發(fā)布時間：2018-08-01 12:45

【摘要】：目的:1.人臍帶來源的間充質干細胞條件培養(yǎng)基(MSC-CM)對肝細胞氧化應激損傷的研究。2.MSC-CM治療肝細胞氧化應激損傷過程中微小RNA(microRNA,miRNA)的差異性表達,探討相關miRNA的調控機制。方法:1.人臍帶來源的間充質干細胞(hUC-MSCs)的分離、培養(yǎng)、鑒定及MSC-CM的制備1.1從臍帶中分離培養(yǎng)細胞;顯微鏡觀察細胞形態(tài);通過流式細胞學表型鑒定和誘導分化能力檢測確定上述細胞具備間充質干細胞(MSCs)的特性。1.2MSCs融合至80%更換培養(yǎng)基,繼續(xù)培養(yǎng)24 h后收集培養(yǎng)基,離心、過濾獲取MSC-CM。2.MSC-CM修復肝細胞氧化應激損傷的研究人正常肝細胞株(L02)經(jīng)1 mMH_2O_2作用3h,構建肝細胞氧化應激損傷模型;損傷模型經(jīng)MSC-CM修復24 h制備肝細胞氧化應激損傷修復模型。使用凋亡、線粒體膜電位(MMP)、周期、細胞活性與凋亡相關蛋白等實驗研究MSC-CM對肝細胞氧化應激損傷模型的影響。將L02細胞分為3組,即損傷組(H_2O_2)、損傷修復(H_2O_2+MSC-CM)與正常對照組(Control)。2.1凋亡與MMP檢測:采用AnnexinV/PI雙染、JC-1單染和流式細胞術檢測MSC-CM對L02細胞凋亡和MMP的影響。2.2細胞周期:使用PI染色和流式細胞術檢測MSC-CM對L02細胞周期的影響。2.3細胞活性檢測:采用CCK8法檢測實驗組L02細胞的吸光度值并繪制增值曲線,研究MSC-CM對L02細胞活性的影響。2.4Western blot方法檢測L02細胞內凋亡相關蛋白Bcl-2,Bax和BMF的表達。2.5凋亡形態(tài)學檢測:Hoechst染色方法檢測L02細胞在損傷與修復過程中細胞核的變化。3.篩選差異性表達miRNA及miRNA功能學驗證3.1根據(jù)MSCs對肝硬化損傷模型拯救機制所行的miRNA微陣列基因芯片雜交分析結果篩選了在肝硬化損傷與修復過程中顯著差異性表達的21種miRNA,RT-qPCR驗證在L02細胞氧化應激損傷與修復過程中miRNA的差異性表達并篩選出差異性表達顯著的4種miRNA(miR143,miR145,miR301a和let7a)。3.2應用生物信息學在線分析軟件預測受miRNA調控的靶基因,根據(jù)文獻確定和凋亡、周期、增殖及細胞代謝相關的靶基因,使用western blot方法驗證在肝細胞氧化應激損傷與修復過程中靶基因的表達。3.3miR143功能學驗證的研究:微陣列與RT-qPCR顯示miR143的差異性表達最為顯著。轉染miR143的模擬物與抑制物,上調或下調L02細胞內miR143的表達,同時設置轉染miRNA的陰性對照組(NC與inhibitor NC)與未經(jīng)轉染的L02細胞(Control)。轉染miR143模擬物即miR143在L02細胞內過表達(mimics);轉染miR143抑制物即下調miR143的表達(inhibitors)。3.3.1RT-qPCR根據(jù)miR143差異性表達驗證miR143是否成功轉染入L02細胞。轉染60 h后行凋亡、細胞活性、MMP、周期及其靶蛋白等檢測,研究miR143的功能。采用流式細胞術研究miR143對L02細胞凋亡、MMP與周期的影響;CCK8方法檢測miR143對肝細胞活性的影響;western blot方法檢測miR143對靶蛋白HK2和ADRB1的影響。3.3.2肝細胞氧化應激損傷與修復過程中miR143功能學驗證的研究:轉染miR143的L02細胞在miR143轉染60 h,經(jīng)0.8 mM H_2O_2損傷2 h制備肝細胞損傷模型,結束損傷后L02細胞自我修復4-6h行凋亡,周期,細胞活性,預測靶蛋白與凋亡相關蛋白等檢測。結果:1.hUC-MSCs的分離、培養(yǎng)與鑒定及MSC-CM的制備:成功分離培養(yǎng)出大量貼壁、集落樣生長的長梭形或紡錘樣的纖維細胞;細胞表面標志物CD44,CD73,CD90與CD105陽性表達,而CD31,CD34與CD45陰性表達;誘導分化鑒定示上述成纖維細胞可向成骨,成軟骨和成脂肪細胞方向定向分化。上述細胞具有MSCs的生物學特性,收集MSC-CM。2.MSC-CM修復肝細胞氧化應激損傷的研究2.1凋亡與MMP檢測:H_2O_2增加細胞凋亡率與MMP去極化比率,MSC-CM會降低上述變化。2.2細胞周期:H_2O_2會降低肝細胞G0/G1期比例,升高S+G2/M期比例;MSC-CM可逆轉上述變化。2.3細胞活性檢測:氧化應急損傷降低細胞活性,加入MSC-CM其明顯上升。2.4Westernblot檢測:H_2O_2組Bcl-2/Bax比值降低,加入MSC-CM其明顯上升。2.5凋亡形態(tài)學檢測:H_2O_2組細胞核為致密濃染或多相碎塊狀致密濃染,有時可見新月體;Control組與MSC-CM修復組無明顯差別。3.差異性表達miRNA的篩選與功能學驗證的研究3.1RT-qPCR 篩選出 miR143,miR145,miR301a 與 let7a。H_2O_2 損傷后上述 miRNA表達升高,MSC-CM救治后表達降低。3.2生物信息學軟件預測靶基因,miR143調控的靶基因為HK2和ADRB1;miRNA145、miRNA301a與let7a調控的靶基因分別為DAB2,NR2C2和ESR2。Western blot分析示靶蛋白的表達均在H_2O_2損傷后降低,MSC-CM作用后升高。3.3 miR143功能學驗證的研究3.3.1 RT-qPCR示mimics組miR143的表達顯著上調,inhibitor組其表達明顯下調。miR143成功轉染入L02細胞且差異性表達。與inhibitor組相比,miR143的過表達(mimics)明顯促進細胞凋亡、MMP的去極化,降低細胞活性;miR143的過表達可引起細胞周期阻滯。除此,mimics組中靶蛋白HK2和ADRB1的表達低于 inhibitor 組。3.3.2肝細胞氧化應激損傷過程中miR143功能學驗證的研究:miR143的過表達顯著增加損傷L02細胞的凋亡比例,降低細胞活性,細胞周期阻滯更加顯著,靶蛋白HK2、ADRB1與Bcl-2/Bax的表達下降;miR143的低表達可降低損傷L02細胞的凋亡比率,增加細胞活性,解除周期阻滯,靶蛋白與Bcl-2/Bax表達上調;損傷條件下,miR143對凋亡,周期及靶蛋白的影響更加顯著。結論:1.成功分離培養(yǎng)出hUC-MSCs,獲取MSC-CM。2.MSC-CM通過調節(jié)凋亡、影響周期與細胞活性逆轉并修復肝細胞氧化應激損傷。3.在肝細胞氧化應激損傷與修復中,miRNA發(fā)揮重要作用。MiR143通過調節(jié)凋亡、周期、細胞活性及靶蛋白HK2、ADRB1的表達等影響肝細胞氧化應激損傷的修復。MiR145、miRNA301a與let7a分別通過調控的靶蛋白DAB2、NR2C2和ESR2的表達參與肝細胞氧化應激損傷與修復進程。
[Abstract]:Objective: Study on the oxidative stress damage in hepatocytes by 1. human umbilical cord derived mesenchymal stem cell conditioned medium (MSC-CM);.2.MSC-CM differential expression of RNA (microRNA, miRNA) in the process of oxidative stress injury of hepatocytes, and to explore the regulation mechanism of related miRNA. Methods: the separation of mesenchymal stem cells (hUC-MSCs) from 1. human umbilical cord derived mesenchymal stem cells (hUC-MSCs) Culture, identification and preparation of MSC-CM 1.1 from the umbilical cord, the cells were isolated from the umbilical cord, and the morphology of the cells was observed under the microscope. The characteristics of the cells with mesenchymal stem cells (MSCs) were fused to 80% replacement medium by flow cytological phenotype identification and induction of differentiation, and the culture medium was collected after 24 h, and the culture medium was collected, centrifugation and filtration. The human normal liver cell line (L02) was repaired by MSC-CM.2.MSC-CM to repair the oxidative stress of liver cells (L02), the oxidative stress damage model of liver cells was constructed by 1 mMH_2O_2 action, and the damage model was repaired by MSC-CM for 24 h to prepare the repair model of oxidative stress injury of hepatocytes. Apoptosis, linear granular membrane potential (MMP), cycle, cell activity and apoptosis related eggs were used. The effect of MSC-CM on oxidative stress damage model of hepatocytes was studied in white test. L02 cells were divided into 3 groups, namely, damage group (H_2O_2), damage repair (H_2O_2+MSC-CM) and normal control group (Control).2.1 apoptosis and MMP detection: AnnexinV/PI double staining, JC-1 single staining and flow cytometry were used to detect MSC-CM to L02 cell apoptosis and MMP Cycle: PI staining and flow cytometry were used to detect the effect of MSC-CM on the cell cycle of L02 cells. The activity of.2.3 cells was detected by CCK8. The effect of MSC-CM on the activity of L02 cells was studied by MSC-CM. The.2.4Western blot method was used to detect the apoptosis related protein Bcl-2. 5 apoptotic morphological detection: Hoechst staining method detection of cell nuclei changes in L02 cells during injury and repair process.3. screening differential expression miRNA and miRNA functional verification 3.1 according to the miRNA microarray gene chip hybridization analysis of MSCs for liver cirrhosis damage model screening results in the process of liver cirrhosis injury and repair process 21 kinds of significant differential expression of miRNA, RT-qPCR verify the differential expression of miRNA during oxidative stress injury and repair of L02 cells and select 4 kinds of miRNA (miR143, miR145, miR301a and let7a).3.2 application bioinformatics online analysis software to pretest the target genes regulated by miRNA, and determine and wither according to literature. Target genes related to death, cycle, proliferation and cell metabolism, and using the Western blot method to verify the.3.3miR143 functional verification of target gene expression in the process of oxidative stress injury and repair of hepatocytes: microarrays and RT-qPCR showed that the difference expression of miR143 was the most significant. MiR143 mimics and inhibitors, up or down L02 The expression of intracellular miR143, and the negative control group transfected with miRNA (NC and inhibitor NC) and untransfected L02 cells (Control). The transfection of miR143 mimics is the overexpression of miR143 in L02 cells (mimics). L02 cells were transfected successfully. After transfection of 60 h, apoptosis, cell activity, MMP, cycle and target protein were detected to study the function of miR143. Flow cytometry was used to study the effect of miR143 on L02 cell apoptosis, MMP and cycle; CCK8 method was used to detect the effect of miR143 on the activity of liver cells; Western blot method was used to detect miR143 against target protein The study of miR143 functional verification during the oxidative stress injury and repair of.3.3.2 liver cells: transfection of miR143 L02 cells to 60 h in miR143 and 2 h by 0.8 mM H_2O_2, and the apoptosis, cycle, cell activity, and prediction of target protein and apoptosis related protein after the injury of L02 cells after the injury. Results: the separation, culture and identification of 1.hUC-MSCs, and the preparation of MSC-CM: a large number of spindle shaped or spindle like fibroblasts were successfully isolated and cultured; the surface markers of CD44, CD73, CD90 and CD105 were positive, and CD31, CD34 and CD45 negative expression, and the induction of differentiation showed that the above fibroblasts were osteogenic. The chondrogenic and adipocyte oriented differentiation. These cells have the biological characteristics of MSCs, collecting MSC-CM.2.MSC-CM to repair oxidative stress damage of liver cells 2.1 apoptosis and MMP detection: H_2O_2 increases the rate of apoptosis and MMP depolarization ratio, MSC-CM will reduce the above-mentioned.2.2 cell cycle: H_2O_2 will reduce the G0/G1 phase of liver cells Proportion, increase the proportion of S+G2/M phase, MSC-CM can reverse the above changes of.2.3 cell activity detection: oxidation emergency injury to reduce cell activity, adding MSC-CM to significantly increase.2.4Westernblot detection: H_2O_2 group Bcl-2/Bax ratio decreased, adding MSC-CM to increase the.2.5 apoptosis morphological detection: H_2O_2 group nuclear dense concentrated or polyphase fragments Dense dense staining and sometimes visible crescent, Control group and MSC-CM repair group had no significant difference in.3. differential expression miRNA screening and functional verification research 3.1RT-qPCR screened miR143, miR145, miR301a and let7a.H_2O_2 injury after the above miRNA expression increased, MSC-CM after treatment to reduce.3.2 bioinformatics software prediction target base The target genes regulated by miR143, HK2 and ADRB1, miRNA145, miRNA301a and let7a are the target genes for DAB2, NR2C2 and ESR2.Western blot respectively. Compared with group inhibitor, the overexpression of miR143 (mimics) significantly promoted apoptosis, MMP depolarization, and reduced cell activity, and the overexpression of miR143 could cause cell cycle arrest compared with the inhibitor group. In addition, the expression of HK2 and ADRB1 of the target protein in mimics group was lower than that of inhibitor group.3.. 3.2 the study of miR143 functional verification during oxidative stress injury of hepatocytes: the overexpression of miR143 significantly increased the apoptosis ratio of L02 cells, reduced cell activity, cell cycle arrest more significantly, the expression of target protein HK2, ADRB1 and Bcl-2/Bax decreased, and the low surface reach of miR143 could reduce the apoptosis ratio of damaged L02 cells and increase the cells. Activity, release cycle block, target protein and Bcl-2/Bax expression up-regulated; under damage conditions, miR143 has more significant effect on apoptosis, cycle and target protein. Conclusion: 1. successfully isolated and cultured hUC-MSCs, obtained MSC-CM.2.MSC-CM by regulating apoptosis, reversing cycle and cell activity and repairing oxidative stress injury of liver cells.3. in liver cell oxygen During the damage and repair of chemical stress, miRNA plays an important role in the repair of.MiR143 by regulating apoptosis, cycle, cell activity and the expression of target protein HK2, ADRB1, and other effects of.MiR145, miRNA301a and let7a through regulated target protein DAB2, NR2C2 and ESR2 expressions involved in oxidative stress injury and repair of liver cells. Cheng.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575

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