原發(fā)性眼眶MALT淋巴瘤患者VCP表達(dá)與患者預(yù)后的相關(guān)性及機制的研究
發(fā)布時間：2018-08-27 11:53
【摘要】：目的：探討含纈酪肽蛋白(valosin-containing protein, VCP)表達(dá)水平與原發(fā)性眼眶MALT淋巴瘤患者預(yù)后的相關(guān)性,及VCP對人淋巴瘤細(xì)胞凋亡及增殖能力的影響。 方法：1.58例原發(fā)性眼眶MALT淋巴瘤患者眶內(nèi)淋巴瘤組織石蠟標(biāo)本,采用免疫組化法檢測VCP表達(dá)水平,并對VCP表達(dá)水平與患者復(fù)發(fā)情況及生存率的相關(guān)性進(jìn)行統(tǒng)計學(xué)分析,用X2檢驗及Fisher精確檢驗來分析VCP蛋白表達(dá)與各臨床因素之間的關(guān)系；Kaplan-Meier法計算復(fù)發(fā)率、生存率,并繪制生存曲線；log-rank法計算組間差異,P0.05為差異具有統(tǒng)計學(xué)意義； 2.根據(jù)免疫組化結(jié)果即VCP蛋白表達(dá)水平,選取10例患者眶內(nèi)腫瘤石蠟組織(5例×2組)抽提總RNA,并應(yīng)用實時熒光定量PCR(RT-qPCR)定量比較2組VCP基因表達(dá)水平的差異； 3.免疫細(xì)胞化學(xué)染色法檢測人淋巴瘤細(xì)胞系Raji細(xì)胞及Daudi細(xì)胞VCP蛋白的表達(dá)。應(yīng)用特異、高效并操作方便的RNAi技術(shù),以VCP作為基因靶點,對Raji細(xì)胞建立有效的RNA干擾方法。利用脂質(zhì)體介導(dǎo)基因si-VCP轉(zhuǎn)染Raji細(xì)胞,RT-qPCR及Western blot分別從1nRNA水平及蛋白水平評估Raji細(xì)胞VCP基因沉默效果； 4.流式細(xì)胞術(shù)檢測VCP基因沉默的人淋巴瘤Raji細(xì)胞凋亡率的變化； 5.MTS法檢測VCP基因沉默對Raji細(xì)胞增殖能力的影響。 結(jié)果：1.58例眼眶MALT淋巴瘤患者中13例(22.4%)患者出現(xiàn)腫瘤復(fù)發(fā)。復(fù)發(fā)時間為10-56個月,其平均值為39.5個月。8例患者(13.8%)死于腫瘤,4例患者(6.9%)死于與腫瘤無關(guān)的其他原因。5年無瘤生存率及總生存率分別是70.7%、79.3%。5年無瘤生存時間為10-60個月,其平均值為54.7個月；5年總生存時間為24-60個月,其平均值為57.2個月。 2.58例原發(fā)性眼眶MALT淋巴瘤患者眶內(nèi)腫瘤組織VCP表達(dá)陽性細(xì)胞百分率為32%到95%。VCP表達(dá)陽性細(xì)胞百分率的中位值為45%,低于或等于45%為VCP表達(dá)level 1,高于45%為VCP表達(dá)level2。58例原發(fā)性眼眶MALT淋巴瘤患者眶內(nèi)腫瘤組織VCP表達(dá)水平與患者復(fù)發(fā)(P=0.003)及腫瘤大小(P=0.008)密切相關(guān)。VCP表達(dá)水平與患者年齡、性別、LDH水平、紅細(xì)胞沉降率(ESR)、血清白蛋白水平、有無B癥狀等臨床特征無明顯相關(guān)性(P0.05)。同時,VCP表達(dá)level1患者5年無瘤生存率及總生存率明顯高于level2患者(P=0.001；P=0.032)。 3.10例患者眶內(nèi)腫瘤組織VCPmRNA表達(dá)水平與VCP蛋白表達(dá)水平一致,根據(jù)免疫組化結(jié)果level2組患者腫瘤組織VCPmRNA表達(dá)明顯高于level1組(P=0.009)。 4.人淋巴瘤細(xì)胞系Raji細(xì)胞及Daudi細(xì)胞VCP蛋白表達(dá)較強。利用脂質(zhì)體進(jìn)行細(xì)胞轉(zhuǎn)染,VCPmRNA水平和蛋白表達(dá)水平分別被抑制65%和57%,基因沉默效果肯定。 5.流式細(xì)胞術(shù)結(jié)果顯示,人淋巴瘤細(xì)胞系Raji細(xì)胞VCP基因沉默后,細(xì)胞凋亡明顯增強。 6.MTS結(jié)果顯示,人淋巴瘤細(xì)胞系Raji細(xì)胞VCP基因沉默后,細(xì)胞增殖能力明顯受到抑制。 結(jié)論：1.VCP是評價原發(fā)性眼眶MALT淋巴瘤患者預(yù)后的良好指標(biāo)。 2.利用脂質(zhì)體介導(dǎo)si-VCP對人淋巴瘤Raji細(xì)胞靶基因VCP的抑制效果明顯,VCPmRNA表達(dá)水平明顯降低,從而使VCP蛋白表達(dá)量明顯下降。 3.VCP基因沉默可明顯促進(jìn)體外培養(yǎng)人淋巴瘤細(xì)胞的凋亡,并降低細(xì)胞增殖能力。VCP在人淋巴瘤細(xì)胞凋亡及增殖過程中起重要作用,可能是原發(fā)性眼眶MALT淋巴瘤治療的新的分子靶點。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.72
本文編號：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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.72
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 徐青,肖利華,何彥津,楊守京,李寧,李敏,高子芬;眼附屬器淋巴瘤組織病理學(xué)及免疫表型研究[J];眼科研究;2003年02期
，本文編號：2207199
本文鏈接：http://sikaile.net/yixuelunwen/yank/2207199.html
最近更新
教材專著
