期新生儿大鼠干细胞的内切内培养
Summary
描述是通过期期新生儿大鼠的四边形注射进行间质质质细胞（MSCs）的腹内移植的协议。这项技术是将干细胞和药物移植到新生儿大鼠肺以评估其疗效的临床可行选择。
Abstract
长时间接触高浓度的氧气会导致炎症和急性肺损伤，这类似于人类支气管肺发育不良（BPD）。在早产儿中，尽管早期使用表面活性剂疗法、最佳通气策略和非侵入性正压通气，但BPD是一个主要并发症。由于肺炎在BPD的发病机制中起着至关重要的作用，皮质类固醇的使用是预防它的潜在治疗方法之一。然而，由于长期不良反应，通常不建议早产儿进行全身皮质类固醇治疗。临床前研究和人类第一阶段临床试验表明，在高氧引起的肺损伤和早产儿中使用间质期频闪细胞（MSCs）是安全和可行的。内切和静脉MSC移植已被证明可以预防新生儿高氧肺损伤。因此，干细胞的腹内给药和表面活性剂和糖皮质激素联合治疗已成为治疗新生儿呼吸系统疾病的新策略。大鼠肺出生时的发育阶段相当于妊娠26~28周时人类肺的发育阶段。因此，新生大鼠适合研究腹内给患呼吸窘迫的早产儿，以评估其疗效。这种中切内注射技术是将干细胞和药物移植到肺部的临床可行选择。
Introduction
治疗患有呼吸窘迫的新生儿时，通常需要补充氧气。然而，婴儿的超氧治疗有长期不利影响。长时间接触高浓度的氧气会导致炎症和急性肺损伤，这类似于人类支气管肺发育不良（BPD）2。BPD 是高氧治疗的主要并发症，尽管早产儿有早期表面活性剂治疗、最佳的通气程序以及使用非侵入性正压通气的增加，但可能会发生这种情况。虽然许多治疗策略已经报告为BPD3，没有已知的治疗可以减少这种并发症。
皮质类固醇的使用是预防BPD的一种潜在治疗方法，因为肺炎在其发病机制中起着至关重要的作用。然而，由于长期不良反应,4，5，通常不建议早产儿进行全身皮质类固醇治疗。4
脑膜状细胞（MSCs）具有多能性特征，可以分化成各种细胞类型，包括骨骼、软骨、脂肪组织、肌肉和肌腱6。MSC具有免疫调节、抗炎和再生作用7，动物研究表明MSCs及其分泌成分在啮齿动物8、9,中高氧引起的肺部损伤中具有治疗作用。内切和静脉MSC移植已被证明可以预防新生儿高氧肺损伤。因此，干细胞的腹内给药和表面活性剂和皮质类固醇联合治疗可能是治疗新生儿呼吸系统疾病的潜在治疗策略。临床前研究在新生大鼠10、11、12,11中使用了干细胞和腺相关病毒的腹内给给。然而，该技术的分步演示和移植干细胞的体内跟踪是不可用的。新生大鼠适合研究腹内给药对患有呼吸窘迫的早产儿的影响，因为大鼠出生时肺的囊期相当于妊娠13的26-28周的人肺的囊期。一种有效的方法，为大鼠气管的药而，是成功进行肺分配的关键。这里介绍的技术允许研究细胞和/或药物的腹内给药，以大鼠为人类模型治疗新生儿肺病。
Protocol
Representative Results
Discussion
患有呼吸窘迫的新生儿通常需要腹内表面活性剂和/或皮质类固醇治疗19。人类第一阶段的临床试验已经证明了早产儿8期中腹间MSC的安全性。这些研究表明，对患有呼吸窘迫的新生儿来说，在血管内服用药物是一个重要的选择。如果模型特征与人类直接相关，动物模型研究最有帮助。定期新生大鼠是早产肺损伤和发育研究的有用模型。然而，新…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了台湾台北美利根生物科技有限公司（A-109-008）的赠款的部分支持。
Materials
| 6-0 silk | Ethicon | 1916G | |
| Alcohol Prep Pad | CSD | 3032 | |
| BD Stemflow hMSC Analysis Kit | BD Biosciences | 562245 | CD markers |
| CMV-Luciferase-EF1α-copGFP BLIV 2.0 Lentivector for In Vivo Imaging | SBI | BLIV511PA-1 | |
| CryoStor10 | BioLife Solutions | 640222 | |
| Human MSCs | Meridigen Biotech Co., Ltd. Taipei, Taiwan | ||
| Infrared light | JING SHANG | JS300T | |
| Isoflurane | Halocarbon | 26675-46-7 | |
| IVIS-200 small animal imaging system | Caliper LifeSciences, Hopkinton, MA | ||
| Luciferin potassium salt | Promega, Madison, WI | ||
| Micro-scissors, straight | Vannas | H4240 | |
| Normal saline | TAIWAN BIOTECH CO., LTD. | 113531 | Isotonic Sodium Chloride Solution |
| Small Hub RN Needle, 30 gauge | Hamilton Company, Reno, NV | 7799-06 | |
| Syringe (100 µl) | Hamilton Company, Reno, NV | 81065 | |
| Xenogen Living Image 2.5 software | Caliper LifeSciences, Hopkinton, MA | N/A |
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