The evaluation of the muscarinicreceptor on the in vitro invasion of the human cholangiocarcinoma cells with Transwell chamber assay / 国际外科学杂志

ABSTRACT

Objective To explore the experiment condition and method for the application of in vitro in vasive Transwell chamber and to observe muscarinicreceptor stimulant and muscarinicreceptor antagonist's influence to cholangiocarcinoma's invasiveness.Methods Two hundred microliter cell suspension of various concentrations(0.5×105/mL,1.0×105/mL,1.5×105/mL and 2.0×105/mL)was added into the upper chamber of the Transwell chamber,and the cells were allowed to penetrate the matrigel for 12,18,24and 48 hours respectively.The numbers was gotten as the invasive cells on the under surface of the membrane.After optimal cell concentration and time were gotten,pilocarpine of various concentrations(0 mmol/L,0.1 mmol/L,0.3 mmol/L and 0.5 mmoL/L)was added into the upper chamber of the Transwell chamber,then the cells on the matrigel were stained and counted.So did the cells when atropine of various concentrations(0.01 mmol/L,0.01 mmol/L,0.05 mmoVL and 0.1 mmol/L)were added into the upper chamher of the Transwell chamber in according to pilocarpine of various concentrations(0 mmol/L,0.3 mmol/L,0.3 mmol/L and 0.3mmol/L).Results With the increase of the time and cell concentrations，the cells couts that penetrated the matrigel increased，while the increase tended to he stable when the culture time exceeded 36 hous and the cell concentration Was over 1.0×105/mL.By adding pilocarpine,there were significant differences between the control and experimental groups(P＜0.05)，but there were no significant differences in experimental groups with various concentrations.There were no significant differences in blank group and experimental groups with atropine added(P＞0.05).When added pilocarpine and atropine,there were significant differences between blank and experimental groups(P＜0.05)，but there were no significant differences in experimental groups with various concentrations.Conclusions Thirty-six hours as invasive time,and one cell concentration 1.0 × 105/mL were optimal to test invasion abilities of cholangiocarcingma cells to different medicines or reagents.There is the possibility that museariniereceptor exists in cholangiocarcinoma cells，and may play an important role in cholangiocarcinoma's invasiveness and metastasis.