E-钙黏蛋白对血管内皮祖细胞和骨髓间充质干细胞之间的黏附作用研究

doi:10.3877/cma.j.issn.2095-1221.2021.06.004

目的

初步探讨E-钙黏蛋白（E-cad）在内皮祖细胞（EPCs）和间充质干细胞（MSCs）之间的黏附作用。

方法

体外C57BL/6J小鼠骨髓腔分离培养EPCs和MSCs，使用Mito Tracker Red标记EPCs，DAPI标记MSCs；采用免疫荧光的方法观察E-cad在细胞内的表达情况；细胞黏附实验观察EPCs与MSCs体外黏附现象；使用E-cad siRNA干扰EPCs和MSCs中E-cad的表达，RT-qPCR和Western blot检测siRNA转染效率；转染成功后在成血管的凝胶上进一步观察二者黏附现象。多组间比较采用单因素方差分析，组间两两比较使用LSD-t检验。

结果

（1）与MSCs、EPCs比较，体外共培养MSCs和EPCs上清液中E-cad表达（378.26±34.47、564.72±41.58比1087.28±101.92）升高，差异均具有统计学意义（P < 0.05）；（2）与Control-siRNA、Mock、Negative相比，E-cad siRNA转染MSCs 48 h后E-cad mRNA表达（0.97±0.07、0.93±0.06、1.00±0.03比0.30±0.05）、E-cad蛋白表达（252.19±11.62、223.70±13.66、257.50±12.08比31.74±4.08）均降低，差异有统计学意义（P < 0.05）；与Control-siRNA、Mock、Negative比较，E-cad siRNA转染EPCs 48 h后E-cad mRNA表达（0.93 ±0.07、0.88±0.08、1.00±0.02比0.36±0.07）、E-cad蛋白表达（429.46±31.87、409.13±26.97、436.80±34.69比54.03±15.05）均降低，差异有统计学意义（P < 0.05）；（3）E-cad siRNA成功转染MSCs和EPCs后，二者黏附现象受到抑制；（4）沉默EPCs和MSCs的E-cad后，二者在成血管凝胶上的黏附及成管现象均受到抑制。

结论

E-cad介导EPCs和MSCs之间的黏附。

关键词: E-钙黏蛋白, 血管内皮祖细胞, 骨髓间充质干细胞, 细胞黏附

Objective

To explore the adhesion of E-cadherin (E-cad) between endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) .

Methods

EPCs and MSCs were isolated and cultured from the bone marrow cavity of C57BL/6Jmice. Use Mito Tracker Red to label EPCs and DAPI to label MSCs. Immunofluorescence was used to examine the E-cad expression of the two cell lines. Cell adhesion assay was used to observe the adhesion of EPCs and MSCs in vitro. E-cad expression in EPCs and MSCs was silenced using E-cad siRNA, and siRNA transfection efficiency was determined by RT qPCR and Western blot. After successful transfection, the adhesion of EPCs and MSCs on the angiogenic Matrigel was observed. One way ANOVA was used for comparison between multiple groups, and LSD-t test was used for pairwise comparison between groups.

Results

(1) Compared with MSCs or EPCs, the expression of E-cad in the supernatant of the co-cultured of MSCs and EPCs in vitro (378.26±34.47, 564.72±41.58 vs 1087.28±101.92) was increased, the difference was statistically significant (P < 0.05) . (2) Compared with Control-siRNA, Mock and Negative, the expression of E-cad mRNA (0.97±0.07, 0.93±0.06, 1.00±0.03 vs 0.30±0.05) and E-cad protein (252.19 ±11.62, 223.70±13.66, 257.50±12.08 vs 31.74±4.08) of MSCs transfected with siRNA for 48 hours were decreased, the difference was statistically significant (P < 0.05) ; Compared with Control-siRNA, Mock and Negative, the expression of E-cad mRNA (0.93 ±0.07、0.88±0.08、1.00±0.02 vs 0.36±0.07) and E-cad protein (429.46±31.87、409.13±26.97、436.80±34.69 vs 54.03±15.05) of EPCs transfected with siRNA for 48 hours were decreased, the difference was statistically significant (P < 0.05) . (3) The adhesion of MSCs and EPCs were significantly inhibited after successful transfection of E-cad siRNA. (4) Adhesion and tube formation were significantly inhibited in the angiogenic Matrigel after E-cad of EPCs and MSCs was silenced.

Conclusions

E-cad could mediate the adhesion between EPCs and MSCs.

Key words: E-cadherin, Endothelial progenitor cells, Mesenchymal stem cells, Cell adhesion

表1 引物序列信息

基因	引物序列（5'-3'）	预期扩增长度（bp）
E-cad	上游GCTGGACCGAGAGAGTTACC	160
	下游AGGCACTTGACCCTGATACG
β-actin（内参）	上游GTGCTATGTTGCTCTAGACTTCG	174
	下游ATGCCACAGGATTCCATACC

表1 引物序列信息

基因	引物序列（5'-3'）	预期扩增长度（bp）
E-cad	上游GCTGGACCGAGAGAGTTACC	160
	下游AGGCACTTGACCCTGATACG
β-actin（内参）	上游GTGCTATGTTGCTCTAGACTTCG	174
	下游ATGCCACAGGATTCCATACC

图1 荧光显微镜下观察体外培养MSCs和EPCs时E-cad的表达（×400）注：a图为MSCs的E-cad表达的荧光图像；b图为EPCs的E-cad表达的荧光图像；c图为体外共培养MSCs和EPCs时E-cad表达的荧光强度和亮度较a和b明显增加；d图为Elisa量化全部的免疫荧光结果定量统计图；^aP < 0.05

图2 E-cad siRNA转染MSCs和EPCs后E-cad mRNA表达注：^aP < 0.05，ns为差异无统计学意义

图3 E-cad siRNA转染MSCs和EPCs后E-cad蛋白的表达注：a、b图为MSCs各组的E-cad蛋白表达；c、d图为EPCs各组的E-cad蛋白表达；^aP < 0.05，ns为差异无统计学意义

图4 DAPI标记的MSCs和未标记的EPCs体外共培养时黏附现象观察（×400）注：a ~ c图为正常细胞；d ~ f图为E-cad siRNA转染的细胞；g ~ i图为Control-siRNA转染的细胞；a、d、g图为DAPI标记MSCs的荧光图像；b、e、h图为MSCs和EPCs的E-cad表达的荧光图像；c图为a和b的融合；f：d和e的融合；i：g和h的融合；红色箭头指示未标记的EPCs；白色箭头指示DAPI标记的MSCs

图5 Mito Tracker Red标记的EPCs和DAPI标记的MSCs在成血管凝胶上的黏附现象观察（×400）注：a、d、g图为Mito Tracker Red标记的EPCs的荧光图像；b、e、h图为DAPI标记的MSCs的荧光图像；c图为a和b的融合；f图为d和e的融合；i图为g和h的融合；a ~ c图为正常的细胞；d ~ f图为E-cad siRNA转染的细胞；g ~ i图为Control-siRNA转染的细胞

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Study on the adhesion of E-cadherin between endothelial progenitor cells and bone marrow mesenchymal stem cells

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Study on the adhesion of E-cadherin between endothelial progenitor cells and bone marrow mesenchymal stem cells