白血病抑制因子 (LIF) 过表达对大熊猫骨髓间充质干细胞生长的影响

doi:10.16829/j.slxb.150902

摘要/Abstract

摘要：

白血病抑制因子 (Leukemia inhibitory factor, LIF) 是一种调节细胞生长、增殖和分化的重要因子。骨髓间充质干细胞 (Bone marrow mesenchymal stem cells, BM‑MSCs) 在组织损伤修复、自身免疫疾病治疗中具有广阔的应用前景，对大熊猫等濒危动物的疾病治疗和遗传保护方面有着巨大的潜力。本研究旨在构建大熊猫LIF过表达载体，应用脂质体转染大熊猫骨髓间充质干细胞 (Panda bone marrow mesenchymal stem cells, PDBM‑MSCs)，研究LIF对PDBM‑MSCs生长的影响。本研究从PDBM‑MSCs cDNA 中扩增LIF基因CDS区，无缝克隆连接至pCDH‑CMV‑MCS‑EF1‑GFP+puro过表达载体上，构建 LIF过表达重组质粒，利用Lipo3000转染重组质粒至PDBM‑MSCs中。研究结果显示，转染48 h后PDBM‑MSCs表达绿色荧光，转染72 h后分别提取细胞总RNA，反转录成cDNA，经q‑PCR检测发现，转染后LIF过表达组相比阴性对照组LIF的mRNA表达水平显著提高。同时，LIF过表达后能够显著上调细胞周期调控基因CCNB2、CDK7在mRNA水平上的表达，显著下调凋亡基因caspase3、P53、P16、P21在mRNA水平上的表达；转染72 h后流式检测显示转染LIF过表达重组质粒的PDBM‑MSCs细胞活率显著高于阴性对照组。本研究成功构建大熊猫LIF过表达载体并转染至PDBM‑MSCs中，显著提高细胞活率、抑制凋亡，表明LIF对PDBM‑MSCs起到促进生长抑制凋亡的作用，为LIF在大熊猫中的作用机制研究奠定了基础。

关键词: 大熊猫, 骨髓间充质干细胞, 白血病抑制因子, 细胞生长

Abstract:

Leukemia inhibitory factor (LIF) is an important factor regulating cell growth, proliferation and differentiation. Bone marrow mesenchymal stem cells (BM-MSCs) have broad application prospects in the repair of tissue damage as well as the treatment of autoimmune diseases, and, as a result, have great potential in the disease treatment and genetic conservation of rare and endangered animals, such as giant pandas. The aim of this study was to construct a giant panda LIF overexpression vector and transfect panda bone marrow mesenchymal stem cells (PDBM-MSCs) with liposome to study the effect of LIF on the growth of PDBM-MSCs. The CDS region of the LIF gene was amplified from the cDNA of PDBM-MSCs, and seamlessly cloned onto the pCDH-CMV-MCS-EF1-GFP+puro overexpression vector to construct the LIF overexpression recombinant plasmid. Lipo3000 was used to transfect recombinant plasmid into PDBM-MSCs. The results showed that PDBM-MSCs expressed green fluorescence 48 h after transfection, and total RNA was extracted and reverse-transcribed into cDNA 72 h after transfection. A q-PCR detection showed that the mRNA expression level of LIF in the LIF overexpression group was significantly higher than that in the negative control group. Moreover, overexpression of LIF could significantly up-regulate the mRNA expressions of CCNB2 and CDK7, and significantly down-regulate the mRNA expressions of apoptosis genes caspase3, P53, P16 and P21. 72 h after transfection, flow detection showed that the viability of PDBM-MSCs transfected with LIF overexpression recombinant plasmid was significantly higher than that of the negative control group. In this study, the panda LIF overexpression vector was successfully constructed and transfected into PDBM-MSCs, which significantly improved cell viability and inhibited apoptosis, indicating that LIF can promote growth and inhibit apoptosis of PDBM-MSCs, laying a foundation for the study of the mechanism of LIF in giant pandas.

Key words: Giant panda, Bone marrow mesenchymal stem cells, Leukemia inhibitor, Cell growth

中图分类号:

Q952.5

李非平, 张梦诗, 王神飞, 胡贤彪, 刘玉良, 侯蓉, 刘项宇, 蔡开来. 白血病抑制因子 (LIF) 过表达对大熊猫骨髓间充质干细胞生长的影响[J]. 兽类学报, 2025, 45(3): 295-301.

Feiping LI, Mengshi ZHANG, Shenfei WANG, Xianbiao HU, Yuliang LIU, Rong HOU, Xiangyu LIU, Kailai CAI. Effect of overexpression of leukemia inhibitory factor (LIF) on the growth of bone marrow mesenchymal stem cells of giant panda[J]. ACTA THERIOLOGICA SINICA, 2025, 45(3): 295-301.

图/表 4

表1 引物序列

Table 1 Primer sequences

引物 Primer	引物序列 Primer sequence	扩增长度 PCR length/bp	退火温度 Tm/℃
LIF	F： TGCCGCCTGTGTAACAAGTA R： CACGGCGATGACCTGCTTAT	135	60
CDK2	F： CCTTTGGAGTCCCTGTTCGTA R： CTTCTAGATAGGAGCACAGCG	231	56
CCNB2	F： GTCGATGTGGAACAGCACAC R： GCGTACTTATTCTTGATGGCGATG	272	55
CCNH	F： GTCGATGTGGAACAGCACAC R： GCGTACTTATTCTTGATGGCGATG	272	56
CDK7	F： TATGGCGTAGGTGTGGACAT R： CTTAGTTTTCAATGCAGGCCAC	162	53
P53	F： TGCTCCGACTGGCGCTAAA R： TCGGAAAATGTCTCCTGGCTC	237	58
P16	F： TCAAGCCGTGAAGGAACGAG R： TGCCACTGGAGTAACACTGC	183	58
P27	F： CGGTGGGTTGATTACTAGGAGC R： GTCCATTCCATGGAGTCAGCG	131	58
P21	F： GAGCTGTGGGTTGTCATGGA R： AGCCATCCATTCCCAACAGG	190	58
Caspase3	F： TGCGGTCTGGGGAACCTAA R： CTGTGTGTTCGGTGTTCTCC	100	57
GAPDH	F： GGCATCGCTCTCAATGACCA R： AGTGGAGGTCAGGAGGTTCT	222	55 ~ 60

图1 PDBM-MSCs 转染后绿色荧光蛋白表达情况及LIF过表达效果. A：PDBM-MSCs 脂质体转染 48 h 后绿色荧光蛋白表达情况 (100 ×).Scale bar = 200 μm.B：脂质体转染后 PDBM-MSCs 的 LIF 在 mRNA 水平上的相对表达量. 不同字母表示组间数据存在统计学差异 (P < 0.05)

Fig. 1 Expression of green fluorescent protein and overexpression of LIF after PDBM-MSCs transfection. A: Expression of green fluorescent protein (GFP) 48 h after transfection with PDBM-MSCs liposomes (100 ×).Scale bar = 200 μm. B: The relative expression level of LIF of PDBM-MSCs at mRNA level after transfection with liposome. Different letters of a and b indicated statistical difference between groups (P < 0.05)

图2 LIF 过表达后PDBM-MSCs周期基因表达. 不同字母表示组间数据存在统计学差异 (P < 0.05)

Fig. 2 PDBM-MSCs cycle gene expression after LIF overexpression. Different letters of a and b indicated statistical difference between groups (P < 0.05)

图3 LIF 过表达后 PDBM-MSCs 细胞凋亡情况. A：LIF 过表达后 PDBM-MSCs 流式凋亡检测；B：LIF 过表达后 PDBM-MSCs活率变化；C：LIF 过表达后 PDBM-MSCs 凋亡基因表达情况，不同字母表示组间数据存在统计学差异 (P < 0.05)

Fig. 3 Apoptosis of PDBM-MSCs after LIF overexpression. A: Flow cytometry of PDBM-MSCs after LIF overexpression; B: Changes of PDBM-MSCs activity after LIF overexpression; C: Apoptotic gene expression of PDBM-MSCs after LIF overexpression. Different letters of a and b indicated statistical difference between groups (P < 0.05)

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