Th17细胞和人髓母细胞瘤的实验研究
摘要
髓母细胞瘤是儿童后颅窝最常见的原发性恶性肿瘤之一,恶性程度高,容易沿蛛网膜下腔转移,预后很差,患儿平均生存期在6个月左右,两年生存率只有9%,故髓母细胞瘤是神经外科的治疗难题之一。且髓母细胞瘤患者大多是儿童,对放、化疗的耐受较成人要差。此外放、化疗还会影响儿童的生长发育,包括手术后缄默症,神经认知障碍,内分泌紊乱,脱发,以及脑组织突变成为恶性胶质瘤的现象。因此探索和寻找新的治疗髓母细胞瘤的治疗方法具有重要的临床意义。目前不少研究发现,肿瘤的发生与免疫系统的变化有很大的关系,肿瘤免疫治疗已成为现代肿瘤治疗的第四种模式,越来越受到医学界的重视,但人们对髓母细胞瘤患者的免疫改变尚未进行过系统的研究。Th17细胞是最近发现的一类T细胞亚群,越来越多的证据表明,Th17细胞在人体肿瘤免疫和自身免疫病中起了重要作用。Th17细胞能分泌包括IL-17在内的大量细胞因子,这些细胞因子均参与了机体的免疫网络,既互相促进又互相抑制,在机体的免疫应答调节中起着重要作用,在许多自身免疫病和肿瘤的发生中也起着不同程度的作用。目前国内外还没有相关的研究报道Th17细胞与髓母细胞瘤关系,基于上述髓母细胞瘤临床治疗与基础研究的研究现状,本研究首次分三个部分对Th17细胞在髓母细胞瘤发生中的作用、免疫改变等进行了较详细的探索。具体研究结果如下：
第一部分目的：了解Th17细胞以及相关的细胞因子IL-17,IL-23在髓母细胞瘤中的表达。
方法：我们收集了外周血,采集自复旦大学华山医院神经外科2006年至2009年的23名经手术、病理证实的髓母细胞瘤患者和对照组17名健康志愿者。本实验应用葡聚糖-泛影葡胺密度梯度离心法分离外周血淋巴细胞,并进行淋巴细胞24小时的培养,然后应用流式细胞仪检测外周血淋巴细胞中的Th17细胞,用酶标记酶联免疫吸附试验的实验手段检测了外周血中Th17细胞的相关细胞因子IL-17和IL-23的浓度。
结果：Th17细胞在外周血CD4+的T细胞中所占的比例用流式细胞仪进行分析计数,与健康对照组比较,髓母细胞瘤组Th17细胞在CD4+的T细胞中所占的比例有所增加,结果进行t检验有统计学意义(2.7±1.4% vs 0.8±0.3%,p<0.01)。髓母细胞瘤患者和健康对照组的血清IL-17和IL-23进行了测定,髓母细胞瘤患者血清的细胞因子浓度和健康对照组比较明显增加,结果进行t检验有统计学意义(IL-17：肿瘤组79.5±37.7 pg/ml对照组15±9.1 pg/ml,p<0.01；IL-23：肿瘤组293±132pg/ml对照组102±55 pg/ml,p<0.01)。
第二部分目的：髓母细胞瘤是小儿神经外科常见的高度恶性肿瘤,具有生长极其迅速,手术不易彻底切除,并有沿脑脊液产生播散性种植的倾向,使本病的治疗很困难,现在有越来越多的证据表明,肿瘤的发生与免疫系统的变化有很大的关系,但髓母细胞瘤患者的免疫改变目前还不为人们所了解。Th17细胞与IL-17以及IL-23关系密切,另外细胞因子RORC为目前已确定的Th17细胞分化所需要的特异性转录因子。故本实验要了解Th17细胞和相关的mRNA(包括IL-17, IL-23, and RORC)在髓母细胞瘤中的表达。
方法：标本采集自复旦大学华山医院神经外科2006年至2009年的13名经手术、病理证实的髓母细胞瘤标本和13例对照组正常小脑组织标本,本实验应用葡聚糖-泛影葡胺密度梯度离心法分离髓母细胞瘤浸润的淋巴细胞,并进行淋巴细胞24小时的培养,然后应用流式细胞仪检测髓母细胞瘤浸润的淋巴细胞中的Th17细胞,用RT-PCR的实验手段检测了髓母细胞瘤浸润的淋巴细胞中Th17细胞相关(?)nRNA(包括IL-17,IL-23p19,和RORC)的表达。
结果：通过流式细胞术发现髓母细胞瘤组Th17细胞在CD4+的T细胞中所占的比例与正常小脑对照组比较有明显的增加(分别是6.7±2.4%和1.7±2.1%,p<0.01)。本实验并用RT-PCR的方法在髓母细胞瘤和正常小脑组织中Th17细胞相关细胞因子的表达水平,IL-17和RORC mRNA在6例中被检测出(共13例),在13个标本中的9例检测出IL-23p19 mRNA,在健康对照组中均未检测出上述细胞因子。
第三部分目的：裸小鼠进行免疫重建后,注入IL-17能否影响肿瘤的生长和荷瘤小鼠的生存期,并检测Th17相关细胞因子IL-6、IL-23及趋化因子CCL-2、CCL-20的表达水平有无改变。
方法：对BALB/c nu/nu雄性裸小鼠进行免疫重建后,建立髓母细胞瘤动物模型,对比加入IL-17组和未加入IL-17组肿瘤的生长趋势,用Western blot和Real-Time PCR检测对比治疗组和对照组中的Th17相关细胞因子IL-6、IL-23及趋化因子CCL-2、CCL-20的表达水平。
结果：裸小鼠注入IL-17组的生存期较对照组延长,肿瘤较对照组生长缓慢,结果均有统计学意义。实验组Th17相关细胞因子IL-6、IL-23及趋化因子CCL-2、CCL-20的表达水平较对照组上升,结果有统计学意义。
全文结论：自本研究结果可得出以下几点结论：(1)Th17细胞在髓母细胞瘤患者外周血淋巴细胞中明显增加,提示Th17细胞在髓母细胞瘤的免疫改变中可能起了一定的作用,可能同肿瘤的发生发展有关。Th17细胞及其相关的细胞因子IL-17,IL-23,与髓母细胞瘤有一定的正相关的关系,可能在髓母细胞瘤患者的免疫改变中起了一定的作用。(2)Th17细胞在髓母细胞瘤外周血淋巴细胞和髓母细胞肿瘤周围的浸润淋巴细胞中都明显增加,提示Th17细胞在髓母细胞瘤的免疫改变中可能起着一定的作用,亦可能同肿瘤的发生发展有关。(3)免疫重建后的裸小鼠中注入IL-17实验结果表明IL-17有可能增加淋巴细胞的抗肿瘤能力,起到减缓肿瘤生长,增加小鼠的生存期的作用。
Medulloblastoma is a commonly malignant tumor of the puerile cerebellum. Patients with medulloblastomas after primary therapy have a particularly poor prognosis, with a median survival of less than 6 months; the 2-year survival rate among these patients is approximately 9%. Medulloblastoma is one of the difficult problems for resolving in neurosurgery. Current therapies have serious short-term and long-term adverse effects, including postoperative mutism, neurocognitive deficits. Recently, a new member of the CD4+ effector T-cell family (Th17 cells) has substantially advanced our understanding of T cell-mediated immunity. These cells are characterized as preferential producers of IL-17 and IL-23 in humans. Although human Th17-cell development is less well understood, IL-23 have been shown to collectively mediate the differentiation of human Th17 cells in vitro. Recently some findings suggest Th17 cells may also play an active role in tumor pathogenesis.
PartⅠObjective:In this study, we want to find the relationship between the Th17 cells and medulloblastoma.
Method and material:Peripheral blood was collected from 23 patients with medulloblastoma and 17 healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque density centrifugation. Then the PBMCs were cultured for 24 hours. We determined the prevalence of Th17 cells in peripheral blood by flow cytometric analysis. The concentrations of IL-17 and IL-23 cytokines in serum were measured by ELISA.
Result:The proportion of peripheral blood Th17 cells in medulloblastoma patients was significantly higher than that in healthy donors (2.7±1.4% vs 0.8±0.3%, p< 0.01, the cytokines concentrations were significantly higher in patients than that in control subjects (IL-17:79.5±37.7 pg/ml vs 15±9.1 pg/ml, p<0.01;IL-23: 293±132pg/ml vs 102±55 pg/ml, p< 0.01).
Part II Objective:To study Th17-related factors (IL-17, IL-23, and RORC) in tumor tissues from patients with medulloblastoma and evaluated the potential association of Th17 cells with human medulloblastoma.
Method:13 tumor tissues were collected during surgery. Tumor-infiltrating lymphcytes were obtained by Ficoll-Hypaque density centrifugation. Control group cotained 13 cases. Then the tumor-infiltrating lymphcytes were cultured for 24 hours. The PBMCs were cultured for 24 hours. We determined the prevalence of Th17 cells in tumor-infiltrating lymphcytes by flow cytometric analysis. Th17-re-lated factors (IL-17, IL-23pl9, and RORC) in tumor-infiltrating lymphcytes and autologous normal tissues was measured by RT-PCR
Result:Our data indicated an increased prevalence of Th17 cells in Medulloblastoma-infiltrating T cells s in comparison to controls (6.7±2.4% vs 1.7±2.1%, p< 0.01). IL-17 and RORC mRNA expressions were synchronically detected in 6 out of the 13 samples. Increased expression of IL-23p19 mRNA was observed in 9 out of 13 specimens. No expression was detected in autologous normal tissues.
PartⅢObjective:To study the activity of IL-17 in immunologic reconstitution BALB/c nu/nu mouse bearing Medulloblastoma and detecte the expression level of IL-23, IL-6, CCL-2, CCL-20 in tumors
Method:IL-17 was added to immunologic reconstitution BALB/c nu/nu mouse bearing Medulloblastoma. The mouse's survival time and the tumors weights were observed. We also detected the expression level of IL-23, IL-6, CCL-2, CCL-20 in tumors by Westle blot and Real-time PCR.
Result:The survival times of IL-17 adding group were prolonged and the growing of the tumor of experimental group was retarded.the expression level of IL-23, IL-6, CCL-2, CCL-20 of IL-17 adding group was ascent.
Conclusion:(1) Th17 cells and relative cytokines IL-17, IL-23 may take a role in the development of medulloblastoma. (2) Th17 cells and related factors (IL-17, IL-23, and RORC) may take a role in the development of medulloblastoma. (3) IL-17 may play a role in the antineoplastic immunity in medulloblastoma.
第一部分目的：了解Th17细胞以及相关的细胞因子IL-17,IL-23在髓母细胞瘤中的表达。
方法：我们收集了外周血,采集自复旦大学华山医院神经外科2006年至2009年的23名经手术、病理证实的髓母细胞瘤患者和对照组17名健康志愿者。本实验应用葡聚糖-泛影葡胺密度梯度离心法分离外周血淋巴细胞,并进行淋巴细胞24小时的培养,然后应用流式细胞仪检测外周血淋巴细胞中的Th17细胞,用酶标记酶联免疫吸附试验的实验手段检测了外周血中Th17细胞的相关细胞因子IL-17和IL-23的浓度。
结果：Th17细胞在外周血CD4+的T细胞中所占的比例用流式细胞仪进行分析计数,与健康对照组比较,髓母细胞瘤组Th17细胞在CD4+的T细胞中所占的比例有所增加,结果进行t检验有统计学意义(2.7±1.4% vs 0.8±0.3%,p<0.01)。髓母细胞瘤患者和健康对照组的血清IL-17和IL-23进行了测定,髓母细胞瘤患者血清的细胞因子浓度和健康对照组比较明显增加,结果进行t检验有统计学意义(IL-17：肿瘤组79.5±37.7 pg/ml对照组15±9.1 pg/ml,p<0.01；IL-23：肿瘤组293±132pg/ml对照组102±55 pg/ml,p<0.01)。
第二部分目的：髓母细胞瘤是小儿神经外科常见的高度恶性肿瘤,具有生长极其迅速,手术不易彻底切除,并有沿脑脊液产生播散性种植的倾向,使本病的治疗很困难,现在有越来越多的证据表明,肿瘤的发生与免疫系统的变化有很大的关系,但髓母细胞瘤患者的免疫改变目前还不为人们所了解。Th17细胞与IL-17以及IL-23关系密切,另外细胞因子RORC为目前已确定的Th17细胞分化所需要的特异性转录因子。故本实验要了解Th17细胞和相关的mRNA(包括IL-17, IL-23, and RORC)在髓母细胞瘤中的表达。
方法：标本采集自复旦大学华山医院神经外科2006年至2009年的13名经手术、病理证实的髓母细胞瘤标本和13例对照组正常小脑组织标本,本实验应用葡聚糖-泛影葡胺密度梯度离心法分离髓母细胞瘤浸润的淋巴细胞,并进行淋巴细胞24小时的培养,然后应用流式细胞仪检测髓母细胞瘤浸润的淋巴细胞中的Th17细胞,用RT-PCR的实验手段检测了髓母细胞瘤浸润的淋巴细胞中Th17细胞相关(?)nRNA(包括IL-17,IL-23p19,和RORC)的表达。
结果：通过流式细胞术发现髓母细胞瘤组Th17细胞在CD4+的T细胞中所占的比例与正常小脑对照组比较有明显的增加(分别是6.7±2.4%和1.7±2.1%,p<0.01)。本实验并用RT-PCR的方法在髓母细胞瘤和正常小脑组织中Th17细胞相关细胞因子的表达水平,IL-17和RORC mRNA在6例中被检测出(共13例),在13个标本中的9例检测出IL-23p19 mRNA,在健康对照组中均未检测出上述细胞因子。
第三部分目的：裸小鼠进行免疫重建后,注入IL-17能否影响肿瘤的生长和荷瘤小鼠的生存期,并检测Th17相关细胞因子IL-6、IL-23及趋化因子CCL-2、CCL-20的表达水平有无改变。
方法：对BALB/c nu/nu雄性裸小鼠进行免疫重建后,建立髓母细胞瘤动物模型,对比加入IL-17组和未加入IL-17组肿瘤的生长趋势,用Western blot和Real-Time PCR检测对比治疗组和对照组中的Th17相关细胞因子IL-6、IL-23及趋化因子CCL-2、CCL-20的表达水平。
结果：裸小鼠注入IL-17组的生存期较对照组延长,肿瘤较对照组生长缓慢,结果均有统计学意义。实验组Th17相关细胞因子IL-6、IL-23及趋化因子CCL-2、CCL-20的表达水平较对照组上升,结果有统计学意义。
全文结论：自本研究结果可得出以下几点结论：(1)Th17细胞在髓母细胞瘤患者外周血淋巴细胞中明显增加,提示Th17细胞在髓母细胞瘤的免疫改变中可能起了一定的作用,可能同肿瘤的发生发展有关。Th17细胞及其相关的细胞因子IL-17,IL-23,与髓母细胞瘤有一定的正相关的关系,可能在髓母细胞瘤患者的免疫改变中起了一定的作用。(2)Th17细胞在髓母细胞瘤外周血淋巴细胞和髓母细胞肿瘤周围的浸润淋巴细胞中都明显增加,提示Th17细胞在髓母细胞瘤的免疫改变中可能起着一定的作用,亦可能同肿瘤的发生发展有关。(3)免疫重建后的裸小鼠中注入IL-17实验结果表明IL-17有可能增加淋巴细胞的抗肿瘤能力,起到减缓肿瘤生长,增加小鼠的生存期的作用。
Medulloblastoma is a commonly malignant tumor of the puerile cerebellum. Patients with medulloblastomas after primary therapy have a particularly poor prognosis, with a median survival of less than 6 months; the 2-year survival rate among these patients is approximately 9%. Medulloblastoma is one of the difficult problems for resolving in neurosurgery. Current therapies have serious short-term and long-term adverse effects, including postoperative mutism, neurocognitive deficits. Recently, a new member of the CD4+ effector T-cell family (Th17 cells) has substantially advanced our understanding of T cell-mediated immunity. These cells are characterized as preferential producers of IL-17 and IL-23 in humans. Although human Th17-cell development is less well understood, IL-23 have been shown to collectively mediate the differentiation of human Th17 cells in vitro. Recently some findings suggest Th17 cells may also play an active role in tumor pathogenesis.
PartⅠObjective:In this study, we want to find the relationship between the Th17 cells and medulloblastoma.
Method and material:Peripheral blood was collected from 23 patients with medulloblastoma and 17 healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque density centrifugation. Then the PBMCs were cultured for 24 hours. We determined the prevalence of Th17 cells in peripheral blood by flow cytometric analysis. The concentrations of IL-17 and IL-23 cytokines in serum were measured by ELISA.
Result:The proportion of peripheral blood Th17 cells in medulloblastoma patients was significantly higher than that in healthy donors (2.7±1.4% vs 0.8±0.3%, p< 0.01, the cytokines concentrations were significantly higher in patients than that in control subjects (IL-17:79.5±37.7 pg/ml vs 15±9.1 pg/ml, p<0.01;IL-23: 293±132pg/ml vs 102±55 pg/ml, p< 0.01).
Part II Objective:To study Th17-related factors (IL-17, IL-23, and RORC) in tumor tissues from patients with medulloblastoma and evaluated the potential association of Th17 cells with human medulloblastoma.
Method:13 tumor tissues were collected during surgery. Tumor-infiltrating lymphcytes were obtained by Ficoll-Hypaque density centrifugation. Control group cotained 13 cases. Then the tumor-infiltrating lymphcytes were cultured for 24 hours. The PBMCs were cultured for 24 hours. We determined the prevalence of Th17 cells in tumor-infiltrating lymphcytes by flow cytometric analysis. Th17-re-lated factors (IL-17, IL-23pl9, and RORC) in tumor-infiltrating lymphcytes and autologous normal tissues was measured by RT-PCR
Result:Our data indicated an increased prevalence of Th17 cells in Medulloblastoma-infiltrating T cells s in comparison to controls (6.7±2.4% vs 1.7±2.1%, p< 0.01). IL-17 and RORC mRNA expressions were synchronically detected in 6 out of the 13 samples. Increased expression of IL-23p19 mRNA was observed in 9 out of 13 specimens. No expression was detected in autologous normal tissues.
PartⅢObjective:To study the activity of IL-17 in immunologic reconstitution BALB/c nu/nu mouse bearing Medulloblastoma and detecte the expression level of IL-23, IL-6, CCL-2, CCL-20 in tumors
Method:IL-17 was added to immunologic reconstitution BALB/c nu/nu mouse bearing Medulloblastoma. The mouse's survival time and the tumors weights were observed. We also detected the expression level of IL-23, IL-6, CCL-2, CCL-20 in tumors by Westle blot and Real-time PCR.
Result:The survival times of IL-17 adding group were prolonged and the growing of the tumor of experimental group was retarded.the expression level of IL-23, IL-6, CCL-2, CCL-20 of IL-17 adding group was ascent.
Conclusion:(1) Th17 cells and relative cytokines IL-17, IL-23 may take a role in the development of medulloblastoma. (2) Th17 cells and related factors (IL-17, IL-23, and RORC) may take a role in the development of medulloblastoma. (3) IL-17 may play a role in the antineoplastic immunity in medulloblastoma.
引文
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