沉默Sp5对mEPMCs中Wnt信号通路相关因子及增殖能力的影响

doi:10.11958/20230521

天津医药 ›› 2023, Vol. 51 ›› Issue (12): 1314-1320.doi: 10.11958/20230521

沉默Sp5对mEPMCs中Wnt信号通路相关因子及增殖能力的影响

柏宇¹(), 兰雪娇¹, 唐璟¹, 文雨¹, 吕明敏¹, 宋庆高²^,^△()

1.遵义医科大学口腔医学院（邮编563000）
2.遵义医科大学附属口腔医院口腔颌面外科

收稿日期:2023-04-17 修回日期:2023-05-19 出版日期:2023-12-15 发布日期:2023-12-22
通讯作者: ^△ E-mail：814641639@qq.com
作者简介:柏宇（1993），女，硕士在读，主要从事唇腭裂方面研究。E-mail：1316981548@qq.com
基金资助:
国家自然科学基金地区科学基金项目(8186040248)

Effects of Sp5 silencing on Wnt signaling pathway related factors and proliferative ability in mEPMCs

BAI Yu¹(), LAN Xuejiao¹, TANG Jing¹, WEN Yu¹, LYU Mingmin¹, SONG Qinggao²^,^△()

1. School of Stomatology, Zunyi Medical University, Zunyi 563000, China
2. Department of Oral and Maxillofacial Surgery, Affiliated Stomatological Hospital of Zunyi Medical University

Received:2023-04-17 Revised:2023-05-19 Published:2023-12-15 Online:2023-12-22
Contact: ^△ E-mail：814641639@qq.com

柏宇, 兰雪娇, 唐璟, 文雨, 吕明敏, 宋庆高. 沉默Sp5对mEPMCs中Wnt信号通路相关因子及增殖能力的影响[J]. 天津医药, 2023, 51(12): 1314-1320.

BAI Yu, LAN Xuejiao, TANG Jing, WEN Yu, LYU Mingmin, SONG Qinggao. Effects of Sp5 silencing on Wnt signaling pathway related factors and proliferative ability in mEPMCs[J]. Tianjin Medical Journal, 2023, 51(12): 1314-1320.

摘要/Abstract

摘要：

目的探讨沉默转录因子特异蛋白5（Sp5）对小鼠胚胎腭突间充质细胞（mEPMCs）中Wnt信号通路相关因子及增殖能力的影响。方法体外分离并培养孕14.5 d C57BL/6J小鼠的mEPMCs，细胞免疫荧光染色鉴定细胞来源；利用慢病毒转染技术沉默mEPMCs中Sp5基因的表达，Western blot验证转染效率。后续实验设空白对照组、空载病毒组、沉默组（Sp5-shRNA组）。各组细胞转染72 h后，采用Western blot及RT-qPCR法分别检测β-连环蛋白（β-catenin）、丝氨酸/苏氨酸激酶（GSK-3β）、Wnt家族成员3a（Wnt3a）、特异性周期蛋白D1（CyclinD1）蛋白及mRNA的表达；CCK-8法检测细胞增殖能力；免疫荧光法检测5-乙炔基-2'脱氧尿嘧啶核苷（EdU）阳性细胞增殖率；流式细胞术检测细胞周期。结果成功分离mEPMCs并沉默Sp5的表达。Western blot及RT-qPCR结果显示，Sp5-shRNA组β-catenin、GSK-3β、Wnt3a、CyclinD1蛋白及mRNA表达水平均高于空白对照组和空载病毒组（P<0.05）；Sp5-shRNA组细胞增殖能力和EdU阳性细胞增殖率均高于空白对照组和空载病毒组（P<0.05）；Sp5-shRNA组mEPMCs处于S期的细胞比例高于空白对照组和空载病毒组（P<0.05）。结论沉默mEPMCs中Sp5可通过对Wnt信号通路的调控来参与腭的发育，促进mEPMCs的增殖。

关键词: Sp转录因子类, Wnt信号通路, 细胞增殖, 腭突间充质细胞, Sp5基因

Abstract:

Objective To investigate the effect of transcription factor specific protein5 (Sp5) silencing on Wnt signaling pathway correlated factors and cell proliferation ability in mouse embryo palatal mesenchymal cells (mEPMCs). Methods mEPMCs of 14.5 d pregnant C57BL/6J mice were isolated and cultured in vitro. Cell source was identified by immunofluorescence staining. Lentivirus transfection technique was used to silence the expression of Sp5 gene in mEPMCs, and the transfection efficiency was verified by Western blot assay. Follow-up experiments were set up with the blank control group, the no-load virus group and the slience group (the Sp5-shRNA group). The protein and mRNA expression levels of β-catenin, GSK-3β, Wnt3a and CyclinD1 were detected by Western blot assay and RT-qPCR after transfection for 72 h in each group. Cell proliferation capacity was detected by CCK-8. The proliferation rate of 5-Ethynyl-2′-deoxyuridine (EdU) positive cells was detected by immunofluorescence assay. Cell cycle was detected by flow cytometry. Results mEPMCs were successfully isolated, and Sp5 expression was silenced. Western blot and RT-qPCR results showed that the protein and mRNA expressions of β-catenin, GSK-3β, Wnt3a and CyclinD1 were significantly higher in the Sp5-shRNA group than those in the blank control group and the no-loaded virus group (P < 0.05). The proliferative ability and the proliferative rate of EdU positive cells were higher in the Sp5-shRNA group than those in the blank control group and the no-loaded virus group (P < 0.05). The proportion of mEPMCs in S phase was higher in the Sp5-shRNA group than that in the blank control group and the no-loaded virus group (P < 0.05). Conclusion Sp5 in silenced mEPMCs can participate in palate development and promote the proliferation of mEPMCs by regulating Wnt signaling pathway.

Key words: Sp transcription factors, Wnt signaling pathway, cell proliferation, embryopalatal mesenchymal cells, Sp5 gene

中图分类号:

R782.22

图/表 13

表1 Sp5基因shRNA干扰靶点序列及病毒滴度

Tab.1 Sp5 gene shRNA interfered with target sequences and viral titers

序列名称	碱基序列	病毒滴度/ （TU/mL）
shRNA4	CCCGTCGGACTTTGCACAGTA	1.5×10⁸
shRNA5	AGTGCGGCAAACGCTTCATGC	1×10⁸
shRNA6	TGGGTTCACCCTCCAGACTTT	1×10⁸
空载病毒对照	TTCTCCGAACGTGTCACGTAA	2×10⁸

表2 各组病毒混合液配制情况

Tab.2 Preparation of virus mixture in each group （μL）

组别	MOI值=10 （病毒原液＋完全培养基）	MOI值=30 （病毒原液＋完全培养基）	MOI值=50 （病毒原液＋完全培养基）
空载病毒组	0.40＋49.55	1.20＋48.75	2.00＋47.95
shRNA4组	0.33＋49.62	1.60＋48.35	2.60＋47.35
shRNA5组	0.80＋49.15	2.40＋47.55	4.00＋45.95
shRNA6组	0.80＋49.15	2.40＋47.55	4.00＋45.95

表3 PCR引物序列

Tab.3 PCR primer sequence

基因名称	引物序列（5′→3′）	产物大小/bp
β-catenin	上游：AGCTGGCCTGGTTTGATACT	125
β-catenin	下游：CCATTCCCACCCTACCAAGT	125
GSK-3β	上游：ACCAGGAGCAGGACATTTCAC	197
GSK-3β	下游：CAGGTGTGTCTCGCCCATT	197
Wnt3a	上游：CCTGGTCTACTACGAGGCCT	201
Wnt3a	下游：ACGTAGCAGCACCAATGGAA	201
CyclinD1	上游：TGCGTGCAGAAGGAGATTGT	241
	下游：AGATGCACAACTTCTCGGCA	241
GAPDH	上游：TCACCATCTTCCAGGAGCGAGAC	303
	下游：TGAGCCCTTCCACAATGCCAAAG	303

图1 倒置相差显微镜下小鼠原代mEPMCs形态（×40，标尺=200 μm）

Fig.1 Morphology of primary mouse palatal mesenchymal cells under inverted phase contrast microscope (×40，scale=200 μm)

图2 小鼠mEPMCs Vimentin及P-CK免疫荧光鉴定（×200，标尺=50 μm）

Fig.2 Identification of primary mouse palatal mesenchymal cells Vimentin and P-CK by fluorescence (×200，scale=50 μm)

图3 转染72 h后各组细胞ZsGreen表达情况（×40，标尺=200 μm）

Fig.3 The expression of ZsGreen in each group 72 h after transfection (×40，scale=200 μm)

图4 Sp5-shRNA对mEPMCs Sp5表达的影响 1：空白对照组；2：空载病毒组；3：shRNA4组；4：shRNA5组；5：shRNA6组。

Fig.4 The influence of Sp5-shRNA on mEPMCs of Sp5 expression

图5 小鼠mEPMCs中β-catenin、GSK-3β、Wnt3a、CyclinD1蛋白印迹图 1：空白对照组；2：空载病毒组；3：Sp5-shRNA组。

Fig.5 Western blot results of mouse palatal mesenchymal cells β-catenin, GSK-3β, Wnt3a and CyclinD1

表4 各组细胞中β-catenin、GSK-3β、Wnt3a、CyclinD1蛋白表达比较

Tab.4 Comparison of β-catenin, GSK-3β, Wnt3a and CyclinD1 protein between three groups of cells （n=3，$\bar{x}±s$）

组别	β-catenin	GSK-3β	Wnt3a	CyclinD1
空白对照组	0.55±0.06	2.26±0.08	0.24±0.03	0.76±0.22
空载病毒组	0.62±0.02	2.67±0.120	0.28±0.04	0.81±0.33
Sp5-shRNA组	1.12±0.22^ab	4.19±0.74^ab	0.34±0.03^ab	1.73±0.04^ab
F	16.640^＊	16.180^＊	14.500^＊	16.760^＊

表5 各组细胞中β-catenin、GSK-3β、Wnt3a、CyclinD1 mRNA表达比较

Tab.5 Expression of β-catenin, GSK-3β, Wnt3a and CyclinD1 mRNA in each group of cells （n=3，$\bar{x}±s$）

组别	β-catenin	GSK-3β	Wnt3a	CyclinD1
空白对照组	0.83±0.09	0.96±0.22	0.90±0.36	0.70±0.24
空载病毒组	1.02±0.02	0.96±0.09	1.03±0.08	1.01±0.01
Sp5-shRNA组	1.26±0.10^ab	1.96±0.39^ab	1.92±0.30^ab	1.61±0.33^ab
F	24.550^＊	14.310^＊	12.370^＊	11.940^＊

表6 各组不同时点细胞增殖能力比较

Tab.6 Comparison of cell proliferation ability between three groups （n=3，$\bar{x}±s$）

组别	OD₄₅₀
组别	24 h	48 h	72 h
空白对照组	0.31±0.05	0.64±0.06	1.24±0.19
空载病毒组	0.33±0.06	0.67±0.05	1.21±0.21
Sp5-shRNA组	0.39±0.03	0.84±0.01^ab	1.81±0.11^ab
F	2.419	16.500^＊	8.457^＊

图6 EdU免疫荧光检测各组细胞增殖情况（×40，标尺=200 μm）

Fig.6 Cell proliferation indexes in each group detected by EdU immunofluorescence(×40，scale=200 μm)

表7 各组细胞的细胞周期分布

Tab.7 Cell cycle distribution of each group of cells （n=3，%，$\bar{x}±s$）

组别	G0/G1期	S期	G2/M期
空白对照组	87.15±0.72	7.65±0.16	5.20±0.82
空载病毒组	87.75±0.33	7.40±0.07	4.85±0.26
Sp5-shRNA组	84.66±0.16^ab	10.48±0.20^ab	4.85±0.28
F	37.200^＊	371.900^＊	0.430

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沉默Sp5对mEPMCs中Wnt信号通路相关因子及增殖能力的影响

Effects of Sp5 silencing on Wnt signaling pathway related factors and proliferative ability in mEPMCs

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