本文選題：RNA干擾 + 小片段雙鏈RNA�。� 參考：《中國醫(yī)科大學(xué)》2006年碩士論文

【摘要】：RNA干擾對(duì)活化的小鼠脾淋巴細(xì)胞分泌TNFα的影響 目的 腫瘤壞死因子(tumor nectosis factor-α,TNF-α)由活化的巨噬細(xì)胞和T淋巴細(xì)胞產(chǎn)生,是一類重要的細(xì)胞因子,TNF-α具有廣泛的生物學(xué)活性。最初對(duì)TNF-α功能的認(rèn)識(shí)僅限于對(duì)腫瘤特異性殺傷作用,后來發(fā)現(xiàn)TNF-α也具有免疫調(diào)節(jié)作用,而且參與某些炎癥反應(yīng)過程。適量的TNF-α可以調(diào)節(jié)機(jī)體免疫功能,對(duì)維持機(jī)體內(nèi)部的穩(wěn)定狀態(tài)及抵御各種致病因子具有重要意義,然而,TNF-α的異常分泌可作為機(jī)體炎癥、損傷及休克的重要介質(zhì)。目前已知與TNF-α有關(guān)的疾病包括:AIDS、貧血、自身免疫性疾病、腫瘤、出血性休克、移植排斥反應(yīng)、結(jié)核病、白血病、糖尿病、類風(fēng)濕性關(guān)節(jié)炎等。TNF-α參與了眾多疾病的發(fā)生發(fā)展過程,因此,在不同水平上阻斷TNF-α的作用,有可能對(duì)與TNF-α有關(guān)的疾病產(chǎn)生治療作用。RNA干擾(RNA interference,RNAi)是利用具有同源性互補(bǔ)序列的雙鏈RNA誘發(fā)序列特異性的轉(zhuǎn)錄后基因沉默現(xiàn)象,它可以通過抑制蛋白表達(dá)來模擬基因敲除技術(shù),RNA干擾在植物、真菌及動(dòng)物細(xì)胞中均存在,在抗腫瘤、抗病毒及研究基因功能等方面都有著廣泛的應(yīng)用前景。抑制效果有明顯的高效性、特異性。本實(shí)驗(yàn)利用Silentgene~(TM) U6試劑盒在體外經(jīng)PCR合成siRNA表達(dá)載體,利用脂質(zhì)體轉(zhuǎn)染入細(xì)胞后,轉(zhuǎn)錄出siRNA引發(fā)干擾。針對(duì)小鼠TNF-α基因選取2個(gè)靶位點(diǎn),測(cè)定并比較其干擾效果,從而選出敏感的抑制位點(diǎn),為今后克隆質(zhì)粒提供最強(qiáng)的干擾序列。本文就位點(diǎn)1干擾作用加以論述。 方法 1.小鼠脾淋巴細(xì)胞的培養(yǎng):C_(57)BL/6小鼠,雄性(中國醫(yī)科大學(xué)動(dòng)物部提供),頸椎脫臼法處死,0℃無菌條件下取脾,尼龍網(wǎng)上研磨,過濾制成細(xì)
[Abstract]:Effect of RNA interference on secretion of TNF 偽 by activated splenic lymphocytes in mice Purpose Tumor nectosis factor- 偽 (TNF- 偽) is produced by activated macrophages and T lymphocytes. TNF- 偽, an important cytokine, has a wide range of biological activities. Initially, the function of TNF- 偽 was limited to tumor-specific killing, but it was later found that TNF- 偽 also had immunomodulatory effect and participated in some inflammatory reaction. Proper amount of TNF- 偽 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- 偽 can be used as an important mediator of inflammation, injury and shock. Diseases known to be related to TNF- 偽 include: AIDS-, anemia, autoimmune diseases, tumors, hemorrhagic shock, transplant rejection, tuberculosis, leukemia, diabetes, rheumatoid arthritis, and so on. TNF- 偽 is involved in the development of many diseases. Therefore, blocking the effect of TNF- 偽 at different levels may have a therapeutic effect on TNF- 偽 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- 偽 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，

本文編號(hào)：1819308