A Study Of HFOB Cells Effected By A549 Cells Or EMT-A549 Cells Based On A Microfluidic Divice

Objective: We aimed to build a co-culture system including Human lung adenocarcinoma A549 cells,fibroblast WI38 cells,and human osteoblast hFOB cells based on the microfluidic technology. On which we can study the effect of the fibroblast WI38 cells that facilitate the lung adenocarcinoma A549 cells to undergo EMT and how the EMT-A549 cells exert an influence on the hFOB cells to enhance the expression of RANKL protein which have the ability to promote the osteoclast differentiation.Both the original A549 cells and the EMT-A549 cells will be compared for the ability to change the expression of RANKL,in order to show reference for clinical work to intervene the EMT process and improve symptom of osteolysis caused by lung cancer matastasis to bone.Method:To construct a co-culture platform including microchanal,cell culture chamber,inlet and outlet based on microfluidic techonolgy.Cell culturing for A549 cell line,WI38 cell line and hFOB cell line will be performed on the chip. The experimental subject will be divided into 3 groups including the control group:which perform a single culturing for hFOB, the experimental group1:which perform a co-culture system including A549 cells and h FOB cells,the experimental group2:a co-culture system for EMT-A549 cells and hFOB cells. After all, immunoflurececce will be used to test the expression of RANKL which located on the membreain of hFOB cells,the transwell invasion assay and western blot will be performed to test the result showed on the chip.Result:1.Human lung adenocarcinoma A549 cells present an obviously EMT transformation after co-cultured with WI38 cells.The expression level of E-Cadherein decreased, while the expression level of Snail increeased.The invasion assay demomstrated that the WI38 cells show an ability which facilitate A549 cells to matastasis.2.After co-cultured with hFOB cells,the EMT-A549 cells show a MET transformation.Western blot assay and invasion assay demonstrated that EMT-A549 cells have the ability to invate to bone.3.Extract all the protein respectively from the control group(single culturing of hFOB),experimental group1(co-culture of hFOB and A549) and experimental group2(co-culture of EMT-A549 and hFOB).Testify the expression level of RANKL by performing western blot assay. The result shows the expression level of RANKL in experimental groups are higher compared with that in the control groupConclusion:This study constructed a platform based on the microfluidic technology to mimic the tumor cells metastasis to bone.It compared the different effection on hFOB cells caused by A549 cells and EMT-A549 cells and demonstrated that both types of A549 cells have the ability to resolve the bone.The experiment provide a new technical mean for clinical research to study how lung cancer or other kinds of tumor matastasis to their target organs.What is more,in vitro evidences for tumor invasion and matastasis can be provided which helps to find new ways for tumor prevention and treatment.