本文關(guān)鍵詞：利用RNA干擾技術(shù)研究PARP-1在低劑量氫醌對(duì)骨髓間充質(zhì)干細(xì)胞毒性中的作用　出處：《吉林大學(xué)》2012年博士論文　論文類型：學(xué)位論文

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【摘要】：苯雖被列為確定的人類致癌物已近30年，但其仍是工業(yè)生產(chǎn)中重要的化工原料和溶劑。氫醌(HQ)是苯在生物體內(nèi)重要的中間代謝產(chǎn)物，流行病學(xué)及動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)HQ慢性毒性作用靶器官為骨髓，而骨髓分為造血和基質(zhì)兩大系統(tǒng)。骨髓間充質(zhì)干細(xì)胞作為多種骨髓基質(zhì)細(xì)胞的前體細(xì)胞是造血微環(huán)境的重要組成部分，是維持造血干細(xì)胞特性及歸巢到骨髓所必需的。聚腺苷二磷酸核糖聚合酶-1是DNA損傷引起的細(xì)胞早期應(yīng)激反應(yīng)的重要因子，參與許多重要的生命活動(dòng)如凋亡、轉(zhuǎn)錄、DNA修復(fù)、細(xì)胞死亡、染色體功能、基因組完整性和DNA甲基化等。RNA干擾是一個(gè)生物基本的過(guò)程，通過(guò)表達(dá)或誘導(dǎo)雙鏈RNA，對(duì)目的基因的特異性轉(zhuǎn)錄后沉默，避免了化學(xué)抑制劑的副作用。 本研究采用貼壁培養(yǎng)和組織塊培養(yǎng)相結(jié)合的方法得到高純度的BMSCs，應(yīng)用流式細(xì)胞儀檢測(cè)細(xì)胞周期并繪制其生長(zhǎng)曲線。取第3代BMSCs進(jìn)行誘導(dǎo)分化，證實(shí)其成骨、成脂肪細(xì)胞多向分化能力。根據(jù)基因序列數(shù)據(jù)庫(kù)中報(bào)道的PARP-1基因序列及短發(fā)夾RNA(shRNA)設(shè)計(jì)原則，分別設(shè)計(jì)和合成用于構(gòu)建RNAi載體的寡核苷酸，構(gòu)建出重組質(zhì)粒pGPU6/GFP/Neo-shRNA，將重組質(zhì)粒轉(zhuǎn)染至大鼠BMSCs，并對(duì)其PAPR-1的抑制效率進(jìn)行檢測(cè)，篩選出抑制率最高的重組質(zhì)粒用于后續(xù)實(shí)驗(yàn)。 由于過(guò)去對(duì)HQ毒性效應(yīng)的體外研究多選用肝細(xì)胞或動(dòng)物的單核細(xì)胞，這些細(xì)胞與人骨髓中細(xì)胞的生物學(xué)特性有較大差異，不能反映HQ對(duì)靶器官的毒性作用，且主要研究高劑量HQ的毒效應(yīng)，因此本研究采用低劑量氫醌染毒，，觀察正常和缺陷BMSCs生物學(xué)性狀變化、DNA受損的遺傳毒作用影響以及PARP-1基因、甲基化相關(guān)基因表達(dá)水平的變化，旨在研究實(shí)際接觸水平下HQ對(duì)BMSCs的毒效應(yīng)，探討PARP-1在BMSCs對(duì)氫醌毒性應(yīng)答中的作用以及PARP-1與甲基化相關(guān)酶基因之間的可能聯(lián)系。 結(jié)論：RNA干擾技術(shù)可以成功抑制PARP-1蛋白在BMSCs中的表達(dá)。在正常生長(zhǎng)環(huán)境下，PARP-l蛋白缺陷對(duì)BMSCs的生長(zhǎng)形態(tài)、生長(zhǎng)速度、細(xì)胞活力無(wú)明顯的影響。低劑量HQ具有遺傳毒作用，PARP-l參與了細(xì)胞對(duì)HQ毒性的應(yīng)答。PARP-1參與了DNA甲基化調(diào)節(jié)，PARP-1催化形成的分支狀長(zhǎng)鏈結(jié)構(gòu)能夠阻止DNA甲基轉(zhuǎn)移酶的作用。 本項(xiàng)研究對(duì)于職業(yè)人群的疾病預(yù)防和治療具有非常重要的意義，其勢(shì)必為找到職業(yè)性有害因素對(duì)機(jī)體損傷的早期敏感的分子生物學(xué)標(biāo)志帶來(lái)推動(dòng)作用。
[Abstract]:Although benzene has been identified as a human carcinogen for nearly 30 years, but it is still an important chemical raw materials in industrial production and solvent. Hydroquinone (HQ) is an important intermediate metabolite of benzene in organism, epidemiological studies and animal experiments found that HQ chronic toxicity target organ for bone marrow, and bone marrow into hematopoietic and stromal two. Bone marrow mesenchymal stem cells as the precursors of various bone marrow stromal cells is an important component of hematopoietic microenvironment, is the maintenance of hematopoietic stem cell homing to the bone marrow and characteristics required. Two poly adenosine phosphate ribose polymerase -1 reaction cell is an important factor in early stress caused by DNA damage, participate in many important life activities such as apoptosis, transcription, DNA repair, cell death, chromosome, genome integrity and DNA methylation,.RNA interference is a fundamental biological process, or induced by expression of double stranded RNA, on The specific post transcriptional silencing of the target gene avoids the side effects of chemical inhibitors.
This study obtained high purity BMSCs by adherent culture and tissue culture method combined with the application of flow cytometry to detect the cell cycle and draw the growth curve. The third generation of BMSCs differentiation, confirmed the osteogenic, adipocytes differentiation. According to the sequence of PARP-1 gene and short reported gene sequence database in the RNA folder (shRNA) design principles, were designed and synthesized oligonucleotides for construction of a RNAi vector, the recombinant plasmid pGPU6/GFP/Neo-shRNA. The recombinant plasmid BMSCs was transfected to rats, and the inhibitory effect on the rate of PAPR-1 testing, screening out the highest inhibition rate of recombinant plasmid was used for subsequent experiments.
The mononuclear cells in vitro study on toxic effects of HQ in the multi selection of liver cell or animal, have different biological characteristics of these cells and human bone marrow cells, can not reflect the toxic effects of HQ on target organs, and the main toxic effects of high dose HQ, therefore this study adopts low dose hydroquinone exposure. The change of normal and defect of BMSCs biological characteristics observation, genetic toxicity effect of DNA damage and PARP-1 gene methylation related gene expression, to study the actual contact toxic effects on BMSCs levels of HQ, PARP-1 in BMSCs on the toxicity of hydroquinone response and the effect of PARP-1 methylation and the possible link between related enzyme genes.
Conclusion: the expression of RNA interference can successfully inhibit PARP-1 protein in BMSCs. Under normal growth conditions, PARP-l protein deficiency on BMSCs growth morphology, growth rate, no obvious affect cell viability. Low dose HQ has genetic toxicity, PARP-l is involved in.PARP-1 cell response to HQ toxicity in DNA methylation regulation, long chain branching structure can prevent PARP-1 catalyzed formation of DNA methyltransferase.
This research is of great significance for disease prevention and treatment of occupational population. It is bound to promote the discovery of occupational hazards and biomarkers of early sensitive molecular injury.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 宋關(guān)斌;李曉娜;;骨髓間充質(zhì)干細(xì)胞對(duì)腫瘤細(xì)胞的生物學(xué)行為的影響[J];東北農(nóng)業(yè)大學(xué)學(xué)報(bào);2010年11期

2 程巧;厲紅元;劉勝春;李凡;任國(guó)勝;;沉默Ku70后PARP1參與DNA雙鏈斷裂修復(fù)[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2011年03期

3 程巧;厲紅元;王小毅;任國(guó)勝;;沉默Ku70促進(jìn)PARP1、ligase3和XRCC1在DNA雙鏈斷裂染色質(zhì)聚集[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2011年14期

4 崔照瓊;張彥;李艷瓊;;RNA干擾技術(shù)應(yīng)用新進(jìn)展[J];安徽醫(yī)藥;2012年03期

5 劉林華;凌曉璇;梁海榮;翟璐;唐煥文;;氫醌誘導(dǎo)TK6細(xì)胞miR-221表達(dá)異常致PARP-1基因下調(diào)的機(jī)制[J];環(huán)境與健康雜志;2011年06期

6 楊淵,張?zhí)K明,方思羽,江紅,許康,常履華;DNA修復(fù)蛋白PARP基因在大鼠缺血腦組織中的表達(dá)[J];中華急診醫(yī)學(xué)雜志;2004年01期

7 陳莉;;核酸干擾技術(shù)的應(yīng)用前景[J];醫(yī)學(xué)研究生學(xué)報(bào);2008年10期

8 房興銳;黃愷;黃丹;王妍;廖玉華;;1型多聚ADP核糖聚合酶激活NF-κB途徑調(diào)節(jié)基質(zhì)金屬蛋白酶活性的機(jī)制研究[J];臨床心血管病雜志;2009年06期

9 高向景;王潔;姚耿東;;氫醌對(duì)人骨髓間充質(zhì)干細(xì)胞蛋白質(zhì)表達(dá)譜的影響[J];環(huán)境與職業(yè)醫(yī)學(xué);2009年05期

10 張冰;何建國(guó);;RNAi技術(shù)及其在基因組學(xué)研究中的應(yīng)用[J];內(nèi)蒙古大學(xué)學(xué)報(bào)(自然科學(xué)版);2007年01期

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