發(fā)布時間：2018-06-12 02:45

  本文選題：HA117 + ATRA��； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：第一部分HA117對人臍帶間充質(zhì)干細胞(hUMSCS)向神經(jīng)元樣細胞分化過程的影響目的:HA117是ATRA小劑量、多次刺激HL-60細胞株后通過抑制消減雜交及基因芯片等技術(shù)得到的一個多藥耐藥基因。前期課題組通過實驗研究及生物信息學(xué)分析,推測HA117可能是一個抗分化相關(guān)基因。因此,本研究目的是觀察HA117對hUMSCS向神經(jīng)元樣細胞分化過程的影響。方法:獲取3代hUMSCS,流式檢測細胞表面特異性抗原,通過成骨、成脂定向分化檢測hUMSCS多向分化能力。然后,轉(zhuǎn)導(dǎo)HA117過表達慢病毒建立HA117過表達hUMSCS,流式測定轉(zhuǎn)染率,神經(jīng)元分化液培養(yǎng)誘導(dǎo)HA117過表達hUMSCS向神經(jīng)元樣細胞方向分化,并設(shè)置空白對照組、陽性對照組及空載對照組,通過觀察各組分化時間,神經(jīng)元樣細胞形態(tài)及Western、q-PCR、免疫熒光技術(shù)檢測各組定向誘導(dǎo)分化后神經(jīng)元標志物的表達水平來評價各組神經(jīng)元樣細胞分化率。結(jié)果:HA117過表達慢病毒轉(zhuǎn)導(dǎo)hUMSCS的轉(zhuǎn)染率為68.9%,hUMSCS加入神經(jīng)元誘導(dǎo)液誘導(dǎo)后,細胞體積變大,未發(fā)現(xiàn)明顯樹突及軸突,神經(jīng)元標志物β-tubulin III、NSE和Nestin的mRNA及蛋白在空白對照組中表達較少,甚至不表達,其表達水平在HA117組明顯低于陽性對照組及空載對照組,P0.05,免疫熒光觀察到HA117組熒光明顯弱于陽性對照組及空載對照組,強于空白對照組。結(jié)論:HA117在hUMSCS向神經(jīng)元定向分化過程中起到抗分化作用。第二部分HA117及SHH通路在先天性巨結(jié)腸中的表達及意義目的:研究HA117及SHH信號通路(SHH、Ptch1、Gli1)在先天性巨結(jié)腸(Hirschsprung's disease,HSCR)中的表達情況,以探討其與HD發(fā)生發(fā)展中的關(guān)系。方法:收集重慶醫(yī)科大學(xué)附屬兒童醫(yī)院2014-1015年經(jīng)手術(shù)確診為HSCR患者的巨結(jié)腸組織標本30例,腸段分吻合段(正常對照組)、擴張段和痙攣段(實驗組),運用實時熒光定量PCR(quantitative real—time PCR,q RT-PCR)、蛋白質(zhì)印跡(Western blot)和免疫組織化學(xué)方法檢測HA117、SHH信號途徑在各段中的表達情況,對其進行定量和定性并比較。結(jié)果:Q-PCR結(jié)果提示:痙攣段HA117 m RNA表達水平為1.52±0.17,明顯高于吻合段(0.28±0.04),差異具有統(tǒng)計學(xué)意義(P0.05)。痙攣段SHH、Ptch1、Gli1 m RNA表達水平分別為0.53±0.11、0.48±0.11、0.62±0.13,明顯低于吻合段(1.23±0.26、1.20±0.22、1.71±0.46),差異具有統(tǒng)計學(xué)意義(P0.05);Western-Blot結(jié)果提示:SHH、Ptch1、Gli1蛋白相對表達水平與m RNA表達水平一致,差異均具有統(tǒng)計學(xué)意義(P0.05);免疫組織化學(xué)(DAB染色)結(jié)果提示:痙攣段SHH、Ptch1、Gli1染色明顯弱于吻合段。結(jié)論:HA117及SHH信號通路可能與腸神經(jīng)系統(tǒng)(ENS)發(fā)育有著密切的聯(lián)系,這可能是HSCR病理發(fā)生的重要作用機制之一。
[Abstract]:Part I effects of HA117 on differentiation of human umbilical cord mesenchymal stem cells into neuron-like cells objective: HA117 is a multidrug resistant gene obtained by suppression subtractive hybridization and gene chip technique after stimulation of HL-60 cell line with low dose ATRA. Through experimental study and bioinformatics analysis, we speculated that HA117 might be an anti differentiation related gene. Therefore, the aim of this study was to observe the effect of HA117 on the differentiation of hUMSCS into neuron-like cells. Methods: three generations of hUMSCSs were obtained and the cell surface specific antigens were detected by flow cytometry. The multidirectional differentiation ability of hUMSCS was detected by osteogenesis and lipopolysaccharide differentiation. Then, HA117 overexpression lentivirus was transduced to establish HA117 overexpression hUMSCSs, transfection rate was measured, neuronal differentiation fluid culture induced HA117 overexpression hUMSCS to differentiate into neuron-like cells, and set up blank control group, positive control group and no-load control group. The differentiation rate of neuron-like cells in each group was evaluated by observing the time of differentiation, morphology of neuron-like cells and the expression level of neuronal markers after induction by immunofluorescence technique. Results the transfection rate of the lentivirus transduction hUMSCS was 68.9%. After the cells were induced with neuronal inducer fluid, the cell volume became larger and no obvious dendrites and axons were found. The mRNA and protein of 尾 -tubulin II I NSE and nestin were less expressed in the control group. The expression level in HA117 group was significantly lower than that in the positive control group and the no-load control group (P0.05). The fluorescence of HA117 group was significantly weaker than that of the positive control group and the no-load control group, and was stronger than that of the blank control group. Conclusion: Hal 117 plays an anti-differentiation role in the process of neuronal directional differentiation of hUMSCS. Part two: expression and significance of HA117 and SHH pathway in Hirschsprungs disease of Hirschsprungs disease in Hirschsprungs disease of Hirschsprungs Methods: thirty histopathological specimens of Hirschsprung's disease (HSCR) from 2014 to 1015 in Children's Hospital affiliated to Chongqing Medical University were collected. Intestinal segments were divided into anastomosed segments (normal control group, dilated segment and spastic segment (experimental group, real-time quantitative real-time PCRQ RT-PCRR, Western blot) and immunohistochemical method to detect the expression of HA117-SHH signal pathway in each segment. Quantitative and qualitative analysis and comparison were carried out. Results the expression level of HA117 m RNA in spastic segment was 1.52 鹵0.17, which was significantly higher than that in anastomotic segment 0.28 鹵0.04, and the difference was statistically significant (P 0.05). The expression level of Gli1 mRNA in spastic segment was 0.53 鹵0.110.48 鹵0.110.62 鹵0.13, which was significantly lower than that in anastomosed segment (1.23 鹵0.261.20 鹵0.221.20 鹵0.221.71 鹵0.46). The difference was statistically significant. The results of Western-Blot showed that the relative expression level of Gli1 protein was the same as the level of mRNA expression. The differences were statistically significant (P 0.05) and immunohistochemical staining (DAB). The results showed that the staining of SHHtch1 Gli1 in spastic segment was significantly weaker than that in anastomotic segment. Conclusion the development of the intestinal nervous system (ENS) may be closely related to the signal pathway of 1: HA117 and SHH, which may be one of the important mechanisms in the pathogenesis of HSCR.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R714.5

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