Study Of Effect And Mechanism Of CD147 On The Invasion And Metastasis Of The Bile Duct Carcinoma

Background:Bile duct cancer is a common neoplasms in hepatobiliary clinic. Athough the resect rate of this tumor is improved markedly, the tumor recurrence rate is still high, which is believed to owe to the invasion and metastasis of the tumor. The metastasis and invasion of the bile duct cancer have disturbed surgery for a long time. For their properties of invasion, the therapeutic efficacy of traditional operation, chemotherapy and radiotherapy is not satisfactory. CD147, also called extracellular metallopoteinase inducer(EMMPRIN)or Basic immunoglobulin(Basigin)or M6, has been give a CD name CD147 now,which is a member of the immunoglobulin superfamily and is a highly glycosylated cell surface protein. CD147 is an moleculer which wild express on the surface of many tumor, It also has been found that expression level of CD147 followed malignant degree of the tumor.CD147 molecule not only stimulate fibroblasts which sorund the tumor to secrete matrix metalloproteinases (MMPs) but also bind to MMPs on the tumor surface. MMPs have been implicated in several aspects of tumor progression, including invasion through basement membranes and interstitial matrices, angiogenesis, and tumor cell growth . Degradation of extracellular matrix components of the basement membrane and interstitial matrix by MMPs is a crucial step in tumor cell invasion and metastasis. In despite of CD147 can stimulates fibroblast to produce MMPs ,It was not been found that CD147 moleculer expressed on the fibroblast. The receptor molecule of CD147 in fibroblasts to enhance MMPs production has nto been clarified yet. Whether CD147 as a molecule to play a role in the invasion and metastasis of bile duct carcimoma has not been identified .Blocking of CD147 molecule expression or functon may be a effective way to inhibit development of tumor .Objective: In order to find out the effect and possible mechanism of CD147 on the invasion and metastasis of the bile duct carcinoma, We constructed a replication deficient recombinant cDNA for CD147 in an antisense orientation and studied the effect on the biological behavior of bile duct cancer transferred with Ad-asCD147 ,The relationship between the CD147 and the invasion and metastasis of bile duct cancer cell is explored too. Methods and results:1. Identifying expression of CD147 ,mmp-2 and mmp-9 on human bile duct cancer cell strain QBC939. Expression of CD147,mmp-2 and mmp-9 in bile duct cancer strain QBC939 was investigated by indirect immunofluorescence and RT-PCR .Results show that CD147,mmp-2 and mmp-9 was positively presented in the QBC939 cell strain .2. Reconstruct a replication deficient recombinant adenoviral vector containing the CD147 cDNA in an antisense orientation.The CD147 gene was amplifide by reverse transcriptage-polymerase chain reaction (RT-PCR) after total RNA being extracted from human bile duct cancer cell strain QBC939 and then cloned it into pTZ57R vector,The recombinant plasmids were cut by two enzymes and obtain CD147 cDNA fragmetn .The pshuttle-cmv-asCD147 vector,an expression plasmid of CD147 gene antisense RNA was constructed after the object CD147 cDNA fragmetn was reversely inserted into pshuttle-cmv vector .Then BJ5183 cell are transformed with Lnearize pshuttle-cmv-asCD147 vector.A recombination event that takes place in the bacterial cells results in the production of recombinant Ad-asCD147. The big fragment which was obtained by Lnearizing Ad-asCD147 was transferred into 293 cells.Finally,Subject the cell suspension to four rounds of freeze/thaw.Ad-asCD147 was harvested in 293 cell line and purified by cesium choride density purification.It was found that the prepared Ad-asCD147 titer was5.012×109PFU/ml 3. Study on Ad-asCD147 mediated antisense CD147 gene in bile duct cancer cell.MTT was used to deteminate the cell growth rate and no reduction in the growth rate was found after infection with Ad-asCD147.The result of DNA counts of QBC939 cell analyzed by the use of flow-cytometry(FCM) was according with that of MTT. The expression of mRNA and p...