脑淋巴引流阻滞后蛛网膜下腔出血兔脑脊液对PC12细胞的损伤作用

Injured effect of cerebrospinal fluid from experimental subarachnoid hemorrhage after cerebral lymphatic blockage on PC12 cells

目的:探讨脑淋巴引流阻滞(CLB)后蛛网膜下腔出血(SAH)兔脑脊液对大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)的损伤作用。方法:建立兔SAH及CLB模型,于SAH模型建立后5d抽取脑脊液加入PC12细胞培养基中,随机将PC12细胞分为空白对照组(无血清F12培养基)、正常脑脊液组、SAH脑脊液组、SAH+CLB脑脊液组,分别于培养0.5h、1h、2h、4h后,通过四甲基偶氮唑盐法(MTT)检测细胞存活率并测定乳酸脱氢酶(LDH)释放率,采用免疫组化法检测细胞凋亡蛋白Bax及热休克蛋白70(HSP70)的表达。结果:MTT、LDH结果显示,与正常脑脊液组比较,SAH脑脊液组和SAH+CLB脑脊液组脑脊液使PC12细胞存活率降低,LDH释放率增加,以后组更为显著;正常脑脊液对PC12细胞无明显损伤作用。在SAH脑脊液组和SAH+CLB脑脊液组均发现PC12细胞Bax及HSP70蛋白表达;Bax蛋白表达后组大于前组,且呈时间依赖性增强;在0.5h和1h,SAH+CLB脑脊液组HSP70蛋白表达强于SAH脑脊液组,而在2h和4h则表达减弱。结论:脑淋巴引流阻滞可加重SAH兔脑脊液对PC12细胞的损伤,表明脑淋巴引流通路在SAH后可能起到内源性保护作用。

AIM： To determine the injured effect of cerebrospinal fluid（CSF） from subarachnoid hemorrhage （SAH） after cerebral lymphatic blockage （CLB） on PC12 cells. METHODS： SAH and CLB models of adult New Zealand rabbits were used. CSF was obtained from experimental animals after 5 d of modeling and was added into cultured PC12 cells. The cells were randomly divided into blank control（F12 Ham＇ s）, normal CSF, SAH CSF, and SAH ＋ CLB CSF groups. At different time points, the survival rate of PCI2 cells was measured by MTT assay. LDH leakage was detected. Expression of Bax and heat - shock protein 70 （HSP70） was determined by immunohistochemical staining. RESULTS ： MTr assay and detection of LDH leakage revealed that the survival rate of PC12 cells was obviously inhibited and the leakage of LDH increased in SAH CSF group and SAH ＋ CLB CSF group. CSF from normal rabbit did not damage the PC12, as compared to blank controls. Above effects were more obvious in SAH ＋ CLB CSF group than those in SAH CSF group. Bax and HSP70 protein expression was found in both SAH CSF group and SAH ＋ CLB CSF group. Expression of Bax protein in SAH ＋ CLB CSF group was stronger than that in SAH CSF group in a time dependent manner. At 0.5 h and 1 h, the expression of HSP70 protein in SAH ＋ CLB CSF group was stronger than that in SAH CSF group, whereas the expression became weaker at 2 h and 4 h in that group. CONCLUSION ： Blockage of cerebral lymphatic drainage pathway deteriorates the damage of CSF from SAH on PC12 ceils, indicating this pathway may acts as an endogenous protective mechanism in SAH.