兰州大学机构库 >口腔医学院
缺氧微环境下Sestrin2对预血管化细胞膜片成血管作用的影响
Alternative TitleSestrin2 promoting the angiogenesis of prevascularized cell sheets under hypoxia microenvironment
任丽娜
Subtype硕士
Thesis Advisor任利玲
2023-05-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline口腔医学
KeywordSestrin2 Sestrin2 组织工程 Tissue engineering 血管化 Vascularization 缺氧 Hypoxia 细胞膜片 Cell sheets 骨髓间充质干细胞 Bone marrow mesenchymal stem cells
Abstract

目的:探究在缺氧微环境下Sestrin2(SESN2)对预血管化细胞膜片体内外成血管能力的影响,以期为工程化组织成功移植提供新的思路。

方法:以慢病毒为载体,转染获得由绿色荧光蛋白(Green fluorescent protein,GFP)标记的稳定高表达SESN2的人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)。使用荧光显微镜以及Western-blot(WB)检测转染效率及SESN2蛋白表达情况。选用全骨髓贴壁法提取兔骨髓间充质干细胞(rabbit Bone mesenchymal stem cells,rBMSCs),经过传代纯化后,将第二代rBMSCs按照2.0×105/孔的密度接种到六孔板中,隔天更换为含有10%胎牛血清、1%青霉素链霉素溶液、1% L-谷氨酰胺以及50 mg/L抗坏血酸的高糖DMEM培养基(锡纸避光),连续培养形成rBMSCs细胞膜片。将转染SESN2的HUVECs(SESN2组)按照5.0×104/cm2的密度接种到rBMSCs细胞膜片,分别在常氧和缺氧条件下培养1、3、7天后进行检测:倒置荧光显微镜下观察细胞排列;细胞增殖/毒性检测试剂盒(Cell counting kit,CCK-8)检测SESN2对HUVECs活力的影响;酶联免疫吸附实验(Enzyme linked immunosorbent assay,Elisa)和实时定量逆转录-聚合酶链式反应(quantitative Reverse transcription-polymerase chain reaction,qRT-PCR)检测血管内皮生长因子-A(Vascular endothelial growth factor-A,VEGF-A)和血小板衍生生长因子-BB(Platelet-derived growth factor subunit-B,PDGF-BB)表达情况;CD31免疫荧光检测观察血管网的形成。进一步将转染SESN2的预血管化细胞膜片移植到裸鼠皮下,分别在1、7、14天进行大体观察、苏木精-伊红染色法(Hematoxylin-eosin staining,HE staining)、CD31免疫荧光检测血管生成情况。

结果:(1)慢病毒转染后,可以成功获得高表达SESN2的HUVECs稳转株,转染效率达到90%以上;CCK-8细胞增殖实验显示SESN2高表达扭转了缺氧对HUVECs增殖的抑制作用,且慢病毒转染并未影响细胞增殖;(2)使用全骨髓贴壁法可以成功提取rBMSCs,流式检测显示间充质干细胞表面标志物CD29阳性表达率95.5%,CD44阳性表达率96.6%,CD34阳性表达率0.57%,CD45阳性表达率0.58%;(3)高密度接种rBMSCs并使用特定培养基连续培养14天,形成了细胞膜片;(4)高表达SESN2可以显著提高预血管化细胞膜片促血管生成相关细胞因子VEGF-A和PDGF-BB的释放,且VEGF-A在成血管早期释放,而在血管生成的晚期则主要释放的是PDGF-BB。CD31免疫荧光染色显示SESN2能在缺氧环境中显著促进HUVECs重排,促进血管网络结构的形成;(5)体内实验显示高表达SESN2可以促进血管网络形成,HE染色结果显示体外预构的血管网络结构与宿主血管发生吻合,形成功能性血管网。

结论:SESN2能有效的提高内皮细胞对缺氧微环境的适应能力,促进血管网络结构的形成。

Other Abstract

Objective: This thesis investigates the effect of Sestrin2 (SESN2) on the vascularization ability of prevascularized cell sheets in vitro and in vivo in a hypoxia microenvironment to provide new ideas for successful engineered tissue transplantation.

Methods: In this study, HUVECs with stable and high expression of SESN2 were firstly obtained by lentiviral transfection and labeled with green fluorescent protein (GFP). The transfection efficiency and SESN2 protein expression were further examined using fluorescence microscopy and Western-blot (WB). Meanwhile, rBMSCs were extracted by the whole bone marrow apposition method and cultured to the second generation, which were inoculated into six-well plates at a density of 2.0×105/hole and replaced with high-sugar DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution (PIS), 1% L-glutamine, and 50 mg/L ascorbic acid on alternate days, and rBMSCs cell sheets were formed in continuous culture. Next, HUVECs transfected with SESN2 (SESN2 group) were inoculated into rBMSCs cell sheets at a density of 5.0×104/cm2 and incubated under normoxia and hypoxia conditions for 1, 3 and 7 days, respectively, and then assayed: cell arrangement was observed under an inverted fluorescence microscope; Cell counting kit (CCK-8) was used to detect the effect of SESN2 on the viability of HUVECs; The enzyme linked immunosorbent assay (Elisa) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor subunit-BB (PDGF-BB); CD31 immunofluorescence assay to observe vascular network formation. Finally, the prevascularized cell sheets transfected with SESN2 were transplanted to the subcutis of nude mice, and angiogenesis was detected by gross observation, hematoxylin-eosin staining (HE staining), and CD31 immunofluorescence at 1, 7, and 14 days, respectively.

Results: (1) A stable transfection strain of HUVECs with high expression of SESN2 was successfully obtained by lentiviral transfection using puromycin screening, and the transfection efficiency was determined to be over 90% by fluorescence microscopy, comparing the number of cells in the same white and green fluorescence fields, and WB results also showed that the protein level of SESN2 was statistically significant compared with the WT and NC groups (P<0.01); CCK-8 cell proliferation assay showed that high expression of SESN2 reversed the inhibitory effect of hypoxia on the proliferation of HUVECs, and lentiviral transfection did not affect cell proliferation. (2) The whole bone marrow apposition method can successfully obtain rabbit bone marrow mesenchymal stem cells, and the flow assay showed 95.5% positive expression of CD29, 96.6% positive expression of CD44, 0.57% positive expression of CD34, and 0.58% positive expression of CD45 (3) High-density inoculation of rBMSCs and continuous culture using sheet-forming medium for 14 days resulted in the formation of cell sheets. (4) High expression of SESN2 significantly increased the release of angiogenesis-related cytokines VEGF-A and PDGF-BB from prevascularized cell membranes, and VEGF-A was released in early angiogenesis, while PDGF-BB was released in late angiogenesis. CD31 immunofluorescence staining showed that SESN2 significantly promoted endothelial cell rearrangement and vascular network structure in hypoxic environment. (5) In vivo experiments showed that high expression of SESN2 could promote vascular network formation, and HE staining showed that the network structure pre-constructed in vitro anastomosed with host vessels to form a functional vascular network.

Conclusion: SESN2 can effectively improve the adaptability of endothelial cells to the hypoxia microenvironment and promote the formation of vascular network structures.

 

MOST Discipline Catalogue医学 - 口腔医学
URL查看原文
Language中文
Other Code262010_220200926950
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/535318
Collection口腔医学院
Affiliation
兰州大学口腔医学院
Recommended Citation
GB/T 7714
任丽娜. 缺氧微环境下Sestrin2对预血管化细胞膜片成血管作用的影响[D]. 兰州. 兰州大学,2023.
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