發(fā)布時間：2018-02-04 08:31

  本文關鍵詞： ROS 臍帶血CD133~+細胞 體外擴增 NAC p38MAPKα 臍帶血CD133~+細胞 體外擴增 NOD/SCID小鼠 p38MAPK ROS CD133~+細胞 造血作用 體外擴增　出處：《華中科技大學》2011年博士論文　論文類型：學位論文

【摘要】：第一部分細胞內(nèi)ROS對臍帶血CD133+細胞體外擴增特性的影響 目的：使用抗氧化劑NAC調(diào)節(jié)體外擴增細胞內(nèi)活性氧水平,評估ROS對臍帶血CD133+細胞體外擴增特性的影響,并初步探討其作用機制。 方法：建立臍帶血CD133+細胞無血清培養(yǎng)體系,CD133+細胞培養(yǎng)過程中分別采用不同濃度的抗氧化劑NAC清除細胞內(nèi)ROS,檢測不同時間點細胞內(nèi)ROS水平,并通過CD133+細胞亞群變化、CFU及CAFC評估擴增后細胞功能。同時檢測擴增后細胞增殖(CFSE)、細胞凋亡、衰老表型及相關基因p16INK4和p21WAF1的表達,探討ROS作用機制。 結果：臍帶血CD133+細胞體外擴增過程中胞內(nèi)ROS升高,并伴隨著HSC自我更新能力損傷�？寡趸瘎㎞AC可以有效清除胞內(nèi)ROS,并且清除程度與藥物劑量呈正相關。其中低劑量處理組(0.1mmol/L)的CD133+細胞擴增倍數(shù)、產(chǎn)生CFU細胞密度、CAFC形成能力均高于對照組(p0.05)。NAC高劑量組(1.0mmol/L)CD133+細胞擴增倍數(shù)低于對照組(p0.01),但CFU及CAFC形成能力強于對照組(p0.01)。NAC可以明顯抑制凋亡率、衰老形成、及衰老相關蛋白的轉錄。高劑量的NAC抑制細胞增殖水平。 結論：ROS參與調(diào)節(jié)CD133+細胞體外擴增,適當?shù)慕档蚏OS可促進擴增細胞干性的維持,過度清除ROS則抑制細胞增殖。ROS主要通過調(diào)控細胞增殖、凋亡及衰老途徑發(fā)揮作用。 第二部分抑制活化的p38 MAPKα促進臍帶血造血干細胞體外擴增 目的：應用p38MAPKα抑制劑及NOD/SCID小鼠人造血干細胞移植模型評估p38MAPKα對臍帶血CD133+細胞體外擴增特性的影響,并初步探討其作用機制。 方法：建立臍帶血CD133+細胞無血清無基質(zhì)培養(yǎng)體系,CD133+細胞培養(yǎng)過程中采用p38MAPKα抑制劑阻斷p38MAPKα活化,檢測不同時間點及不同處理組細胞內(nèi)p38MAPK活性,并通過CD133+細胞亞群變化、CFU、CAFC、細胞遷移率及CXCR4表達水平評估擴增后細胞特性,運用NOD/SCID小鼠人造血干細胞移植模型評估擴增后細胞長期造血重建能力。同時檢測擴增后細胞CFSE增殖、細胞凋亡、衰老表型及相關基因p16INK4和p21WAF1的表達,探討p38MAPKα作用機制。 結果：細胞體外擴增過程中p38MAPKa被激活,并伴隨著HSC自我更新能力損傷。p38MAPKα抑制劑抑制p38MAPKα活化后,促進了臍帶血CD133+細胞體外擴增,代表早期干祖細胞的CD133+細胞亞群、CD133+CD38-細胞亞群、CD133+CXCR4+細胞亞群、CFU-GEMM及CAFC相較于對照組均明顯增加(p0.01)。NOD/SCID小鼠人造血干細胞移植模型中,p38MAPKa抑制劑組的移植物植入率及移植物嵌合率均高于對照組(p0.01)。機制檢測顯示p38MAPKα抑制劑可以明顯抑制凋亡率、衰老形成、及衰老相關蛋白的轉錄,促進細胞向髓系祖分化,對細胞分裂增殖無明顯作用。 結論：p38MAPKα參與調(diào)節(jié)CD133+細胞體外擴增,抑制p38MAPKα激活可促進HSC擴增,并維持HSC干性。主要通過抑制細胞凋亡及衰老途徑發(fā)揮作用。 第三部分ROS與p38MAPKα在臍帶血CD133+細胞體外擴增中的相互關系的研究 目的：分別單用或聯(lián)合應用p38MAPKa抑制劑SB203580及抗氧化劑NAC調(diào)節(jié)體外擴增,比較與評估不同作用靶點對臍帶血CD133+細胞體外擴增特性的影響,探討ROS與p38MAPK在臍帶血CD133+細胞體外擴增中的相互關系。 方法：分別采用不加藥物、單用p38MAPKa抑制劑SB203580、單用抗氧化劑NAC、聯(lián)合應用p38MAPKα抑制劑SB203580及抗氧化劑NAC干預體外擴增,監(jiān)測胞內(nèi)ROS水平及p38MAPK活化狀態(tài),通過CD133+細胞亞群變化、CFU及CAFC評估并比較各處理組擴增后細胞特性差異。 結果：兩藥聯(lián)用下ROS抑制作用最強,下降到同期對照組的15.3±2.1%,SB203580處理組為52.0±7.8%,NAC組為63.4±9.0%,SB抑制效果優(yōu)于NAC。p38MAPKα抑制劑及抗氧化劑NAC對p38MAPKa活化均有抑制作用。相對于初始細胞,SB組擴增效率最高,CD133+細胞擴增了21.93±1.36倍,NAC組擴增了14.50±1.19倍,對照組僅擴增了10.13±0.57倍,而藥物聯(lián)用組幾乎沒有擴增。CFU及CAFC結果均顯示SB組優(yōu)于NAC組。 結論：p38MAPKα作為靶點調(diào)控HSC體外擴增效果優(yōu)于ROS；胞內(nèi)ROS調(diào)控造血生成；ROS-p38MAPK通路上并非單向傳遞信息,存在p38MAPK至ROS的反饋循環(huán)。
[Abstract]:The effect of ROS on the in vitro amplification of umbilical cord blood CD133+ cells in part 1
Objective: to regulate the in vitro expansion of reactive oxygen species (ROS) in vitro by using antioxidant NAC, and to evaluate the effect of ROS on the in vitro amplification characteristics of umbilical cord blood CD133+ cells, and to preliminarily explore its mechanism.
Methods: umbilical cord blood CD133+ cells in serum free medium, cultured CD133+ cells in vitro were treated by different concentration of antioxidants NAC removal of intracellular ROS, detect the level of ROS in cells at different time points, and the changes of CD133+ cell subsets, CFU cell function after amplification and CAFC amplification detection evaluation. At the same time after cell proliferation (CFSE) expression, apoptosis, senescence phenotype and related genes p16INK4 and p21WAF1, to explore the mechanism of ROS.
Results: the amplification of intracellular ROS in elevated cord blood CD133+ cells in vitro, and with HSC self-renewal ability damage. The antioxidant NAC can effectively remove intracellular ROS, and clear level is positive correlation with dosage. The low dose treatment group (0.1mmol/L) expansion of CD133+ cells, CFU cell density, CAFC formation ability were higher than the control group (P0.05.NAC) high dose group (1.0mmol/L) CD133+ cell proliferation ratio is lower than the control group (P0.01), but CFU and CAFC have strong ability to form in the control group (P0.01).NAC can inhibit the apoptosis rate of aging, transcription and senescence associated protein. High dose of NAC inhibited cell proliferation.
Conclusion: ROS is involved in the regulation of CD133+ cell expansion in vitro, decreasing ROS appropriately can promote the maintenance of the expansion cell's dry. Excessive clearance of ROS inhibits cell proliferation..ROS plays a role in regulating cell proliferation, apoptosis and senescence.
The second part inhibits activation of p38 MAPK alpha in promoting in vitro expansion of hematopoietic stem cells from umbilical cord blood
Objective: To evaluate the effect of p38MAPK alpha on the in vitro amplification characteristics of umbilical cord blood CD133+ cells by using p38MAPK alpha inhibitor and NOD/SCID mouse hematopoietic stem cell transplantation model, and preliminarily explore its mechanism.
Methods: umbilical cord blood CD133+ cells in serum free matrix culture system of cultured CD133+ cells in vitro by p38MAPK alpha inhibitor p38MAPK alpha activation, p38MAPK activity was detected at different time points and different treatment groups, and the changes of CD133+ cell subsets, CFU, CAFC, cell migration and expression of CXCR4 cells after amplification level evaluation using NOD/SCID mice artificial blood stem cell transplantation model to assess the amplified cells long-term hematopoietic reconstitution ability. At the same time detection amplified CFSE cell proliferation, apoptosis, and expression of senescence related genes p16INK4 and p21WAF1, to explore the mechanism of p38MAPK alpha.
Results: during the amplification of activated p38MAPKa cells in vitro, and with HSC self-renewal ability damage.P38MAPK alpha inhibitors inhibit p38MAPK alpha activation, promotes the umbilical cord blood CD133+ cells in vitro, representing early stem / progenitor cells of CD133+ cell subsets, CD133+CD38- cell subsets, CD133+CXCR4+ cell subsets, CFU-GEMM and CAFC compared with the control group were significantly increased (P0.01).NOD/SCID mice hematopoietic stem cells transplantation model in p38MAPKa inhibitor group graft implantation rate and graft chimerism rate were higher than the control group (P0.01). The mechanism showed that p38MAPK inhibitors can significantly inhibit the apoptosis rate of aging, transcription and senescence associated protein, promote cells to the spinal cord progenitor differentiation, had no obvious effect on cell proliferation.
Conclusion: p38MAPK alpha is involved in regulating CD133+ cell expansion in vitro, inhibiting p38MAPK alpha activation, promoting HSC amplification and maintaining HSC dry. It mainly plays a role in inhibiting cell apoptosis and aging.
The study of the relationship between the third part of ROS and p38MAPK alpha in the expansion of umbilical cord blood CD133+ cells in vitro
Purpose: used singly or combined with p38MAPKa inhibitor SB203580 and NAC antioxidant regulation in vitro, evaluate and compare the effect of different target amplification characteristics of cord blood CD133+ cells in vitro and explore the relationship between ROS and p38MAPK in umbilical cord blood CD133+ cells.
Methods: using without drugs, with p38MAPKa inhibitor SB203580, with antioxidant NAC, amplification of the combined application of p38MAPK alpha inhibitor SB203580 and antioxidant NAC intervention in vitro activation level of ROS and p38MAPK by monitoring intracellular changes of CD133+ cell subsets, CFU and CAFC to evaluate and compare the differences in cell characteristics after amplification of each treatment group.
Results: two drugs combined with ROS had the strongest inhibitory effect, decreased to the control group in the same period of 15.3 + 2.1%, 52 + 7.8% SB203580 treatment group, NAC group is 63.4 + 9%, the inhibiting effect of SB is better than NAC.p38MAPK alpha inhibitors and antioxidant NAC had inhibitory effect on activation of p38MAPKa cells in SB group. Compared with the initial, the amplification efficiency is highest, CD133+ cells was 21.93 + 1.36 times, group NAC was 14.50 + 1.19 times, the control group only was 10.13 + 0.57 times, and the drug combination group almost no amplification of.CFU and CAFC showed that the SB group than in NAC group.
Conclusion: p38MAPK alpha as a target for HSC amplification is superior to ROS in vitro, and ROS in cells regulate hematopoiesis. ROS-p38MAPK pathway is not a one-way transmission of information, and there is a feedback loop from p38MAPK to ROS.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R329

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相關期刊論文 前2條

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本文編號：1489818