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顺铂是一种用于治疗恶性肿瘤的有效铂类化疗药物。然而顺铂对机体许多组织器官,如肾脏、肝脏、神经系统以及内耳等都具有毒性损害作用。FLIVO是一种能够穿越机体组织屏障进入到机体每个细胞并用荧光标记处于凋亡活动状态半胱天冬酶的亲脂性注射用示踪剂。本研究应用袢利尿剂(利尿酸钠,40mg/kg,Ⅰ.V.)暂时性破坏南美栗鼠的血-迷路屏障,促使顺铂(0.8 mg/kg,I.P.)经蜗管外壁屏障消除处进入耳蜗从而引起耳蜗毛细胞凋亡。受试南美栗鼠在联合应用利尿酸钠和顺铂后6小时和18小时终止实验。为了检测顺铂引起的发生在耳蜗和中枢以及肝肾等器官的细胞凋亡,在终止实验前经颈静脉注入100μlFLIVO探测液体并使之随着血液循环60分钟以探测出现在全身各个脏器的凋亡细胞。终止实验时,对麻醉动物常规施行心脏灌流磷酸盐缓冲液5分钟,再经心脏灌流10%福尔马林磷酸盐固定液,然后分别取出耳蜗、耳蜗核、听皮层、海马以及肝肾组织并浸入上述固定液继续固定6小时。在解剖显微镜下分离取出全耳蜗基底膜并制备成全耳蜗基底膜铺片,耳蜗核、听皮层、海马、肝脏和肾脏则常规制备成冰冻切片。在共聚焦显微镜下,观察FLIVO在上述各个器官标记出的凋亡细胞。在正常南美栗鼠各个组织器官中,均未发现FLIVO标记的凋亡信号;在用药后6小时,仅在耳蜗外毛细胞及肾组织中检测出凋亡细胞,但在其它器官也未发现FLIVO标记的凋亡细胞;与用药后6小时相比,在用药后18小时,所有的耳蜗外毛细胞和耳蜗底回大部分内毛细胞都呈现出荧光标记的凋亡信号,出乎意料的是,尽管耳蜗腹侧核神经元出现了大量凋亡神经元,但耳蜗背侧核的神经元却未检测到明显的凋亡信号;值得注意的是,凋亡信号还出现在更为核心的中枢海马神经元和听皮层神经元。此外,肾脏组织和肝脏组织在用药后18小时也出现大量的凋亡细胞。这些结果表明,联合应用利尿酸钠和顺铂不仅导致大量耳蜗毛细胞凋亡,而且顺铂的神经毒性作用还造成了耳蜗腹侧核大量神经元的凋亡和耳蜗背侧核和海马及听皮层的部分神经元凋亡,顺铂同样导致大量的肾脏细胞和肝脏细胞凋亡,出现在上述各个脏器的细胞凋亡现象与顺铂的耳毒性作用、神经毒性作用、肾毒性作用及肝毒性作用完全一致。
Cisplatin is a potent platinum-based chemotherapeutic drug used to treat malignant tumors. Cisplatin, however, has toxic effects on many tissues and organs of the body, such as the kidneys, liver, nervous system and inner ear. FLIVO is a lipophilic injectable tracer that penetrates each cell of the body through the body tissue barrier and fluoresces to label an apoptotic active caspase. In this study, diuretics (sodium leucovorin, 40 mg / kg, Ⅰ.V.) were used to transiently destroy the blood-labyrinth barrier of S. chinensis and promote cisplatin (0.8 mg / kg, IP) The cochlea causes cochlear hair cell apoptosis. The test chinchillas terminated the experiment at 6 and 18 hours after the combination of sodium and sodium urate. In order to detect cisplatin-induced apoptosis in the cochlear and central nervous system, liver and kidney and other organs, 100 μl of FLIVO detection fluid was injected through the jugular vein before termination of the experiment and allowed to circulate through the bloodstream for 60 minutes to detect the presence of Apoptotic cells. At the end of the experiment, the animals were routinely perfused with cardioversion phosphate buffer for 5 minutes and then perfused with 10% formalin phosphate solution in the heart, and then cochlear, cochlear nucleus, auditory cortex, hippocampus and liver and kidney tissues were removed Immersed in the fixative continued for 6 hours. The whole cochlear basal membrane was isolated and dissected from the cochlear basal membrane, and the whole cochlear basal membrane membrane, cochlear nucleus, auditory cortex, hippocampus, liver and kidney were prepared and frozen. Under confocal microscopy, FLIVO was observed in each of the above organs labeled apoptotic cells. No FLIVO-labeled apoptotic signal was detected in all tissues and organs of normal South American chinchillas; apoptotic cells were detected only in cochlear outer hair cells and renal tissues 6 hours after treatment, but FLIVO markers were not found in other organs Of apoptotic cells; all of the inner hair cells of cochlear outer hair cells and most of the inner hair cells of cochlear bottom showed fluorescently labeled apoptotic signals 18 hours after treatment, compared with 6 hours after treatment. Unexpectedly, Although a large number of apoptotic neurons were present in the ventral nuclear nucleus of the cochlea, no obvious apoptotic signal was detected in the dorsal nucleus of the cochlea; it is noteworthy that apoptotic signals also appeared in the more central central hippocampus Neurons and cortical neurons. In addition, a large number of apoptotic cells also appeared in kidney and liver tissues 18 hours after treatment. These results indicate that combination of sodium and cisplatin not only leads to apoptosis of a large number of cochlear hair cells, but also the neurotoxic effect of cisplatin also causes apoptosis of a large number of neurons in ventral cochlear ventral and cochlear dorsal nucleus and hippocampus Cortical neurons apoptosis, cisplatin also lead to a large number of renal cells and liver cells apoptosis, appear in the above organ apoptosis phenomenon and cisplatin ototoxicity, neurotoxicity, nephrotoxicity and liver Toxicity is exactly the same.