B淋巴细胞激活因子BAFF及其特异性受体BAFF-R在非霍奇金B细胞淋巴瘤中的作用、作用机制和临床意义的研究

发布时间：2018-07-11 17:12

本文选题：非霍奇金B细胞淋巴瘤 + B淋巴细胞激活因子　；参考：《浙江大学》2015年博士论文

【摘要】：第一部分：B-NHL细胞中BAFF及其特异性受体BAFF-R的表达和定位的研究研究目的：明确B-NHL细胞系Raji、Jeko-1、Pfeiffer细胞中BAFF和BAFF-R的表达和定位。研究方法：采用RT-PCR和Western blot技术分别检测B-NHL细胞系Raji、Jeko-1、Pfeiffer细胞中BAFF及BAFF-R在mRNA和蛋白水平的表达；细胞免疫荧光化学法和激光共聚焦技术检测BAFF、BAFF-R在Jeko-1、Pfeiffer细胞中的定位；流式细胞术检测B-NHL细胞系Raji、Jeko-1、Pfeiffer细胞表面BAFF及BAFF-R的表达情况。研究结果： RT-PCR、Western blot和流式细胞术检测结果发现B-NHL细胞系Raji、Jeko-1、 Pfeiffer细胞在mRNA和蛋白水平均有不同程度表达BAFF和BAFF-R, Jeko-1和Pfeiffer细胞的表达量相对较高,Raji细胞的表达量相对低。免疫荧光定位检测证实BAFF和BAFF-R表达在细胞膜上。结论： B-NHL细胞膜上表达BAFF和BAFF-R,其中Jeko-1和Pfeiffer细胞的表达量高于Raji细胞。第二部分：BAFF体外促进B-NHL细胞增殖及其相关机制的研究研究目的：探索rhBAFF(重组人BAFF)对B-NHL细胞增殖的影响及其作用机制。实验方法： MTT法检测Raji、Jeko-1-Pfeiffer细胞经rhBAFF刺激前后的细胞活力；Western blot技术检测rhBAFF作用后Jeko-1和Pfeiffer细胞中NF-κB、PI3K/AKT、ERK等信号通路相关分子和BCL-2家族凋亡相关蛋白表达的变化。研究结果： MTT结果显示Raji、Jeko-1、Pfeiffier细胞经500ng/ml rhBAFF作用48h后细胞存活率分别为125.28±10.86%,170.89±15.73%,140.77±7.50%,分别是对照组的1.25、1.71、1.41倍(各组均P0.05)；同时也可以看到不同浓度(3.90625、7.8125、15.625、31.25、62.5、125、250、500ng/ml) rhBAFF作用Jeko-1细胞48h后,细胞存活率随BAFF浓度增高而升高(分别为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%),呈现浓度依赖的细胞增殖。 Western blot结果显示经不同浓度rhBAFF处理Jeko-1、Pfeiffer细胞后P52, p-IκBα、p-AKT、p-ERK水平和核蛋白P65均呈上调趋势,IκBα水平呈下调趋势；抗凋亡蛋白BCL-2、BCL-XL、MCL-1水平呈现上调趋势,凋亡前体蛋白BAX水平则呈现下调趋势。结论： BAFF对Raji、Jeko-1、Pfeiffer细胞具有明显的促增殖作用,其机制与活化NF-κB、PI3K/AKT、ERK信号通路和抗凋亡蛋白增加有关。第三部分：B-NHL患者外周血BAFF和APRIL水平及其临床意义实验目的：明确B-NHL患者外周血血清中BAFF和APRIL表达水平及相互关系以及与临床特征和病理组织中Ki67表达量之间的关系,明确患者血清中BAFF表达水平和IL-10表达水平的关系。研究方法：收集77例2014.1-2014.12间来自浙江大学医学院附属第二医院的B-NHL患者和65例健康体检者的外周血血清,收集患者临床资料如患者年龄、性别、疾病分期,B症状,淋巴瘤结外累及数量,血清LDH, B2MG表达水平,ECOG评分,IPI疾病分层和淋巴结病理组织中淋巴瘤细胞CD分子的表达和相关生物分子的表达；ELISA法检测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检验分析BAFF、APRIL、IL-10在初发、复发/难治性B-NHL和健康志愿者间水平的差异,采用Spearman相关性分析和卡方检验分析BAFF、APRIL与临床特征的关系以及BAFF和APRIL、IL-10水平的相关性。研究结果： 48例初发B-NHL,患者中BAFF平均水平为1739.91±1013.07pg/ml,显著高于健康对照组的969.25±346.30pg/ml(P0.05),29例复发／难治性B-NHL患者的BAFF水平平均值为3526.01±2080.98pg/ml,明显高于初发患者和健康对照组(P0.05)。初发患者BAFF水平分层与临床特征的卡方检验结果显示高BAFF水平组患者中高ECOG评分、高IPI评分、高LDH水平人数明显多于低BAFF水平组(P0.05). Spearman相关性分析显示BAFF水平与Ann Arbor分期、血清B2MG水平、ECOG评分和IPI评分呈正相关性,相关系数分别为0.323、0.381、0.390和0.442(P0.05)。外周血血清中IL-10水平在34例初发B-NHL患者、25例复发/难治性患者和32例健康对照者中的中位值分别为20.19pg/ml、6.89pg/ml和0.OOpg/ml,患者组与对照组间具有显著性差异(P0.05),BAFF和IL-10分层后卡方检验发现,无论初发还是复发患者,高IL-10水平组患者中BAFF水平增高人数明显多于低IL-10水平组(P0.05),相关性分析进一步证实了二者的正相关性,在初发患者和复发/难治性患者中相关系数分别为0.363和0.620,P值分别为0.030和0.001。 39例初发患者外周血血清中APRIL平均水平为3.44±2.57ng/ml,显著高于对照组1.33±0.86ng/ml, P0.05;而20例复发/难治性患者APRIL水平平均值为3.45±2.98ng/ml,与初发患者无明显差异(P0.05)。相关性分析也未发现APRIL水平与临床特征和BAFF水平有相关性。结论： B-NHL患者外周血血清中BAFF水平明显升高,尤其在复发/难治性患者中更高,BAFF水平与患者ECOG评分、IPI评分、LDH水平、Ann Arbor分期、B2MG、 IL-10呈正相关,提示BAFF水平越高,疾病的严重程度越高；而APRIL水平虽有升高,并未显示其在疾病程度评价中的作用。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.1

【参考文献】