本文選題：內(nèi)皮祖細胞 + 黃芪多糖��； 參考：《貴陽中醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的：1.觀察黃芪多糖(Astragalus Polysaccharides,APS)體外干預(yù)培養(yǎng)對大鼠骨髓源性內(nèi)皮祖細胞(Endothelial Progenitor Cells,EPCs)數(shù)量、遷移、黏附、增殖、周期分布及分化的影響。2.觀察APS體外干預(yù)大鼠骨髓源性EPCs共培養(yǎng)對人臍靜脈內(nèi)皮細胞(Human Umbilical Vein Endothelial Cells,HUVEC)增殖的影響。方法：1.取雄性Wistar大鼠予以水合氯醛腹腔注射麻醉,脫頸處死,將其股骨、脛骨、胸骨、肱骨取出,用PBS緩沖液沖洗骨髓腔,采用密度梯度離心法分離大鼠骨髓源性單個核細胞,培養(yǎng)7天以后收集貼壁細胞。采用多波長激光共聚焦顯微鏡對貼壁細胞進行鑒定,采用流式細胞術(shù)檢測貼壁細胞的表型。收集大鼠骨髓源性EPCs,將其隨機分為6組：設(shè)立對照組；其他組為APS各濃度組(共5組),于DMEM(低糖)培養(yǎng)基中加入終濃度為0.05mg/ml、0.1mg/ml、0.2mg/ml、 0.4mg/ml、0.8mg/ml的APS培養(yǎng)24h,其中APS濃度為0.4mg/ml組予以不同時間(Oh、6h、12h、24h、48h)進行培養(yǎng)。倒置顯微鏡觀察每組大鼠骨髓源性EPCs并進行計數(shù)；Transwell小室培養(yǎng)檢測大鼠骨髓源性EPCs的遷移數(shù)量；黏附能力實驗檢測大鼠骨髓源性EPCs的黏附數(shù)量；MTT增殖實驗檢測大鼠骨髓源性EPCs的增殖；流式細胞術(shù)檢測大鼠骨髓源性EPCs的細胞周期的分布和分化。2.分離培養(yǎng)大鼠骨髓源性EPCs,7天后收集貼壁細胞,取樣進行染色和流式鑒定,待細胞表達符合標(biāo)準(zhǔn)后,建立Transwell共培養(yǎng)體系,設(shè)立對照組,將大鼠骨髓源性EPCs與HUVEC予以DMEM(低糖)培養(yǎng)基共培養(yǎng),并予以APS各濃度(0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml、0.8mg/ml)干預(yù)共培養(yǎng)24h后,MTT增殖實驗檢測APS各濃度干預(yù)共培養(yǎng)后HUVEC的增殖；其中APS濃度為0.4mg/ml組予以干預(yù)共培養(yǎng)不同時間(Oh、6h、12h、24h、48h),MTT增殖實驗檢測共培養(yǎng)各時間點HUVEC的增殖。結(jié)果：(1)APS體外干預(yù)培養(yǎng)對大鼠骨髓源性EPCs數(shù)量的影響：APS能明顯增加大鼠骨髓源性EPCs的數(shù)量,并且呈一定的量效和時效關(guān)系,APS濃度為0. Olmg/ml時,其數(shù)量較對照組開始增加(P0.05),APS濃度為0.4mg/ml時達到最佳效應(yīng)(P0.01)；APS濃度為0.4mg/ml干預(yù)培養(yǎng)12h后,其數(shù)量明顯增加(P0.01),24h達到最佳效應(yīng)(P0.01),48h稍有下降,但仍然優(yōu)于對照組(P0.01)。(2)APS體外干預(yù)培養(yǎng)對大鼠骨髓源性EPCs遷移能力的影響：APS能明顯提高大鼠骨髓源性EPCs的遷移能力,并且呈一定的量效和時效關(guān)系,AAPS濃度為0.1mg/ml時,其遷移數(shù)量較對照組開始增加(P0.05),APS濃度為0.4mg/ml時達到最佳效應(yīng)(P0.01);APS濃度為0.4mg/ml干預(yù)培養(yǎng)6h后,其遷移數(shù)量開始增加(P0.05),24h達到最佳效應(yīng)(P0.01),48h稍有下降,但仍然優(yōu)于對照組(P0.01)。(3)APS體外干預(yù)培養(yǎng)對大鼠骨髓源性EPCs增殖能力的影響：APS能明顯提高大鼠骨髓源性EPCs增殖的能力,并且呈一定的量效和時效關(guān)系,APS濃度為0.05mg/ml時,其增殖能力較對照組開始增加(P0.05),APS濃度為0.4mg/ml時達到最佳效應(yīng)(P0.01);APS濃度為0.4mg/ml干預(yù)培養(yǎng)6h后,其增殖能力開始增加(P0.05),24h達到最佳效應(yīng)(P0.01),48h稍有下降,但仍然優(yōu)于對照組(P0.01)。(4) APS體外干預(yù)培養(yǎng)對大鼠骨髓源性EPCs周期分布的影響：APS濃度為0.1mg/ml時,G0/G1期大鼠骨髓源性EPCs的比例開始減少(P0.05),而S期和G2期的大鼠骨髓源性EPCs比例開始增加(P0.05),APS濃度為0.4mg/ml時達最佳效應(yīng)(P0.01)。(5) APS體外干預(yù)培養(yǎng)對大鼠骨髓源性EPCs分化的影響：APS濃度為0.2mg/ml時開始促進大鼠骨髓源性EPCs向內(nèi)皮細胞系方向分化,其表達單核/巨噬細胞表而標(biāo)志(CD14+和CD64+)細胞百分比與對照組相比降低(P0.05或P0.01),而內(nèi)皮細胞特異性標(biāo)志(vWF+)細胞百分比與對照組相比升高(P0.05),APS濃度為0.4mg/ml時達最佳效應(yīng)(P0.01)。(6)Transwell間接共同培養(yǎng)條件下APS干預(yù)大鼠骨髓源性EPCs對HUVEC增殖能力的影響：在APS不同濃度和不同時間體外干預(yù)下呈一定的量效和時效關(guān)系。APS體外干預(yù)培養(yǎng)HUVEC與對照組相比HUVEC增殖能力明顯增加(P0.01)；大鼠骨髓源性EPCs和HUVEC共培養(yǎng)與對照組相比HUVEC增殖能力增加(P0.01);APS體外干預(yù)大鼠骨髓源性EPCs和HUVEC共培養(yǎng)與APS體外干預(yù)培養(yǎng)HUVEC相比HUVEC增殖能力明顯增加(P0.01)。APS (0.4mg/ml)干預(yù)大鼠骨髓源性EPCs共培養(yǎng)不同時間與對照組相比HUVEC增殖能力增加(P0.05或P0.01),而且呈一定的時間依賴性,于24h達到最佳效應(yīng)。結(jié)論：(1)APS體外干預(yù)大鼠骨髓源性EPCs能增加其數(shù)量,促進其遷移、黏附、增殖,且呈一定的濃度及時間依賴性。(2)APS體外干預(yù)大鼠骨髓源性EPCs能使其G0/G1期的細胞比例減少,而S期和G2期細胞比例增加。(3)APS體外干預(yù)大鼠骨髓源性EPCs能使其表達的單核/巨噬細胞表而標(biāo)志(CD14+、CD64+)細胞百分比降低,內(nèi)皮細胞特異性標(biāo)志(vWF+)細胞百分比明顯升高。(4)APS體外干預(yù)大鼠骨髓源性EPCs與HUVEC共培養(yǎng)能明顯促進HUVEC的增殖,且呈一定的濃度及時間依賴性變化。
[Abstract]:Objective: 1. to observe the effect of Astragalus Polysaccharides (APS) on the quantity, migration, adhesion, proliferation, periodic distribution and differentiation of Endothelial Progenitor Cells (EPCs) in vitro, and the effect of.2. observation on human umbilical vein endothelial cells (Human Um) in vitro (Human Um) in vitro. The effects of the proliferation of bilical Vein Endothelial Cells, HUVEC). Methods: 1. the male Wistar rats were injected with chloral hydrate in the abdominal cavity, and the femur, tibia, sternum, humerus were removed, and the bone marrow cavity was washed with PBS buffer, and the bone marrow derived mononuclear cells were separated by density gradient centrifugation and collected for 7 days after culture. Mural cells were identified by multi wavelength laser confocal microscopy. The phenotype of adherent cells was detected by flow cytometry. The rat bone marrow derived EPCs was collected and divided into 6 groups randomly: the control group was set up; the other groups were APS concentration groups (5 groups), and the final concentration in the DMEM (low sugar) medium was 0.05mg/ml, 0.1mg/ml, The APS of 0.2mg/ml, 0.4mg/ml and 0.8mg/ml was cultured in 24h, in which the concentration of APS was cultured in group 0.4mg/ml (Oh, 6h, 12h, 24h, 48h). The bone marrow origin of rats in each group was observed and counted by inverted microscope. The adhesion quantity of PCs; MTT proliferation test to detect the proliferation of rat bone marrow derived EPCs; flow cytometry to detect the distribution of cell cycle of bone marrow derived EPCs in rats and differentiation and differentiation of.2. to culture rat bone marrow derived EPCs. 7 days later, the adherent cells were collected and sampled for staining and flow identification. After the cell expression was conformed to the standard, a common Transwell was established. The rat bone marrow derived EPCs and HUVEC were co cultured with DMEM (low sugar) medium, and the concentration of APS (0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml) was co cultured for 24h, and MTT proliferation experiment was conducted to detect the proliferation of APS each concentration. A total of different time (Oh, 6h, 12h, 24h, 48h) and MTT proliferation test were used to detect the proliferation of HUVEC in each time point. Results: (1) the effect of APS in vitro intervention on the number of bone marrow derived EPCs in rats: APS can significantly increase the number of bone marrow derived EPCs in rats, and a certain dose effect and aging relationship, when APS concentration is 0. As compared with the control group (P0.05), the optimal effect (P0.01) was reached when the concentration of APS was 0.4mg/ml; APS concentration increased significantly (P0.01) after 0.4mg/ml intervention for 12h, 24h reached the best effect (P0.01), and 48h had a slight decrease, but it was still better than the control group (P0.01). (2) the effect of in vitro intervention on the ability of bone marrow derived migration in rats APS could significantly improve the migration ability of rat bone marrow derived EPCs, and showed a certain dose effect and aging relationship. When the concentration of AAPS was 0.1mg/ml, the number of migration began to increase compared with the control group (P0.05). The APS concentration was 0.4mg/ml when the concentration was 0.4mg/ml, and the concentration of APS was 0.4mg /ml, and the number of migration began to increase. To the best effect (P0.01), 48h was slightly decreased, but still better than the control group (P0.01). (3) the effect of APS in vitro intervention on the proliferation of bone marrow derived EPCs in rats: APS could significantly improve the ability of rat bone marrow derived EPCs proliferation, and showed a certain dose effect and aging system. When APS concentration was 0.05mg/ml, its proliferation ability began to begin with the control group. Increase (P0.05), APS concentration was 0.4mg/ml to reach the best effect (P0.01); APS concentration was 0.4mg/ml intervention culture 6h, the proliferation ability began to increase (P0.05), 24h reached the best effect (P0.01), 48h slightly decreased, but still better than the control group (P0.01). (4) At 0.1mg/ml, the proportion of bone marrow derived EPCs in G0/G1 rats began to decrease (P0.05), while the proportion of bone marrow derived EPCs in S and G2 rats began to increase (P0.05), and APS concentration was 0.4mg/ml. (P0.01). (5) the effect of intervention on bone marrow derived differentiation of rats in vitro The source EPCs differentiated into the endothelial cell line, and the percentage of the expression of mononuclear / macrophage markers (CD14+ and CD64+) decreased (P0.05 or P0.01) compared with the control group (P0.05 or P0.01), while the percentage of endothelial cell specific markers (vWF+) increased (P0.05) and APS concentration was 0.4mg/ml (P0.01). (6) Transwell indirect The effect of APS intervention on the proliferation of HUVEC in rat bone marrow EPCs under common culture conditions: a certain dose effect and aging relationship under the intervention of different concentrations and different times of APS in vitro.APS increased the proliferation ability of HUVEC in vitro compared with the control group (P0.01), and the co culture and control of rat bone marrow derived EPCs and HUVEC were compared with the control group (P0.01). The proliferation ability of the group was increased compared with the HUVEC (P0.01), and the proliferation ability of the bone marrow derived EPCs and HUVEC in the rat in vitro was significantly increased (P0.01).APS (P0.01).APS (0.4mg/ml), compared with the APS in vitro, and the proliferation ability of the bone marrow derived EPCs co culture of rats was increased at different times. The time dependence of 24h reached the best effect. Conclusion: (1) in vitro, APS intervention in rat bone marrow derived EPCs can increase its number, promote its migration, adhesion, proliferation, and have a certain concentration and time dependence. (2) APS in vitro intervention in rat bone marrow derived EPCs can reduce the proportion of cells in the G0/ G1 phase, while the proportion of S phase and G2 phase increases. 3) APS in vitro intervention in rat bone marrow derived EPCs could reduce the percentage of the expression of mononuclear / macrophage (CD14+, CD64+) cells and increase the percentage of endothelial cell specific markers (vWF+) cells. (4) APS in vitro intervention in rat bone marrow derived EPCs and HUVEC co culture can significantly promote the proliferation of HUVEC, and present a certain concentration in time. Inter dependent change.
【學(xué)位授予單位】：貴陽中醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

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5 楊兆華;內(nèi)皮祖細胞（EPCs）對移植性動脈硬化的作用及其機制研究[D];復(fù)旦大學(xué);2009年

6 王航;過表達PDGFR-β的EPCs在損傷血管修復(fù)中作用研究[D];第三軍醫(yī)大學(xué);2012年

7 崔斌;eNOS基因修飾EPCs移植與損傷內(nèi)膜修復(fù)的實驗研究[D];第三軍醫(yī)大學(xué);2008年

8 譚虎;vWF A3-GPI強化EPCs富集及其在損傷血管壁修復(fù)中作用的實驗研究[D];第三軍醫(yī)大學(xué);2008年

9 況春燕;STIM1/TRPC1復(fù)合體對EPCs修復(fù)損傷血管能力的影響[D];第三軍醫(yī)大學(xué);2011年

10 喻楊;血管損傷修復(fù)中CCNI對EPCs功能的影響及其機制研究[D];第三軍醫(yī)大學(xué);2009年

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