發(fā)布時(shí)間：2018-02-14 11:51

  本文關(guān)鍵詞： 骨髓增生異常綜合征 microRNA-378 細(xì)胞凋亡 BCL2L2 CDC40　出處：《重慶醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的檢測(cè)骨髓增生異常綜合征(myelodysplastic syndrome,MDS)患者骨髓標(biāo)本中miR-378的表達(dá)水平,構(gòu)建mi R-378過表達(dá)重組慢病毒載體,通過體內(nèi)外實(shí)驗(yàn)去探討miR-378過表達(dá)對(duì)人MDS細(xì)胞株SKM-1增殖和凋亡的影響,并進(jìn)一步探索miR-378在MDS細(xì)胞中發(fā)揮作用所調(diào)控的靶基因及其機(jī)制。方法1、收集20例MDS患者新鮮骨髓標(biāo)本,其中RA 5例,RARS 3例,RCMD 2例,RAEB 5例,5q-綜合征1例,sAML 1例,MDS-U 3例。此外還收集13例健康人的骨髓標(biāo)本作為對(duì)照。梯度離心分離出骨髓標(biāo)本中的單個(gè)核細(xì)胞,提取總RNA,再利用qRT-PCR檢測(cè)兩組骨髓標(biāo)本中miR-378的表達(dá)。2、構(gòu)建miR-378過表達(dá)重組慢病毒載體(LV-miR-378)及陰性對(duì)照病毒載體(LV-control),分別用LV-miR-378和LV-control去感染SKM-1細(xì)胞,流式細(xì)胞術(shù)檢測(cè)感染效率,qRT-PCR明確各組細(xì)胞中miR-378的表達(dá)。用CCK-8法研究miR-378對(duì)MDS細(xì)胞增殖的作用,Annexin V/7-AAD雙染法檢測(cè)各組細(xì)胞的凋亡率,流式細(xì)胞術(shù)觀察細(xì)胞周期情況。最后利用Western blot檢測(cè)內(nèi)外源凋亡通路相關(guān)凋亡因子的表達(dá)。3、分別將感染了lv-mir-378的skm-1細(xì)胞、lv-control感染的skm-1細(xì)胞及未經(jīng)任何處理的skm-1細(xì)胞接種于nod/scid小鼠皮下,以建立mds荷瘤小鼠模型。觀察各組小鼠的荷瘤生長(zhǎng)情況,測(cè)量移植瘤的體積和重量,并用tunel法檢測(cè)瘤細(xì)胞的原位凋亡情況。4、應(yīng)用生物信息學(xué)軟件預(yù)測(cè)mir-378潛在的靶基因,構(gòu)建候選靶基因mrna3'utr野生型和突變型的雙熒光素酶載體,分別與mir-378過表達(dá)質(zhì)粒及陰性對(duì)照質(zhì)粒共轉(zhuǎn)染293t細(xì)胞,并用雙熒光素酶報(bào)告基因試劑盒檢測(cè)并計(jì)算相對(duì)熒光素酶活性。最后用pcr和westernblot技術(shù)研究mir-378表達(dá)上調(diào)對(duì)靶基因表達(dá)的影響。結(jié)果1、實(shí)時(shí)熒光定量pcr結(jié)果顯示:mir-378在mds患者中的表達(dá)水平低于正常對(duì)照組(p0.05)。成功地構(gòu)建了高表達(dá)mir-378的mds細(xì)胞模型,流式細(xì)胞術(shù)檢測(cè)慢病毒感染效率大于70%,qrt-pcr檢測(cè)到lv-mir-378慢病毒轉(zhuǎn)染組的mir-378表達(dá)水平明顯高于陰性對(duì)照病毒轉(zhuǎn)染組和未經(jīng)處理的空白對(duì)照組(p0.05)。2、cck-8結(jié)果顯示,上調(diào)mir-378的表達(dá)可以明顯抑制skm-1細(xì)胞的增殖活性,mir-378過表達(dá)組的od值與陰性對(duì)照組和未感染組相比明顯降低(p0.05)。過表達(dá)mir-378慢病毒感染組的細(xì)胞凋亡率為[(12.90±3.72)%],明顯高于陰性對(duì)照病毒感染組[(3.21±1.92)%]和未感染組[(2.78±1.04)%]。此外,上調(diào)skm-1細(xì)胞中mir-378的表達(dá)使g1/g0期細(xì)胞比例增加,s期細(xì)胞比例減少(p0.05)。我們還發(fā)現(xiàn)mir-378過表達(dá)可以同時(shí)激活內(nèi)外源凋亡通路,使cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9及Bax蛋白的表達(dá)水平增高。3、miR-378過表達(dá)組的移植瘤體積和重量均小于陰性對(duì)照組和空白對(duì)照組(P0.05),TUNEL法結(jié)果顯示miR-378過表達(dá)組瘤組織的原位細(xì)胞凋亡率[(37.08±7.65)%]明顯高于陰性對(duì)照組[(17.49±3.13)%,P=0.001]和空白對(duì)照組[(16.93±2.95)%,P=0.001]。4、兩個(gè)靶基因預(yù)測(cè)網(wǎng)站都提示抗凋亡蛋白基因BCL2L2是miR-378的潛在靶基因。雙熒光素酶實(shí)驗(yàn)結(jié)果提示,與對(duì)照組相比,miR-378質(zhì)粒與BCL2L2 3'UTR野生型質(zhì)粒共轉(zhuǎn)染組的熒光素酶活性明顯下降(P0.05)。上調(diào)MDS細(xì)胞中miR-378的表達(dá)后,BCL2L2mRNA水平無明顯變化(P0.05),而BCL2L2蛋白表達(dá)明顯降低。此外,我們還發(fā)現(xiàn)miR-378可以降低miR-378的另一個(gè)靶基因CDC40蛋白的表達(dá)。結(jié)論miR-378在MDS中呈低表達(dá),上調(diào)mi R-378的表達(dá)可以抑制MDS細(xì)胞的生長(zhǎng),促進(jìn)MDS細(xì)胞凋亡及造成細(xì)胞周期阻滯。其發(fā)揮作用的機(jī)制可能與靶向作用于BCL2L2和CDC40有關(guān)。
[Abstract]:Objective to detect the myelodysplastic syndrome (myelodysplastic, syndrome, MDS) the expression level of miR-378 in patients with bone marrow specimens, construction of MI over expression of R-378 recombinant lentiviral vectors in vitro and in vivo to investigate the effect of overexpression of miR-378 on proliferation and apoptosis of human MDS cell line SKM-1, and to further explore the target genes regulated by miR-378 play a role and its mechanism in MDS cells. Methods 1, 20 MDS patients were collected fresh bone marrow specimens, including 5 cases of RA, 3 cases RARS, 2 cases RCMD, 5 cases of RAEB, 1 cases of 5q- syndrome, 1 cases of sAML, 3 cases of MDS-U. In addition, bone marrow samples were collected from 13 healthy people as control. The gradient isolated mononuclear cells in bone marrow samples, extraction of total RNA, using qRT-PCR to detect miR-378 expression of.2 in bone marrow samples of two groups, the construction of miR-378 over expression of recombinant lentiviral vector (LV-miR-378) and negative control vector (LV-control), LV-miR-378 and LV-control were used to infect SKM-1 cells. The infection efficiency was assessed by flow cytometry, the expression of miR-378 qRT-PCR in the cells were clear. To study the effect of miR-378 CCK-8 on MDS cell proliferation and apoptosis of Annexin V/7-AAD double staining method to detect cell rate, cell cycle was measured by flow cytometry. The expression of.3 Western blot detection of apoptosis related protein locus, were infected with lv-mir-378 skm-1 cells, lv-control infected skm-1 cells and skm-1 cells without any treatment inoculation in nod/scid mice skin, in order to establish MDS mice model. The growth of tumor bearing mice were observed, measured the tumor volume and weight the TUNEL method was used to detect tumor cells in situ apoptosis of.4, using bioinformatics software predicted the potential target gene of mir-378, construct the candidate target gene mrna3'utr in the wild Dual luciferase vector type and mutant, and overexpression of mir-378 plasmid and negative control plasmid were transfected into 293T cells, and dual luciferase reporter kit to detect and calculate the relative luciferase activity. Finally, using PCR and Westernblot technology research on the influence of mir-378 expression on target gene expression. Results 1, real-time fluorescence quantitative the result of PCR showed that the expression level of mir-378 in patients with MDS than those of the normal control group (P0.05). The successful construction of the model of MDS cells with high expression of mir-378 is greater than 70%, the efficiency of flow cytometry to detect slow virus infection, qRT-PCR detected lv-mir-378 lentiviral transfection group mir-378 expression levels were significantly higher than that in the negative control group and transfection the untreated control group (P0.05).2, CCK-8 results showed that the upregulation of the expression of mir-378 can significantly inhibit the proliferation of skm-1 cells, overexpression of mir-378 group The OD value decreased obviously compared with the negative control group and non infection group (P0.05). Expression of apoptosis mir-378 lentivirus infection group was [(12.90 + 3.72) detected in the negative control group was significantly higher than that of virus infection [(3.21 + 1.92) detected and uninfected Group [(2.78 + 1.04) in addition. The expression of mir-378, skm-1 in the cells increased the percentage of g1/g0 phase, decreased the proportion of cells in S phase (P0.05). We also found that overexpression of mir-378 can also activate exogenous apoptosis pathway, cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax protein expression increased.3, overexpression of miR-378 group transplanted tumor volume and weight are less than the negative control group and blank control group (P0.05), TUNEL assay showed that cell apoptosis in miR-378 overexpression group tumor rate [(37.08 + 7.65) when it was significantly higher than that of negative control group [(17.49 + 3.13)%, P=0.001] and blank control group [(16.9 3 + 2.95)%, P=0.001].4, two target gene prediction sites showed that the anti apoptosis protein BCL2L2 gene is a potential target gene of miR-378. Dual luciferase results showed that, compared with the control group, miR-378 plasmid and BCL2L2 plasmid were co transfected with wild-type 3'UTR luciferase activity group significantly decreased (P0.05). The expression of miR-378 increased MDS in cells, no significant changes in BCL2L2mRNA level (P0.05), BCL2L2 protein expression was significantly reduced. In addition, we also found that miR-378 can reduce the expression of another miR-378 target gene CDC40 protein. Conclusion the expression of miR-378 is lower in MDS, MI up-regulated the expression of R-378 can inhibit the growth of MDS cells, promoting MDS cell apoptosis and caused cell cycle arrest. Its function and possible mechanism of targeting BCL2L2 and CDC40.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R551.3

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本文編號(hào)：1510641