| Part I:Experimental study of effect of local short time use of high concentration cisplatin solution on malignant bone tumor cellBackground At present, surgical treatment is one of major therapeutic methods of malignant spinal tumor. But because of the existence of complex and important anatomical structure around spinal tumor, such as spine cord, nerves, vessels and so on, it is difficult for spinal tumor to be extensively or radically resected like limbs'. For a long time intralesional excision is the universal clinical surgical methods. With the development of the spinal tumor staging system, operation technology perfection and treatment concept renovation, scholars have put forward the concept of TES(total en bloc spondylectomy). The basic principle of the TES is that the tumor is resected along the normal tissues (marginal excision or wide excision), thereby reducing the local recurrence rate and improve the patient's long-term survival. However, even in TES, in order to protect spine cord, the spinal tumor must be resected extremely carefully, and sometimes intralesional operation will be unavoidable. Especially for violations of a wider range of tumors, there is still the risk of tumor spread. These potential risks can lead to contamination of tumor cells in the surgical field, thereby increasing the chance of recurrence. So when we perform TES, in addition to improving surgical technique to avoid tumor intrasac operation, we should find a method that can kill malignant tumor cells in surgical field and reduce the recurrence of spinal tumor.Objective To investigate the killing effect of local short time use of high concentration cisplatin solution on malignant bone tumor cell, and to find the appropriate drug concentration.Methods Distilled water, different concentration distilled water cisplatin solution and normal saline cisplatin solution (500μg/ml,250μg/ml,125μg/ml,62.5μg/ml,31.25μg/ml) are used to process cultured osteosarcoma cell U2-OS for 5 minutes. At 0k,24h,48h,72h,96h, Absorbance value (A) is determined by MTT assay. Set zero with blank control group. Calculate tumor cell inhibition rate after using different drug and analyze them statistically. Changes of cell morphology and quantity are observed with inverted microscopy at 0h,24h,48h,72h,96h. Distilled water, different concentration distilled water cisplatin solution (500μg/ml,250μg/ml,125μg/ml,62.5μg/ml,31.25μg/ml) are used to process cultured lung adenocarcinoma cell A-549 and breast adenocarcinoma cell MCF-7 for 5 minutes. At 0h,24h,48h,72h,96h, Absorbance value (A) is determined by MTT assay. Set zero with blank control group. Calculate tumor cell inhibition rate after using different drug and analyze them statistically.Results At Oh, there is no significant difference in tumor cell inhibition rates of osteosarcoma cell U2-OS of distilled water group and different concentration distilled water cisplatin groups. And there is no significant difference in tumor cell inhibition rates of distilled water cisplatin groups which concentrations are 500μg/ml,250μg/ml,125μg/ml,62.5μg/ml respectively at 72h and 96h (P>0.05). But these tumor cell inhibition rates are significantly higher than distilled water cisplatin group which concentration is 31.25μg/ml and pure distilled water (P<0.05), and the tumor cell inhibition rates are all more than 95%. At 72h and 96h, there is hardly any osteosarcoma cell to survive after using high concentration distilled water cisplatin (500μg/ml,250μg/ml,125μg/ml,62.5μg/ml) examined under inverted microscope. But many osteosarcoma cells can survive after using distilled water. The tumor cell inhibition rates of normal saline cisplatin groups are significantly lower than distilled water cisplatin groups. At 0h,24h,48h,72h,96h, with the extension of time, the tumor cell inhibition rates of different concentration distilled water cisplatin(500μg/ml,250μg/ml,125μg/ml,62.5μg/ml,31.25μg/ml) and distilled water to lung adenocarcinoma cell A-549 and breast adenocarcinoma cell MCF-7 increase gradually. At 96h, the tumor cell inhibition rates of distilled water cisplatin groups which concentrations are 500μg/ml,250μg/ml,125μg/ml and 62.5μg/ml to lung adenocarcinoma cell A-549 and breast adenocarcinoma cell MCF-7 is significantly higher than distilled water cisplatin group which concentration is 31.25μg/ml and distilled water group, and the tumor cell inhibition rates are all more than 95%.Conclusion The killing effect of local short time use of high concentration distilled water cisplatin solutions which concentrations are 500μg/ml,250μg/ml,125μg/ml and 62.5μg/ml on osteosarcoma cell, lung adenocarcinoma cell and breast adenocarcinoma is strong. The tumor cell inhibition rates are all more than 95%. In clinical practice, we can choose distilled water cisplatin solution which concentration is from 62.5μg/ml to 125μg/ml to irrigate operating field. Thus we can kill the tumor cell in operating field, and decrease the recurrence of malignant tumor.PartⅡ:Safety study of high concentration distilled water cisplatin solution used locally around rat spinal cordBackground Systemic use of cisplatin may lead to the damage of kidney, bone marrow, liver, gonad, thyroid, and other organs. In recent years, more and more scholars began to focus on the damage of cisplatin to spinal cord. According to the literatures, high-dose cisplatin chemotherapy can cause demyelination of central nervous system, including the spinal cord. The neurotoxicity of cisplatin can involve the dorsal root ganglia and cause dysesthesia. Some scholars have studied the nerve toxicity and ultrastructural changes caused by intrathecal injection of cisplatin in rat spinal cord and found that high dose cisplatin can cause spinal cord injury and ultrastructural changes, while the small dose cisplatin will not cause nerve toxicity. In this experiment, as it is not systemic use, but only short-term local administration, side effects of cisplatin may be low. However, due to spinal cord existence around tumors, the spinal cord injury should be considered when the drugs are used. And spinal tumors may invade the dural and intraoperative rupture of the dural sac is sometimes inevitable, thus the spinal cord may be exposed to the drug directly. In this case, whether cisplatin would cause significant spinal cord injury, the present study remains less.Objective To investigate the effect of high concentration cisplatin solution locally used around the rat spinal cord on the rat spinal cord.Methods 40 Wistar rats of 300-350g are randomly divided into two groups,20 in each group, respectively, the experimental group of distilled water cisplatin (DW), the control group of normal saline (NS). Each group is divided into 2 groups,10 in each, namely, the dural sac intact group (A) and the dural sac partial excision group (B). After anesthetized, the 7th,8th,9th arch of the thoracic spine were removed and dura was exposed. Dural sac partial excision group requires careful removal of part of dura and arachnoid. In the operating area of rat laminectomy model,2ml distilled water cisplatin solution which concentration is 500μg/ml or normal saline were injected, soaking for 10 minutes. The solution was reserved. Then the incision was sutured. Dural sac of partial excision group should be carefully sutured to avoid leakage of cerebrospinal fluid. All groups are examined with SEP in preoperation, after laminectomy, after dural partial excision, after injection of drugs, and 3 weeks after operation respectively, and record the latency and amplitude of SEP. All groups are evaluated the lower motional function with modified Tarlov score in preoperation, postoperation 1st,3rd,7th,14th,21rd day. All rats are executed after 3 weeks, and the spinal cords are acquired. The morphological changes of spinal cords are observed in light microscope and electron microscope. The gene expressions of caspase-3 and bax are observed by immunohistochemical staining. All experimental results are statistically analyzed, and the differences are compared among all groups.Results The function of spinal cord of the experimental rats were detected with SEP and data are collected in preoperation,after laminectomy,after dural partial excision,after injection of drugs,and 3 weeks after operation, and the waveform received are ideal. The SEP latency and amplitude after injection of drugs and post-operation 3 weeks with high concentration distilled water cisplatin solution, compared with normal saline, have no significant difference. And The SEP latency and amplitude with high concentration distilled water cisplatin solution in dural intact and partial excision groups have also no statistical difference after injection of drugs and post-operation 3 weeks. The modified Tarlov scores of the rats with high concentration distilled water cisplatin solution, compared with normal saline, have no significant difference. And the modified Tarlov score with high concentration distilled water cisplatin solution in dural intact and partial excision groups have no statistical difference. Under the light microscopy, gray matter neurons of the spinal cord of NSA, NSB, DWA, DWB groups show intact shape, clear and complete nucleus, rich in Nissl bodies, organelles full, clear and complete vessel wall morphology, glial cell nuclear integrity; white matter shows nerve fiber arrangement rules, the connective tissue arranged closely, no significant demyelination. There is no significant difference among experimental groups. Under the electron microscopy, neurons show complete cell membranes, clear, uniform and non-agglutination nuclear chromatin, rough endoplasmic reticulum and mitochondria integrity. Concentric lamellar myelin sheath is arranged in dense order, no delamination and disintegration. There is no significant difference among experimental groups. The expression results of caspase-3 and bax in each group are analyzed by ANOVA(P> 0.05). The expression results of caspase-3 and bax in each group show no significant difference.Conclusion There is no significant effect of high concentration distilled water cisplatin used around the rat spinal cord on the SEP latency and amplitude of rat spinal cord, modified Tarlov scores and morphology by the light and electron microscopy. And the gene expressions of caspase-3 and bax in rat spinal cord aren't obviously upregulated. So it is relatively safe when high concentration distilled water cisplatin is used around the spinal cord, even though spinal dural sac is ruptured.
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