Transwell Invasion Assay Determination Of M Receptor Cholangiocarcinoma Cell Invasion

Objective:To explore the experiment condition and method for the application of in vitro invasive Transwell chamber and to observe muscarinicreceptor stimulant and muscarinicreceptor antagonist's influence to cholangiocarcinoma's invasiveness. Methods: 200μl cell suspension of various concentrations was added into the upper chamber of the Transwell chamber, and the cells were allowed to penetrate the matrigel for 6,12,18 and 24 hours respectively. The numbers was gotten as the invasive cells on the under surface of the membrane. After optimum cell concentration and time was gotten, pilocarpine of various concentrations(0 0.1 0.30.5 mmol/l) were 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 0.01 0.05 0.1 mmol/l) were added into the upper chamber of the Transwell chamber in according to pilocarpine of various concentrations(0 0.3 0.3 0.3 mmol/l). Results:The invasive number of tumor cells was still low at 12 hours after the incubation, and increased with the time goes on, and after 36 hours tended to be smooth. Besides, the invasive rate went high as the cells concentration added increaed. while not dramatically when above 1.0×10^5/ml. When added pilocarpine, there were significant difference between blank and experimental groups (P<0.05),but there were no significant difference in experimental groups with various concentrations. There were no significant difference in blank group and experimental groups with atropine added (P>0.05). When added pilocarpine and atropine, there were difference between blank and experimental groups (P<0.05),but there were no difference in experimental groups with various concentrations. Conclusions:36 hours were supposed to most exemplifies cholangiocarcinoma cells' invasiveness as invasive time, to cell concentration, is 1.0×10^5/ml, which was considered had minimal impact on the observation to invasiveness. There is the possibility that muscarinicreceptor exists in cholangiocarcinoma cells, which were accounted to cholangiocarcinoma' invasiveness and metastasis.