枸杞多糖通过调节血脑屏障抑制大鼠胶质瘤的生长

Lycium barbarum polysaccharide inhibits the growth of rat glioma by regulating the blood-brain barrier

目的:探讨枸杞多糖(lycium bararum polysaccharide,LBP)对大鼠胶质瘤生长的抑制作用及其可能的作用机制。方法:用400、200、100、50和25 mg·kg-1·d-1的LBP和饮用水(对照组)喂养大鼠[分别称为LBP A组、LBP B组、LBP C组、LBP D组、LBP E组和F组(对照组)],然后通过手术建立大鼠胶质瘤C6细胞的原位肿瘤模型。观察胶质瘤大鼠的生存期,并测量肿瘤体积;应用FCM法检测胶质瘤大鼠血液中CD3+CD8+T细胞所占百分比,免疫组织化学法检测大鼠胶质瘤组织中CD3和CD8的表达。胶质瘤大鼠股静脉注射伊文思蓝溶液后,观察LBP对大鼠血脑屏障通透性的影响,在扫描电子显微镜下观察大鼠脑组织超微结构的变化。实时荧光定量PCR和蛋白质印迹法分别检测大鼠胶质瘤组织中CD8、膜联蛋白A1(annexin A1,ANXA1)mRNA及ANXA1蛋白的表达。结果:LBP A组、LBP B组和LBP C组胶质瘤大鼠的存活时间长于F组(对照组)(P值均<0.05),LBP A组、LBP B组和LBP C组大鼠胶质瘤体积均小于LBP D组、LBP E组和F组(对照组)(P值均<0.05)。LBP C组胶质瘤大鼠血液中CD3+CD8+T细胞所占百分比[(18.9±1.4)%]高于F组(对照组)[(11.5±0.7)%,P<0.01]。LBP C组大鼠胶质瘤组织中CD3+T细胞和CD8+T细胞数多于F组(对照组)(P值均<0.01)。伊文思蓝溶液进入LBP C组大鼠脑组织的含量增多,毛细血管内皮细胞皱缩,基膜厚薄不均,部分断裂。LBP C组大鼠胶质瘤组织中CD8 mRNA的表达水平上调(P<0.01),而ANXA1 mRNA及蛋白表达水平均明显下调(P值均<0.01)。结论:LBP可以抑制胶质瘤生长,延长胶质瘤大鼠的生存期,其机制可能是LBP通过调节血脑屏障促使CD8+T细胞进入颅内,抑制肿瘤生长。

Objective： To investigate the inhibitory effect of lycium barbarum polysaccharides （LBPs） on growth of glioma in rats and its possible mechanism. Methods： The rats were divided into LBP A group, LBP B group, LBP C group, LBP D group, LBP E group and F group （control group） to be fed with 400, 200, 100, 50 and 25 mg.kg-1.d-1 LBP and drinking water, respectively. The model of rat C6 glioma in situ was established through surgery. The survival of the glioma-bearing rats was observed, and the tumor volume was examined. The percentage of CD3＋CD8＋T cells in rat blood was detected by FCM method. The expressions of CD3 and CD8 in glioma tissues in rats were detected by immunohistochemistry. After injection of Evans blue solution through femoral vein, the effect of LBP on the permeability of blood-brain barrier in rats was observed, and the change of ultrastructure of brain tissues was observed under a scanning electron microscope. The expressions of CD8 and annexin A1 （ANXA1） mRNAs and ANXA1 protein in glioma tissues in rats were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. Results： The survival of glioma-bearing rats in LBP A, LBP B and LBP C groups was superior to that in F group （all P 〈 0.05）. The tumor volume of glioma-bearing rats in LBP A, LBP B and LBP C groups was smaller than that in F group （all P 〈 0.05）. The percentage of CD3＋CD8＋ T cells in blood of glioma-bearing rats in LBP C group was higher than that in F group [（1 8.9±1.4）% vs （11.5±0.7）%, P 〈 0.01]. The number of CD3＋ and CD8＋ T cells in glioma tissues in rats in LBP C group was higher than that in F group （both P 〈 0.01）. The amount of Evans blue solution into the brain tissues in LBP C group was increased, the capillary endothelial cells were shrinkable, and the basement membrane was uneven with partial fracture. The expression level of CD8 mRNA in glioma tissues in rats was up-regulated （P 〈 0.01）, whereas the expression levels of ANXA1 mRNA and protein were down-regulated （both P 〈 0.01）. Conclusion： LBPs can inhibit the growth of glioma and prolong the survival of glioma-bearing rats. The mechanism may be related to the inhibitory effect of LBPs on the growth of tumor by regulating the blood-brain barrier and promote the invasion of CD8＋T cells into the brain.