Notch信號通路調(diào)控人脂肪間充質(zhì)干細(xì)胞向內(nèi)皮細(xì)胞分化的研究
發(fā)布時間：2018-07-18 15:40
【摘要】：目的:用VEGF165誘導(dǎo)人脂肪間充質(zhì)干細(xì)胞(human adipose-derived mesenchymal stem cells,h ADSCs)向內(nèi)皮細(xì)胞分化,通過γ-內(nèi)分泌酶抑制劑(DAPT)對誘導(dǎo)分化進(jìn)行干預(yù),檢測細(xì)胞的分化程度、Notch信號的表達(dá)、細(xì)胞遷移能力及成血管能力的變化,探究Notch信號通路在VEGF所誘導(dǎo)的h ADSCs向內(nèi)皮細(xì)胞分化中的作用。方法:1、脂肪組織來源于遵義醫(yī)學(xué)院附屬醫(yī)院整形外科住院的3例患者,經(jīng)病人同意并簽署知情同意書后,手術(shù)室無菌條件下收集標(biāo)本,采取器械剪切、酶消化及離心法提取h ADSCs,用含胎牛血清的DMEM培養(yǎng)基培養(yǎng)細(xì)胞并傳代,倒置顯微鏡下觀察細(xì)胞形態(tài),流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志CD29、CD31、CD34及CD44的表達(dá);取傳代至第3代的h ADSCs,用VEGF165誘導(dǎo)其向內(nèi)皮細(xì)胞分化,14天后流式檢測內(nèi)皮細(xì)胞表面抗原的表達(dá)。2、取傳代至第3代的h ADSCs分為2組:對照組:h ADSCs;誘導(dǎo)組:h ADSCs+50ng/m L的VEGF165。實時熒光定量PCR法及Western bolt電泳法檢測兩組細(xì)胞培養(yǎng)至7天及14天時Notch1,Notch14及Dll4的m RNA和蛋白的表達(dá)。3、取傳代至第3代的h ADSCs分為3組:對照組:傳至第三代的h ADSCs;誘導(dǎo)組:h ADSCs+VEGF165(50ng/m L);干預(yù)組:VEGF165(50ng/m L)+h ADSCs+DAPT(三個不同濃度2.5ug/ml、10ug/ml、25ug/ml);各組細(xì)胞培養(yǎng)至7天及14天后,分別用流式細(xì)胞儀檢測內(nèi)皮細(xì)胞表面特異性抗原VE cadherin和CD31表達(dá)程度。用Transwell小室檢測上述細(xì)胞的細(xì)胞遷移能力。4、取傳代至第3代的h ADSCs分為3組,分組及處理情況同細(xì)胞遷移實驗,將上述三組細(xì)胞培養(yǎng)至7天和14天后,分別取約2×104個細(xì)胞移植于鋪好Matrigel基質(zhì)膠中的96孔板上,37℃培養(yǎng)箱中孵育6h后顯微鏡拍照并分析體外血管生成能力。5、取傳代至第3代的h ADSCs分為3組,分組及處理情況同細(xì)胞遷移實驗,將上述三組細(xì)胞分別培養(yǎng)7天后,取各組對應(yīng)的新的細(xì)胞培養(yǎng)液和融化的Matrigel基質(zhì)膠混合,再與三組細(xì)胞分別混勻,植入裸鼠側(cè)肋部皮下,7天后頸椎脫臼處死裸鼠并取出種植的Matrigel膠,觀察膠內(nèi)血管生成情況,切片做CD31免疫化學(xué)染色,分析體內(nèi)血管生成能力。結(jié)果:1、從人的脂肪組織中分離出間充質(zhì)干細(xì)胞,細(xì)胞形態(tài)多為梭形或星形,呈放射集落樣生長。流式細(xì)胞儀檢測細(xì)胞表型分析表明,傳至第三代的脂肪間充質(zhì)干細(xì)胞表型CD29(96.31%)、CD44(98.91%)呈陽性表達(dá),內(nèi)皮細(xì)胞表型CD31、CD34陰性表達(dá);向內(nèi)皮細(xì)胞誘導(dǎo)14天后CD31(88.38%)、CD34(92.81%)均呈陽性表達(dá),而CD29、CD44的陽性表達(dá)率降低。2、q PCR結(jié)果顯示:對照組和誘導(dǎo)組的細(xì)胞中Notch1、Notch4及Dll4的m RNA均有表達(dá),誘導(dǎo)組的相對表達(dá)量高于對照組,誘導(dǎo)組中誘導(dǎo)14天的相對表達(dá)量比7天的表達(dá)高,差異均有統(tǒng)計學(xué)意義(P0.05);Western blot結(jié)果顯示:誘導(dǎo)組和對照組細(xì)胞中Notch1、Notch4及Dll4的蛋白均有表達(dá),誘導(dǎo)組的蛋白相對表達(dá)量高于對照組,誘導(dǎo)組中14天的相對表達(dá)量比7天的表達(dá)高,差異均有統(tǒng)計學(xué)意義(P0.05)。3、流式檢測內(nèi)皮細(xì)胞表面特異性抗原VE-cadherin和CD31的陽性率的趨勢相同;對照組陽性率最低,均低于1%;誘導(dǎo)組和干預(yù)組中14天的陽性率高于7天組;誘導(dǎo)組陽性率高于干預(yù)組;干預(yù)組中低濃度DAPT組的陽性率高于高濃度DAPT組,差異有統(tǒng)計學(xué)意義(P0.05)。細(xì)胞遷移實驗中,誘導(dǎo)組的細(xì)胞遷移能力最強(qiáng),干預(yù)組次之,對照組細(xì)胞遷移能力最弱;誘導(dǎo)組和干預(yù)組中14天的細(xì)胞遷移能力較7天強(qiáng),干預(yù)組中低濃度DAPT組的細(xì)胞遷移能力較高濃度組的遷移能力強(qiáng),差異有統(tǒng)計學(xué)意義(P0.05)。4、體外基質(zhì)膠Matrigel中的血管生成實驗中,對照組中的細(xì)胞間成分離狀態(tài),未見血管形成;誘導(dǎo)組可見形成閉合的多邊形結(jié)構(gòu)或形成復(fù)雜的網(wǎng)狀結(jié)構(gòu);干預(yù)組中見較多細(xì)胞排列呈線,或少數(shù)形成網(wǎng)狀結(jié)構(gòu)。誘導(dǎo)組的血管生成能力強(qiáng)于干預(yù)組;干預(yù)組中低濃度DAPT組的血管生成能力較高濃度組強(qiáng),差異均有統(tǒng)計學(xué)意義(P0.05)。5、三組細(xì)胞在裸鼠體內(nèi)血管生成能力的強(qiáng)弱關(guān)系與體外血管生成實驗結(jié)果一致,CD31免疫化學(xué)染色見形成的管腔內(nèi)壁有內(nèi)皮細(xì)胞著色,證實所成管網(wǎng)為血管網(wǎng);誘導(dǎo)組有大量管腔形成,干預(yù)組形成的管腔數(shù)少于誘導(dǎo)組,(P0.05);干預(yù)組中,低濃度DAPT組的管腔數(shù)多于高濃度DAPT組,(P0.05);對照組偶見管腔形成。結(jié)論:(1)在h ADSCs向內(nèi)皮細(xì)胞分化過程中,VEGF能上調(diào)Notch信號通路的表達(dá)。(2)阻斷Notch信號通路能抑制hADSCs向內(nèi)皮細(xì)胞分化。(3)阻斷Notch信號通路能抑制h ADSCs所分化成的內(nèi)皮細(xì)胞的遷移能力及在體內(nèi)、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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622
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