利用RNA干扰技术研究PARP-1在低剂量氢醌对骨髓间充质干细胞毒性中的作用

发布时间：2018-01-04 01:32

本文关键词：利用RNA干扰技术研究PARP-1在低剂量氢醌对骨髓间充质干细胞毒性中的作用　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：苯虽被列为确定的人类致癌物已近30年，但其仍是工业生产中重要的化工原料和溶剂。氢醌(HQ)是苯在生物体内重要的中间代谢产物，流行病学及动物实验发现HQ慢性毒性作用靶器官为骨髓，而骨髓分为造血和基质两大系统。骨髓间充质干细胞作为多种骨髓基质细胞的前体细胞是造血微环境的重要组成部分，是维持造血干细胞特性及归巢到骨髓所必需的。聚腺苷二磷酸核糖聚合酶-1是DNA损伤引起的细胞早期应激反应的重要因子，参与许多重要的生命活动如凋亡、转录、DNA修复、细胞死亡、染色体功能、基因组完整性和DNA甲基化等。RNA干扰是一个生物基本的过程，通过表达或诱导双链RNA，对目的基因的特异性转录后沉默，避免了化学抑制剂的副作用。本研究采用贴壁培养和组织块培养相结合的方法得到高纯度的BMSCs，应用流式细胞仪检测细胞周期并绘制其生长曲线。取第3代BMSCs进行诱导分化，证实其成骨、成脂肪细胞多向分化能力。根据基因序列数据库中报道的PARP-1基因序列及短发夹RNA(shRNA)设计原则，分别设计和合成用于构建RNAi载体的寡核苷酸，构建出重组质粒pGPU6/GFP/Neo-shRNA，将重组质粒转染至大鼠BMSCs，并对其PAPR-1的抑制效率进行检测，筛选出抑制率最高的重组质粒用于后续实验。由于过去对HQ毒性效应的体外研究多选用肝细胞或动物的单核细胞，这些细胞与人骨髓中细胞的生物学特性有较大差异，不能反映HQ对靶器官的毒性作用，且主要研究高剂量HQ的毒效应，因此本研究采用低剂量氢醌染毒，，观察正常和缺陷BMSCs生物学性状变化、DNA受损的遗传毒作用影响以及PARP-1基因、甲基化相关基因表达水平的变化，旨在研究实际接触水平下HQ对BMSCs的毒效应，探讨PARP-1在BMSCs对氢醌毒性应答中的作用以及PARP-1与甲基化相关酶基因之间的可能联系。结论：RNA干扰技术可以成功抑制PARP-1蛋白在BMSCs中的表达。在正常生长环境下，PARP-l蛋白缺陷对BMSCs的生长形态、生长速度、细胞活力无明显的影响。低剂量HQ具有遗传毒作用，PARP-l参与了细胞对HQ毒性的应答。PARP-1参与了DNA甲基化调节，PARP-1催化形成的分支状长链结构能够阻止DNA甲基转移酶的作用。本项研究对于职业人群的疾病预防和治疗具有非常重要的意义，其势必为找到职业性有害因素对机体损伤的早期敏感的分子生物学标志带来推动作用。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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