Notch信号通路调控人脂肪间充质干细胞向内皮细胞分化的研究
发布时间：2018-07-18 15:40
【摘要】：目的:用VEGF165诱导人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells,h ADSCs)向内皮细胞分化,通过γ-内分泌酶抑制剂(DAPT)对诱导分化进行干预,检测细胞的分化程度、Notch信号的表达、细胞迁移能力及成血管能力的变化,探究Notch信号通路在VEGF所诱导的h ADSCs向内皮细胞分化中的作用。方法:1、脂肪组织来源于遵义医学院附属医院整形外科住院的3例患者,经病人同意并签署知情同意书后,手术室无菌条件下收集标本,采取器械剪切、酶消化及离心法提取h ADSCs,用含胎牛血清的DMEM培养基培养细胞并传代,倒置显微镜下观察细胞形态,流式细胞仪检测细胞表面标志CD29、CD31、CD34及CD44的表达;取传代至第3代的h ADSCs,用VEGF165诱导其向内皮细胞分化,14天后流式检测内皮细胞表面抗原的表达。2、取传代至第3代的h ADSCs分为2组:对照组:h ADSCs;诱导组:h ADSCs+50ng/m L的VEGF165。实时荧光定量PCR法及Western bolt电泳法检测两组细胞培养至7天及14天时Notch1,Notch14及Dll4的m RNA和蛋白的表达。3、取传代至第3代的h ADSCs分为3组:对照组:传至第三代的h ADSCs;诱导组:h ADSCs+VEGF165(50ng/m L);干预组:VEGF165(50ng/m L)+h ADSCs+DAPT(三个不同浓度2.5ug/ml、10ug/ml、25ug/ml);各组细胞培养至7天及14天后,分别用流式细胞仪检测内皮细胞表面特异性抗原VE cadherin和CD31表达程度。用Transwell小室检测上述细胞的细胞迁移能力。4、取传代至第3代的h ADSCs分为3组,分组及处理情况同细胞迁移实验,将上述三组细胞培养至7天和14天后,分别取约2×104个细胞移植于铺好Matrigel基质胶中的96孔板上,37℃培养箱中孵育6h后显微镜拍照并分析体外血管生成能力。5、取传代至第3代的h ADSCs分为3组,分组及处理情况同细胞迁移实验,将上述三组细胞分别培养7天后,取各组对应的新的细胞培养液和融化的Matrigel基质胶混合,再与三组细胞分别混匀,植入裸鼠侧肋部皮下,7天后颈椎脱臼处死裸鼠并取出种植的Matrigel胶,观察胶内血管生成情况,切片做CD31免疫化学染色,分析体内血管生成能力。结果:1、从人的脂肪组织中分离出间充质干细胞,细胞形态多为梭形或星形,呈放射集落样生长。流式细胞仪检测细胞表型分析表明,传至第三代的脂肪间充质干细胞表型CD29(96.31%)、CD44(98.91%)呈阳性表达,内皮细胞表型CD31、CD34阴性表达;向内皮细胞诱导14天后CD31(88.38%)、CD34(92.81%)均呈阳性表达,而CD29、CD44的阳性表达率降低。2、q PCR结果显示:对照组和诱导组的细胞中Notch1、Notch4及Dll4的m RNA均有表达,诱导组的相对表达量高于对照组,诱导组中诱导14天的相对表达量比7天的表达高,差异均有统计学意义(P0.05);Western blot结果显示:诱导组和对照组细胞中Notch1、Notch4及Dll4的蛋白均有表达,诱导组的蛋白相对表达量高于对照组,诱导组中14天的相对表达量比7天的表达高,差异均有统计学意义(P0.05)。3、流式检测内皮细胞表面特异性抗原VE-cadherin和CD31的阳性率的趋势相同;对照组阳性率最低,均低于1%;诱导组和干预组中14天的阳性率高于7天组;诱导组阳性率高于干预组;干预组中低浓度DAPT组的阳性率高于高浓度DAPT组,差异有统计学意义(P0.05)。细胞迁移实验中,诱导组的细胞迁移能力最强,干预组次之,对照组细胞迁移能力最弱;诱导组和干预组中14天的细胞迁移能力较7天强,干预组中低浓度DAPT组的细胞迁移能力较高浓度组的迁移能力强,差异有统计学意义(P0.05)。4、体外基质胶Matrigel中的血管生成实验中,对照组中的细胞间成分离状态,未见血管形成;诱导组可见形成闭合的多边形结构或形成复杂的网状结构;干预组中见较多细胞排列呈线,或少数形成网状结构。诱导组的血管生成能力强于干预组;干预组中低浓度DAPT组的血管生成能力较高浓度组强,差异均有统计学意义(P0.05)。5、三组细胞在裸鼠体内血管生成能力的强弱关系与体外血管生成实验结果一致,CD31免疫化学染色见形成的管腔内壁有内皮细胞着色,证实所成管网为血管网;诱导组有大量管腔形成,干预组形成的管腔数少于诱导组,(P0.05);干预组中,低浓度DAPT组的管腔数多于高浓度DAPT组,(P0.05);对照组偶见管腔形成。结论:(1)在h ADSCs向内皮细胞分化过程中,VEGF能上调Notch信号通路的表达。(2)阻断Notch信号通路能抑制hADSCs向内皮细胞分化。(3)阻断Notch信号通路能抑制h ADSCs所分化成的内皮细胞的迁移能力及在体内、外的血管生成能力。
[Abstract]:Objective: to induce human adipose mesenchymal stem cells (human adipose-derived mesenchymal stem cells, H ADSCs) to differentiate into endothelial cells by VEGF165, and to interfere with induced differentiation through gamma endocrine enzyme inhibitor (DAPT). The differentiation degree of cells, the expression of Notch signal, cell migration ability and vascular ability change, and the Notch letter are explored. The role of H ADSCs induced by VEGF in the differentiation of ADSCs into endothelial cells. Methods: 1, adipose tissue was derived from 3 patients hospitalized in plastic surgery in Affiliated Hospital of Zunyi Medical College. After the patients agreed and signed informed consent books, the specimens were collected under aseptic conditions in the operation room, and H ADSCs was extracted by apparatus, enzyme digestion and centrifugation. The DMEM medium of fetal bovine serum was cultured and subcultured. The cell morphology was observed under inverted microscope. Flow cytometry was used to detect the expression of CD29, CD31, CD34 and CD44 on the surface of the cell. It was passed to the third generation of H ADSCs, induced to differentiate into the endothelial cells by VEGF165, and 14 days later, the expression.2 of the endothelial cell surface antigen was detected by flow cytometry. The 3 generation of H ADSCs were divided into 2 groups: the control group: H ADSCs; the induced group: VEGF165. real-time VEGF165. of H ADSCs+50ng/m L and Western bolt electrophoresis method to detect two groups of cells to be cultured to 7 days and 14 days. Guide group: H ADSCs+VEGF165 (50ng/m L); intervention group: VEGF165 (50ng/m L) +h ADSCs+DAPT (three different concentrations 2.5ug/ml, 10ug/ml, 25ug/ml). Cells in each group were cultured for 7 days and 14 days later to detect the surface specific antigen of endothelial cells and the degree of expression by flow cytometry. .4, H ADSCs was divided into 3 groups, grouped and treated with cell migration, and three groups of cells were cultured to 7 days and 14 days later. 2 x 104 cells were transplanted on the 96 foramen of the paving Matrigel matrix respectively. After incubating for 6h in the 37 temperature incubator, the microscopes were photographed and the angiogenesis ability of.5 in vitro was analyzed. Third generations of H ADSCs were divided into 3 groups, grouped and treated with cell migration experiments. The above three groups of cells were cultured for 7 days, and the corresponding new cell culture fluid and melted Matrigel matrix gum were mixed, and then mixed with the three groups of cells and implanted subsubcutaneously in the lateral ribs of nude mice. The nude mice were killed and removed for 7 days after the cervical dislocations. Matrigel glue was planted to observe the angiogenesis in the glue, and the section was stained with CD31 immunochemistry to analyze the angiogenesis ability of the body. Results: 1, the mesenchymal stem cells were isolated from the human adipose tissue. The cell morphology was mostly spindle or star, and the cell phenotype analysis showed that the cell phenotype analysis showed that it was passed to the third generation. The phenotypic CD29 (96.31%) and CD44 (98.91%) of the adipose mesenchymal stem cells were positive, the endothelial cell phenotype was CD31, and the CD34 negative expression, CD31 (88.38%) and CD34 (92.81%) expressed in the endothelial cells for 14 days, while the positive expression rate of CD29, CD44 was reduced.2, and Q PCR results showed that in the cells of the control group and the induction group The relative expression of the induced group was higher than that of the control group. The relative expression of the induced group was higher than the 7 day expression in the induction group, and the difference was statistically significant (P0.05). The Western blot results showed that the proteins of Notch1, Notch4 and Dll4 in the induction and control groups were expressed, and the relative expression of protein in the induced group was higher than that of the control group. The expression of protein in the induction group and the control group was higher than that of the control group. The relative expression of the induced group was higher than that of the control group. The results of Western blot showed that the relative expression of protein in the induced group was higher than that of the control group. The relative expression of 14 days in the induction group was higher than that of the 7 day, and the difference was statistically significant (P0.05).3. The positive rate of VE-cadherin and CD31 was the same in the flow detection endothelial cell surface specific antigen; the positive rate of the control group was the lowest, all lower than 1%; the positive rate of the induction group and the dry pre group was higher than that of the 7 day group; the positive rate of the induction group was higher than that of the 7 day group. The positive rate of middle low concentration DAPT group in the intervention group was higher than that of the high concentration DAPT group, the difference was statistically significant (P0.05). In the cell migration experiment, the cell migration ability of the induction group was the strongest, the intervention group was the lowest, the cell migration ability of the control group was the weakest; the cell migration ability of the induction group and the intervention group was stronger than the 14 day in the induction group and the low concentration in the intervention group. The migration ability of the high concentration group of the DAPT group was strong, and the difference was statistically significant (P0.05).4. In the angiogenesis experiment of the matrix Matrigel in vitro, the cells in the control group were separated and no blood vessels were formed; the induced group could form a closed polygon structure or form a complex reticular structure; the intervention group was found to form a complex network structure. The angiogenic ability of the induced group was stronger than that in the intervention group; the low concentration DAPT group of the intervention group had a higher concentration of angiogenic capacity in the low concentration DAPT group, and the difference was statistically significant (P0.05).5. The relationship between the vascular viability of the three groups of cells in the nude mice and the experimental formation of the angiogenesis in vitro According to the results, CD31 immunochemistry staining showed that the inner wall of the lumen was stained with endothelial cells, which proved that the pipe network was vascular network, the induced group had a large number of lumen formation, the number of lumen formed in the intervention group was less than that of the induced group, (P0.05); in the intervention group, the number of the lumen in the low concentration DAPT group was more than the high concentration DAPT group, (P0.05); and the control group saw the formation of the lumen. Conclusion: (P0.05) 1) VEGF can up-regulate the expression of Notch signaling pathway during the differentiation of H ADSCs into endothelial cells. (2) blocking the Notch signaling pathway can inhibit the differentiation of hADSCs into the endothelial cells. (3) blocking the Notch signaling pathway can inhibit the migration ability of the endothelial cells differentiated by H ADSCs and the angiogenesis in the body.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622
[Abstract]:Objective: to induce human adipose mesenchymal stem cells (human adipose-derived mesenchymal stem cells, H ADSCs) to differentiate into endothelial cells by VEGF165, and to interfere with induced differentiation through gamma endocrine enzyme inhibitor (DAPT). The differentiation degree of cells, the expression of Notch signal, cell migration ability and vascular ability change, and the Notch letter are explored. The role of H ADSCs induced by VEGF in the differentiation of ADSCs into endothelial cells. Methods: 1, adipose tissue was derived from 3 patients hospitalized in plastic surgery in Affiliated Hospital of Zunyi Medical College. After the patients agreed and signed informed consent books, the specimens were collected under aseptic conditions in the operation room, and H ADSCs was extracted by apparatus, enzyme digestion and centrifugation. The DMEM medium of fetal bovine serum was cultured and subcultured. The cell morphology was observed under inverted microscope. Flow cytometry was used to detect the expression of CD29, CD31, CD34 and CD44 on the surface of the cell. It was passed to the third generation of H ADSCs, induced to differentiate into the endothelial cells by VEGF165, and 14 days later, the expression.2 of the endothelial cell surface antigen was detected by flow cytometry. The 3 generation of H ADSCs were divided into 2 groups: the control group: H ADSCs; the induced group: VEGF165. real-time VEGF165. of H ADSCs+50ng/m L and Western bolt electrophoresis method to detect two groups of cells to be cultured to 7 days and 14 days. Guide group: H ADSCs+VEGF165 (50ng/m L); intervention group: VEGF165 (50ng/m L) +h ADSCs+DAPT (three different concentrations 2.5ug/ml, 10ug/ml, 25ug/ml). Cells in each group were cultured for 7 days and 14 days later to detect the surface specific antigen of endothelial cells and the degree of expression by flow cytometry. .4, H ADSCs was divided into 3 groups, grouped and treated with cell migration, and three groups of cells were cultured to 7 days and 14 days later. 2 x 104 cells were transplanted on the 96 foramen of the paving Matrigel matrix respectively. After incubating for 6h in the 37 temperature incubator, the microscopes were photographed and the angiogenesis ability of.5 in vitro was analyzed. Third generations of H ADSCs were divided into 3 groups, grouped and treated with cell migration experiments. The above three groups of cells were cultured for 7 days, and the corresponding new cell culture fluid and melted Matrigel matrix gum were mixed, and then mixed with the three groups of cells and implanted subsubcutaneously in the lateral ribs of nude mice. The nude mice were killed and removed for 7 days after the cervical dislocations. Matrigel glue was planted to observe the angiogenesis in the glue, and the section was stained with CD31 immunochemistry to analyze the angiogenesis ability of the body. Results: 1, the mesenchymal stem cells were isolated from the human adipose tissue. The cell morphology was mostly spindle or star, and the cell phenotype analysis showed that the cell phenotype analysis showed that it was passed to the third generation. The phenotypic CD29 (96.31%) and CD44 (98.91%) of the adipose mesenchymal stem cells were positive, the endothelial cell phenotype was CD31, and the CD34 negative expression, CD31 (88.38%) and CD34 (92.81%) expressed in the endothelial cells for 14 days, while the positive expression rate of CD29, CD44 was reduced.2, and Q PCR results showed that in the cells of the control group and the induction group The relative expression of the induced group was higher than that of the control group. The relative expression of the induced group was higher than the 7 day expression in the induction group, and the difference was statistically significant (P0.05). The Western blot results showed that the proteins of Notch1, Notch4 and Dll4 in the induction and control groups were expressed, and the relative expression of protein in the induced group was higher than that of the control group. The expression of protein in the induction group and the control group was higher than that of the control group. The relative expression of the induced group was higher than that of the control group. The results of Western blot showed that the relative expression of protein in the induced group was higher than that of the control group. The relative expression of 14 days in the induction group was higher than that of the 7 day, and the difference was statistically significant (P0.05).3. The positive rate of VE-cadherin and CD31 was the same in the flow detection endothelial cell surface specific antigen; the positive rate of the control group was the lowest, all lower than 1%; the positive rate of the induction group and the dry pre group was higher than that of the 7 day group; the positive rate of the induction group was higher than that of the 7 day group. The positive rate of middle low concentration DAPT group in the intervention group was higher than that of the high concentration DAPT group, the difference was statistically significant (P0.05). In the cell migration experiment, the cell migration ability of the induction group was the strongest, the intervention group was the lowest, the cell migration ability of the control group was the weakest; the cell migration ability of the induction group and the intervention group was stronger than the 14 day in the induction group and the low concentration in the intervention group. The migration ability of the high concentration group of the DAPT group was strong, and the difference was statistically significant (P0.05).4. In the angiogenesis experiment of the matrix Matrigel in vitro, the cells in the control group were separated and no blood vessels were formed; the induced group could form a closed polygon structure or form a complex reticular structure; the intervention group was found to form a complex network structure. The angiogenic ability of the induced group was stronger than that in the intervention group; the low concentration DAPT group of the intervention group had a higher concentration of angiogenic capacity in the low concentration DAPT group, and the difference was statistically significant (P0.05).5. The relationship between the vascular viability of the three groups of cells in the nude mice and the experimental formation of the angiogenesis in vitro According to the results, CD31 immunochemistry staining showed that the inner wall of the lumen was stained with endothelial cells, which proved that the pipe network was vascular network, the induced group had a large number of lumen formation, the number of lumen formed in the intervention group was less than that of the induced group, (P0.05); in the intervention group, the number of the lumen in the low concentration DAPT group was more than the high concentration DAPT group, (P0.05); and the control group saw the formation of the lumen. Conclusion: (P0.05) 1) VEGF can up-regulate the expression of Notch signaling pathway during the differentiation of H ADSCs into endothelial cells. (2) blocking the Notch signaling pathway can inhibit the differentiation of hADSCs into the endothelial cells. (3) blocking the Notch signaling pathway can inhibit the migration ability of the endothelial cells differentiated by H ADSCs and the angiogenesis in the body.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622
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