RNA干扰对小鼠脾淋巴细胞分泌TNF-α的影响

发布时间：2018-04-29 09:14

  本文选题：RNA干扰 + 小片段双链RNA　； 参考：《中国医科大学》2006年硕士论文

【摘要】：RNA干扰对活化的小鼠脾淋巴细胞分泌TNFα的影响 目的 肿瘤坏死因子(tumor nectosis factor-α,TNF-α)由活化的巨噬细胞和T淋巴细胞产生,是一类重要的细胞因子,TNF-α具有广泛的生物学活性。最初对TNF-α功能的认识仅限于对肿瘤特异性杀伤作用,后来发现TNF-α也具有免疫调节作用,而且参与某些炎症反应过程。适量的TNF-α可以调节机体免疫功能,对维持机体内部的稳定状态及抵御各种致病因子具有重要意义,然而,TNF-α的异常分泌可作为机体炎症、损伤及休克的重要介质。目前已知与TNF-α有关的疾病包括:AIDS、贫血、自身免疫性疾病、肿瘤、出血性休克、移植排斥反应、结核病、白血病、糖尿病、类风湿性关节炎等。TNF-α参与了众多疾病的发生发展过程,因此,在不同水平上阻断TNF-α的作用,有可能对与TNF-α有关的疾病产生治疗作用。RNA干扰(RNA interference,RNAi)是利用具有同源性互补序列的双链RNA诱发序列特异性的转录后基因沉默现象,它可以通过抑制蛋白表达来模拟基因敲除技术,RNA干扰在植物、真菌及动物细胞中均存在,在抗肿瘤、抗病毒及研究基因功能等方面都有着广泛的应用前景。抑制效果有明显的高效性、特异性。本实验利用Silentgene~(TM) U6试剂盒在体外经PCR合成siRNA表达载体,利用脂质体转染入细胞后,转录出siRNA引发干扰。针对小鼠TNF-α基因选取2个靶位点,测定并比较其干扰效果,从而选出敏感的抑制位点,为今后克隆质粒提供最强的干扰序列。本文就位点1干扰作用加以论述。 方法 1.小鼠脾淋巴细胞的培养:C_(57)BL/6小鼠,雄性(中国医科大学动物部提供),颈椎脱臼法处死,0℃无菌条件下取脾,尼龙网上研磨,过滤制成细
[Abstract]:Effect of RNA interference on secretion of TNF 伪 by activated splenic lymphocytes in mice Purpose Tumor nectosis factor- 伪 (TNF- 伪) is produced by activated macrophages and T lymphocytes. TNF- 伪, an important cytokine, has a wide range of biological activities. Initially, the function of TNF- 伪 was limited to tumor-specific killing, but it was later found that TNF- 伪 also had immunomodulatory effect and participated in some inflammatory reaction. Proper amount of TNF- 伪 can regulate the immune function of the body and play an important role in maintaining the stable state of the body and resisting various pathogenic factors. However, the abnormal secretion of TNF- 伪 can be used as an important mediator of inflammation, injury and shock. Diseases known to be related to TNF- 伪 include: AIDS-, anemia, autoimmune diseases, tumors, hemorrhagic shock, transplant rejection, tuberculosis, leukemia, diabetes, rheumatoid arthritis, and so on. TNF- 伪 is involved in the development of many diseases. Therefore, blocking the effect of TNF- 伪 at different levels may have a therapeutic effect on TNF- 伪 related diseases. RNAi is a sequence-specific post-transcriptional gene silencing phenomenon induced by double-stranded RNA with homologous complementary sequences. It can mimic gene knockout by inhibiting protein expression. RNA interference exists in plants, fungi and animal cells. It has a wide application prospect in anti-tumor, anti-virus and gene function. The inhibitory effect has obvious high efficiency and specificity. In this study, siRNA expression vector was synthesized by PCR using Silentgenetin TMU6 kit in vitro. After transfection with liposome into cells, siRNA was transcribed to induce interference. Two target sites for mouse TNF- 伪 gene were selected to determine and compare their interference effects, and then the sensitive inhibition sites were selected, which provided the strongest interference sequence for cloning plasmid in the future. The interference of site 1 is discussed in this paper. Method 1. Culture of splenic lymphocytes of mice:% CStue 57 / BL / 6 mice, male (provided by the Animal Department of China Medical University, cervical spine dislocated method, executed under aseptic conditions at 0 鈩，

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