目的 基于生物信息分析與動物實驗探討人臍帶間充質干細胞（hUCMSCs）與機體血小板穩(wěn)態(tài)的關系。方法 運用DisGeNET、GeneCards、STRING 11.5等數(shù)據(jù)庫，分析hUCMSCs誘發(fā)血小板減少的靶點及通路。將SD大鼠隨機區(qū)組分為5組：陰性對照組（0.9%氯化鈉注射液）、溶媒對照（80mg·kg^-1白蛋白）組和hUCMSCs高、中、低劑量（1.0×107、5×106、2.5×106個·kg^-1，分別為臨床等效劑量的10.0、5.0、2.5倍）組，每組6只，雌雄各半。每周按10mL·kg^-1尾iv1次，共4次，與臨床擬用方法一致。全自動血液分析儀分析血小板相關的指標：血小板計數(shù)（PLT）、血小板壓積（PCT）、血小板分布寬度（PDW）、平均血小板體積（MPV）；剖取大鼠胸骨，取骨髓，制備骨髓涂片，瑞特-吉姆薩復合染液染色，光學顯微鏡下計數(shù)全片巨核細胞總數(shù)、產(chǎn)血小板巨核細胞數(shù)；測量脾臟總長度；蘇木精-伊紅（HE）和過碘酸-雪夫（PAS）染色脾臟作組織病理學檢查；實時熒光定量PCR（qRT-PCR）分析肝臟促血小板生成素（TPO）、血小板生成素受體（Mpl）、Krüppel樣因子1（Klf1）、Friend白血病整合素1（FLI1）、GATA結合蛋白1（GATA1）mRNA變化。結果 hUCMSCs與血小板減少相關的靶點有209個，作用于158條信號通路，與血小板生成最密切的通路為造血細胞譜通路，包含了20個靶點。以平均連接度篩選出14個核心靶點：IL6、TNFRSF11A、CD34、KIT、IL4、CSF2、CSF3、IL3、IL2RA、TPO、EPO、TFRC、CD44、IL11。CD44既是核心靶點又是hUCMSCs陽性表面標志物。與陰性對照、溶媒對照組比較，hUCMSCs高、中、低劑量組PLT、PCT顯著降低（P＜0.05），PDW、MPV差異無統(tǒng)計學意義；hUCMSCs組全片巨核細胞總數(shù)、產(chǎn)血小板巨核細胞數(shù)差異無統(tǒng)計學意義；hUCMSCs高、中劑量組脾臟長度顯著增加（P＜0.05）；PAS染色見hUCMSCs高、中劑量組脾臟血小板儲存增加；hUCMSCs高、中、低劑量組肝臟組織TPO、Mpl、KLF1 mRNA表達顯著增加（P＜0.05），FLI1、GATA1 mRNA表達變化不顯著。結論 CD44⁺hUCMSCs可能通過促脾臟吞噬血小板，增加血小板在脾臟中儲存，使血液中PLT、PCT減少，從而擾亂血小板穩(wěn)態(tài)。

Objective To explore the relationship between human umbilical cord mesenchymal stem cells (hUCMSCs) and platelet homeostasis based on biological information analysis and animal experiments. Methods DisGeNET, GeneCards and STRING 11.5 databases were used to analyze the targets and pathways of hUCMSCs induced thrombocytopenia. SD rats were randomly divided into five groups: Negative control group (0.9% sodium chloride injection), solvent control group (80 mg·kg^-1 albumin) and hUCMSCs high, medium and low dose groups (1.0×10⁷, 5×10⁶, 2.5×10⁶·kg^-1, which were 2.5, 5.0 and 10.0 times of the clinical equivalent dose, respectively) with 6 in each group, half male and half female. 10 mL·kg^-1 tail iv was applied once a week for four times, which was consistent with the proposed clinical method. Platelet count (PLT), platelet pressure (PCT), platelet distribution width (PDW), and mean platelet volume (MPV) were analyzed by automatic hematology analyzer. The rat sternum was dissected and bone marrow was extracted. Bone marrow smears were prepared and stained with Reiter-Giemsa composite dye solution. The total number of megakaryocytes and platelet-producing megakaryocytes in the whole film were counted under the optical microscope. Total spleen length was measured. The spleen was stained with HE and PAS for histopathological examination. Realtime fluorescence quantitative PCR (qRT-PCR) was used to analyze the mRNA changes of liver thrombopoietin (TPO), thrombopoietin receptor (Mpl), Kruppel-like factor 1 (Klf1), Friend leukemia integrin 1 (FLI1) and GATA binding protein 1 (GATA1). Results hUCMSCs had 209 targets related to thrombocytopenia, acting on 158 signal pathways. The most closely related pathway to platelet formation was the hematopoietic cell lineage, which contains 20 targets. Based on the average connectivity, 14 core targets were screened: IL6, TNFRSF11A, CD34, KIT, IL4, CSF2, CSF3, IL3, IL2RA, TPO, EPO, TFRC, CD44 and IL11. CD44 was both a core target and a positive surface marker of hUCMSCs used in this study. Compared with negative control and solvent control group, PLT and PCT of hUCMSCs high, medium and low dose groups were significantly decreased (P<0.05), and PDW and MPV differences were not statistically significant. There was no significant difference in the total number of megakaryocytes and the number of platelet-producing megakaryocytes in hUCMSCs group. Spleen length in hUCMSCs high-dose and medium-dose groups was significantly increased (P<0.05). PAS staining showed increased platelet storage in spleen of hUCMSCs high-dose and medium-dose groups. The mRNA level of TPO, Mpl and KLF1 in liver tissues of hUCMSCs high, medium and low dose groups were significantly increased (P<0.05), while the mRNA level of FLI1 and GATA1 were not significantly changed. Conclusion CD44⁺ hUCMSCs may promote the spleen to phagocytize platelets, increase the storage of platelets in the spleen, reduce PLT and PCT in the blood, and thus disturb the platelet homeostasis.

R329.2

重慶市技術創(chuàng)新與應用發(fā)展專項（cstc2020jscx-lyggX0004）