【作者】 熊文艳；

【导师】 陆志刚； 李玉华；

【作者基本信息】 南方医科大学 ， 内科学， 2011， 硕士

【摘要】 研究背景恶性血液肿瘤(HematoIogical malignancy)主要包括白血病、恶性淋巴瘤以及多发性骨髓瘤,前两类肿瘤发病率排在目前人类恶性肿瘤的前十位。其中白血病在恶性肿瘤所致的死亡率中,占第6位(男)和第8位(女)；儿童及35岁以下成人中,则居第1位,且发病率逐年升高,严重威胁着人类健康和生命。白血病分急性白血病(AL)和慢性白血病(CL),我国AL比CL多见(约5.5：1),其中以急性髓系白血病AML最多(1.62/10万)。急性白血病发病时骨髓中异常的原始细胞及幼稚细胞(白血病细胞)大量增殖并抑制正常造血,广泛浸润肝、脾、淋巴结等各种脏器,表现为贫血、出血、感染和浸润等征象。近年来随着支持治疗的加强、多药联合方案的应用、大剂量化疗和造血干细胞移植(HSCT)的推广,AML患者化疗完全缓解率(CR)可达75%左右,但仍有20%-30%AML经正规化疗后未能获得缓解,称为难治。即使CR后约50%患者会出现复发,一旦复发,患者的预后极差。目前普遍认为白血病难治和复发的原因主要包括：①白血病细胞对凋亡通路的抵制；②白血病细胞高表达多药耐药基因／蛋白；③白血病细胞处于G0期静止状态,对化疗药物不敏感；由于细胞对药物不敏感或耐药,导致大剂量化疗药物的使用,临床上相当一部份患者死于化疗药物的毒副作用而非白血病进展。因此,难治和复发是导致AML治疗失败的最根本的原因,也是目前治疗白血病的一个难题。Eps8 (EGF receptor pathway substrate NO.8)为表皮生长因子受体通路底物8,1993年由Fazioli等人于鼠科纤维母细胞NIH3T3所发现并提出此概念。Eps8广泛分布在不同形态的细胞中,包括间质细胞、上皮细胞,亦有少量存在于造血细胞中。Eps8主要分布于细胞质、核膜及细胞膜周围,是一个接受器型的酪氨酸激酶受体(RTKs),它本身除了受EGFR所磷酸化外,还受其他RTKs磷酸化而激活。Eps8与EGFR结合后,使得细胞的DNA合成和癌化能力增加,此外Eps8也是一个骨架蛋白,Eps8与F-actin及Cortractin结合使细胞骨架重组,细胞膜发生皱襞,促进细胞迁移。Eps8的结构包括磷酸化酪氨酸结合区(phosphotyrosine binding protein, PTB), SH3区和SAM-PNT区(sterile alpha-pointed)。近年来Eps8在实体瘤中的研究表明,Eps8在实体肿瘤中异常高表达,并通过各种调节机制促使肿瘤细胞的迅速生长和迁移,与肿瘤的恶性程度相关,是肿瘤预后不良的指标之一。例如：Eps8通过提高粘附分子(FAK)的表达诱导结肠癌细胞的浸润生长和迁移；Eps8在口腔鳞癌高表达并通过调节Rac1的活性提高癌细胞的侵袭和迁移的能力；Eps8 mRNA在胰腺腺管癌表达显著升高,并与肿瘤转移正相关,Eps8在维持细胞的骨架结构和细胞间的连接起着重要作用,但是,Eps8过度表达则促使细胞恶性转化；Eps8通过降低宫颈癌细胞对化疗药物的敏感性从而影响宫颈癌的预后；光辉霉素(Mithramycin MTM)通过降低Eps8的表达来降低人类上皮癌细胞的增殖和迁移。然而,Eps8在恶性血液肿瘤中的表达如何,国内外尚未见报道。因此,探索Eps8在恶性血液细胞株及恶性血液病中表达情况及研究Eps8在恶性血液细胞株中表达意义显得十分必要。目的以Hela细胞为阳性对照,利用普通RT-PCR检测7种恶性血液肿瘤细胞株He1a、KG1a、K562、HL-60、ARH-77、8226、Raji、Jurkat中Eps8 mRNA的表达情况；构建实时荧光定量PCR检测Eps8基因的方法,进一步利用RQ-PCR (Relative Quantification-PCR)和免疫印迹法(Western Blot)检测7种恶性血液肿瘤细胞株中Eps8表达情况及分析其意义；药物干扰KGla细胞,台盼蓝拒染色法检测药物对KGla细胞的杀伤抑制率,RQ-PCR和Western Blot法检测药物作用浓度及药物作用时间对Eps8表达情况的影响。方法培养He1a、KG1a、K562、HL-60、ARH-77、8226、Raji、Jurkat细胞,利用TRIzol法提取细胞总RNA。选用β-actin作为内参,NCBI里查找eps8基因,参照参考文献及引物设计原则,选定目的基因引物序列,利用普通RT-PCR检测Eps8表达情况,Hela细胞作为阳性对照组,检测健康供着外周血单个核细胞(PBMNCs)及KG1a、K562、HL-60、ARH-77、8226、Raji、Jurkat细胞Eps8表达情况。建立RQ-PCR方法,随机选取样品cDNA模板按10010-1 10-210-3104浓度梯度稀释,用SYBR Green法检验目的基因与内参基因扩增效率,对稀释梯度倍数的log值与ΔCT (ΔCT=CTEps8-CTGAPDH)作直线关系图,斜率小于0.1即可认为两基因扩增效率一致。利用SYBR GreenⅠRQ-PCR法及Western Blot法检测各细胞株Eps8表达差异。利用PE荧光探针法标记KG1a、K562细胞,流式细胞仪检测CD34阳性细胞的表达率。选用蒽环类抗肿瘤药物柔红霉素(DNR)作用KG1a细胞株,台盼蓝拒染法检测DNR对KG1a细胞的杀伤抑制率；(1)以未加药物组为对照组,测DNR浓度分别为0.05μg/ml、0.1μg/ml、0.2μg/ml、0.4μg/ml、0.8μg/ml24小时对KG1a的杀伤抑制率,并利用RQ-PCR和Western Blot检测对照组和0.05μg/ml、0.1μg/ml、0.4μg/ml组的Eps8表达情况。(2)以未加药物组为对照组,实验组药物浓度分别为0.1μg/ml、0.2μg/ml、0.4μg/ml,加药后24h、48h、72h测得DNR对KG1a的杀伤抑制率,并利用RQ-PCR法和Western Blot法检测0.1μg/ml、0.4μg/ml组的Eps8表达情况；荧光定量PCR数据应用2-△△CT进行处理,计算各样本平均CT值、ACT值(ΔCt=CtEps8-CtGAPDH)、2-ΔΔCT值(△△Ct=△Ct目的样本一△Ct内参样本),2-ΔΔCT值用于表示目的值相对于参照值的倍数,Western Blot蛋白条带采用ImageJ软件分析,相对蛋白表达量为Eps8光密度值/β-actin光密度值(ODEps8/ODβ-actin)。统计学分析,采用SPSS13.0统计软件进行数据处理,计量数据以均数±标准差x±s)表示。所用统计方法单因素方法分析(One-Way ANOVA),方差齐时,组间多重比较采用LSD方法；方差不齐时,采用Welch检验,组间多重比较采用Dunnett T3检验；析因设计的方差分析,组间多重比较采用SNK方法；P<0.05表示差异有统计学意义。结果总RNA抽样经1.5%琼脂糖凝胶电泳鉴定,可见28S和18S和5S明显的3条条带,证明抽提的RNA质量完好,无DNA污染。RT-PCR扩增后终产物经1.5%琼脂糖凝胶电泳,在紫外线下观察,可看到与目的片段大小一致的165bp(eps8)、及510bp(β-actin)的电泳条带。两基因扩增效率的判断,结果所得直线y=-0.065x+1.743斜率绝对值为0.065(斜率绝对值<0.1),说明两基因的扩增效率基本相似,实验结果可以用2-△ACT法相对定量分析。即RQ-PCR检验方法成立。各细胞间Eps8 mRNA△CT值总体均数间有显著性差异(F=2082.917,P<0.001),KG1a为BMMNCs的3177.48倍(P<0.001),ARH-77为BMMNCs的377.40倍(P=0.002),8226为BMMNCs的1/10(P=0.025),Raji为BMMNCs的3/100(P=0.001)。其余3种恶性血液细胞株与BMMNCs差异无统计学意义(K562,Jurkat,HL-60)(P=0.831,P=0.960,P=0.969)。同时Western Blot检验的结果示KG1a、ARH-77细胞株中Eps8阳性表达,其余细胞株及BMMNCs组未见阳性条带。实验重复5次,结果一致。KG1a、K562细胞CD34阳性表达率分别为(98.3±1.5)%、(1.3±0.6)%。DNR浓度0.05μg/ml、0.1μg/ml、0.4μg/ml、0.8μg/ml24小时对KG1a的抑制率分别为(12.36±4.07)%,(26.83±3.68)%,(60.38±3.57)%,(81.44±3.38)%。各浓度对KG1a 24h的杀伤抑制率有显著意义(F=317.11,P<0.001),且随着药物浓度的增加杀伤率提高(P<0.05)。不同药物浓度对Eps8 mRNA的表达组间差异有显著性意义(F=1069.725,P<0.001),药物浓度为0.05μg/ml、0.1μg/ml、0.4μg/ml组Eps8 mRNA分别为对照组的1/5(P=0.023)、1/20(P=0.011)、1/100(P=0.004),且0.1μg/ml组较0.05μg/ml组Eps8表达下降(P=0.005) 0.4μg/ml较0.05μg/ml组表达下降(P<0.001)。不同药物浓度组间,Eps8蛋白表达有显著性差异(F=99.802,P<0.001),以对照组相比,浓度为0.05g/ml、0.1g/ml、0.4μg/ml组Eps8蛋白表达下调(P<0.001, P<0.001, P<0.001),0.05μg/ml组较0.1μg/ml组Eps8表达下调(P=0.044),0.4μg/ml组较0.1μg/ml组Eps8表达明显下调(P<0.001)。随着药物浓度的增加,DNR对KG1a 24h的杀伤抑制作用增加,Eps8 mRNA和蛋白表达相应降低。药物浓度为0.1μg/ml、0.2μg/ml、0.4μg/ml对KG1a 24h、48h、72h的杀伤抑制率分别(26.91±2.70)%、(45.65±0.54)%、(74.79±1.37)%、(43.15±1.78)%、(61.29±1.97)%、(85.70±1.45)%、(61.28±1.35)%、(88.59±0.76)%、(95.76±0.95)%。DNR作用浓度和作用时间对KG1a细胞的杀伤抑制率、Eps8 mRNA和Eps8蛋白表达均存在交互效应(F=42.33, P<0.001; F=25.916, P<0.001; F=12.786, P=0.000)。且在同一浓度作用下随着作用时间的延长Eps8表达下降(P<0.05),同一作用时间随着药物浓度的增加Eps8表达也下降(P<0.05)。即随着药物浓度的增加和作用时间的延长,DNR对KG1a的杀伤抑制作用增加,Eps8表达下降。结论建立实时荧光定量PCR检测Eps8 mRNA表达方法。2-△△CT分析方法能较好地应用于基因表达相对差异的比较。KG1a及ARH-77细胞株Eps8 mRNA显著高于BMMNCs,8226及Raji细胞株Eps8 mRNA较BMMNCs低表达,其余选用细胞株与BMMNCs的Eps8mRNA表达差异无统计学意义。Eps8蛋白在KG1a及ARH-77阳性表达,其余细胞株与PBMNCs均未检测阳性表达。Eps8在早期造血干细胞来源的细胞株中高表达,推断其对部分恶性血液肿瘤细胞的早期分化起一定作用。DNR作用KG1a细胞发现随药物浓度的增加和作用时间的延长Eps8表达下降,推断DNR对KG1a的杀伤机制之一可能是通过降低Eps8的表达来实现的。更多还原

【Abstract】 BackgroundHematological malignancy including leukemia, malignant lymphoma and multiple myeloma, the incidence of the former two types cancer is currently ranked in the top ten human malignant tumors.Where the mortality rate of leukemia in the malignancy, accounting for 6 (male) and 8 (female); children and adults under the age of 35, the No.1 ranking. And the incidence increased year by year, it is a serious threat to human health and life, leukemia include Acute leukemia (AL) and chronic leukemia (CL), in china the incidence of AL is more than CL (about 5.5:1), of which the most is acute myeloid leukemia AML (1.62/100000)in china. When human get leukemia,the acute leukemia blast cells and immature abnormal cells (leukemia cells) will be full of the bone marrow and they proliferate rapidly, and they inhibit normal hematopoiesis and extensively infiltrative the liver, spleen, lymph nodes and other organs. Manifested as anemia, bleeding, infection and signs of infiltration.In recent years, with the strengthening of supportive care, the application of multi-drug joint programs, high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) in the promotion, AML patients with chemotherapy, complete remission rate (CR) up to 75%, but there are still 20%～30% AML failed to obtain through formal remission after chemotherapy, known as refractory. Even though about 50% patients relapse after CR, if relapse, patients with poor prognosis. It is now generally considered that the basic reasons of leukemia refractory and relapsed are:①the first reason is leukemia cells resist apoptosis pathway;②the second reason is leukemia cells with high expression of multidrug resistance;③The third reason is most leukemia cells stay in the Go quiescent state and they are not sensitive to chemotherapy.Due to leukemia cells are not sensitive and resistant to drugs, in clinical treatment the use of high-dose chemotherapy, a considerable part of the patients died of toxic effects of chemotherapy rather than the progress of leukemia.Therefore, the most failure fundamental reasons for the treatment AML are refractory and relapsed, It is aslo a problem for treatment AML.Eps8 (EGF receptor pathway substrate NO.8) epidermal growth factor receptor pathway substrate 8, Fazioli et al found it expreesed in NIH3T3 murine fibroblasts and proposed the concept in 1993. Eps8 widely distribute in different forms of cells, including stromal cells, epithelial cells, there is also a small amount of hematopoietic stem cells. And main branch in the cytoplasm, nuclear membrane and the membrane surrounding, it is a receptor-type tyrosine kinase receptor (RTKs), in addition to it is phosphorylated by EGFR completely apart, but also phosphorylated and activated by other RTKs. Eps8 with the EGFR binding, making cells to be the cancer, DNA synthesis and increased capabilities, in addition to Eps8 is a scaffold protein, Eps8 with F-actin and Cortractin combination cytoskeletal reorganization, membrane folds occur, and promote cell migration.The structure of Eps8 includes phosphotyrosine binding domain (phosphotyrosine binding protein, PTB), SH3 and the SAM-PNT zone area (sterile alpha-pointed).Many studies have shown that Eps8 is highly expressed in the solid tumors, and through various mechanisms to promote tumor cell migration and rapid growth, and progression of tumor metastasis,it is a bad prognostic indicator.For example, in colon cancer, Eps8 Facilitates celluar growth and motility of colon cancer cell by incressing the expression and activity of Focal Adhesion Kniase (FAK). Oral squamous cell carcinoma cells with high expression of Eps8,and by regulating the activity of Racl increased cell invasion and migration capacity. Pancreatic ductal carcinoma, the Eps8 mRNA expression was significantly increased and positively correlated with tumor metastasis, and Eps8 play an important role in maintaining cell cytoskeleton structure and cell-cell junctions. Besides, Eps8 is involved in but not essential,for cell migration, overexpression makes cells to malignant cell transformation.Eps8 decreases chemosensitivity and affects survival of cervical patients. Mithramycin (MTM) inhibits human epithelial carcinoma cell proliferation and migration involving downregulation of Eps8 expression.However, what it the Eps8 express in Hematologic Neoplasms cell lines? there are no people to study, therefore, It is very important to explore the Eps8 express in hematological malignant cell lines and hematological malignancy and its relationship with prognosis.ObjectionSelecting Hela cell as a positive control, using RT-PCR to detect Eps8 mRNA expression of seven kinds of hematological malignant cell lines about KGla、K562、HL-60、ARH-77、8226、Raji、Jurkat, and to determine the Eps8 mRNA expressions in hematological malignant cell lines.Builded real-time fluorescence quantitative PCR method for detect Eps8 genes, further used of RQ-PCR (Relative Quantification-PCR) and immunoblotting (Western Blot) to dectect the expression of Eps8 in the 7 kinds of hematological malignant cell lines.Drugs interfered KGla cell, using placenta Blue resist staining live cells to get the Inhibition rate of anti-KGla.RQ-PCR and Western Blot assayed with different concentrations of the drug effected the expression of Eps8 KGla cells.MethodsCulturing Hela、KG1a、K562、HL-60、ARH-77、8226、Raji、Jurkat cells, using TRIzol extracted total cellular RNA. searched for Eps8 gene in NCBI, designed primers, usedβ-actin as internal reference, used RT-PCR to detect the expression of Eps8 in the 7 kinds of cell lines, Hela cells as a positive control group, healthy human peripheral blood mononuclear cells (PBMNCs) as the control group.Established RQ-PCR method, selected randomly one cDNA template samples, diluted at 100 10-1 10-2 10-3 10-4 concentration gradient, using SYBER GreenⅠtested the objective gene and reference gene amplification efficiency, to make a straight line graph about the log value of dilution gradient andΔCT (ΔCT= CTEps8-CTGAPDH), the slope is less than 0.1 can be considered the two genes have the same amplification efficiency. Using SYBR Green I RQ-PCR and Western Blot to detected the Eps8 expression in the cell lines and normal bone marrow mononuclear cells (BMMNCs), and analysis the difference between them.Using fluorescent probe PE labeled KGla, K562 cells, flow cytometry tested the expression of CD34 positive cells.Used anthracycline antitumor drugs daunorubicin (DNR) to kill KG1a, (1)DNR concentration was 0.05μg/ml,0.1μg/ml,0.4μg/ml,0.8μg/ml, after 24 hours we used placenta Blue resist staining live cells to count the inhibition rate DNR to anti-KG1a.Using RQ-PCR and Western Blot assayed the Eps8 expressionof the 0.05μg/ml、0.1μg/ml、0.4μg/ml groups. (2)non-adding Drug group as control group, experimental group of drug concentrations were 0.1μg/ml,0.2μg/ml,0.4μg/ml, respectively, after dosing measured 24h,48h,72h to get the DNR inhibition rate of anti-KG1a and Using RQ-PCR and Western Blot assayed 0.1μg/ml,0.4μg/ml group Eps8 expression. Applicating 2-ΔΔCT to processe the quantitative PCR data, calculated of the average CT value of each sample,ΔCT values (ΔCt=CtEps8-CtGAPDH).Calculated of 2-ΔΔCT(ΔΔCt=ΔCt purpose of the sample -ΔCt internal reference sample), the purpose of the digital value used to represent a multiple of the value relative to the reference. Weatern Blot protein bands using ImageJ software analysised, protein expression was Eps8 optical density/β-actin optical density (ODEps8/ODβ-actin).Statistical analysis, applied SPSS13.0 statistical software to deal with the data, measurement data use Mean±deviation state(x±s). compared differences among the groups use One-Way-ANONA. when homogeneity, using LSD test to compare multiple between groups, Heterogeneity of variance, used Welch test,and used Dunnett T3 test to compare multiple between groups; P<0.05 indicated significant difference.ResultsSelected one sample of total RNA,by 1.5% agarose gel electrophoresis, showing 28s and 18s and 5s three roads with a clear, Proved the quality of the RNA, no DNA contamination the final RT-PCR products by 1.5% agarose gel electrophoresis, observed in ultraviolet light can be seen with the same fragment size of 165bp (Eps8), and 510bp (β-actin) of electrophoretic bands.Gene amplification efficiency of the two genes, the results from the slope of a straight line y=-0.065x+1.743,absolute value was 0.065 (slope of absolute value <0.1), indicated that the amplification efficiency of the two genes are similar.The results could be used 2-ΔΔCT relative quantitative analysis method. RQ-PCR tested method is established.There were significant differences in the cells population means (F=2082.917, P<0.001), KG1a is 3177.48-fold relative to BMMNCs (P<0.001), ARH-77 is 377.40-fold relative to BMMNCs (P=0.002),8226 is 1/10 relative to BMMNCs (P= 0.025), Raji is 3/100 relative to BMMNCs (P=0.001). The remaining three kinds of malignant blood cells relative to BMMNCs have no significant difference (K562, Jurkat, HL-60) (P=0.831, P=0.960, P=0.969). Western Blot tested results also show that the Eps8 expression in KG1a,and ARH-77 cell lines, and the other cells; and BMMNCs had no positive bands. Experiment was repeated 5 times independently and got the same results.The CD34 positive rates of KGla, K562 cells respectively were positive (98.3±1.5)%, (1.3±0.6)%.The concentrations of DNR respectively were 0.05μg/ml,0.1μg/ml,0.4μg/ml, 0.8μg/ml to kill KG1a after 24 hours, the inhibition rates were respectively (12.36±4.07)%, (26.83±3.68)%, (60.38±3.57)%, (81.44±3.38)%.there was significant difference that different concentrations of DNR on the inhibition of anti-KG1a (F= 317.11, P<0.001), and when the concentrations increased the inhibition of anti-KG1a increased (P<0.05). The difference of expression of Eps8 mRNA between the groups was significant by different concentration drug (F=1069.725, P<0.001), Comparison with the no drug group (0μg/ml), the role of drug concentration 0.05μg/ml group Eps8 mRNA expression is about 1/5 (P=0.023); drug concentration O.1μg/ml group expression is about 1/20 (P= 0.011); the role of drug concentration was 0.4μg/ml group expression is about 1/100 (P=0.004).Western Blot results showed that different concentration groups, Eps8 expression was significantly different (F= 99.802, P<0.001), compared with the control group, the Eps8 protein downregulated in the concentration of 0.05g/ml, 0.1g/ml,0.4μg/ml group (P<0.001, P<0.001, P<0.001),0.05μg/ml group was lower than 0.1μg/ml group (P=0.044). 0.4μg/ml was group lower than 0.1μg/ml group (P<0.001). and when the concentration was 0.4μg/ml Eps8 expression was significantly decreased. In short, with the drug concentration increased, the inhibition of anti-KG1a 24h after by DNR 24h raised, The expression of Eps8 mRNA and protein were corresponding lower. The concentrations of DNR respectively were 0.1μg/ml、0.2μg/ml、0.4μg/ml to kill KG1a after 24h、48h、72h the inhibition rate of anti-KGla respectively were (26.91±2.70)%, (45.65±0.54)%, (74.79±1.37)%, (43.15±1.78)%, (61.29±1.97)%, (85.70±1.45)%, (61.28±1.35)%, (88.59±0.76)%, (95.76±0.95)%. Different DNR concentration and different time for the inhibition rate of anti-KG1a, the expression of Eps8 mRNA and protein had exists interaction (F=42.33, P<0.001; F=25.916, P<0.001; F=12.768, P=0.000). Under the same concentration Eps8 decreased with the time (P<0.05), the same reaction time Eps8 decreased with the drug concentration (P<0.05). In short, with the DNR concentration and the time, the inhibition rate of anti-KG1a decreased, Eps8 expression decreased.ConclusionEstablished the method of SYBR GreenⅠRQ-PCR to assay the Eps8 mRNA expression.2-ΔΔCT analytical method to analyze gene expression in different cells is a very good relative method.Compared with normal BMMNCs, Eps8 mRNA expression in KG1a and ARH-77 were highly expressed,8226 and Raji cell lines were low expression, the difference about Eps8 mRNA expression between the others selected cell lines was not statistically significant.Eps8 protein expressed in KG1a and ARH-77, and the other cell lines showed no expression and BMMNCs, Eps8 were high expression in same hematopoietic stem cells cell lines, Eps8 plays a role in early differentiation in some malignant tumor cells.With the drug concentration increased and treatment time prolonged, the expression of Eps8 decreased, Maybe DNR inhibits KG1a cell proliferation involving downregulation of Eps8 expression.更多还原