原发性眼眶MALT淋巴瘤患者VCP表达与患者预后的相关性及机制的研究
发布时间：2018-08-27 11:53
【摘要】：目的：探讨含缬酪肽蛋白(valosin-containing protein, VCP)表达水平与原发性眼眶MALT淋巴瘤患者预后的相关性,及VCP对人淋巴瘤细胞凋亡及增殖能力的影响。 方法：1.58例原发性眼眶MALT淋巴瘤患者眶内淋巴瘤组织石蜡标本,采用免疫组化法检测VCP表达水平,并对VCP表达水平与患者复发情况及生存率的相关性进行统计学分析,用X2检验及Fisher精确检验来分析VCP蛋白表达与各临床因素之间的关系；Kaplan-Meier法计算复发率、生存率,并绘制生存曲线；log-rank法计算组间差异,P0.05为差异具有统计学意义； 2.根据免疫组化结果即VCP蛋白表达水平,选取10例患者眶内肿瘤石蜡组织(5例×2组)抽提总RNA,并应用实时荧光定量PCR(RT-qPCR)定量比较2组VCP基因表达水平的差异； 3.免疫细胞化学染色法检测人淋巴瘤细胞系Raji细胞及Daudi细胞VCP蛋白的表达。应用特异、高效并操作方便的RNAi技术,以VCP作为基因靶点,对Raji细胞建立有效的RNA干扰方法。利用脂质体介导基因si-VCP转染Raji细胞,RT-qPCR及Western blot分别从1nRNA水平及蛋白水平评估Raji细胞VCP基因沉默效果； 4.流式细胞术检测VCP基因沉默的人淋巴瘤Raji细胞凋亡率的变化； 5.MTS法检测VCP基因沉默对Raji细胞增殖能力的影响。 结果：1.58例眼眶MALT淋巴瘤患者中13例(22.4%)患者出现肿瘤复发。复发时间为10-56个月,其平均值为39.5个月。8例患者(13.8%)死于肿瘤,4例患者(6.9%)死于与肿瘤无关的其他原因。5年无瘤生存率及总生存率分别是70.7%、79.3%。5年无瘤生存时间为10-60个月,其平均值为54.7个月；5年总生存时间为24-60个月,其平均值为57.2个月。 2.58例原发性眼眶MALT淋巴瘤患者眶内肿瘤组织VCP表达阳性细胞百分率为32%到95%。VCP表达阳性细胞百分率的中位值为45%,低于或等于45%为VCP表达level 1,高于45%为VCP表达level2。58例原发性眼眶MALT淋巴瘤患者眶内肿瘤组织VCP表达水平与患者复发(P=0.003)及肿瘤大小(P=0.008)密切相关。VCP表达水平与患者年龄、性别、LDH水平、红细胞沉降率(ESR)、血清白蛋白水平、有无B症状等临床特征无明显相关性(P0.05)。同时,VCP表达level1患者5年无瘤生存率及总生存率明显高于level2患者(P=0.001；P=0.032)。 3.10例患者眶内肿瘤组织VCPmRNA表达水平与VCP蛋白表达水平一致,根据免疫组化结果level2组患者肿瘤组织VCPmRNA表达明显高于level1组(P=0.009)。 4.人淋巴瘤细胞系Raji细胞及Daudi细胞VCP蛋白表达较强。利用脂质体进行细胞转染,VCPmRNA水平和蛋白表达水平分别被抑制65%和57%,基因沉默效果肯定。 5.流式细胞术结果显示,人淋巴瘤细胞系Raji细胞VCP基因沉默后,细胞凋亡明显增强。 6.MTS结果显示,人淋巴瘤细胞系Raji细胞VCP基因沉默后,细胞增殖能力明显受到抑制。 结论：1.VCP是评价原发性眼眶MALT淋巴瘤患者预后的良好指标。 2.利用脂质体介导si-VCP对人淋巴瘤Raji细胞靶基因VCP的抑制效果明显,VCPmRNA表达水平明显降低,从而使VCP蛋白表达量明显下降。 3.VCP基因沉默可明显促进体外培养人淋巴瘤细胞的凋亡,并降低细胞增殖能力。VCP在人淋巴瘤细胞凋亡及增殖过程中起重要作用,可能是原发性眼眶MALT淋巴瘤治疗的新的分子靶点。
[Abstract]:Objective: To investigate the correlation between the expression of valosin-containing protein (VCP) and the prognosis of primary orbital MALT lymphoma, and the effect of VCP on the apoptosis and proliferation of human lymphoma cells.
Methods:1.58 paraffin specimens of primary orbital MALT lymphoma were used to detect the expression of VCP by immunohistochemistry. The correlation between the expression of VCP and the recurrence and survival rate was analyzed statistically. The expression of VCP and the clinical factors were analyzed by X2 test and Fisher exact test. The recurrence rate, survival rate and survival curve were calculated by Kaplan-Meier method, and the difference between groups was statistically significant by log-rank method (P 0.05).
2. According to the expression of VCP protein, total RNA was extracted from paraffin-embedded tissues of orbital tumors of 10 patients (5 cases by 2 groups). The expression of VCP gene was quantitatively analyzed by real-time fluorescence quantitative PCR (RT-qPCR).
3. Immunocytochemical staining was used to detect the expression of VCP protein in Raji cells and Daudi cells of human lymphoma cells. An effective RNA interference method was established for Raji cells by using specific, efficient and convenient RNA technology with VCP as a gene target. Liposome-mediated gene si-VCP was transfected into Raji cells, and RT-qPCR and Western blot were performed from 1n to 1n respectively. RNA level and protein level were used to evaluate the effect of VCP gene silencing on Raji cells.
4. flow cytometry was used to detect the apoptosis rate of human lymphoma Raji cells with VCP gene silencing.
5.MTS assay was used to detect the effect of VCP gene silencing on the proliferation of Raji cells.
Results: Of 58 patients with orbital MALT lymphoma, 13 (22.4%) had recurrence. The mean recurrence time was 39.5 months (10-56 months). 8 patients (13.8%) died of the tumor, 4 patients (6.9%) died of other causes unrelated to the tumor. The 5-year tumor-free survival rate and overall survival rate were 70.7% and 79.3% respectively. The mean value of -60 months was 54.7 months, and the total survival time of 5 years was 24-60 months, with an average of 57.2 months.
The percentage of VCP positive cells was 32% to 95% in 58 cases of primary orbital MALT lymphoma. The median percentage of VCP positive cells was 45%, which was lower than or equal to 45%.
1. The expression of VCP in orbital tumors was closely related to the recurrence (P = 0.003) and tumor size (P = 0.008) of patients with primary orbital MALT lymphoma. The expression of VCP was related to age, sex, LDH level, erythrocyte sedimentation rate (ESR), serum albumin level and B symptoms. Meanwhile, the 5-year tumor-free survival rate and overall survival rate of patients with VCP expression level 1 were significantly higher than those of patients with level 2 (P = 0.001; P = 0.032).
3.The expression of VCP mRNA in orbital tumor tissues of 10 patients was consistent with that of VCP protein. According to immunohistochemical results, the expression of VCP mRNA in tumor tissues of Level 2 group was significantly higher than that of Level 1 group (P=0.009).
4. The expression of VCP protein in human lymphoma cell line Raji and Daudi cells was strong. The expression of VCP mRNA and protein were inhibited by 65% and 57% respectively by liposome transfection. The gene silencing effect was confirmed.
5. The results of flow cytometry showed that VCP gene silencing in human lymphoma cell line Raji increased the apoptosis significantly.
6. MTS results showed that VCP gene silencing significantly inhibited the proliferation of human lymphoma Raji cells.
Conclusion: 1.VCP is a good prognostic marker for patients with primary orbital MALT lymphoma.
2. Liposome-mediated si-VCP can inhibit VCP in Raji cells of human lymphoma, and the expression level of VCP mRNA is significantly decreased, so that the expression of VCP protein is significantly decreased.
3. VCP gene silencing can significantly promote the apoptosis of cultured human lymphoma cells in vitro and reduce the proliferation of cells. VCP plays an important role in the apoptosis and proliferation of human lymphoma cells, and may be a new molecular target for the treatment of primary orbital MALT lymphoma.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.72
本文编号：2207199
[Abstract]:Objective: To investigate the correlation between the expression of valosin-containing protein (VCP) and the prognosis of primary orbital MALT lymphoma, and the effect of VCP on the apoptosis and proliferation of human lymphoma cells.
Methods:1.58 paraffin specimens of primary orbital MALT lymphoma were used to detect the expression of VCP by immunohistochemistry. The correlation between the expression of VCP and the recurrence and survival rate was analyzed statistically. The expression of VCP and the clinical factors were analyzed by X2 test and Fisher exact test. The recurrence rate, survival rate and survival curve were calculated by Kaplan-Meier method, and the difference between groups was statistically significant by log-rank method (P 0.05).
2. According to the expression of VCP protein, total RNA was extracted from paraffin-embedded tissues of orbital tumors of 10 patients (5 cases by 2 groups). The expression of VCP gene was quantitatively analyzed by real-time fluorescence quantitative PCR (RT-qPCR).
3. Immunocytochemical staining was used to detect the expression of VCP protein in Raji cells and Daudi cells of human lymphoma cells. An effective RNA interference method was established for Raji cells by using specific, efficient and convenient RNA technology with VCP as a gene target. Liposome-mediated gene si-VCP was transfected into Raji cells, and RT-qPCR and Western blot were performed from 1n to 1n respectively. RNA level and protein level were used to evaluate the effect of VCP gene silencing on Raji cells.
4. flow cytometry was used to detect the apoptosis rate of human lymphoma Raji cells with VCP gene silencing.
5.MTS assay was used to detect the effect of VCP gene silencing on the proliferation of Raji cells.
Results: Of 58 patients with orbital MALT lymphoma, 13 (22.4%) had recurrence. The mean recurrence time was 39.5 months (10-56 months). 8 patients (13.8%) died of the tumor, 4 patients (6.9%) died of other causes unrelated to the tumor. The 5-year tumor-free survival rate and overall survival rate were 70.7% and 79.3% respectively. The mean value of -60 months was 54.7 months, and the total survival time of 5 years was 24-60 months, with an average of 57.2 months.
The percentage of VCP positive cells was 32% to 95% in 58 cases of primary orbital MALT lymphoma. The median percentage of VCP positive cells was 45%, which was lower than or equal to 45%.
1. The expression of VCP in orbital tumors was closely related to the recurrence (P = 0.003) and tumor size (P = 0.008) of patients with primary orbital MALT lymphoma. The expression of VCP was related to age, sex, LDH level, erythrocyte sedimentation rate (ESR), serum albumin level and B symptoms. Meanwhile, the 5-year tumor-free survival rate and overall survival rate of patients with VCP expression level 1 were significantly higher than those of patients with level 2 (P = 0.001; P = 0.032).
3.The expression of VCP mRNA in orbital tumor tissues of 10 patients was consistent with that of VCP protein. According to immunohistochemical results, the expression of VCP mRNA in tumor tissues of Level 2 group was significantly higher than that of Level 1 group (P=0.009).
4. The expression of VCP protein in human lymphoma cell line Raji and Daudi cells was strong. The expression of VCP mRNA and protein were inhibited by 65% and 57% respectively by liposome transfection. The gene silencing effect was confirmed.
5. The results of flow cytometry showed that VCP gene silencing in human lymphoma cell line Raji increased the apoptosis significantly.
6. MTS results showed that VCP gene silencing significantly inhibited the proliferation of human lymphoma Raji cells.
Conclusion: 1.VCP is a good prognostic marker for patients with primary orbital MALT lymphoma.
2. Liposome-mediated si-VCP can inhibit VCP in Raji cells of human lymphoma, and the expression level of VCP mRNA is significantly decreased, so that the expression of VCP protein is significantly decreased.
3. VCP gene silencing can significantly promote the apoptosis of cultured human lymphoma cells in vitro and reduce the proliferation of cells. VCP plays an important role in the apoptosis and proliferation of human lymphoma cells, and may be a new molecular target for the treatment of primary orbital MALT lymphoma.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.72
【参考文献】
相关期刊论文 前1条
1 徐青,肖利华,何彦津,杨守京,李宁,李敏,高子芬;眼附属器淋巴瘤组织病理学及免疫表型研究[J];眼科研究;2003年02期
，本文编号：2207199
本文链接：https://www.wllwen.com/yixuelunwen/yank/2207199.html
最近更新
教材专著
