iPSCs诱导的造血干细胞对内皮细胞血管生成作用的影响及相关作用机制研究

doi:10.3877/cma.j.issn.2095-1221.2021.04.001

目的

体外实验探讨诱导多能干细胞（iPSCs）诱导的造血干细胞（HSCs）对内皮细胞血管生成作用的影响，及其相关的作用机制。

方法

将iPSCs经体外诱导分化，形成造血干细胞（iPS-HSCs）。实验分为两组，分别为单纯人脐静脉内皮细胞（HUVECs）组和iPS-HSC+ HUVEC组（iPS-HSCs与HUVEC进行共培养）。在基质胶中观察iPS-HSCs对内皮细胞血管生成作用的影响，并采用Western blot检测血管内皮生长因子（VEGF）及血管生成素1 （Ang-1）蛋白的相对表达水平。两组间比较采用t检验。

结果

iPS-HSCs与HUVECs共培养后可促进内皮细胞的管腔形成，并提高VEGF和Ang-1的蛋白表达水平。与单纯HUVEC组相比，iPS-HSC+HUVEC组网络结点数量（10.33±1.53比28.67±3.51）升高，差异具有统计学意义（t = 8.292，P < 0.01）；iPS-HSC+HUVEC组VEGF的相对表达水平（0.86±0.09比1.44±0.11）升高，差异具有统计学意义（t = 43.715，P < 0.01）；iPS-HSC+HUVEC组Ang-1的相对表达水平（0.46±0.01比0.68±0.05）升高，差异具有统计学意义（t = 52.255，P < 0.01）。

结论

iPS细胞诱导的HSC能够促进内皮细胞的血管生成作用，其促进血管生成的作用可能与VEGF及Ang-1蛋白上调有关。

关键词: 诱导多能干细胞, 造血干细胞, 血管生成, 作用机制

Objective

To investigate the effects and the mechanism of induced pluripotent stem cells (iPSCs) induced hematopoietic stem cells on endothelial cell angiogenesis in vitro experiments.

Methods

iPS cells were induced to differentiate into hematopoietic stem cells (iPS-HSCs) in vitro. The experiment was divided into two groups: human umbilical vein endothelial cells (HUVECs) group and iPS-HSC+HUVEC group. The effects of iPS-HSCs on the angiogenesis of endothelial cells were observed in the Matrigel, and the relative expression levels of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) protein were detected by Western blot.

Results

The co-culture of iPS-HSCs and HUVECs could significantly promote the tube formation of endothelial cells, and the protein expression levels of VEGF and Ang-1 were significantly increased. Compared with HUVEC group, the number of branch points in iPS-HSC+HUVEC group (10.33±1.53 vs 28.67±3.51) was significantly increased (t = 8.292, P < 0.01) ; the relative expression level of VEGF in iPS-HSC+HUVEC group (0.86±0.09 vs 1.44±0.11) was significantly increased (t = 43.715, P < 0.01) ; the relative expression level of Ang-1 in iPS-HSC+HUVEC group (0.46±0.01 vs 0.68±0.05) was significantly higher (t =52.255, P < 0.01) .

Conclusion

The hematopoietic stem cells induced by iPS cells can promote the angiogenesis of endothelial cells, which may be related to the up-regulation of VEGF and Ang-1 proteins.

Key words: Induced pluripotent stem cells, Hematopoietic stem cells, Angiogenesis, Mechanism

表1 相关基因引物序列

基因	（5'→3'）	预期扩增产物长度（bp）
Oct3/4	上游GATCCTCGGACCTGGCTAA	592
	下游ACGAGGGTTTCTGCTTTGC	592
Sox2	上游ACAGTTACGCGCACATGAA	424
	下游GGTAGTGCTGGGACATGTGA	424
Lin28	上游GGGTCTGGAATCCATCCGTG	302
	下游GGGTAGGGCTGTGGATTTCT	302

表1 相关基因引物序列

基因	（5'→3'）	预期扩增产物长度（bp）
Oct3/4	上游GATCCTCGGACCTGGCTAA	592
	下游ACGAGGGTTTCTGCTTTGC	592
Sox2	上游ACAGTTACGCGCACATGAA	424
	下游GGTAGTGCTGGGACATGTGA	424
Lin28	上游GGGTCTGGAATCCATCCGTG	302
	下游GGGTAGGGCTGTGGATTTCT	302

图2 倒置显微镜下观察iPSCs克隆（×40）。2a为iPSCs传代形成的1号ESCs样克隆，2b为2号ESCs样克隆

图3 倒置显微镜下观察iPSCs碱性磷酸酶染色结果（×40）

图4 iPSC RT-PCR产物的电泳检测条带图

图5 倒置荧光显微镜下观察iPSCs形态（免疫荧光染色，×40）

图6 倒置显微镜下白光观察iPSCs向HSCs诱导分化的克隆形态变化（无染色，×40）

图7 iPSCs诱导分化的HSCs表型流式检测图

图8 管腔形成检测HUVEC组、iPSC-HSC+HUVEC组和iPSC-HSC+HUVEC+VEGF抗体组内皮细胞的血管生成能力

图9 Western blot检测HUVEC组和iPSC-HSC + HUVEC组内皮细胞VEGF、Ang-1蛋白的表达

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Influence of hematopoietic stem cells induced from iPS cells on endothelial cell angiogenesis and related mechanisms

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Influence of hematopoietic stem cells induced from iPS cells on endothelial cell angiogenesis and related mechanisms