缓释BCNU对GL261胶质瘤治疗作用分析

目的 研究卡氮芥( BCNU)聚乳酸-羟基乙酸(PLGA)缓释微球(BCNU-PLGA)对GL261胶质瘤模型的治疗作用及化疗耐药机制.方法 利用溶剂挥发萃取法制备BCNU-PLGA缓释微球.将36只C57BL/6小鼠随机等分为假手术组、治疗组与非治疗组,借助动物立体定向仪,将体外培养小鼠GL261胶质瘤细胞接种于小鼠颅内,治疗组于术后第2 w立体定位植入BCNU-PLGA缓释片剂,观察小鼠生存期,MRI了解瘤体变化.获取标本行HE染色,并行胶原纤维酸性蛋白(GFAP)、甲基鸟嘌呤DNA甲基转移酶(MGMT)免疫组化检查检测其表达情况,相关数据采用统计学方法进行分析.结果 治疗组与对照组生存期有明显差异(P＜0.05),治疗组与对照组凋亡MGMT无明显差异(P＞0.05).GFAP表达为阳性.结论 BCNU-PLGA局部缓释制剂可延长荷瘤小鼠生存期,且不改变其MGMT表达.     

三种鼠脑胶质瘤模型的建立与比较

目的 ;建立GL261胶质瘤细胞C57BL/6小鼠、C6胶质瘤细胞SD大鼠及BALB/c小鼠皮下动物模型,比较其肿瘤生长特点。方法借助动物立体定向仪,将体外培养小鼠GL261、大鼠C6胶质瘤细胞分别接种于C57BL/6小鼠及SD大鼠右侧尾状核区,C6胶质瘤细胞接种于BALB/c小鼠左前肢皮下。接种后观察不同种实验鼠的生存状态及肿瘤的生长特性,颅内模型于接种后7d、14d、21d、28d进行MRI检查,皮下模型测量体积,并绘制生长曲线。解剖标本,做组织病理学和胶质纤维酸性蛋(GFAP)免疫组化检查。结果 GL261胶质瘤细胞C57BL/6小鼠模型较之后两种模型在组织病理学上接近人脑胶质瘤,而且颅内生长稳定,成瘤率高,未见颅外转移病灶,实验周期短,重复性好。结论 GL261胶质瘤细胞C57BL/6小鼠模型,其肿瘤生长特性及病理特征与人脑胶质瘤相似,可作为临床胶质瘤基础研究的理想模型。     

氧增强放疗对大鼠胶质瘤细胞凋亡相关基因Bcl-2和Bax影响

目的 探讨氧增强放疗对胶质瘤凋亡相关基因Bcl-2、Bax 影响的差异.方法 建立大鼠胶质瘤动物模型,治疗组10 只,高压氧处理后放疗;对照组10 只,常规放疗.免疫组化检测凋亡相关基因Bcl-2、Bax 的表达.结果 治疗组Bcl-2 表达明显低于对照组,组间差异有显著性(P ＜ 0.05);治疗组Bax 的表达明显高于对照组,组间差异有显著性(P ＜0.05).结论 氧增强放疗可以更有效地抑制Bcl-2、增加Bax 的表达.     

WISP-2siRNA对人脑胶质瘤U251细胞增殖、凋亡的影响

目的 探讨siRNA沉默WISP-2基因表达对人脑胶质瘤细胞U251增殖、凋亡的影响.方法 脂质体介导siRNA转染人脑胶质瘤细胞U251后,检测U251细胞的增殖、凋亡的变化,并用Western blot法检测WISP-2、Bcl-2、Bax蛋白的表达.结果 WISP-2 siRNA能有效抑制U251细胞株的生长,诱导凋亡,siRNA干扰组WISP-2蛋白表达水平为(18.67±1.40)％,明显低于空白对照组(70.18±1.82)％和阴性对照组(69.41±1.77)％,且差异有统计学意义(P＜0.01);siRNA干扰组Bcl-2、Bax蛋白表达水平为(29.67±1.47)％、(31.62±1.32)％,明显低于空白对照组和阴性对照组,且差异有统计学意义(P＜0.01).结论 WISP-2 siRNA抑制WISP-2 mRNA和蛋白表达后,能有效抑制胶质瘤细胞的增殖,增加U251细胞凋亡,可能通过下调Bcl-2/Bax蛋白表达的比值来诱导细胞凋亡.     

白芍总苷对EAE大鼠中枢神经系统炎症浸润细胞凋亡及Bcl-2、Bax表达的影响

目的 探讨白芍总苷（TGP）对实验性自身免疫性脑脊髓炎（EAE）大鼠中枢神经系统（CNS）炎症浸润细胞凋亡及Bcl-2、Bax表达的影响.方法 建立大鼠EAE模型,将大鼠随机分为正常对照组、模型组、TGP组、泼尼松组,TGP组免疫后第1天起每天经口灌服白芍总苷悬浊液0.2 g/kg,泼尼松组给予泼尼松,正常对照组、模型组给予同体积生理盐水溶液,第17天处死,病理切片观察CNS炎症细胞浸润情况,TUNEL法检测浸润细胞凋亡情况,免疫组化检测浸润细胞Bcl-2、Bax蛋白的表达.结果 泼尼松组、TGP组与模型组相比,中枢神经系统炎症浸润细胞的数目减少,凋亡率增高.TGP组与模型组相比,Bcl-2表达下降,Bax的表达上调,Bcl-2/Bax的比值下降.泼尼松组与模型组相比,Bcl-2/Bax的比值下降.结论 TGP能减轻EAE的病情,其机制可能是下调Bcl-2的表达,上调Bax蛋白的表达,降低Bcl-2/Bax比值,促进EAE大鼠CNS炎症浸润细胞的凋亡,减少CNS炎症细胞的浸润.     

CDC2敲低治疗人脑胶质瘤的实验研究

目的 提供CDC2作为胶质瘤发生发展相关新分子的实验依据.方法 构建针对目的 基因的shRNA逆转录病毒表达载体;对体外培养的人脑胶质瘤细胞及其裸小鼠移植瘤进行转染,观察与目标基因相关的表型变化;荧光实时定量PCR检测mRNA表达,Western印迹检测蛋白表达;流式细胞仪检测细胞周期和凋亡的变化.结果 shRNA表达载体使人脑胶质瘤细胞株SHG44 CDC2的mRNA、蛋白表达显著下调,同时出现了明显的细胞G2/M期阻滞,细胞生长抑制,凋亡增加.裸小鼠皮下移植瘤明显缩小;肿瘤颅内移植裸小鼠生存期明显延长.结论 CDC2基因过表达是胶质瘤发生发展病因分子之一,敲低其表达可使肿瘤的恶性增殖得到控制.     

PUMA在人脑胶质瘤中表达及其与Bcl-2、Bax相关性的研究

目的 探讨促凋亡基因PUMA在脑胶质瘤中的表达,及其与Bcl-2、Bax蛋白表达的相关性.方法 应用免疫印迹法(Western blot)检测10例正常脑组织及52例脑胶质瘤组织中PUMA、Bcl-2、Bax蛋白的表达,通过计算吸光度比值求得各蛋白相对表达量并统计分析结果.结果 (1)PUMA蛋白、Bax、Bcl-2蛋白表达在Ⅱ、Ⅲ、Ⅳ级胶质瘤中与正常脑组织差异有统计学意义(P＜0.01),Ⅰ级胶质瘤与对照组表达差异无统计学意义(P＞0.05).(2)病理分级与PUMA、Bax蛋白和Bcl-2蛋白表达相关(rPUMA=-0.925,rBax=-0.931,rBcl-2=0.945,P＜0.001).(3)PUMA和Bax、Bcl-2蛋白的表达相关(r1=0.963,r2=-0.937 P＜0.001).结论 PUMA可能是胶质瘤的一个重要的预后标记物.PUMA可通过促进肿瘤细胞凋亡,对脑胶质瘤的发生、发展起作用;PUMA可能与凋亡相关基因Bax、Bcl-2在脑胶质瘤的凋亡中分别起协同和拮抗作用.     

雷公藤内酯醇对EAE大鼠中枢神经系统炎症浸润细胞凋亡及Bcl-2、Bax的影响

目的 观察雷公藤内酯醇(Tri)对实验性自身免疫性脑脊髓炎(EAE)大鼠中枢神经系统炎症浸润细胞凋亡及凋亡相关蛋白Bcl-2、Bax的影响. 方法 采用四肢足掌皮内注射完全福氏佐剂一豚鼠全脊髓匀浆(CFA-GPSCH),并辅以注射百日咳疫苗,诱导大鼠建立EAE模型.将大鼠随机分为模型组(EAE组)和腹腔注射Tri治疗组(Tri组),免疫后第11天Tri组腹腔注射Tri 40μg/(kg·d),EAE组给予等体积的生理盐水腹腔注射,第17天处死,观察中枢神经系统炎症细胞浸润程度,TUNEL法检测浸润细胞凋亡情况,免疫组化检测浸润细胞Bcl-2、Bax蛋白的表达. 结果 Tri组与EAE组相比,其中枢神经系统炎症浸润细胞的数目减少,凋亡率增高,Bcl-2表达下降,Bax的表达无明显变化,Bcl-2/Bax的比值下降. 结论 Tri可能是通过抑制EAE大鼠中枢神经系统血管周围炎症浸润细胞Bcl-2的表达,降低Bcl-2/bax的比值,从而提高炎症浸润细胞的凋亡率,减少炎症浸润细胞数目,减轻EAE的病情.     

亚低温对大鼠局灶性脑缺血神经细胞凋亡及Bcl—2、Bax的影响

目的 研究不同时程的亚低温对大鼠大脑中动脉闭塞（MCAO）模型的神经细胞凋亡及Bcl-2、Bax的影响。方法 利用MCAO模型,缺血3小时后再灌注。缺血后即刻分别进行不同时程（0．5、1、3小时）的亚低温治疗,再灌注后24小时取脑切片作TUNEL染色及Bcl-2、Bax免疫组化染色。结果 （1）亚低温1、3小时组的凋亡细胞、Bax细胞数与对照组比较均明显降低（P＜0．05,P＜0．01）。（2）亚低温3小时组的Bcl－2细胞阳性率与对照组相比明显增加（P＜0．05）。（3）凋亡细胞阳性率与Bcl-2／Bax比值呈负相关关系（r＝－0．9759,P＜0．01）。结论 缺血性脑损伤的病理过程与细胞凋亡有关,Bcl-2／Bax比值能决定细胞凋亡的发生。亚低温能增强Bcl－2并降低Bax基因的表达,有抑制细胞凋亡的作用,是保护缺血神经元的重要方法之一。     

过表达钙整合素结合蛋白CIB诱导SHG44胶质瘤细胞凋亡的实验研究

目的 观察CIB基因对人胶质瘤SHG44细胞体外生长和细胞凋亡的影响.方法 构建重组质粒pcDNA3-CIB,转染人胶质瘤细胞株SHG44,MTT观察各组细胞的体外生长情况,流式细胞术(FCM)测定各组细胞的细胞周期和凋亡细胞数量,Western-blot检测CIB转染后凋亡相关蛋白的表达变化.结果 RT-PCR、Western印迹显示pcDNA3-CIB转染组CIB的mRNA及CIB蛋白表达水平明显高于对照组;与对照组细胞相比,转染CIB的SHG44-CIB组细胞牛长明显减缓(P＜0.01),SHG44-CIB组细胞G_1、S期细胞减少,G_2期细胞增多,凋亡细胞增加至19.2%;凋亡相关蛋白Bcl-2表达下调,Bax表达上调.结论 CIB在体外明显抑制SHG44细胞的生长并诱导其发生凋亡,CIB诱导的凋亡可能与Bcl-2及Bax表达的变化相关.     

GDNF mediates glioblastoma-induced microglia attraction but not astrogliosis

High-grade gliomas are the most common primary brain tumors. Their malignancy is promoted by the complex crosstalk between different cell types in the central nervous system. Microglia/brain macrophages infiltrate high-grade gliomas and contribute to their progression. To identify factors that mediate the attraction of microglia/macrophages to malignant brain tumors, we established a glioma cell encapsulation model that was applied in vivo. Mouse GL261 glioma cell line and human high-grade glioma cells were seeded into hollow fibers (HF) that allow the passage of soluble molecules but not cells. The glioma cell containing HF were implanted into one brain hemisphere and simultaneously HF with non-transformed fibroblasts (controls) were introduced into the contralateral hemisphere. Implanted mouse and human glioma- but not fibroblast-containing HF attracted microglia and up-regulated immunoreactivity for GFAP, which is a marker of astrogliosis. In this study, we identified GDNF as an important factor for microglial attraction: (1) GL261 and human glioma cells secret GDNF, (2) reduced GDNF production by siRNA in GL261 in mouse glioma cells diminished attraction of microglia, (3) over-expression of GDNF in fibroblasts promoted microglia attraction in our HF assay. In vitro migration assays also showed that GDNF is a strong chemoattractant for microglia. While GDNF release from human or mouse glioma had a profound effect on microglial attraction, the glioma-induced astrogliosis was not affected. Finally, we could show that injection of GL261 mouse glioma cells with GDNF knockdown by shRNA into mouse brains resulted in reduced tumor expansion and improved survival as compared to injection of control cells.     

替莫唑胺及甲泼尼龙对人胶质瘤细胞放射治疗敏感性的影响

目的探讨替莫唑胺（TMZ）和甲泼尼龙（MP）对人胶质瘤细胞系U251放射治疗敏感性的影响,以期为临床优化治疗方案提供依据。方法经体外传代培养的U251细胞根据治疗方案的不同,分为对照组、放射治疗（R）组、放射治疗＋替莫唑胺（R＋TMZ）组、放射治疗＋甲泼尼龙（R＋MP）组、放射治疗＋替莫唑胺＋甲泼尼龙（R＋TMZ＋MP）组,分别予以放射治疗（2Gy／24h）、替莫唑胺、甲泼尼龙及三者联合治疗。采用磺酰罗丹明B（SRB）比色法、流式细胞术和Western blotting法分析U251细胞增殖率和凋亡率,观察凋亡相关蛋白Bax和Bcl-2表达变化。结果放射线照射联合甲泼尼龙治疗后,U251细胞存活率明显升高（均P=0．000）;但经体外培养24和48h后,三者联合治疗组（R＋TMZ＋MP组）U251细胞增殖率显著高于放射治疗联合替莫唑胺组（P=0．000）。除对照组外,不同抗肿瘤治疗组U251细胞凋亡率均呈现升高趋势（P=0．000）,但放射治疗联合甲泼尼龙组细胞凋亡率显著低于其他各组（均P=0．000）。经放射线照射联合替莫唑胺和三者联合治疗后,U251细胞Bax蛋白表达水平升高（P=0．000）,而放射线照射联合甲泼尼龙和三者联合治疗,Bcl-2蛋白表达水平升高;其中以放射治疗联合替莫唑胺组Bax／Bcl-2比值最高（P=0．000）,放射治疗联合甲泼尼龙组最低（P=0．000）。结论甲泼尼龙可以诱导人胶质瘤细胞产生放射抵抗,而替莫唑胺对甲泼尼龙诱导的放射抵抗具有增敏作用。提示：胶质母细胞瘤患者在应用甲泼尼龙减轻放射治疗不良反应过程中所诱导的放射治疗抵抗可通过同时应用替莫唑胺而抵消。     

兔颅脑爆炸伤后早期行高压氧治疗对Cyt C、Bax、Bcl-2表达的影响

目的 研究早期高压氧治疗(HBOT)对兔颅脑爆炸伤后脑皮质细胞色素C(Cty C)、Bax、Bcl-2的影响.方法 新西兰大白兔150只随机分为正常对照组(n=10)、创伤组(n=70)、HBOT组(n=70).创伤组和HBOT组建立兔颅脑爆炸伤模型,正常对照组不行爆炸伤.伤后1、6、12、24 h和3、7、14d,采用免疫组化方法观察3组兔脑皮质中cyt C、Bax、Bcl-2 的表达情况.结果 与正常对照组比较,创伤组伤后1h即出现CytC表达上升,伤后12 h达高峰,伤后24 h开始下降,但维持较高水平:HBOT组CytC的表达在伤后早期较创伤组下降明显(均P＜0.05).与正常对照组比较,创伤组Bax和Bcl-2 的表达在伤后1、6、12、24 h和3d均明显上调(均P＜0.05);HBOT组Bax的表达在伤后早期较创伤组降低(P＜0.05),Bcl-2的表达则明显上调(P＜0.05).结论 早期高压氧治疗对颅脑爆炸伤后的脑保护作用可能与抑制Bax表达,诱导Bcl-2表达上调从而阻止伤后早期线粒体释放Cyt C有关.     

替尼泊苷诱导脑胶质瘤细胞凋亡的检测指标评估

目的：观察化疗药物替尼泊苷在脑胶质瘤化疗过程中诱导的细胞凋亡,探讨进行临床疗效有效监控的评价指标。方法：从40例脑胶质瘤组织中分离胶质瘤细胞,用替尼泊苷对培养的胶质瘤细胞分别干预24、48和72h。用流式细胞技术测定胶质瘤细胞线粒体膜电位（MMP）、细胞周期、Bcl-2和Bax水平。结果：随着替尼泊苷干预时间的延长,MMP降低的百分率和细胞周期凋亡峰比例均增加,Bcl-2／Bax的比值下降。结论：替尼泊苷在脑胶质瘤化疗过程中诱导细胞凋亡的效果显著。在凋亡检测中MMP为最敏感和快捷的检测指标。     

Microglial Exosome miR-7239-3p Promotes Glioma Progression by Regulating Circadian Genes

Glioma-associated microglial cells, a key component of the tumor microenvironment, play an important role in glioma progression. In this study, the mouse glioma cell line GL261 and the mouse microglia cell line BV2 were chosen. First, circadian gene expression in glioma cells co-cultured with either M1 or M2 microglia was assessed and the exosomes of M2-polarized and unpolarized BV-2 microglia were extracted. Subsequently, we labeled the exosomes with PKH67 and treated GL261 cells with them to investigate the exosome distribution. GL261 cell phenotypes and related protein expression were used to explore the role of M2 microglial exosomes in gliomas. Then a specific miR-7239-3p inhibitor was added to verify miR-7239-3p functions. Finally, the mouse subcutaneous tumorigenic model was used to verify the tumorigenic effect of M2 microglial exosomes in vivo. Our results showed that in gliomas co-cultured with M2 microglia, the expression of the BMAL1 protein was decreased (P < 0.01), while the expression of the CLOCK protein was increased (P < 0.05); opposite results were obtained in gliomas co-cultured with M1 microglia. After treatment with M2 microglial exosomes, the apoptosis of GL261 cells decreased (P < 0.001), while the viability, proliferation, and migration of GL261 cells increased. Increased expression of N-cadherin and Vimentin, and decreased E-cadherin expression occurred upon treatment with M2 microglial exosomes. Addition of an miR-7239-3p inhibitor to M2 microglial exosomes reversed these results. In summary, we found that miR-7239-3p in the glioma microenvironment is recruited to glioma cells by exosomes and inhibits Bmal1 expression. M2 microglial exosomes promote the proliferation and migration of gliomas by regulating tumor-related protein expression and reducing apoptosis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12264-020-00626-z.     

Stimulation of TLR9 with CpG ODN enhances apoptosis of glioma and prolongs the survival of mice with experimental brain tumors

Toll-like receptors (TLRs) recognize a set of conserved molecular structures, so called pathogen-associated molecular patterns, which allow them to sense and initiate innate and adaptive immune responses. In this study, we examined the expression of TLRs in both human and murine glioma. We then analyzed the change in TLR expression after treatment with synthetic phosphorothioate oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG ODNs), strong activators of both innate and adaptive immunity. In addition, we investigated the in vivo effect of CpG injection into C57BL/6 mice implanted with syngeneic GL261 glioma. Our results indicate that TLR9 is overexpressed in human and murine glioma cell lines and CpG stimulation prolongs the survival of mice with experimental brain tumors. CpGs induce TLR9 down-regulation, followed by apoptosis of GL261 cells in vitro as well as in vivo. Furthermore, the effects of CpG stimulation appear to enhance the antigen presenting capacity of microglia, shift the immune response toward CD8(+) T cells, and decrease the number of CD4(+)CD25(+) regulatory T cells. Taken together, our data support the role of CpG in glioma immunotherapy and provide a rationale for further clinical development of CpG therapy in patients with malignant glioma.