞外分泌蛋白，筛选出高表达蛋白POSTN。
流式细胞检测方法为：将羊膜间充质干细胞在PBS中洗涤2-4次，羊膜间充质细胞悬浮液与FITC-CD73，CD44，CD29，HLA-DR，CD31，CD45的荧光抗体在避光常温孵育20-40分钟，然后PBS洗涤2-4次并重悬，上机测定抗原的阳性率。
条件培养基培养方法为：诱导后的人羊膜间充质干细胞培养第二代融合度为80%-90%时传代，分别以2×106-2×108个细胞种至75cm2培养瓶中，24-48小时后用PBS洗2-4次，将培养基换成无血清培养基，24小时收集无血清培养基，-80℃冰箱保存。
本发明的有益效果及特点：
本发明鉴定了羊膜间充质干细胞外分泌蛋白组，并对结果进行了分析整合，筛选出精原干细胞增殖积极因子POSTN，对后续羊膜干细胞旁分泌作用的研究提供了详实的基础和新的思路。POSTN为精原干细胞体外增殖提供了新的细胞因子，为更好的挖掘羊膜干细胞外分泌蛋白的特点提供了方向。
附图说明
图1是羊膜间充质干细胞流式细胞技术鉴定干细胞表面标志物；
图2是羊膜间充质干细胞分化潜能的鉴定（成脂、成骨）；
图3是ELISA实验验证筛选出羊膜间充质干细胞高表达的外分泌蛋白POSTN、THBS1；
图4 是MTS证明POSTN促进精原干细胞体外增殖；
图5是不同浓度POSTN作用后，EdU染色阳性细胞所占比例。
具体实施方式
以下结合说明书附图更详细的说明本发明。
本发明是从羊膜上皮干细胞和羊膜间充质干细胞外分泌蛋白的鉴定入手，借助生物信息学分析，从中筛选出羊膜间充质高表达的外分泌蛋白POSTN。ELISA实验证实筛选结果，利用纯品蛋白刺激精原干细胞，MTS和EDU实验发现该蛋白能有效的促进精原干细胞增殖。本发明涉及干细胞与再生医学、蛋白质组在基础医学领域的深入研究与临床的转化应用，重点应用于羊膜干细胞在促进精原干细胞增殖中的积极因子POSTN与男性无精子症临床研究的推广与发展。
实施例1
1.人羊膜间充质干细胞条件培养基的收取
材料与方法:
蛋白酶抑制剂(碧云天)
胰酶（trypsin） BI
超滤离心管（Merck Millipore; Amicon Ultra-50, Ultracel-3k）
BCA试剂盒(碧云天)
Ⅳ型胶原酶(sigma)
成骨/成脂/成软骨试剂盒(赛业生物)
（1）所述羊膜间充质干细胞是由以下方法得到的：
取健康剖腹产产妇的羊膜组织，浸泡在生理盐水中30min，用PBS反复冲洗去除粘液。用眼科手术剪将羊膜剪成约1mm×1mm小碎块，加入10ml 0.25%trypsin-EDTA，37℃消化30 min后加入含血清的培养液，离心去上清，如此反复消化两次，再用PBS洗2-3次，尽可能去除上皮细胞。用细胞筛过滤后剩余的羊膜组织加入IV型胶原酶37℃消化60 min，加入含血清的培养液终止消化，混匀后用200目细胞筛过滤，400g离心10min后重悬得到hAMSCs，将细胞接种于DMEM/F12培养基（添加10%胎牛血清和青链霉素）放置于5% CO2的37℃培养箱中培养。
（2）对三个不同产妇来源羊膜间充质干细胞进行流式细胞检测：
流式细胞仪对第三代羊膜间充质干细胞表面相对特异性抗原进行检测。将羊膜间充质干细胞和羊膜上皮干细胞在PBS中洗涤三次，羊膜间充质细胞悬浮液与FITC-CD73，CD44，CD29，HLA-DR，CD31，CD45的荧光抗体在避光常温孵育30分钟，然后PBS洗涤三次并重悬。上机测定抗原的阳性率，结果显示CD44、CD73、CD29阳性细胞比例均达99%以上，CD31、CD45、HLA-DR阳性细胞比例均小于1%，见附图1。
（3）对三个不同来源人羊膜间充质干细胞进行成骨、成脂分化诱导：
对步骤(1)的三个不同来源的羊膜间充质干细胞，分别利用成骨、成脂诱导培养基相应进行成骨、成脂分化，按照试剂盒说明书诱导操作，结果显示hAMSCs在成脂肪细胞分化培养液中诱导3周后，用油红O染色呈红色，表明hAMSCs可以向脂肪细胞方向分化；hAMSCs在成骨诱导后，用茜素红染色呈红色，表明hAMSCs可以向成骨细胞方向分化，见附图2。
（4）所述羊膜间充质干细胞条件培养基是由以下方法得到的：
羊膜间充质干细胞培养第二代融合度为80%时传代，分别以2x106个细胞种至75cm2培养瓶中。24小时后用PBS洗三遍，将培养基换成无血清培养基。24小时收集无血清培养基，负八十度冰箱保存。
实施例2
质谱分析条件培养基中的羊膜间充质干细胞外分泌蛋白。蛋白注释采用GeneOntology（GO）分析。GO分析是一种重要的生物信息学分析方法，用于描述基因和基因表达产物的各种特征。GO注释分为三类：生物进程，细胞组成和分子功能，这三种分类方式分别从不同角度阐释了蛋白的生物学功能。我们对hAMSC分泌蛋白在GO二级注释中的分布进行了统计。结果显示，hAMSCs-CM中的外分泌蛋白参与细胞的增殖、分化、代谢、免疫、信号通路等生物过程。从中筛选出高表达蛋白POSTN。ELISA验证羊膜间充质干细胞中的高表达筛选蛋白。
材料与方法:
ELISA试剂盒(上海酶联)
结果：
取待测样品和试剂盒标准品于室温平衡1-2h。将50ul条件培养基加入反应孔中。根据试剂盒说明书对标准品进行稀释，各取50ul加入反应孔内用以制备标准曲线。之后立即加入生物素标记抗体50ul。贴上封口膜，37℃孵育1h。弃去孔内液体，在每孔内加入提前配置好的洗涤液，弃去洗涤液，用吸水纸拍干。重复此操作3次。之后，向每孔中加入亲和链酶素-HRP 80ul，37℃孵育30min。重复上述洗板步骤3次。最后，分别向每孔中加入底物A、B各50ul，37℃孵育10min。反应结束后，迅速加入50ul终止液。利用酶标仪测定450nm下的OD值。利用标准曲线计算待测样品的浓度。如图3所示，ELISA实验验证羊膜间充质干细胞培养液上清分泌蛋白POSTN和THBS1表达量高，符合预期。
实施例3
MTS和EDU实验发现该蛋白能有效的促进精原干细胞增殖。
利用MTS实验检测细胞的增殖情况，按照每孔2.5×104个细胞接种于96孔板中。24h后，将不同浓度POSTN分别加入对应孔中。每隔24h，在待检孔中加入10µl MTS，随后37°C孵育2h，利用酶标仪测量490nm波长时的数值。
为了确定hAMSCs-CM中的两个候选蛋白POSTN是否与细胞增殖有关，分别将不同浓度（0、10、50、100、200ng/mL）的POSTN蛋白纯品添加至细胞增殖培养液中，分别在0、24、48、72、96 h用MTS法检测细胞增殖情况。如图4所示，结果显示POSTN作用浓度越高，对细胞增殖的促进作用越强。从48小时起，与对照组相比POSTN浓度为50、100、200ng/mL时细胞增殖比例显著增加。
利用EdU染色的方法进一步确认不同浓度（0、10、50、100、200ng/mL）POSTN对细胞增殖的影响。以每孔3×104个细胞接种至培养皿，实验组分别加入不同浓度的POSTN，对照组加入等体积的DMSO，培养48 h后更换细胞培养液为含EdU的培养基，孵育2 h，吸弃培养基，用4%多聚甲醛进行细胞固定化处理，用2 mg/mL甘氨酸脱色，加入渗透剂（0.5%Triton-100）后行Apollo染色，室温下置于摇床上避光孵育30 min，用二苯甲亚胺, 三氯化氢 2-(4-乙基苯基)-5-(4-甲基-1-哌嗪基)-2,5-二-1H-苯并咪唑(2’-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5’-bi-1H-benzimidazole, trihydrochloride，Hoechst33342)染料进行细胞核染色，之后在共聚焦显微镜下观察，拍照并记录每个视野内的阳性细胞数目。细胞增殖率=（EdU阳性细胞数/ Hoechst 33342阳性细胞数）×100%。统计结果显示，100ng/mL与200ng/mLPOSTN组EdU染色阳性细胞所占比例显著高于对照组，显著促进细胞增殖，见图5。
本申请基于羊膜间充质干细胞条件培养基中外分泌蛋白的鉴定，进一步分析整合质谱数据。最终选定并验证POSTN促进人精原干细胞增殖。因此，羊膜干细胞外分泌蛋白POSTN对于精原干细胞增殖的促进作用，在临床上男性不育的治疗及男性生育力保存中产生良好的作用。羊膜外分泌蛋白组的进一步研究解析，可以在临床中更精准的应用。
以上所述，仅为本发明的优选实施例，并非是对本发明任何形式上的和实质上的限制，应当指出，对于本技术领域的普通技术人员，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，均应包含的本发明的保护范围之内。
Claims (5)
1.人羊膜间充质干细胞外分泌蛋白POSTN在制备促进精原干细胞增殖制品中的应用。
2.根据权利要求1所述的人羊膜间充质干细胞外分泌蛋白POSTN在制备促进精原干细胞增殖制品中的应用，其特征在于：所述制品为药品、保健品和食品。
3.一种如权利要求1所述的人羊膜间充质干细胞外分泌蛋白POSTN的鉴定方法，其特征在于：步骤为：
1）人羊膜间充质干细胞条件培养基的收取：在产妇的羊膜组织中提取人羊膜间充质干细胞，对提取的羊膜间充质干细胞进行流式细胞检测；满足阳性率条件后，人羊膜间充质干细胞进行成骨、成脂分化诱导：获得诱导后的人羊膜间充质干细胞；对诱导后的羊膜间充质干细胞采用条件培养基培养，收集条件培养基；
2）质谱分析条件培养基中羊膜间充质干细胞外分泌蛋白，筛选出高表达蛋白POSTN。
4.根据权利要求3所述的人羊膜间充质干细胞外分泌蛋白POSTN的鉴定方法，其特征在于：流式细胞检测方法为：将羊膜间充质干细胞在PBS中洗涤2-4次，羊膜间充质细胞悬浮液与FITC-CD73，CD44，CD29，HLA-DR，CD31，CD45的荧光抗体在避光常温孵育20-40分钟，然后PBS洗涤2-4次并重悬，上机测定抗原的阳性率。
5.根据权利要求3所述的人羊膜间充质干细胞外分泌蛋白POSTN的鉴定方法，其特征在于：条件培养基培养方法为：诱导后的人羊膜间充质干细胞培养第二代融合度为80%-90%时传代，分别以2×106-2×108个细胞种至75cm2培养瓶中，24-48小时后用PBS洗2-4次，将培养基换成无血清培养基，24小时收集无血清培养基，-80℃冰箱保存。
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