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目的:同一晚期卵巢癌患者,对组织来源和腹水来源的exosomes进行方法和数量的比较,探讨获得卵巢癌患者exosomes的最佳途径.方法:收集晚期卵巢癌患者卵巢癌组织,分离癌细胞进行培养,留上清液,取同一患者的腹水,通过超速离心和超滤等方法提取上清液及腹水中的exosomes,电镜下检测;Western Blot测定exosomes上HSP70,HSP90,MHCⅠ类分子和MHCⅡ类分子的表达;用BCA蛋白测定法测定exosomes的蛋白浓度,计算蛋白总含量.结果:腹水离心时间比上清液离心时间长,所有细胞培养上清液和癌细胞阳性的腹水均能分离出exosomes,癌细胞阴性的腹水中未分离出exosomes;两种来源的exosomes共同表达MHCⅠ类分子、HSP70和HSP90,均不表达MHCⅡ类分子,腹水中分离出来的exosomes蛋白含量比细胞培养上清液的含量高(P<0.05).结论:腹水中癌细胞阳性的患者,腹水是exosomes的比较理想的来源;无腹水或腹水中癌细胞阴性的患者,exosomes可来源于卵巢癌组织.
OBJECTIVE: To compare the method and quantity of exosomes derived from tissue and ascites from patients with advanced ovarian cancer and to explore the best ways to obtain exosomes from patients with ovarian cancer.Methods: The ovarian cancer tissues were collected from patients with advanced ovarian cancer and cultured to separate the cancer cells The supernatant and ascites were extracted by ultracentrifugation and ultrafiltration. The exosomes were detected by electron microscopy. The expression of HSP70, HSP90, MHC class I molecules and MHC class II molecules on exosomes were detected by Western Blot .The protein concentration of exosomes was determined by BCA protein assay and the total protein content was calculated.Results: Centrifugal time of ascites was longer than that of supernatant, exosomes could be isolated from all cell culture supernatants and ascites of cancer cells, The exosomes were not isolated from the ascites of the cancerous cells. Both exosomes expressed MHC class I molecules, HSP70 and HSP90, neither expressed MHC class II molecules. The exosomes protein content in ascites was higher than that in the cell culture supernatant (P0.05) .Conclusion: Ascites is the ideal source of exosomes in patients with positive ascites cancer cells; no ascites or cancer cells in ascites Of patients, exosomes may be derived from ovarian cancer.