脂肪來源的間充質(zhì)干細胞體外轉(zhuǎn)分化內(nèi)皮細胞及鈣調(diào)蛋白拮抗劑W7調(diào)控研究
發(fā)布時間：2018-08-07 16:46
【摘要】：研究背景：通過組織工程技術(shù)實現(xiàn)缺損組織或器官的修復與替代具有重要的臨床意義。而理想的種子細胞是組織工程的基礎(chǔ)。對于缺血性疾病而言,利用干細胞的多向分化潛能進行血管重構(gòu)來改善局部缺血的治療模式,是近年來許多學者關(guān)注的重點。作為候選細胞的內(nèi)皮祖細胞,骨髓來源間充質(zhì)干細胞,衛(wèi)星細胞等,由于取材困難、獲取率低、分化能力和細胞數(shù)量隨體外傳代培養(yǎng)逐漸下降等應(yīng)用受限,此外更受到倫理學的質(zhì)疑。脂肪來源的間充質(zhì)干細胞(human adipose-derived mesenchymal stem cells, hADSCs)是從成體脂肪組織中分離出來的一類成體干細胞,具有和骨髓來源的基質(zhì)干細胞相似的生物學特性和多系分化潛能,并因其不侵犯相關(guān)倫理及取材方面的優(yōu)勢,有望成為種子細胞的熱門選擇。為此,建立合理的hADSCs體外培養(yǎng)體系、向內(nèi)皮細胞的誘導分化、以及適當載體的尋找都亟待深入研究。 研究目的：第一部分研究：在無血清培養(yǎng)條件下,將人脂肪來源的間充質(zhì)干細胞(hADSCs)分化為內(nèi)皮細胞,為血管組織工程化種子細胞的來源和血管新生模型的建立提供基礎(chǔ)。第二部分研究：以(6-氨乙基)-5-氯-1-萘磺酰胺(W7)研究鈣調(diào)蛋白拮抗劑對人脂肪間充質(zhì)干細胞(hADSCs)向內(nèi)皮細胞分化過程中細胞表型、細胞內(nèi)游離鈣離子濃度變化、成血管功能以及ERK/MAPK信號通路的作用。第三部分研究：研究人脂肪來源的間充質(zhì)干細胞與新型可降解多孔生物材料---聚乳酸-聚乙二醇共聚物(Poly(lactic acid)-poly(ethylene glycol), PLA-PEG)的相容性,及hADSCs在材料上轉(zhuǎn)分化為內(nèi)皮細胞(endothelial cells, ECs)和血管形成的能力。 研究方法：第一部分：從健康人體抽脂術(shù)獲得的脂肪進行hADSCs原代培養(yǎng),用流式細胞術(shù)檢測CD45、CD44、CD14、CD29及CD105,并分化至成骨和脂肪細胞,以免疫組化方法檢測其干細胞的分化能力；然后用含40ng/ml VEGF和lOng/ml bFGF的無血清分化培養(yǎng)基誘導hADSCs分化為內(nèi)皮細胞,在第15、30、50d通過流式細胞術(shù)檢測vWF和VE-Cadherin兩種特異性抗原表達,并用透射電鏡檢測WP小體以及成血管實驗來檢測是否分化為內(nèi)皮細胞。第二部分：以含40ng/ml VEGF和lOng/mlbFGF的干細胞無血清分化培養(yǎng)基培養(yǎng)P3 hADSCs,將實驗分為空白分化對照組(不含W7的分化培養(yǎng)基),A23187陽性對照組(含50μmol/L鈣離子載體A23187的分化培養(yǎng)基)、高(30μmol/L)、中(20μmol/L)、低(10μmol/L)三個不同W7劑量的實驗組,及預先將hADSCs用ERK抑制劑V0126 (40μmol/L)處理24h的高(30μmol/L)、中(20μmol/L)、低(10μmol/L)三個不同W7劑量的對照組。hADSCs加藥8d后,用流式細胞術(shù)檢測其vWF和VE-Cadherin表型變化,以激光共聚焦顯微鏡檢測Fluo-3標記的細胞胞質(zhì)內(nèi)游離鈣離子濃度變化。同時將未加抑制劑只用藥物處理24h的細胞及加入抑制劑干預24h后用藥物處理24h的細胞種植至Matrigel膠上,觀察細胞成血管能力。利用Western blot技術(shù)分析不同濃度藥物處理8d后細胞ERK和p-ERK變化。第三部分：選取孔徑為60-80μm和180-200μm的PEG-PLA材料,并計算不同孔徑該材料14d內(nèi)的吸水率和降解率。利用本室已建立的人脂肪間充質(zhì)干細胞的培養(yǎng)方法,將hADSCs種植在不同孔徑的材料上,計算接種率。利用DAPI免疫熒光標記及掃描電鏡(scanning electron microscope, SEM).觀察hADSCs在材料上的生長及分布情況,并采用CCK-8法繪制hADSCs在材料上的增殖曲線。用含40ng/mlVEGF和lOng/mlbFGF的分化培養(yǎng)基處理材料上的hADSCs,50d后利用流式細胞術(shù)檢測vWF和VE-Cadherin及免疫熒光檢測F1k-1觀察hADSCs在材料上分化為ECs的能力。 研究結(jié)果：在第一部分中,分離得到的細胞流式細胞術(shù)分析結(jié)果為,CD45(-)CD14(-)CD44(+)CD29(+)CD105(+),表明該細胞為人脂肪來源的間充質(zhì)干細胞,并且可分化成骨成脂細胞。隨著分化時間的延長,vWF和VE-Cadherin的表達不斷增加,并且可在電鏡下觀察到WP小體的出現(xiàn)以及在光鏡下觀察到分化50d的hADSCs具有成血管的能力。第二部分中,分化至8d時,與空白分化對照組相比,隨著W7濃度的降低,hADSCs轉(zhuǎn)分化后的細胞VE-Cadherin和vWF的表達量顯著上升(P0.01),胞質(zhì)內(nèi)游離的鈣離子濃度升高(P0.01)。藥物作用24h后,藥物干預組的培養(yǎng)細胞均有管腔樣血管結(jié)構(gòu)形成,空白分化對照組的細胞無血管管型形成。與空白分化對照組相比,不同藥物干預組細胞ERK表達水平?jīng)]有顯著性改變(P0.05),而隨著W7濃度的降低p-ERK表達水平明顯升高(P0.01)。在第三部分中,利用已知文獻的方法觀察孔徑分別為60-80μm和180-200μm的PEG-PLA材料14d的吸水率分別為372.73%0和245.31%,降解率為9.09%和6.25%。種植hADSCs后,SEM下觀察細胞呈長梭形依附于多孔材料表面。比較不同孔徑的材料對細胞的影響發(fā)現(xiàn),60-80μm孔徑的材料細胞接種率為99.00±0.71%,大于180-200μm孔徑材料上的細胞接種率92.00±1.22%；但生長曲線卻顯示在180-200μm孔徑材料上細胞具有更快的增殖速度。選擇180-200μm孔徑的材料進行EC分化實驗發(fā)現(xiàn),分化50d后,FCM檢測vWF和VE-cadherin的陽性率分別為78.5±1.50和83.3±2.00,免疫熒光化學檢測Flk-1顯示大多數(shù)細胞表面均表達Flk-1。 研究結(jié)論：成功從人體脂肪中分離得到間充質(zhì)干細胞,并且在無血清條件下可將其分化為內(nèi)皮細胞。適當濃度的鈣離子拮抗劑W7可以顯著促進hADSCs向內(nèi)皮細胞分化,其機制為通過促進細胞內(nèi)游離鈣離子濃度的增加,激活細胞分化過程中的ERK/MAPK信號通路。hADSCs較適合于在孔徑大小為180-200μm PEG-PLA材料上生長和增殖,并可在材料上有效分化為內(nèi)皮細胞。 下一步待研究工作：增加W7 10μmol/L以下濃度及30μmol/L以上濃度的多個劑量組,觀察其對hADSCs轉(zhuǎn)分化內(nèi)皮的影響,進一步確定W7的最適劑量。嘗試調(diào)節(jié)PLA-PEG共聚段中二者的共聚比或共聚物的分子量大小,以爭取獲得最優(yōu)材料。設(shè)計動物體內(nèi)試驗,將該材料及細胞應(yīng)用于缺血模型,從而觀察其在體內(nèi)的各項結(jié)果參數(shù)是否適合臨床需求。
[Abstract]:Background: the reconstruction and replacement of tissue or organs by tissue engineering has important clinical significance. The ideal seed cells are the basis of tissue engineering. For ischemic disease, vascular remodeling by using the multidirectional differentiation potential of stem cells to improve the therapeutic pattern of ischemia is a lot of recent years. As a candidate cell, endothelial progenitor cells, bone marrow derived mesenchymal stem cells, satellite cells, and so on, are limited due to the difficulty in obtaining material, low acquisition rate, the differentiation ability and the number of cells decreasing with the gradual decline of the external transmission culture, in addition to the ethical question. Mesenchymal stem cells derived from fat (human adipose-der) Ived mesenchymal stem cells (hADSCs), a class of adult stem cells isolated from adult adipose tissue, has a similar biological and multi lineage differentiation potential with bone marrow derived stromal cells, and is expected to be a hot choice for seed cells because of its non invasion of ethical and material advantages. The induction of hADSCs in vitro, the differentiation into endothelial cells, and the search for suitable vectors are urgently needed to be further studied.
The first part of the study: the first part of the study: to differentiate human adipose derived mesenchymal stem cells (hADSCs) into endothelial cells in serum-free culture, and provide the basis for the origin of vascular tissue engineering seed cells and the establishment of a angiogenesis model. The second part studies the study of calcic eggs with (6- amethyl) -5- chloride -1- naphthalamyl sulfonamide (W7) The effect of white antagonists on the cell phenotype, intracellular free calcium ion concentration, vascular function and ERK/MAPK signaling pathway in human adipose mesenchymal stem cells (hADSCs) differentiation into endothelial cells. Third part study: the study of human adipose derived mesenchymal stem cells and new biodegradable porous biomaterials - polylactic acid poly B The compatibility of the diol copolymer (Poly (lactic acid) -poly (ethylene glycol), PLA-PEG), and the conversion of hADSCs on the material into endothelial cells (endothelial cells, ECs) and angiogenesis.
Research methods: the first part: hADSCs primary culture obtained from healthy human liposuction, CD45, CD44, CD14, CD29 and CD105 were detected by flow cytometry and differentiated into osteoblasts and adipocytes, and the differentiation ability of stem cells was detected by immunohistochemical method; then the serum free differentiation culture containing 40ng/ml VEGF and lOng/ml bFGF was used. The culture medium induced hADSCs to differentiate into endothelial cells. Two specific antigens of vWF and VE-Cadherin were detected by flow cytometry in 15,30,50d. The WP corpuscles and vascular experiments were used to detect the differentiation into endothelial cells by transmission electron microscopy. The second part was serum-free differentiation and culture of the stem cells containing 40ng/ml VEGF and lOng/mlbFGF. The base culture of P3 hADSCs was divided into blank differentiation control group (without W7 differentiation medium), A23187 positive control group (including 50 micron mol/L calcium carrier A23187 differentiation medium), high (30 mu mol/L), medium (20 mu mol/L), low (10 micron), three different W7 dosages, and hADSCs ERK inhibitor (40 mu) pretreatment High (30 u mol/L), medium (20 mol/L), and low (10 u mol/L) three different W7 doses of.HADSCs added 8D, the phenotypic changes of vWF and VE-Cadherin were detected by flow cytometry. The change of intracellular free calcium concentration in the cytoplasm of the Fluo-3 labeled cells was detected by laser confocal microscopy. And after adding inhibitor to 24h, the cells of 24h were grown on Matrigel glue to observe the vascular capacity of the cells. The Western blot technique was used to analyze the changes of ERK and p-ERK in the cells after 8D treatment with different concentrations. The third part: select the PEG-PLA material with a pore size of 60-80 u m and 180-200 micron m. Water absorption and degradation rate. Using the culture method of human adipose mesenchymal stem cells established in this room, hADSCs was planted on different pore size materials to calculate the inoculation rate. The growth and distribution of hADSCs on the material were observed by DAPI immunofluorescent labeling and scanning electron microscopy (scanning electron microscope, SEM), and the CCK-8 method was used. The proliferation curve of hADSCs on material. HADSCs on the material was treated with 40ng/mlVEGF and lOng/mlbFGF differentiation medium. 50D was used to detect vWF and VE-Cadherin and immunofluorescence detection F1k-1 by flow cytometry to observe the ability of hADSCs to differentiate into ECs on the material.
Results: in the first part, the results of cell flow cytometry, CD45 (-) CD14 (-) CD44 (+) CD29 (+) CD105 (+), indicate that the cells are mesenchymal stem cells derived from human fat and can differentiate into osteoblast. With the extension of differentiation, the expression of vWF and VE-Cadherin is increasing and can be used in electron microscopy. The appearance of WP body and the ability of hADSCs to differentiate 50D under light microscopy were observed under the light microscope. In the second part, the expression of VE-Cadherin and vWF in the cells after hADSCs transdifferentiation was significantly increased (P0.01), and the concentration of free calcium ions in the cytoplasm was raised as compared with the blank differentiation control group. High (P0.01). After the drug action of 24h, the cultured cells in the drug intervention group had a lumen like vascular structure, and the cells in the blank differentiation control group had no vascular type formation. Compared with the blank differentiation control group, the expression level of ERK was not significantly changed in the different drug intervention groups (P0.05), but with the decrease of W7 concentration, the expression level of p-ERK was obvious. Increase (P0.01). In the third part, the water absorption of PEG-PLA material 14d with the pore size of 60-80 m and 180-200 micron m, respectively, was 372.73%0 and 245.31%, the degradation rate was 9.09% and 6.25%. planted hADSCs, and the cells were observed on the surface of the porous material with a long shuttle shape under SEM, and the cells with different pore sizes were compared to the cells. The results showed that the inoculation rate of the material cells with 60-80 m aperture was 99 + 0.71%, and the cell inoculation rate on the material larger than 180-200 mu m was 92 + 1.22%, but the growth curve showed that the cells had faster proliferation rate on the 180-200 micron m pore material. The material selected for the aperture of 180-200 mu m was found by EC differentiation experiment, and after the differentiation of 50D, the FCM examination was found. The positive rates of vWF and VE-cadherin were 78.5 (+ 1.50) and 83.3 (+ 2.00) respectively. Flk-1 was detected by immunofluorescence.
It is concluded that mesenchymal stem cells can be isolated from human body fat and differentiate into endothelial cells in serum-free conditions. The appropriate concentration of calcium antagonist W7 can significantly promote the differentiation of hADSCs into endothelial cells. The mechanism is to activate the cell differentiation process by promoting the increase of intracellular free calcium concentration. The ERK/MAPK signal pathway.HADSCs is more suitable for growth and proliferation on the size of 180-200 micron m PEG-PLA, and can effectively differentiate into endothelial cells on the material.
The next step to study: to increase the concentration of W7 10 mu below mol/L and more than 30 mol/L concentration, observe its effect on the hADSCs transdifferentiation endothelium and further determine the optimum dosage of W7. Try to adjust the copolymerization ratio or the molecular weight of the copolymer in the PLA-PEG copolymerization segment, in order to obtain the optimal material. In the internal test, the material and cells were applied to the ischemic model to observe whether the parameters of the results in vivo were suitable for clinical needs.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329
本文編號：2170694
[Abstract]:Background: the reconstruction and replacement of tissue or organs by tissue engineering has important clinical significance. The ideal seed cells are the basis of tissue engineering. For ischemic disease, vascular remodeling by using the multidirectional differentiation potential of stem cells to improve the therapeutic pattern of ischemia is a lot of recent years. As a candidate cell, endothelial progenitor cells, bone marrow derived mesenchymal stem cells, satellite cells, and so on, are limited due to the difficulty in obtaining material, low acquisition rate, the differentiation ability and the number of cells decreasing with the gradual decline of the external transmission culture, in addition to the ethical question. Mesenchymal stem cells derived from fat (human adipose-der) Ived mesenchymal stem cells (hADSCs), a class of adult stem cells isolated from adult adipose tissue, has a similar biological and multi lineage differentiation potential with bone marrow derived stromal cells, and is expected to be a hot choice for seed cells because of its non invasion of ethical and material advantages. The induction of hADSCs in vitro, the differentiation into endothelial cells, and the search for suitable vectors are urgently needed to be further studied.
The first part of the study: the first part of the study: to differentiate human adipose derived mesenchymal stem cells (hADSCs) into endothelial cells in serum-free culture, and provide the basis for the origin of vascular tissue engineering seed cells and the establishment of a angiogenesis model. The second part studies the study of calcic eggs with (6- amethyl) -5- chloride -1- naphthalamyl sulfonamide (W7) The effect of white antagonists on the cell phenotype, intracellular free calcium ion concentration, vascular function and ERK/MAPK signaling pathway in human adipose mesenchymal stem cells (hADSCs) differentiation into endothelial cells. Third part study: the study of human adipose derived mesenchymal stem cells and new biodegradable porous biomaterials - polylactic acid poly B The compatibility of the diol copolymer (Poly (lactic acid) -poly (ethylene glycol), PLA-PEG), and the conversion of hADSCs on the material into endothelial cells (endothelial cells, ECs) and angiogenesis.
Research methods: the first part: hADSCs primary culture obtained from healthy human liposuction, CD45, CD44, CD14, CD29 and CD105 were detected by flow cytometry and differentiated into osteoblasts and adipocytes, and the differentiation ability of stem cells was detected by immunohistochemical method; then the serum free differentiation culture containing 40ng/ml VEGF and lOng/ml bFGF was used. The culture medium induced hADSCs to differentiate into endothelial cells. Two specific antigens of vWF and VE-Cadherin were detected by flow cytometry in 15,30,50d. The WP corpuscles and vascular experiments were used to detect the differentiation into endothelial cells by transmission electron microscopy. The second part was serum-free differentiation and culture of the stem cells containing 40ng/ml VEGF and lOng/mlbFGF. The base culture of P3 hADSCs was divided into blank differentiation control group (without W7 differentiation medium), A23187 positive control group (including 50 micron mol/L calcium carrier A23187 differentiation medium), high (30 mu mol/L), medium (20 mu mol/L), low (10 micron), three different W7 dosages, and hADSCs ERK inhibitor (40 mu) pretreatment High (30 u mol/L), medium (20 mol/L), and low (10 u mol/L) three different W7 doses of.HADSCs added 8D, the phenotypic changes of vWF and VE-Cadherin were detected by flow cytometry. The change of intracellular free calcium concentration in the cytoplasm of the Fluo-3 labeled cells was detected by laser confocal microscopy. And after adding inhibitor to 24h, the cells of 24h were grown on Matrigel glue to observe the vascular capacity of the cells. The Western blot technique was used to analyze the changes of ERK and p-ERK in the cells after 8D treatment with different concentrations. The third part: select the PEG-PLA material with a pore size of 60-80 u m and 180-200 micron m. Water absorption and degradation rate. Using the culture method of human adipose mesenchymal stem cells established in this room, hADSCs was planted on different pore size materials to calculate the inoculation rate. The growth and distribution of hADSCs on the material were observed by DAPI immunofluorescent labeling and scanning electron microscopy (scanning electron microscope, SEM), and the CCK-8 method was used. The proliferation curve of hADSCs on material. HADSCs on the material was treated with 40ng/mlVEGF and lOng/mlbFGF differentiation medium. 50D was used to detect vWF and VE-Cadherin and immunofluorescence detection F1k-1 by flow cytometry to observe the ability of hADSCs to differentiate into ECs on the material.
Results: in the first part, the results of cell flow cytometry, CD45 (-) CD14 (-) CD44 (+) CD29 (+) CD105 (+), indicate that the cells are mesenchymal stem cells derived from human fat and can differentiate into osteoblast. With the extension of differentiation, the expression of vWF and VE-Cadherin is increasing and can be used in electron microscopy. The appearance of WP body and the ability of hADSCs to differentiate 50D under light microscopy were observed under the light microscope. In the second part, the expression of VE-Cadherin and vWF in the cells after hADSCs transdifferentiation was significantly increased (P0.01), and the concentration of free calcium ions in the cytoplasm was raised as compared with the blank differentiation control group. High (P0.01). After the drug action of 24h, the cultured cells in the drug intervention group had a lumen like vascular structure, and the cells in the blank differentiation control group had no vascular type formation. Compared with the blank differentiation control group, the expression level of ERK was not significantly changed in the different drug intervention groups (P0.05), but with the decrease of W7 concentration, the expression level of p-ERK was obvious. Increase (P0.01). In the third part, the water absorption of PEG-PLA material 14d with the pore size of 60-80 m and 180-200 micron m, respectively, was 372.73%0 and 245.31%, the degradation rate was 9.09% and 6.25%. planted hADSCs, and the cells were observed on the surface of the porous material with a long shuttle shape under SEM, and the cells with different pore sizes were compared to the cells. The results showed that the inoculation rate of the material cells with 60-80 m aperture was 99 + 0.71%, and the cell inoculation rate on the material larger than 180-200 mu m was 92 + 1.22%, but the growth curve showed that the cells had faster proliferation rate on the 180-200 micron m pore material. The material selected for the aperture of 180-200 mu m was found by EC differentiation experiment, and after the differentiation of 50D, the FCM examination was found. The positive rates of vWF and VE-cadherin were 78.5 (+ 1.50) and 83.3 (+ 2.00) respectively. Flk-1 was detected by immunofluorescence.
It is concluded that mesenchymal stem cells can be isolated from human body fat and differentiate into endothelial cells in serum-free conditions. The appropriate concentration of calcium antagonist W7 can significantly promote the differentiation of hADSCs into endothelial cells. The mechanism is to activate the cell differentiation process by promoting the increase of intracellular free calcium concentration. The ERK/MAPK signal pathway.HADSCs is more suitable for growth and proliferation on the size of 180-200 micron m PEG-PLA, and can effectively differentiate into endothelial cells on the material.
The next step to study: to increase the concentration of W7 10 mu below mol/L and more than 30 mol/L concentration, observe its effect on the hADSCs transdifferentiation endothelium and further determine the optimum dosage of W7. Try to adjust the copolymerization ratio or the molecular weight of the copolymer in the PLA-PEG copolymerization segment, in order to obtain the optimal material. In the internal test, the material and cells were applied to the ischemic model to observe whether the parameters of the results in vivo were suitable for clinical needs.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329
【參考文獻】
相關(guān)期刊論文 前8條
1 趙麗華;莫菊彩;王晨明;;聚乳酸-聚乙二醇共聚物的研究概述[J];海峽藥學;2009年07期
2 劉國鋒;楊大平;郭鐵芳;郝晨光;馬輝;王冬艷;;血管組織工程支架材料的研究進展[J];哈爾濱醫(yī)科大學學報;2007年03期
3 岳慧敏;張磊;王韞芳;梁峰;管利東;李紹青;閆舫;南雪;白慈賢;林峰;顏永年;裴雪濤;;人骨髓來源的間充質(zhì)干細胞在聚乳酸羥基乙酸材料上增殖及向內(nèi)皮細胞分化的研究[J];科學通報;2006年10期
4 管利東;李紹青;王韞芳;岳慧敏;劉大慶;何麗娟;白慈賢;閆舫;南雪;施雙雙;裴雪濤;;人脂肪間充質(zhì)干細胞體外血管內(nèi)皮細胞分化[J];科學通報;2006年13期
5 范曉麗;鄒遠文;;組織工程血管構(gòu)建中支架材料的特征[J];中國組織工程研究與臨床康復;2009年29期
6 岳慧敏,梁峰,王韞芳,裴雪濤;組織工程血管研究現(xiàn)狀[J];自然科學進展;2005年05期
7 廖雪雁;梁爭論;周紹兵;張華遠;李孝紅;鄧先模;李河民;;聚乳酸-聚乙二醇共聚物的生物學效應(yīng)研究[J];中國免疫學雜志;2006年03期
8 曹瑩;孟艷;孫昭;廖聯(lián)明;韓欽;李靜;劉艷寧;趙春華;;脂肪來源成體干細胞分化為內(nèi)皮細胞的潛能[J];中國醫(yī)學科學院學報;2005年06期
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