布比卡因与左旋布比卡因对大肠癌细胞的作用及其与内质网应激通路的相关性研究

发布时间：2018-03-10 16:48

本文选题：布比卡因　切入点：左旋布比卡因　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究1mM布比卡因与1mM左旋布比卡因培养24-48小时后人大肠腺癌细胞系Caco-2的各项肿瘤细胞活性指标以及内质网应激通路标志蛋白的表达水平,包括:癌细胞迁移能力,核蛋白Ki67,膜联蛋白V(Annexin V)和碘化丙啶(PI),78kDa葡萄糖调节蛋白(Grp78)和C/EBP同源蛋白(CHOP)。方法:本实验于体外培养人大肠腺癌细胞株Caco-2并将细胞随机分为四组:空白组(N组);载体对照组(V组)-1mM DPBS液;布比卡因组(B组)-1mM盐酸布比卡因;左旋布比卡因组(L组)-1mM盐酸左旋布比卡因。各组在相同条件下培养24小时后(划痕实验取24小时与48小时两个时间点),使用细胞划痕实验以检测癌细胞的迁移能力;使用免疫荧光技术计算Ki67阳性率比较组间细胞周期情况;使用流式细胞术检测膜联蛋白V和碘化丙啶的表达情况以研究大肠癌细胞的凋亡水平;使用蛋白免疫印迹法和免疫荧光实检测内质网应激通路标志蛋白Grp78和CHOP的表达水平与核聚集现象。结果:在连续48小时体外培养过程中,布比卡因组(p0.01)与左旋布比卡因组(p0.001)的大肠癌细胞迁移速度显著慢于空白组与载体对照组;在24小时培育后,B组与L组的癌细胞胞核中Ki67的阳性率(B组为54.93±7.64,L组为57.23±8.04)显著低于N组(92.33±6.74)和V组(98.66±1.17)(p0.01);通过流式细胞术测得24小时培育后,布比卡因与左旋布比卡因处理并未引起大肠癌细胞的显著凋亡;通过Western Blot和免疫荧光技术测得相比N组和V组,Grp78在B组和L组中表达明显降低(p0.05),而CHOP在B组和L组中表达明显增高(p0.05),同时CHOP在荧光图中呈现显著的核聚集现象。结论:在体外培养的条件下,1mM布比卡因或左旋布比卡因24-48小时的处理可使人大肠腺癌细胞的迁移能力与增殖能力受到同等水平的显著抑制但不能诱发凋亡,其分子机制可能与酰胺类局麻药物对癌细胞内质网应激通路的强化有关。但须注意的是体内外细胞培育理化环境存在差异,关于局麻药物对大肠癌细胞的抑制作用及其机制有待进一步研究。
[Abstract]:Objective: to study the tumor cell activity and the expression level of endoplasmic reticulum stress pathway markers in human colorectal adenocarcinoma cell line Caco-2 after culture of 1 mm bupivacaine and 1 mm levobupivacaine for 24-48 hours, including the ability of cancer cell migration. The nucleoprotein Ki67, annexin V) and GRP 78 kDa glucose regulatory protein (GRP 78) and C / EBP homologous protein CHOPP78) were cultured in vitro, and the cells were randomly divided into four groups: blank group N group; vector group N; vector. Control group (group V): 1 mm DPBS solution; Bupivacaine group (group B) and bupivacaine hydrochloride (group B); The levobupivacaine group was treated with L-bupivacaine hydrochloride (L-1mm) for 24 hours under the same conditions (24 hours and 48 hours after scratch test), and cell scratch test was used to detect the migration ability of cancer cells. Immunofluorescence technique was used to calculate the positive rate of Ki67 and flow cytometry was used to detect the expression of connexin V and pyridine iodide in order to study the apoptosis level of colorectal cancer cells. Western blotting and immunofluorescence were used to detect the expression level of Grp78 and CHOP, and the nuclear aggregation phenomenon of endoplasmic reticulum stress pathway markers. Results: during 48 hours of continuous culture in vitro, The migration rate of colorectal cancer cells in bupivacaine group (P 0.01) and levobupivacaine group (P 0.001) was significantly slower than that in blank group and vector control group. After 24 hours incubation, the positive rates of Ki67 in the nuclei of cancer cells in group B and group L were 54.93 卤7.64 渭 g / L and 57.23 卤8.04, respectively, which were significantly lower than those in group N (92.33 卤6.74) and group V (98.66 卤1.17p0.01), and were measured by flow cytometry for 24 hours. Bupivacaine and levobupivacaine did not induce significant apoptosis in colorectal cancer cells. Compared with group N and group V, the expression of CHOP in group B and L was significantly lower than that in group N and group V by Western Blot and immunofluorescence, while the expression of CHOP in group B and L was significantly higher than that in group B and L. Meanwhile, CHOP showed a significant nuclear aggregation in fluorescence images. Conclusion: under the condition of culture in vitro, 1 mm bupivacaine or levobupivacaine for 24-48 hours can significantly inhibit the migration and proliferation of human intestinal adenocarcinoma cells, but can not induce apoptosis. The molecular mechanism may be related to the enhancement of the endoplasmic reticulum stress pathway in cancer cells induced by amides, but it should be noted that there are differences in the physical and chemical environment of cell culture in vivo and in vitro. The inhibitory effect of local anesthetic on colorectal cancer cells and its mechanism need further study.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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