本文選題：非霍奇金B(yǎng)細(xì)胞淋巴瘤 + B淋巴細(xì)胞激活因子��； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：第一部分：B-NHL細(xì)胞中BAFF及其特異性受體BAFF-R的表達(dá)和定位的研究 研究目的： 明確B-NHL細(xì)胞系Raji、Jeko-1、Pfeiffer細(xì)胞中BAFF和BAFF-R的表達(dá)和定位。 研究方法： 采用RT-PCR和Western blot技術(shù)分別檢測B-NHL細(xì)胞系Raji、Jeko-1、Pfeiffer細(xì)胞中BAFF及BAFF-R在mRNA和蛋白水平的表達(dá)；細(xì)胞免疫熒光化學(xué)法和激光共聚焦技術(shù)檢測BAFF、BAFF-R在Jeko-1、Pfeiffer細(xì)胞中的定位；流式細(xì)胞術(shù)檢測B-NHL細(xì)胞系Raji、Jeko-1、Pfeiffer細(xì)胞表面BAFF及BAFF-R的表達(dá)情況。 研究結(jié)果： RT-PCR、Western blot和流式細(xì)胞術(shù)檢測結(jié)果發(fā)現(xiàn)B-NHL細(xì)胞系Raji、Jeko-1、 Pfeiffer細(xì)胞在mRNA和蛋白水平均有不同程度表達(dá)BAFF和BAFF-R, Jeko-1和Pfeiffer細(xì)胞的表達(dá)量相對較高,Raji細(xì)胞的表達(dá)量相對低。免疫熒光定位檢測證實(shí)BAFF和BAFF-R表達(dá)在細(xì)胞膜上。 結(jié)論： B-NHL細(xì)胞膜上表達(dá)BAFF和BAFF-R,其中Jeko-1和Pfeiffer細(xì)胞的表達(dá)量高于Raji細(xì)胞。 第二部分：BAFF體外促進(jìn)B-NHL細(xì)胞增殖及其相關(guān)機(jī)制的研究 研究目的： 探索rhBAFF(重組人BAFF)對B-NHL細(xì)胞增殖的影響及其作用機(jī)制。 實(shí)驗(yàn)方法： MTT法檢測Raji、Jeko-1-Pfeiffer細(xì)胞經(jīng)rhBAFF刺激前后的細(xì)胞活力；Western blot技術(shù)檢測rhBAFF作用后Jeko-1和Pfeiffer細(xì)胞中NF-κB、PI3K/AKT、ERK等信號通路相關(guān)分子和BCL-2家族凋亡相關(guān)蛋白表達(dá)的變化。 研究結(jié)果： MTT結(jié)果顯示Raji、Jeko-1、Pfeiffier細(xì)胞經(jīng)500ng/ml rhBAFF作用48h后細(xì)胞存活率分別為125.28±10.86%,170.89±15.73%,140.77±7.50%,分別是對照組的1.25、1.71、1.41倍(各組均P0.05)；同時(shí)也可以看到不同濃度(3.90625、7.8125、15.625、31.25、62.5、125、250、500ng/ml) rhBAFF作用Jeko-1細(xì)胞48h后,細(xì)胞存活率隨BAFF濃度增高而升高(分別為113.53±4.17%,137.16±4.79%,145.34±7.38%,150.90±7.67%,159.67±6.32%,160.31±9.12%,163.20±3.61%,170.89±15.73%),呈現(xiàn)濃度依賴的細(xì)胞增殖。 Western blot結(jié)果顯示經(jīng)不同濃度rhBAFF處理Jeko-1、Pfeiffer細(xì)胞后P52, p-IκBα、p-AKT、p-ERK水平和核蛋白P65均呈上調(diào)趨勢,IκBα水平呈下調(diào)趨勢；抗凋亡蛋白BCL-2、BCL-XL、MCL-1水平呈現(xiàn)上調(diào)趨勢,凋亡前體蛋白BAX水平則呈現(xiàn)下調(diào)趨勢。 結(jié)論： BAFF對Raji、Jeko-1、Pfeiffer細(xì)胞具有明顯的促增殖作用,其機(jī)制與活化NF-κB、PI3K/AKT、ERK信號通路和抗凋亡蛋白增加有關(guān)。 第三部分：B-NHL患者外周血BAFF和APRIL水平及其臨床意義 實(shí)驗(yàn)?zāi)康模?明確B-NHL患者外周血血清中BAFF和APRIL表達(dá)水平及相互關(guān)系以及與臨床特征和病理組織中Ki67表達(dá)量之間的關(guān)系,明確患者血清中BAFF表達(dá)水平和IL-10表達(dá)水平的關(guān)系。 研究方法： 收集77例2014.1-2014.12間來自浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院的B-NHL患者和65例健康體檢者的外周血血清,收集患者臨床資料如患者年齡、性別、疾病分期,B癥狀,淋巴瘤結(jié)外累及數(shù)量,血清LDH, B2MG表達(dá)水平,ECOG評分,IPI疾病分層和淋巴結(jié)病理組織中淋巴瘤細(xì)胞CD分子的表達(dá)和相關(guān)生物分子的表達(dá)；ELISA法檢測B-NHL患者及健康志愿者外周血血清中BAFF、APRIL、IL-10的水平；采用Mann-Whitney U檢驗(yàn)分析BAFF、APRIL、IL-10在初發(fā)、復(fù)發(fā)/難治性B-NHL和健康志愿者間水平的差異,采用Spearman相關(guān)性分析和卡方檢驗(yàn)分析BAFF、APRIL與臨床特征的關(guān)系以及BAFF和APRIL、IL-10水平的相關(guān)性。研究結(jié)果： 48例初發(fā)B-NHL,患者中BAFF平均水平為1739.91±1013.07pg/ml,顯著高于健康對照組的969.25±346.30pg/ml(P0.05),29例復(fù)發(fā)／難治性B-NHL患者的BAFF水平平均值為3526.01±2080.98pg/ml,明顯高于初發(fā)患者和健康對照組(P0.05)。初發(fā)患者BAFF水平分層與臨床特征的卡方檢驗(yàn)結(jié)果顯示高BAFF水平組患者中高ECOG評分、高IPI評分、高LDH水平人數(shù)明顯多于低BAFF水平組(P0.05). Spearman相關(guān)性分析顯示BAFF水平與Ann Arbor分期、血清B2MG水平、ECOG評分和IPI評分呈正相關(guān)性,相關(guān)系數(shù)分別為0.323、0.381、0.390和0.442(P0.05)。 外周血血清中IL-10水平在34例初發(fā)B-NHL患者、25例復(fù)發(fā)/難治性患者和32例健康對照者中的中位值分別為20.19pg/ml、6.89pg/ml和0.OOpg/ml,患者組與對照組間具有顯著性差異(P0.05),BAFF和IL-10分層后卡方檢驗(yàn)發(fā)現(xiàn),無論初發(fā)還是復(fù)發(fā)患者,高IL-10水平組患者中BAFF水平增高人數(shù)明顯多于低IL-10水平組(P0.05),相關(guān)性分析進(jìn)一步證實(shí)了二者的正相關(guān)性,在初發(fā)患者和復(fù)發(fā)/難治性患者中相關(guān)系數(shù)分別為0.363和0.620,P值分別為0.030和0.001。 39例初發(fā)患者外周血血清中APRIL平均水平為3.44±2.57ng/ml,顯著高于對照組1.33±0.86ng/ml, P0.05;而20例復(fù)發(fā)/難治性患者APRIL水平平均值為3.45±2.98ng/ml,與初發(fā)患者無明顯差異(P0.05)。相關(guān)性分析也未發(fā)現(xiàn)APRIL水平與臨床特征和BAFF水平有相關(guān)性。 結(jié)論： B-NHL患者外周血血清中BAFF水平明顯升高,尤其在復(fù)發(fā)/難治性患者中更高,BAFF水平與患者ECOG評分、IPI評分、LDH水平、Ann Arbor分期、B2MG、 IL-10呈正相關(guān),提示BAFF水平越高,疾病的嚴(yán)重程度越高；而APRIL水平雖有升高,并未顯示其在疾病程度評價(jià)中的作用。
[Abstract]:Part one: expression and localization of BAFF and its specific receptor BAFF-R in B-NHL cells
The purpose of the study is:
The expression and location of BAFF and BAFF-R in B-NHL cell line Raji, Jeko-1 and Pfeiffer cells were identified.
Research methods:
RT-PCR and Western blot were used to detect the expression of BAFF and BAFF-R at mRNA and protein levels in Raji, Jeko-1, Pfeiffer cells of B-NHL cell lines; cell immunofluorescence chemistry and laser confocal technique were used to detect BAFF, BAFF-R was located in the cells. The expression of BAFF and BAFF-R on the surface of the cell.
The results of the study:
The results of RT-PCR, Western blot and flow cytometry showed that the B-NHL cell line Raji, Jeko-1, Pfeiffer cells expressed BAFF and BAFF-R in varying degrees in mRNA and protein levels, and the expression of Jeko-1 and Pfeiffer cells was relatively high, and the expression of the cells was relatively low. Up.
Conclusion:
BAFF and BAFF-R were expressed on the B-NHL cell membrane, and the expression levels of Jeko-1 and Pfeiffer cells were higher than those of Raji cells.
The second part: BAFF promotes B-NHL cell proliferation and its related mechanisms in vitro.
The purpose of the study is:
Objective to explore the effect of rhBAFF (recombinant human BAFF) on the proliferation of B-NHL cells and its mechanism.
Experimental methods:
MTT assay was used to detect the cell viability of Raji, Jeko-1-Pfeiffer cells before and after rhBAFF stimulation, and Western blot technique was used to detect the changes in the expressions of NF- kappa B, PI3K/AKT, and apoptosis related proteins in Jeko-1 and Pfeiffer cells after rhBAFF action.
The results of the study:
MTT results showed that the cell survival rate of Raji, Jeko-1, Pfeiffier cells after 48h was 125.28 + 10.86%, 170.89 + 15.73%, 140.77 + 7.50%, respectively, the 1.25,1.71,1.41 times of the control group (P0.05), and the rhBAFF action Jeko- (3.90625,7.8125,15.625,31.25,62.5125250500ng/ml) could also be seen. After 1 cell 48h, the cell survival rate increased with the increase of BAFF concentration (113.53 + 4.17%, 137.16 + 4.79%, 145.34 + 7.38%, 150.90 + 7.67%, 159.67 +, 160.31 + 9.12%, 160.31 + 4.79%), showing a concentration dependent cell proliferation.
Western blot showed that Jeko-1 was treated with different concentrations of rhBAFF, P52, p-I kappa B a, p-AKT, p-ERK level and nuclear protein P65 were up-regulated after Pfeiffer cells, and the level of I kappa was down downward trend, and the anti apoptotic protein showed an upward trend, while the level of apoptotic precursor protein showed a downward trend.
Conclusion:
BAFF has an obvious proliferation promoting effect on Raji, Jeko-1 and Pfeiffer cells, and its mechanism is related to the activation of NF- kappa B, PI3K/AKT, ERK signal pathway and anti apoptotic protein.
The third part: the level of BAFF and APRIL in peripheral blood of B-NHL patients and its clinical significance.
Objective:
The relationship between the expression level of BAFF and APRIL in the serum of B-NHL patients and the relationship with the clinical features and the expression of Ki67 in the pathological tissue were identified, and the relationship between the level of BAFF expression and the level of IL-10 expression in the serum of the patients was clearly defined.
Research methods:
The peripheral blood serum of 77 B-NHL patients and 65 health examiners from the Second Affiliated Hospital of Zhejiang University medical college were collected, and the patients' clinical data such as age, sex, disease staging, B symptoms, the number of B, the level of LDH, B2MG expression, ECOG score, IPI disease stratification and lymph node pathology were collected. The expression of CD molecules of lymphoma cells in the tissue and the expression of related biomolecules; the level of BAFF, APRIL and IL-10 in the peripheral blood serum of B-NHL patients and healthy volunteers by ELISA method; and Mann-Whitney U test for the analysis of BAFF, APRIL, IL-10 in primary, recurrent / refractory B-NHL and healthy volunteers. Sex analysis and chi square test were used to analyze the relationship between BAFF, APRIL and clinical characteristics, as well as the correlation between BAFF and APRIL and IL-10 levels.
48 cases of primary B-NHL, the average level of BAFF in the patients was 1739.91 + 1013.07pg/ml, significantly higher than that in the healthy control group (969.25 + 346.30pg/ml). The average value of BAFF in 29 cases of recurrent / refractory B-NHL patients was 3526.01 + 2080.98pg/ml, obviously higher than that of the primary and health care group (P0.05). The level of BAFF level and the clinical characteristics of the primary patients were significantly higher than those in the healthy control group. The results of chi square test showed that the high ECOG score, high IPI score and high LDH level in the high BAFF level group were significantly more than those of the low BAFF level group (P0.05). The Spearman correlation analysis showed that the level of BAFF and Ann Arbor, the B2MG level of the serum, the ECOG score and the score were 0.442, respectively. ).
The level of IL-10 in peripheral blood serum was in 34 primary B-NHL patients, 25 cases of recurrent / refractory patients and 32 healthy controls were 20.19pg/ml, 6.89pg/ml and 0.OOpg/ml respectively. There was significant difference between the patient group and the control group (P0.05). The chi square test after BAFF and IL-10 stratification found that high IL-10 water was found in both initial and recurrent patients. In the patients in the flat group, the number of BAFF increased significantly more than the low IL-10 level group (P0.05). The correlation analysis further confirmed the positive correlation between the two, and the correlation coefficient was 0.363 and 0.620 in the primary and recurrent / refractory patients, and the P value was 0.030 and 0.001., respectively.
The average APRIL level in peripheral blood serum of 39 primary patients was 3.44 + 2.57ng/ml, which was significantly higher than that in the control group, 1.33 + 0.86ng/ml, P0.05, while the average APRIL level of 20 recurrent / refractory patients was 3.45 + 2.98ng/ml, and there was no significant difference from the primary patients (P0.05). The correlation analysis also did not find that the level of APRIL was associated with the clinical features and BAFF levels. Sex.
Conclusion:
The level of BAFF in peripheral blood serum of B-NHL patients was significantly higher, especially in recurrent / refractory patients. The level of BAFF was positively correlated with patients' ECOG score, IPI score, LDH level, Ann Arbor staging, B2MG, IL-10, suggesting that the higher the BAFF level, the higher the severity of the disease; although APRIL level increased, it did not show its evaluation of the degree of disease. The role of it.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R733.1

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相關(guān)期刊論文 前1條

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本文編號：2115987