【摘要】：目的:骨髓微環(huán)境中的間充質(zhì)干細(xì)胞(MSC)可以誘導(dǎo)腫瘤細(xì)胞生長(zhǎng)和存活,并形成適合腫瘤繁殖及耐藥的微環(huán)境。急性髓系白血病(AML)復(fù)發(fā)和耐藥的關(guān)鍵在于白血病細(xì)胞與MSC之間的相互作用。白血病干細(xì)胞(LSCs)與MSC的粘附及相互作用,可以直接促進(jìn)細(xì)胞的自我更新、增殖、分化停滯,并“保護(hù)”白血病細(xì)胞免受化療藥物的毒性作用。micro RNA(微小RNA,mi RNA)作為一種非編碼的小RNA分子,能夠在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。mi RNA在生物發(fā)育過(guò)程中發(fā)揮著重要作用,如細(xì)胞分化及凋亡、腫瘤生成等。研究mi RNA的表達(dá)及功能,對(duì)防治腫瘤性疾病具有重要意義。有關(guān)mi RNA在AML發(fā)病中的研究近幾年剛剛起步,多從表達(dá)量高低的角度闡述,與發(fā)病機(jī)制相關(guān)的功能性研究較少,mi RNA在AML中MSC誘導(dǎo)的耐藥中作用的研究尚未見報(bào)道。我們?cè)缙诠ぷ靼l(fā)現(xiàn),AML細(xì)胞系KG1a與MSC粘附后,對(duì)化療藥物的反應(yīng)下降,細(xì)胞凋亡減少。RT-PCR和Western結(jié)果證實(shí)與細(xì)胞周期有關(guān)的基因C-Myc表達(dá)上調(diào)。本研究擬在將AML細(xì)胞系與人骨髓MSC共培養(yǎng)的基礎(chǔ)上通過(guò)mi RNA芯片尋找調(diào)控C-Myc的mi RNAs,并將篩選出的mi RNAs進(jìn)行表達(dá)量驗(yàn)證。為后續(xù)功能驗(yàn)證做準(zhǔn)備。方法:1.培養(yǎng)AML細(xì)胞系KG1a,準(zhǔn)備15例健康供者的骨髓原代細(xì)胞。2.分離和培養(yǎng)人骨髓MSC。3.建立共培養(yǎng)體系,與MSC共培養(yǎng)48h后CD33陽(yáng)選出KG1a(樣品編號(hào)4D,5E,6F),以AML細(xì)胞系KG1a單獨(dú)培養(yǎng)(樣品編號(hào)1,2,3)作為對(duì)照。4.將上述樣品分裝6管加Trizol處理,送公司進(jìn)行樣本質(zhì)量檢測(cè)并制成基因芯片,根據(jù)全基因組芯片結(jié)果尋找調(diào)控C-Myc的mi RNAs。5.重復(fù)步驟1,2,3;RT-PCR法檢測(cè)兩種培養(yǎng)條件下篩選出的mi RNAs表達(dá)量變化情況。結(jié)果:1.基因芯片樣本檢驗(yàn)合格。2.根據(jù)芯片結(jié)果初步尋找調(diào)控C-Myc的mi RNAs:let-7a,mi R-17-3p,mi R-34c,mi R-135a,mi R-494;U6作為內(nèi)參。3.RT-PCR法檢測(cè)結(jié)果:let-7a表達(dá)上升3.39倍,mi R-34c表達(dá)上升621.67倍,mi R-135a表達(dá)上升13.00倍,mi R-494表達(dá)上升17.51倍,mi R-17-3p表達(dá)下降3.70倍。4.mi R-17-3p可能參與調(diào)控C-Myc表達(dá)。結(jié)論:AML細(xì)胞粘附于MSC后,通過(guò)一系列mi RNA的變化引起C-Myc表達(dá)的上調(diào),而mi R-17-3p可能參與調(diào)控C-Myc的表達(dá)。
[Abstract]:Aim: mesenchymal stem cells (MSC) in bone marrow microenvironment can induce the growth and survival of tumor cells and form a microenvironment suitable for tumor reproduction and drug resistance. The key to the recurrence and drug resistance of (AML) in acute myeloid leukemia lies in the interaction between leukemia cells and MSC. The adhesion and interaction between (LSCs) and MSC can directly promote the self-renewal, proliferation and differentiation stagnation of leukemic cells, and "protect" leukemic cells from the toxicity of chemotherapeutic drugs. Micro RNA (micro RNA,) Mi RNA), as a small non-coding RNA molecule, can regulate the expression of genes at the post-transcriptional level. Mi RNA plays an important role in biological development, such as cell differentiation and apoptosis, tumorigenesis and so on. It is of great significance to study the expression and function of mi RNA in the prevention and treatment of tumor diseases. The study of mi RNA in the pathogenesis of AML has just started in recent years. Most of them are expounded from the point of view of expression level. Few functional studies related to the pathogenesis of, mi RNA have not been reported on the role of, mi RNA in MSC-induced drug resistance in AML. Our early work found that after the adhesion of AML cell line KG1a to MSC, the response to chemotherapeutic drugs decreased and apoptosis decreased. The results of RT-PCR and Western confirmed that the expression of C-Myc, which is related to cell cycle, was up-regulated. In this study, on the basis of co-culture of AML cell line and human bone marrow MSC, the mi RNAs, regulating C-Myc was found by mi RNA chip and the expression of mi RNAs was verified. Prepare for subsequent functional verification. Methods: 1. Primary bone marrow cells from 15 healthy donors were prepared by culturing AML cell line KG1a,. 2. Isolation and culture of human bone marrow MSC.3. The co-culture system was established. After co-culture with MSC for 48 h, KG1a (sample number 4D, 5E, 6F) was selected. AML cell line KG1a was cultured alone (sample number 1, 2, 3) as control. 4. The above samples were divided into 6 tubes and treated with Trizol, and sent to the company for sample quality test and made into gene chip. According to the results of the whole genome chip, the mi RNAs.5. regulating C-Myc was found. Repeat step 1, 2, 3 RT PCR was used to detect the expression of mi RNAs screened under the two culture conditions. Result: 1. The sample of gene chip is up to standard. 2. According to the results of the chip, the mi RNAs:let-7a,mi R 鈮，

本文編號(hào)：2476679