Influence of silencing ZHX3 on BMP2/smad pathway in BMSCs

Li Yanqin; Zhang Miaomiao; Min Heming; Wang Yan; Qin Shujian; Yuan Jing

doi:10.13406/j.cnki.cyxb.001038

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Influence of silencing ZHX3 on BMP2/smad pathway in BMSCs

更新时间：2024-01-17

- Influence of silencing ZHX3 on BMP2/smad pathway in BMSCs
- Journal of Chongqing Medical University Vol. 41, Issue 10, Pages: 990-994(2016)
- 作者机构：
  
  1. 锦州医科大学生命科学研究院
  2. 锦州医科大学解剖学教研室
  3. 锦州医科大学细胞生物学教研室
  4. 锦州医科大学附属第一医院神经内科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001038
  CLC： R687;R318.08
- Published：2016，
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[1]李艳芹,张苗苗,闵鹤鸣,王艳,秦书俭,袁静.ZHX3基因沉默对BMSCs中BMP2/smad通路的影响[J].重庆医科大学学报,2016,41(10):990-994.

Li Yanqin, Zhang Miaomiao, Min Heming, et al. Influence of silencing ZHX3 on BMP2/smad pathway in BMSCs. [J]. Journal of Chongqing Medical University 41(10):990-994(2016)
[1]李艳芹,张苗苗,闵鹤鸣,王艳,秦书俭,袁静.ZHX3基因沉默对BMSCs中BMP2/smad通路的影响[J].重庆医科大学学报,2016,41(10):990-994. DOI： 10.13406/j.cnki.cyxb.001038.

Li Yanqin, Zhang Miaomiao, Min Heming, et al. Influence of silencing ZHX3 on BMP2/smad pathway in BMSCs. [J]. Journal of Chongqing Medical University 41(10):990-994(2016) DOI： 10.13406/j.cnki.cyxb.001038.

摘要

目的:探讨转录抑制因子——锌指蛋白和同源框3（zinc fingers and homeoboxes 3

ZHX3）基因沉默对骨髓间充质干细胞（bone marrow stem cells

BMSCs）中BMP2/smad通路相关因子骨形成蛋白2（bone morphogenetic protein 2

BMP2）、磷酸化smad1（phospho-smad1（或pho-smad1））表达的影响。方法:构建ZHX3低表达慢病毒载体转染大鼠BMSCs

同时设阴性对照空载病毒转染BMSCs及空白对照不做任何处理的BMSCs。采用荧光显微镜下测定细胞转染率、免疫印迹技术鉴定转染效果、免疫细胞化学和免疫印迹技术定性定量检测ZHX3沉默时BMP2、pho-smad1和smad4的表达情况。细胞经成骨诱导液处理后进行碱性磷酸酶和茜素红染色检测各组细胞成骨能力。结果:1复苏培养的细胞具有BMSCs形态。2转染后

ZHX3低表达组和空载体对照组均表达绿色荧光

转染率分别为（93.18±4.41）%和（92.56±4.35）%

空白对照组不表达荧光

未见转染阳性细胞（F=1 123.464

P<0.05）。3免疫细胞化学及免疫印迹结果显示

BMP2、pho-smad1、smad4于3组细胞内均可见阳性表达

但ZHX3低表达组的BMP2、pho-smad1水平（0.383 0±0.006 3

0.329 1±0.025 8）明显高于空白对照组（0.306 2±0.011 6

0.209 0±0.017 0）和空载体对照组（0.291 2±0.022 2

0.180 0±0.018 7）

P<0.05;而ZHX3低表达组smad4的表达（0.107 4±0.004 3）显著低于空白对照组（0.380 1±0.023 6）和空载体对照组（0.353 7±0.016 7）

P<0.05。4空白对照组、空载体对照组、ZHX3低表达组均可见碱性磷酸酶阳性表达的细胞及矿化结节

但后者数量均小于前两者。结论:ZHX3基因低表达可延迟BMSCs体外成骨能力

可能通过调控BMP2/smad通路中的smad4来发挥作用。

Abstract

Objective:To investigate the effects of silencing zinc fingers and homeoboxes 3(ZHX3) on expressions of bone morphogenetic proteins 2(BMP2)

phospho-smad1(or pho-smad1)in BMP2/smad pathway in bone marrow mesenchymal stem cells(BMSCs).Methods:ZHX3 low expression vector was constructed to transfect the rat BMSCs. At the same time

BMSCs transfected with no-load virus was taken as negative control group and BMSCs without any treatment as blank control group. Fluorescence microscope was used to determinate cell transfection rate. Immunocytochemistry method was used to identify the success of the transfection. Immunocytochemistry and Western blot were used to detect the protein expressions of BMP2

phospho-smad1

smad4 when silencing ZHX3 gene. Osteogenesis ability of cell after inducement of osteogenesis solution was detected by alkaline phosphatase and alizarin red staining. Results:1 Cells expressing BMSCs phenotype could be obtained by recovery culturing. 2After transfection

transfection rate of the ZHX3 low expression group and negative control group was(93.18±4.41)% and(92.56±4.35)% respectively

but no transfection cell was observed in blank control group(F=1 123.464

P<0.05). 3 According to the results of immunocytochemistry and Western blot

positive expressions of BMP2

pho-smad1

smad4 were observed in three groups

but their expressions were higher in ZHX3 low expression group(0.383 0±0.006 3

0.329 1±0.025 8)than in blank control group(0.306 2±0.011 6

0.209 0±0.017 0)group and negative control group(0.291 2 ±0.022 2

0.180 0 ±0.018 7

P <0.05). Expression of smad4 was lower in ZHX3 low expression group(0.107 4±0.004 3)than in blank control group(0.380 1±0.023 6)and negative control group(0.353 7±0.016 7

P<0.05). 4Positive expression of nalkaline phosphatase was observed in cells in all groups

however

its quantity was higher in blank control group and negative control group than in ZHX3 low expression group. Conclusion :ZHX3 gene silencing may delay BMSCs in vitro osteogenesis

which may through the regulation of smad4 in BMP/smad pathway.

关键词

锌指蛋白和同源框3基因沉默骨髓间充质干细胞骨形成蛋白smad

Keywords

zinc fingers and homeoboxes 3gene silencingmesenchymal stem cellsbone morphogenetic proteinssmad

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