本文關鍵詞：利用RNA干擾技術研究PARP-1在低劑量氫醌對骨髓間充質干細胞毒性中的作用　出處：《吉林大學》2012年博士論文　論文類型：學位論文

  更多相關文章： RNA干擾 PARP-1 骨髓間充質干細胞 氫醌 大鼠

【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)生產中重要的化工原料和溶劑。氫醌(HQ)是苯在生物體內重要的中間代謝產物，流行病學及動物實驗發(fā)現HQ慢性毒性作用靶器官為骨髓，而骨髓分為造血和基質兩大系統。骨髓間充質干細胞作為多種骨髓基質細胞的前體細胞是造血微環(huán)境的重要組成部分，是維持造血干細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損傷引起的細胞早期應激反應的重要因子，參與許多重要的生命活動如凋亡、轉錄、DNA修復、細胞死亡、染色體功能、基因組完整性和DNA甲基化等。RNA干擾是一個生物基本的過程，通過表達或誘導雙鏈RNA，對目的基因的特異性轉錄后沉默，避免了化學抑制劑的副作用。 本研究采用貼壁培養(yǎng)和組織塊培養(yǎng)相結合的方法得到高純度的BMSCs，應用流式細胞儀檢測細胞周期并繪制其生長曲線。取第3代BMSCs進行誘導分化，證實其成骨、成脂肪細胞多向分化能力。根據基因序列數據庫中報道的PARP-1基因序列及短發(fā)夾RNA(shRNA)設計原則，分別設計和合成用于構建RNAi載體的寡核苷酸，構建出重組質粒pGPU6/GFP/Neo-shRNA，將重組質粒轉染至大鼠BMSCs，并對其PAPR-1的抑制效率進行檢測，篩選出抑制率最高的重組質粒用于后續(xù)實驗。 由于過去對HQ毒性效應的體外研究多選用肝細胞或動物的單核細胞，這些細胞與人骨髓中細胞的生物學特性有較大差異，不能反映HQ對靶器官的毒性作用，且主要研究高劑量HQ的毒效應，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、甲基化相關基因表達水平的變化，旨在研究實際接觸水平下HQ對BMSCs的毒效應，探討PARP-1在BMSCs對氫醌毒性應答中的作用以及PARP-1與甲基化相關酶基因之間的可能聯系。 結論：RNA干擾技術可以成功抑制PARP-1蛋白在BMSCs中的表達。在正常生長環(huán)境下，PARP-l蛋白缺陷對BMSCs的生長形態(tài)、生長速度、細胞活力無明顯的影響。低劑量HQ具有遺傳毒作用，PARP-l參與了細胞對HQ毒性的應答。PARP-1參與了DNA甲基化調節(jié)，PARP-1催化形成的分支狀長鏈結構能夠阻止DNA甲基轉移酶的作用。 本項研究對于職業(yè)人群的疾病預防和治療具有非常重要的意義，其勢必為找到職業(yè)性有害因素對機體損傷的早期敏感的分子生物學標志帶來推動作用。
[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.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329

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