本文選題：淋巴瘤　切入點(diǎn)：Raji細(xì)胞　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的運(yùn)用轉(zhuǎn)染慢病毒介導(dǎo)的si RNA干擾的方法,抑制伯基特淋巴瘤Raji細(xì)胞中PARP-1基因的表達(dá),研究其對腫瘤細(xì)胞增殖與凋亡的影響,探討PARP-1基因在淋巴瘤中的作用機(jī)制,為惡性淋巴瘤的靶向治療提供理論和實(shí)驗(yàn)依據(jù)。方法(1)細(xì)胞培養(yǎng):將人伯基特淋巴瘤Raji細(xì)胞株培養(yǎng)于含1%青霉素和1%鏈霉素、15%的胎牛血清的RPMI1640培養(yǎng)液中,并置于37℃、5%CO2的培養(yǎng)箱中培養(yǎng),以及后續(xù)的細(xì)胞傳代、換液、凍存、復(fù)蘇。采用細(xì)胞活力數(shù)90%、對數(shù)生長期的細(xì)胞用于本實(shí)驗(yàn)。(2)分組:將細(xì)胞隨機(jī)分為三組:空白對照組;陰性對照組(NC-si RNA):轉(zhuǎn)染PARP-1小干擾RNA陰性對照序列的細(xì)胞;實(shí)驗(yàn)組:轉(zhuǎn)染PARP-1小干擾RNA的細(xì)胞。(3)轉(zhuǎn)染慢病毒介導(dǎo)的si RNA抑制PARP-1基因在淋巴瘤細(xì)胞中的表達(dá)。(4)運(yùn)用實(shí)時熒光定量PCR的方法分別對空白對照組、陰性對照組和實(shí)驗(yàn)組細(xì)胞中PARP-1 m RNA的表達(dá)情況進(jìn)行檢測,并計算出其抑制效率。(5)運(yùn)用CCK-8的方法分別于12h、24h、36h、48h檢測三組細(xì)胞的增殖活性。(6)運(yùn)用流式細(xì)胞術(shù)分別于細(xì)胞培養(yǎng)12h、24h、36h、48h后檢測三組細(xì)胞的凋亡率。結(jié)果(1)熒光顯微鏡下見到綠色熒光視為轉(zhuǎn)染成功,轉(zhuǎn)染后我們于24h、48h、72h分別進(jìn)行熒光計數(shù),比較綠色熒光細(xì)胞的數(shù)量,隨著時間的延長,綠色熒光的表達(dá)量逐漸增高,于72h綠色熒光的表達(dá)量最高,NC-si RNA組、PARP-1—si RNA組Raji細(xì)胞的轉(zhuǎn)染效率達(dá)80%以上,用來進(jìn)行后續(xù)實(shí)驗(yàn)的研究。(2)運(yùn)用實(shí)時熒光定量PCR的方法檢測三組細(xì)胞中si RNA對PARP-1 m RNA的表達(dá)水平。以對照組細(xì)胞中PARP-1基因的m RNA表達(dá)水平為100%,NC-si RNA組和實(shí)驗(yàn)組細(xì)胞中PARP-1基因m RNA表達(dá)水平分別為(94.8±0.5)%、(54.6±0.3)%。與未轉(zhuǎn)染的空白對照組相比,轉(zhuǎn)染無關(guān)序列的NC組對PARP-1 m RNA的表達(dá)無影響,差異無統(tǒng)計學(xué)意義(P0.05),而實(shí)驗(yàn)組PARP-1 m RNA的表達(dá)明顯受抑制,差異具有顯著統(tǒng)計學(xué)意義(P0.01)。(3)CCK-8法檢測細(xì)胞增殖活性,結(jié)果顯示,隨著時間的延長,細(xì)胞增殖活性抑制率逐漸升高,在48h時,三組細(xì)胞增殖活性抑制率分別為(22±0.4)%、(24±0.2)%、(58±0.7)%,對照組與陰性對照組相比,差異無統(tǒng)計學(xué)意義(P0.05),而實(shí)驗(yàn)組Raji細(xì)胞的增殖活性明顯受抑制,與對照組相比,差異有統(tǒng)計學(xué)意義(P0.05)。(4)運(yùn)用流式細(xì)胞術(shù)檢測細(xì)胞凋亡率,結(jié)果顯示,隨著時間的延長,凋亡率逐漸增加,于48h時細(xì)胞凋亡率增高,實(shí)驗(yàn)組細(xì)胞凋亡率達(dá)到(37±0.4)%,而空白對照組、陰性對照組細(xì)胞凋亡率分別為(11.4±0.3)%、(13.8±0.3)%,實(shí)驗(yàn)組與對照組相比,差異具有顯著統(tǒng)計學(xué)意義(P0.01),而陰性對照組與空白對照組相比,差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論抑制PARP-1基因在淋巴瘤Raji細(xì)胞中的表達(dá),可抑制細(xì)胞的增殖,促進(jìn)細(xì)胞的凋亡。其作用機(jī)制較為復(fù)雜,可能為不僅通過調(diào)控轉(zhuǎn)錄因子對惡性腫瘤的增殖產(chǎn)生抑制作用,而且通過激活“死亡底物”—Caspase促進(jìn)其凋亡,起到促進(jìn)細(xì)胞凋亡的作用,該方法可以提高治療腫瘤細(xì)胞的針對性和敏感性,為惡性淋巴瘤的靶向治療提供理論和實(shí)驗(yàn)依據(jù)。
[Abstract]:Methods Si RNA interference by lentivirus mediated transfection, inhibit the expression of PARP-1 gene in Burkitt's lymphoma Raji cells, to study its effect on tumor cell proliferation and apoptosis, to explore the mechanism of PARP-1 gene in lymphoma, malignant lymphoma of the target and provide theoretical and experimental basis to the treatment method (1). Cell culture: the human Burkitt lymphoma cell line Raji cultured in 1% and 1% penicillin streptomycin, 15% fetal bovine serum RPMI1640 culture medium, and at 37 DEG C, the cultivation box of 5%CO2, and the subsequent cell passage, cryopreservation, resuscitation fluid change, cell viability. The number 90%, logarithmic growth the cells used in this experiment. (2) group: the cells were randomly divided into three groups: control group; negative control group (NC-si RNA): transfection of PARP-1 small interfering RNA sequence of negative control cells; experimental group: transfection of PARP-1 small interfering RNA (cell. 3) Si RNA with lentivirus mediated inhibition of PARP-1 gene expression in lymphoma cells. (4) using the method of real-time fluorescence quantitative PCR respectively on the blank control group, negative control group and the experimental group the expression of PARP-1 in cells of M RNA were detected, and calculate the inhibition rate (5). Using the method of CCK-8 in 12h, 24h, 36h, proliferation of 48h were detected in three groups of cells. (6) the use of flow cytometry in cell culture 12h, 24h, 36h, 48h after the detection of apoptotic cells in three groups. Results (1) see green fluorescence under the fluorescence microscope as a successful transfection. After transfection, we at 24h, 48h, 72h respectively for fluorescence count, number of green fluorescent cells, with the extension of time, the expression of green fluorescence increased gradually, the expression of 72h in green fluorescence is the highest, NC-si RNA group, PARP-1 Si Group RNA Raji cell transfection efficiency was more than 80%, to 榪涜鍚庣畫瀹為獙鐨勭爺絀，

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