α7烟碱型受体兴奋剂、拮抗剂对胆管癌细胞侵袭能力的影响及机制

Effect of α7 nicotinic receptor agonist and antagonist on invasion ability of cholangiocarcinoma cells

目的观察α7烟碱型受体兴奋剂尼古丁、拮抗剂银环蛇毒素(α-BTX)对胆管癌细胞侵袭能力的影响,并探讨机制。方法取对数期胆管癌细胞,去血清培养12 h,取200μL浓度分别为0.5×105、1.0×105、1.5×105、2.0×105/mL的胆管癌细胞加入Transwell上室,下室加入10%血清的RPMI1640培养液600μL,于12、24、36、48 h观察侵袭细胞数。结果随细胞浓度及作用时间的增加,侵袭细胞数逐渐增加,但当浓度超过1.5×105/mL,时间超过36 h后,侵袭细胞数增长缓慢,据此筛选出1.5×105/mL细胞浓度、作用时间36 h用于后续实验。①取1.0×105/mL的胆管癌细胞加入Transwell上室,分为实验1、2、3、4组,分别加入尼古丁0、1×10-2、1×10-3、1×10-4g/L,培养36 h后0.1%结晶紫染色并于倒置显微镜下计数侵袭细胞数。②取1.0×105/mL的胆管癌细胞加入Transwell上室,分为实验1、2、3、4组,分别加入尼古丁0、1×10-2、1×10-2、1×10-2g/L,α-BTX分别为2、2、4、6μg/mL,培养36 h后0.1%结晶紫染色并于倒置显微镜下计数侵袭细胞数。结果单纯尼古丁作用后穿过基质胶的细胞数增多,实验1、2、3、4组细胞侵袭数分别为(17.90±1.70)、(25.56±1.02)、(26.03±1.32)、(23.22±1.24)个,实验1组与后三组比较,P均<0.05;尼古丁联合α-BTX共同作用后穿过基质胶的细胞数减少,实验1、2、3、4组细胞侵袭数分别为(16.89±1.27)、(18.16±0.89)、(18.20±1.46)、(18.15±2.01)个,实验1组与后三组比较,P均<0.05。结论尼古丁可明显增强胆管癌细胞的侵袭能力,α-BTX可明显抵消尼古丁所产生的促侵袭能力,其机制可能通过改变α7烟碱型受体活性而发挥作用。

Abstract： Objective To observe the effect of α7 nicotinic receptor agonist nicotine, antagonist-bungarotoxin （α- BTX） on the invasion ability of cholangioearcinoma cells and to discuss the mechanism. Methods We extracted the logarith- mic phase eholangiocarcinoma cells for serum-free culture for 12 h. The eholangiocarcinoma cells at the concentration of 0.5 x 10^5, 1.0 x 10^5, 1.5 x 10^5 and 2.0 x 10^5/mL were separately added into Transwell upper chamber, 600 μL of the RP- MI1640 culture solution at 10% serum was added into the lower chamber, then we observed the number of the invasive cells at 12 h, 24 h, 36 h, and 48 h. The results increased with the increase of cell concentration and action time, and the results also gradually increased with the number of invasive cells, but when the concentration exceeded 1.5 x 10^5/mL and the action time exceeded 36 h, the number of the invasive cells increased slowly; accordingly, 1.5 x 10^5/mL cell concentration was screened, 36 h of reaction time for subsequent experiments. ① 1.0 x 105/mL cholangiocarcinoma cells were taken to be used in the experimental groups 1, 2, 3 and 4. Then we added nicotine O, O. 1 x 10^-2, 1 x 10 ^-3 and 1 x 10^-4 g/L into the upper chamber of Transwell respectively, after being cultured for 36 h and stained with O. 1% crystal violet, we counted the number of the invasive cells by using inverted microscope. ② 1.0 x 10^5/mL eholangioeareinoma cells were taken to add into the up- per chamber of the Transwell, and then were used for experimental groups 1, 2, 3 and 4. Then we added nicotine O, O. 1 x10^-2, 1 ×10-2, 1×10^-2 g/L, and ot-BTX 2, 2, 4, 6 μg/mL respectively. After being culture for 36 h and stained with 0. 1% crystal violet, we counted the number of the invasive cells by using inverted microscope. Results It showed an in- crease in the number of cells to pass Matrigel after the action of pure nicotine, the numbers of the cell invasion in the experi- mental groups 1,2, 3 and 4 were（ 17.90 ± 1.70）, （25.56 ±1.02）, （26.03 ± 1.32） and（23.22 ± 1.24）, respectively. When we compared the experimental group 1 with experimental groups 2, 3 and 4, all P 〈 0.05. It showed a decrease in the number of cells to pass Matrigel after the action of nicotine combining with ct-BTX. The numbers of the cell invasion in the experimental groups 1,2,3 and 4 were （ 16.89 ± 1.27）, （ 18.16 ± 0.89）, （ 18.20 ±1.46） and （ 18.15 ±2.01 ）, respective- ly. When we compared the experimental group 1 with experimental groups 2, 3 and 4, all P 〈 0.05. Conclusions Nicotine can significantly enhance the invasion ability of cholangiocarcinoma cells, and ct-BTX can significantly offset the invasiveness generated by the nicotine, its mechanism may play its role by changing the α7 nicotinic receptor activity.