Z-VRPR-FMK对弥漫大B细胞淋巴瘤裸鼠移植瘤生长的影响及相关机制的研究

发布时间：2018-06-30 06:46

本文选题：淋巴瘤 + MALT1　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:通过观察粘膜相关淋巴组织淋巴瘤易位基因1(mucosa-associated lymphoid tissue lymphoma translocation gene 1,MALT1)抑制剂Z-VRPR-FMK对弥漫大B细胞淋巴瘤(Diffuse large B cell lymphoma,DLBCL)裸鼠移植瘤生长的影响和核因子κB(Nuclear factor kappa B,NF-κB)通路相关的信号分子异常,探讨MALT1在DLBCL生长中的作用,为发现DLBCL治疗的新靶点提供理论依据。方法:培养生发中心细胞样弥漫大B细胞淋巴瘤(germinal center B cell like-Diffuse large B cell lymphoma,GCB-DLBCL)和活化后B细胞样弥漫大B细胞淋巴瘤(Activated B cell like-Diffuse large B cell lymphoma,ABC-DLBCL)细胞株OCI-LY1和OCI-LY10,建立DLBCL裸鼠移植瘤模型;将成瘤裸鼠随机分为对照组和抑制剂组。抑制剂组用Z-VRPR-FMK、对照组用等量生理盐水腹腔注射。定期测量并记录裸鼠体重和肿瘤体积,绘制移植瘤生长曲线图;实时荧光定量PCR(Real time PCR)检测p65基因在核酸水平的表达,免疫组化染色检测P65蛋白表达,蛋白印记和免疫组织化学染色检测MALT1、A20、MMP2和MMP9蛋白的表达。对上述检查结果进行统计学分析。结果:1.裸鼠成瘤:DLBCL细胞总成瘤率68%(17/25),其中OCI-LY1细胞株成瘤率为60%(9/15),成瘤的时间为18.16±3.36天,OCI-LY10细胞株成瘤率为80%(8/10),成瘤的时间为13.63±3.02天。2.裸鼠和移植瘤的生长:OCI-LY1细胞株荷瘤鼠对照组和抑制剂组之间体重和瘤块体积变化都没有统计学差异,(P均大于0.05)。两组OCI-LY10细胞移植瘤鼠体重在处理后逐渐上升,对照组在第9天开始逐渐下降,而抑制剂组体重则持续上升,在第11天和第13天两组体重有统计学差异,P分别为0.028和0.016。3.P65表达:OCI-LY1细胞移植瘤中,抑制剂组p65的mRNA和蛋白核表达均低于对照组,但差异无统计学意义(P=0.060),OCI-LY10细胞移植瘤中p65核酸和蛋白表达水平都较对照组显著降低(P=0.000,P=0.028)。4.MALT1、A20、MMP2和MMP9蛋白表达:免疫组化染色显示MALT1,A20,MMP2和MMP9阳性信号均定位于细胞质。两种DLBCL移植瘤的抑制剂处理组MALT1,MMP2,MMP9蛋白阳性表达信号较对照组强,A20表达则相反。Western blot检测和分析显示:两组OCI-LY1细胞株移植瘤比较:在抑制剂组MALT1蛋白表达显著降低(P=0.013),而A20蛋白表达则升高(P=0.005),两组间MMP9、MMP2表达水平无明显差异(P=0.234,P=0.071);两组OCI-LY10细胞移植瘤相比:在抑制剂组MALT1、MMP2、MMP9蛋白表达水平均显著较低(P依次为0.042,0.002,0.006),A20蛋白表达显著升高(P=0.000)。结论:1.ABC样DLBCL中存在MALT1蛋白的异常表达,并与肿瘤中NF-κB的持续活化密切相关,MALT1可能成为ABC样DLBCL治疗的有效靶点。2.Z-VRPR-FMK能有效地抑制DLBCL中MALT1蛋白的表达,可能通过解除其对A20蛋白的裂解作用,从而抑制NF-κB的活化。3.ABC样DLBC中,MMP2和MMP9是NF-κB活化后上调表达的靶分子,其表达影响肿瘤的生长。
[Abstract]:Objective: to investigate the effects of Z-VRPR-FMK, a translocation gene 1 (mucosa-associated lymphoid tissue lymphoma translocation gene 1) -MALT1 inhibitor, on the growth of diffuse large B cell lymphomatous lymphoma (DLBCL) xenografts in nude mice and the correlation between nuclear factor kappa B (NF- 魏 B) pathway and nuclear factor 魏 B (NF- 魏 B) pathway. Abnormal signal molecules, To explore the role of MALT1 in the growth of DLBCL and to provide theoretical basis for finding new targets for DLBCL therapy. Methods: OCI-LY1 and OCI-LY10 were cultured from germinal center cell-like diffuse large B-cell lymphoma (germinal center B cell like-Diffuse large B cell lymphoma1) and Activated B cell like-Diffuse large B cell lymphoma1 (OCI-LY1) and OCI-LY10. Nude mice were randomly divided into control group and inhibitor group. The inhibitor group was treated with Z-VRPR- FMK and the control group with the same amount of normal saline intraperitoneal injection. The body weight and tumor volume of nude mice were measured and recorded regularly, the growth curve of transplanted tumor was drawn, the expression of p65 gene at nucleic acid level was detected by Real time PCR, and the expression of p65 protein was detected by immunohistochemical staining. Protein imprinting and immunohistochemical staining were used to detect the expression of MMP2 and MMP9. The results were statistically analyzed. The result is 1: 1. The tumorigenic rate of OCI-LY1 cell line was 60% (9 / 15), the tumorigenesis time of OCI-LY10 cell line was 18.16 卤3.36 days (80%, 8 / 10), and the tumorigenic time of OCI-LY10 cell line was 13.63 卤3.02 days, and the tumorigenic rate of OCI-LY10 cell line was 68% (17 / 25). The tumorigenic rate of OCI-LY1 cell line was 60% (9 / 15), and the tumorigenic time was 18.16 卤3.36 days. There was no significant difference in body weight and tumor volume between the control group and the inhibitor group (P > 0.05). The weight of OCI-LY10 transplanted tumor mice gradually increased after treatment, the control group began to decrease gradually on the 9th day, while the weight of the inhibitor group continued to increase. On the 11th and 13th days, there were significant differences in body weight between the two groups (P = 0.028 and 0.016.3.P65, respectively). The expression of p65 mRNA and protein in the inhibitor group was lower than that in the control group. The expression of p65 nucleic acid and protein in OCI-LY10 cells was significantly lower than that in the control group (P0. 000). 4. The expression of MMP2 and MMP9 protein in MALT1A20 MMP2 and MMP9 protein: the positive signals of MALT1A20 MMP2 and MMP9 were localized in cytoplasm by immunohistochemical staining. The positive expression of MALT1 mMP2mMP9 protein in the two DLBCL transplanted tumor groups was significantly lower than that in the control group. Western blot analysis showed that the expression of MALT1 protein was significantly lower in the inhibitor group than that in the control group (Pn0.013), and the expression of MALT1 protein in the two groups was significantly lower than that in the OCI-LY1 cell line. However, the expression of A20 protein increased (P0. 005), and there was no significant difference between the two groups in the expression of MMP9 and MMP2 (P0. 234, P0. 071). The expression of A20 protein in OCI-LY10 cells was significantly lower than that in the inhibitor group (P = 0. 042, 0. 002, 0. 006, P = 0. 0000), and the expression of A20 protein in OCI-LY10 cells was significantly lower than that in the control group (P = 0. 042, 0. 002P = 0. 0000). Conclusion: 1. There is abnormal expression of MALT1 protein in ABC-like DLBCL, and it is closely related to the continued activation of NF- 魏 B in tumor. MALT1 may be an effective target for ABC-like DLBCL treatment. Z-2.VRPR-FMK can effectively inhibit the expression of MALT1 protein in DLBCL. It is possible to inhibit the activation of NF- 魏 B by removing the cleavage of A20 protein. 3. MMP2 and MMP9 in ABC-like DLBC are the target molecules that up-regulate the expression of NF- 魏 B, and their expression affects the growth of tumor.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1

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