CN114010660A - 一种ASP联合hAD-MSCs治疗POI的实验方法 - Google Patents
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Abstract
本发明涉及一种ASP联合hAD‑MSCs治疗POI的实验方法，包括以下步骤：ASP含药血清的制备；不同浓度ASP含药血清加入培养基中培养hAD‑MSCs；hAD‑MSCs移植；5)实验分组；电镜观察各级卵泡和子宫内膜超微结构变化；免疫组化测定颗粒细胞表面标志Fshr和卵母细胞表面标志DDX4表达；Elisa法测定卵巢和子宫组织抗氧化酶与氧化产物ROS、SOD、等分泌情况；13)Western‑blot测定和磷酸化水平变化情况；Tunel染色测定卵泡颗粒细胞和卵母细胞的凋亡情况；免疫荧光双标记测定卵巢组织FOXO3a表达；16)合笼实验。本发明优点在于：采用当归多糖联合hAD‑MSCs提高干细胞的增殖，可以改善放射性POI的微环境，促进干细胞迁移归巢及局部的存活率，提高干细胞的对放射性POI治疗效果，具有很好的推广前景。
Description
技术领域
本发明涉及一种ASP联合hAD-MSCs治疗POI的实验方法。
背景技术
放疗(Radiotherapy RT)所致卵巢功能不全(premature ovarian insufficiencyPOI)。RT是多种癌症的常见治疗方式，所有癌症患者中大约有70％的疾病治疗过程中接受了RT。放射线可以直接或间接作用造成机体的多器官损伤，其中，代谢旺盛、更新较快的生殖细胞最容易被射线损伤。既往研究表明，当卵巢暴露于辐射时射线通过以下几种作用损伤生殖细胞：1)卵母细胞会通过细胞死亡途径丢失；2)颗粒细胞损伤导致性腺激素产生受损；3)卵巢血管和间质受到损害。这些损伤导致POI，甚至卵巢早衰(premature ovarianfailure POF)，POF是POI的终末阶段。POI是女性40岁之前出现月经稀发、闭经，并有两次或两次以上基础卵泡刺激素(follicle stimulating hormone FSH)≥25U/L，雌激素水平下降。临床上主要表现为全身各个系统并发症，如骨质疏松、心血管疾病、老年性痴呆等，并伴随生殖器官的萎缩。严重危害女性的身心健康。另外，放射线不仅损伤卵巢，子宫暴露于放射线也会导致子宫内膜、子宫肌层和血管结构的不可逆转的损害，从而进一步导致POI/POF的发生。不同报道中癌症患者经过RT后POI的发生率，文献检索发现POI的患病率从2.1％到82.2％不等。POI的发生与治疗剂量、个体年龄、放疗范围和个体卵巢储备有关，并且除盆腔以外，腹部、头颅及全身均可以导致POI，但盆腔放疗对卵巢功能的损伤最为严重，而且既往研究证实卵巢功能障碍的发生率与放疗剂量显著相关。终末期的POI即POF是不可逆的，但是早期发现并及时有效治疗可能会延迟甚至改善病情。因此，寻找一种有效而安全的保护策略对于年轻的癌症患者和医师都是至关重要的。
RT所致POI发生机制：研究发现，放射线导致组织中水分子裂解直接产生活性氧自由基,通过诱发吞噬细胞呼吸爆发等作用间接产生活性氧自由基，使组织细胞的氧化还原平衡破坏，产生氧化胁迫，直接损伤蛋白质、核酸、脂质等生物大分子，或扰乱细胞的信号传导途径，对组织细胞造成可逆或不可逆的损伤。实际上吞噬细胞行使功能的物质基础就是活性氧产物,它们不仅能对组织细胞直接作用导致机体炎症发生,还能通过局部生成的趋化因子放大炎症反应。氧自由基的产生导致毛细血管膜的通透性增强，照射后最突出的病理变化就是早期岀现渗出性水肿，其渗出液含有溶解的低纤维蛋白，它的长期存在并持续刺激成纤维细胞增生并分泌胶原蛋白，可导致纤维化形成。因此，清除活性氧自由基是降低辐射反应的有效方法之一。卵巢对辐射高度敏感，损伤机制通过电离和活性氧类的形成导致卵泡细胞凋亡、氧化应激、卵巢萎缩、皮质纤维化和血管损伤。这些变化导致炎性细胞的积累和一系列炎性因子的释放。这些炎性因子促进大量氧自由基、活性氧(ROS)产生和细胞毒性发生。这可以归因于微环境中炎性和纤维化介质的增多、表观遗传变化和正常氧代谢的破坏，导致卵泡DNA直接损伤、卵泡萎缩和卵巢储备降低，最终引起卵巢激素的产生减少而过早出现更年期。申请人在前期研究中发现，放射性损伤导致卵巢炎性因子产生增加，ROS积累，大鼠性周期及性激素紊乱，卵泡闭锁，卵巢功能受损伤。结合研究背景和前期研究基础，申请者提出科学问题：通过拮抗放疗所致的氧化和炎性损伤，清除体内的氧自由基，是否能有效地抵抗放射线对卵巢的损害、防止POI的发生？
FOXO3a的国内外研究进展：当机体受到放射线损伤发生氧化应激时，累积的ROS通过激活多种信号通路，使FOXO3a发生磷酸化/去磷酸化翻译后修饰，从而调控FOXO3a的活性与功能。而这一过程是相当复杂，有些甚至是矛盾的。FOXO3a是重要的转录因子，能促进氧化应激、抑制代谢、从而引起细胞凋亡。FOXO3a调节其下游基因的机制取决于细胞类型及其周围环境的刺激，不同的条件对FOXO3a活性和目标选择产生深远的影响，其范围可能从触发细胞死亡到通过诱导抗氧化应激因子的表达促进存活。FOXO3a活性调节的经典机制是由PI3K/AKT途径介导的，它是PI3K/Akt信号通路的直接靶标，它的功能受到磷酸化的关键调节，磷酸化的PI3K激活AKT，被激活的AKT进而磷酸化FOXO3a，磷酸化的FOXO3a保留在细胞质中，进一步被泛素化降解，丧失正常的转录活性，细胞凋亡将会降低。研究表明：卵母细胞内FOXO3a磷酸化水平增加，可以促进原始卵泡和初级卵泡的发育；相反，当AKT途径受到抑制时，大部分去磷酸化的FOXO3a进入了细胞核并加速细胞凋亡。换过来说，AKT和它的下游分子也能被FOXO3a进行负调节，它们的表达状态能促使细胞凋亡，去磷酸化的FOXO3a既可通过调控Fas/FasL、Bim、Caspases等凋亡相关蛋白表达诱导细胞凋亡,又可通过调控细胞周期蛋白如p27kip、CyclinD等使细胞周期停滞在G0/G1期。证据表明，FOXO3a的调控可以促进多种细胞的凋亡，如：造血干细胞、神经元细胞、间充质干细胞等。卵母细胞核中FOXO3a的过度表达可通过上调caspase-3和caspase-8，促凋亡蛋白以及Bim，FasL和P27kip1来导致卵母细胞凋亡。干细胞DNA修复与FOXO3a凋亡通路的调控密切相关，FOXO3a可促进干细胞的增殖即有助于维持干细胞的功能，调控PI3K-FOXO3信号通路可以促进间充质干细胞移植治疗卵巢去势模型大鼠的卵巢功能。综上所述：FOXO3a是POI的可能的治疗靶点，联合干细胞可促进卵巢功能的修复。
目前为止放疗所致POI临床上还没有确切有效的方法彻底的恢复卵巢功能。在肿瘤治疗中用于保护卵巢功能的是促性腺激素释放激素激动剂，但是存在点火效应和延误治疗的风险，效果尚不确切，而且价格昂贵，因此临床上应用受限；激素补充治疗仅能缓解症状，不能根治，而且还有严重副作用；保存成熟的卵子，胚胎和卵巢组织样本来保护生育能力，并非所有患者都是保留生育能力的候选人，而且保存的组织存在癌症转移的风险。除了保留生育力外，很多患者还需要采取其他的方法来确保具有足够的激素水平以维持全身健康。MSCs具有自我更新和多向分化潜能，可维持、修复、改善或再生受损的组织和器官的潜能，因此它为恢复POI卵巢功能及生育能力带来了希望。目前，国内外关于MSCs在放射损伤治疗中的研究已有大量文献报道，MSCs分泌大量干细胞因子可以明显改善放疗小鼠骨髓细胞凋亡，减少辐射诱导的内皮细胞基质金属蛋白酶的表达，从而使血管功能恢复正常，血管结构损伤减轻。而且MSCs移植也可以减轻放疗所致的全身炎症，促进放疗所致肺损伤、肠损伤、皮肤损伤的恢复。同时也证明在肿瘤病人自体MSCs移植的安全性。胎儿源人羊膜间充质干细胞(Human Amniotic Mesenchymal stem cell hAD-MSCs)因具有以下优点:1)不存在供体年龄依赖性；2)具有高度的增殖活性；3)易于获得；4)细胞丰富；5)与来源于骨髓或其他组织间充质干细胞相比，具有更强的免疫抑制作用。已经成为相比其他来源MSCs更好的替代物作为治疗疾病用于实验和临床研究。
中医药治疗POI能够降低西药的副作用并且提高临床效果，早在神农本草纲目中就有记载中医中药用于该疾病的治疗。。中医认为卵巢功能衰竭的病因多为肾虚为主，兼夹肝郁、瘀血阻滞，病属虚实夹杂，以虚为主。ASP是当归主要药物活性成分，有补肾填精、调补气血、养血疏肝功效。自古以来就用于月经不调、闭经等症。
从中医学角度来讲，辐射属于一种热毒、火毒，火热为阳邪，易伤机体阴液。ASP能清除辐射损伤机体的热毒、火毒，具有抗氧化、抗炎性损伤，减少细胞凋亡，对辐射损伤心、肝、脾、肺、肾功能、免疫系统和血液系统均有防护作用；ASP可增强细胞DNA损伤修复能力来提高细胞的辐射耐受性,通过AKT/FOXO3a通路，抑制小鼠氧化应激，改善卵巢早衰小鼠内分泌功能；ASP可以改善辐射大鼠的“伤津耗气，破血动血”的病症，增加PI3K和AKT的蛋白含量促进骨髓抑制小鼠骨髓干细胞增殖；ASP与MSCs联合治疗脑血管疾病，改善新血管形成，脑的变化和行为能力的变化显示ASP对MSCs移植治疗脑血管疾病有增强作用；ASP可抑制MSCs在炎性微环境中生物学特性的变化，激活PI3K/AKT信号通路来促进MSC的增殖和分化，促进MSCs在特定微环境中的修复能力。ASP从多环节抗炎症、减少炎症物质渗出，对于已有渗出则活血化瘀，疏通微循环，从而减少刺激成纤维细胞增生物质的来源，起到抑制胶原蛋白合成及抗纤维化作用。ASP的这些药理作用都有望成为hAD-MSCs移植治疗放射性POI提供良好的微环境，促进hAD-MSCs在放疗损伤微环境中的增殖、归巢、植活、分化，这将成为解决MSCs移植的瓶颈问题提出新方法。
MSCs移植治疗RT所致POI成为目前研究的热点，但是其迁移归巢率及局部存活率低限制其治疗效果。ASP是当归的主要活性成分，它具有养血活血、抗辐射损伤作用。本研究采用ASP体内处理对RT所致POI模型进行整体调节，改善POI局部的微环境，为hAD-MSCs向RT损伤所致POI微环境迁移及其在局部存活创造良好的条件；ASP血清培养hAD-MSCs促进其增殖及定向分化，它联合hAD-MSCs治疗POI可能的机制模型：ASP和hAD-MSCs协同调控靶标FOXO3a磷酸化，抑制下游凋亡相关因子的表达，从正负反馈双向上调PI3K/AKT磷酸化表达，减少卵母和颗粒细胞的凋亡，共同增强hAD-MSCs对RT所致POI的修复作用。与单独的ASP或hAD-MSCs治疗相比，ASP与hAD-MSCs的结合是否具有更好的抗凋亡作用，更好的卵巢功能恢复能力呢？为此，结合前期研究基础和干细胞在临床运用瓶颈设计本课题，采用传统中药ASP联合现代生物技术干细胞相结合，探索促进hAD-MSCs增殖、迁移归巢的关键技术，这将为未来干细胞治疗放疗所致损伤性疾病的研究提供新思路。
发明内容
本发明的目的在于提供一种ASP联合hAD-MSCs治疗POI的实验方法，以解决上述背景技术中提出的问题。
为解决上述技术问题，本发明提供的技术方案为：一种ASP联合hAD-MSCs治疗POI的实验方法，具体包括以下步骤：
1)ASP含药血清的制备：当归多糖含药当归用量按照临床用量换算成大鼠的用量，当归多糖按当归的用量同比换算成相对剂量；当归多糖组按254.2mg/kg/d腹腔注射1周，注射完后2小时处死大鼠，心脏取血，常规方法收集血清，56℃30min，灭活补体，0.22μm滤过除菌，-20℃保存备用；
2)不同浓度ASP含药血清加入培养基中培养hAD-MSCs，探索最佳浓度ASP含药物血清浓度，并对hAD-MSCs进行鉴定、纯化；
3)hAD-MSCs移植：采用尾静脉和卵巢局部两种移植路径，hAD-MSCs移植方式分为一次移植，放疗后4周移植一次、两次移植放疗开始和放疗后4周各移植一次；植入PKH26标记hAD-MSCs4×106/0.6mlPBS；
4)实验分组：
a.对照组：腹腔注射与ASP组等时等量的生理盐水；
b.假手术组：同hAD-MSCs卵巢移植组，麻醉下手术切开腹部，暴露卵巢，向卵巢注射等量PBS；
c.戊酸雌二醇组：给予0.3mg/kg/d腹腔注射，稀释药物生理盐水同ASP组；
d.模型组：构建放疗模型，方法同前；
e.ASP:放疗开始腹腔注射ASP254.2mg/kg/d，共4周；
f.hAD-MSCs组：移植PKH26标记含ASP药物血清培养hAD-MSCs4×106/0.6mlPBS，经尾静脉和卵巢两种途径移植，分一次移植组和两次移植组；
g.hAD-MSCs+ASP组：腹腔注射ASP联合PKH26标记ASP含药血清培养hAD-MSCs移植，腹腔注射ASP同e组，hAD-MSCs移植同f组；
h.hAD-MSCs对照组+ASP组：腹腔注射ASP联合移植PKH26标记无ASP含药血清培养hAD-MSCs，移植方法和ASP使用同g组；
i.hAD-MSCs+ASP组+LY294002阻断剂，阻断hAD-MSCs联合ASP上调PI3K/AKT/FOXO3a磷酸化信号通路的表达，对其作用机制进行验证；
5)一般情况：每天8:00-9:00AM进行阴道脱落细胞涂片检查，对大鼠动情周期进行观察；选择消毒棉签插入大鼠阴道约0.5cm处，并轻轻转动1圈，取出后在洁净载玻片上均匀涂抹，晾干，巴氏染色，每2周统计正常性周期大鼠的比例；每天观察大鼠的饮食，毛发，活动及二便等情况，并记录体重；
6)卵巢和子宫系数：hAD-MSCs移植后适应性喂养一周脱颈椎法处死大鼠，取出卵巢、子宫，新鲜称重，计算卵巢和子宫重量指数；同时将心、肝、肺、骨髓、卵巢和子宫等器官制备冷冻和石蜡组织切片，显微镜下计数卵巢上各级卵泡和黄体数，测量子宫肌层和子宫内膜的厚度；
7)卵巢功能：放疗当天，放疗后每两周经尾静脉取血测量大鼠雌二醇E2、促黄体生成素LH、FSH、抗苗勒管激素AMH、孕激素P、雄激素T；
8)激光共聚焦和活体成像了解干细胞在各器官分布情况；
9)电镜观察各级卵泡和子宫内膜超微结构变化；
10)免疫组化测定颗粒细胞表面标志Fshr和卵母细胞表面标志DDX4表达；
11)Elisa法测定卵巢和子宫组织抗氧化酶与氧化产物ROS、SOD、MDA、GSH-Px；炎性指标白介素-6、白介素-1β、肿瘤坏死因子-αTNF-α；血管内皮生长因子、碱性成纤维细胞生长因子-2、胰岛素样生长因子-1、肝细胞生长因子HGF的分泌情况；
12)Western-blot测定卵巢组织FOXO3a、PI3K、AKT、Bim、FasL、Fas、P27kip1、caspase-3/Cleaved-caspase-3、caspase-8/Cleaved-caspase-8总蛋白和磷酸化水平变化情况；
13)Tunel染色测定卵泡颗粒细胞和卵母细胞的凋亡情况；
14)免疫荧光双标记测定卵巢组织FOXO3a表达，鉴定FOXO3a的上下游目标的表达，对Bim、FasL、P27kip1抗体的双重免疫荧光染色；
15)合笼实验：每组选取S-D大鼠4只，与经验证具有正常受精功能的雄鼠，按雌：雄比例2:1进行交配，观察幼鼠的数量，死亡率，畸形率，体重，了解对生殖结局的影响；进一步评价其疗效和安全性。
本发明优点在于：采用当归多糖联合hAD-MSCs提高干细胞的增殖，可以改善放射性POI的微环境，促进干细胞迁移归巢及局部的存活率，提高干细胞的对放射性POI治疗效果，具有很好的推广前景。
附图说明
图1是本发明的技术路线图。
具体实施方式
下面用具体实施例说明本发明，并不是对本发明的限制。
实施例
一种ASP联合hAD-MSCs治疗POI的实验方法，具体包括以下步骤：
具体包括以下步骤：
1)ASP含药血清的制备：当归多糖含药当归用量按照临床用量换算成大鼠的用量，当归多糖按当归的用量同比换算成相对剂量；当归多糖组按254.2mg/kg/d腹腔注射1周，注射完后2小时处死大鼠，心脏取血，常规方法收集血清，56℃30min，灭活补体，0.22μm滤过除菌，-20℃保存备用；
2)不同浓度ASP含药血清加入培养基中培养hAD-MSCs，探索最佳浓度ASP含药物血清浓度，并对hAD-MSCs进行鉴定、纯化；
3)hAD-MSCs移植：采用尾静脉和卵巢局部两种移植路径，hAD-MSCs移植方式分为一次移植，放疗后4周移植一次、两次移植放疗开始和放疗后4周各移植一次；植入PKH26标记hAD-MSCs4×106/0.6mlPBS；
4)实验分组：
e.对照组：腹腔注射与ASP组等时等量的生理盐水；
f.假手术组：同hAD-MSCs卵巢移植组，麻醉下手术切开腹部，暴露卵巢，向卵巢注射等量PBS；
g.戊酸雌二醇组：给予0.3mg/kg/d腹腔注射，稀释药物生理盐水同ASP组；
h.模型组：构建放疗模型，方法同前；
e.ASP:放疗开始腹腔注射ASP254.2mg/kg/d，共4周；
f.hAD-MSCs组：移植PKH26标记含ASP药物血清培养hAD-MSCs4×106/0.6mlPBS，经尾静脉和卵巢两种途径移植，分一次移植组和两次移植组；
g.hAD-MSCs+ASP组：腹腔注射ASP联合PKH26标记ASP含药血清培养hAD-MSCs移植，腹腔注射ASP同e组，hAD-MSCs移植同f组；
h.hAD-MSCs对照组+ASP组：腹腔注射ASP联合移植PKH26标记无ASP含药血清培养hAD-MSCs，移植方法和ASP使用同g组；
i.hAD-MSCs+ASP组+LY294002阻断剂，阻断hAD-MSCs联合ASP上调PI3K/AKT/FOXO3a磷酸化信号通路的表达，对其作用机制进行验证；
5)一般情况：每天8:00-9:00AM进行阴道脱落细胞涂片检查，对大鼠动情周期进行观察；选择消毒棉签插入大鼠阴道约0.5cm处，并轻轻转动1圈，取出后在洁净载玻片上均匀涂抹，晾干，巴氏染色，每2周统计正常性周期大鼠的比例；每天观察大鼠的饮食，毛发，活动及二便等情况，并记录体重；
6)卵巢和子宫系数：hAD-MSCs移植后适应性喂养一周脱颈椎法处死大鼠，取出卵巢、子宫，新鲜称重，计算卵巢和子宫重量指数；同时将心、肝、肺、骨髓、卵巢和子宫等器官制备冷冻和石蜡组织切片，显微镜下计数卵巢上各级卵泡和黄体数，测量子宫肌层和子宫内膜的厚度；
7)卵巢功能：放疗当天，放疗后每两周经尾静脉取血测量大鼠雌二醇E2、促黄体生成素LH、FSH、抗苗勒管激素AMH、孕激素P、雄激素T；
8)激光共聚焦和活体成像了解干细胞在各器官分布情况；
9)电镜观察各级卵泡和子宫内膜超微结构变化；
10)免疫组化测定颗粒细胞表面标志Fshr和卵母细胞表面标志DDX4表达；
11)Elisa法测定卵巢和子宫组织抗氧化酶与氧化产物ROS、SOD、MDA、GSH-Px；炎性指标白介素-6、白介素-1β、肿瘤坏死因子-αTNF-α；血管内皮生长因子、碱性成纤维细胞生长因子-2、胰岛素样生长因子-1、肝细胞生长因子HGF的分泌情况；
12)Western-blot测定卵巢组织FOXO3a、PI3K、AKT、Bim、FasL、Fas、P27kip1、caspase-3/Cleaved-caspase-3、caspase-8/Cleaved-caspase-8总蛋白和磷酸化水平变化情况；
13)Tunel染色测定卵泡颗粒细胞和卵母细胞的凋亡情况；
14)免疫荧光双标记测定卵巢组织FOXO3a表达，鉴定FOXO3a的上下游目标的表达，对Bim、FasL、P27kip1抗体的双重免疫荧光染色；
15)合笼实验：每组选取S-D大鼠4只，与经验证具有正常受精功能的雄鼠，按雌：雄比例2:1进行交配，观察幼鼠的数量，死亡率，畸形率，体重，了解对生殖结局的影响；进一步评价其疗效和安全性。
以上所述，仅为本发明较佳的具体实施方式，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，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (1)
1.一种ASP联合hAD-MSCs治疗POI的实验方法，其特征在于，具体包括以下步骤：
1)ASP含药血清的制备：当归多糖含药当归用量按照临床用量换算成大鼠的用量，当归多糖按当归的用量同比换算成相对剂量；当归多糖组按254.2mg/kg/d腹腔注射1周，注射完后2小时处死大鼠，心脏取血，常规方法收集血清，56℃30min，灭活补体，0.22μm滤过除菌，-20℃保存备用；
2)不同浓度ASP含药血清加入培养基中培养hAD-MSCs，探索最佳浓度ASP含药物血清浓度，并对hAD-MSCs进行鉴定、纯化；
3)hAD-MSCs移植：采用尾静脉和卵巢局部两种移植路径，hAD-MSCs移植方式分为一次移植，放疗后4周移植一次、两次移植放疗开始和放疗后4周各移植一次；植入PKH26标记hAD-MSCs4×106/0.6mlPBS；
4)实验分组：
a.对照组：腹腔注射与ASP组等时等量的生理盐水；
b.假手术组：同hAD-MSCs卵巢移植组，麻醉下手术切开腹部，暴露卵巢，向卵巢注射等量PBS；
c.戊酸雌二醇组：给予0.3mg/kg/d腹腔注射，稀释药物生理盐水同ASP组；
d.模型组：构建放疗模型，方法同前；
e.ASP:放疗开始腹腔注射ASP254.2mg/kg/d，共4周；
f.hAD-MSCs组：移植PKH26标记含ASP药物血清培养hAD-MSCs4×106/0.6mlPBS，经尾静脉和卵巢两种途径移植，分一次移植组和两次移植组；
g.hAD-MSCs+ASP组：腹腔注射ASP联合PKH26标记ASP含药血清培养hAD-MSCs移植，腹腔注射ASP同e组，hAD-MSCs移植同f组；
h.hAD-MSCs对照组+ASP组：腹腔注射ASP联合移植PKH26标记无ASP含药血清培养hAD-MSCs，移植方法和ASP使用同g组；
i.hAD-MSCs+ASP组+LY294002阻断剂，阻断hAD-MSCs联合ASP上调PI3K/AKT/FOXO3a磷酸化信号通路的表达，对其作用机制进行验证；
5)一般情况：每天8:00-9:00AM进行阴道脱落细胞涂片检查，对大鼠动情周期进行观察；选择消毒棉签插入大鼠阴道约0.5cm处，并轻轻转动1圈，取出后在洁净载玻片上均匀涂抹，晾干，巴氏染色，每2周统计正常性周期大鼠的比例；每天观察大鼠的饮食，毛发，活动及二便等情况，并记录体重；
6)卵巢和子宫系数：hAD-MSCs移植后适应性喂养一周脱颈椎法处死大鼠，取出卵巢、子宫，新鲜称重，计算卵巢和子宫重量指数；同时将心、肝、肺、骨髓、卵巢和子宫等器官制备冷冻和石蜡组织切片，显微镜下计数卵巢上各级卵泡和黄体数，测量子宫肌层和子宫内膜的厚度；
7)卵巢功能：放疗当天，放疗后每两周经尾静脉取血测量大鼠雌二醇E2、促黄体生成素LH、FSH、抗苗勒管激素AMH、孕激素P、雄激素T；
8)激光共聚焦和活体成像了解干细胞在各器官分布情况；
9)电镜观察各级卵泡和子宫内膜超微结构变化；
10)免疫组化测定颗粒细胞表面标志Fshr和卵母细胞表面标志DDX4表达；
11)Elisa法测定卵巢和子宫组织抗氧化酶与氧化产物ROS、SOD、MDA、GSH-Px；炎性指标白介素-6、白介素-1β、肿瘤坏死因子-αTNF-α；血管内皮生长因子、碱性成纤维细胞生长因子-2、胰岛素样生长因子-1、肝细胞生长因子HGF的分泌情况；
12)Western-blot测定卵巢组织FOXO3a、PI3K、AKT、Bim、FasL、Fas、P27kip1、caspase-3/Cleaved-caspase-3、caspase-8/Cleaved-caspase-8总蛋白和磷酸化水平变化情况；
13)Tunel染色测定卵泡颗粒细胞和卵母细胞的凋亡情况；
14)免疫荧光双标记测定卵巢组织FOXO3a表达，鉴定FOXO3a的上下游目标的表达，对Bim、FasL、P27kip1抗体的双重免疫荧光染色；
15)合笼实验：每组选取S-D大鼠4只，与经验证具有正常受精功能的雄鼠，按雌：雄比例2:1进行交配，观察幼鼠的数量，死亡率，畸形率，体重，了解对生殖结局的影响；进一步评价其疗效和安全性。
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