The Expression Of TfR In Glioma And Its Construction And Identification Of Plasmid As A Report Gene In Molecular Imaging Of MR

Objective Glioma is one of the most common tumor toneurosurgery, it is difficult to be resected completely, not sensitive tochemotherapy and radiotherapy, easy to recur, we cannot reach goodtherapeutic efficacy and make early diagnosis through common CT, MRand other imageological means,we also have difficulty in doing zooperalresearch in vivo.Transferrin receptor(TfR) is a kind of transmembranceglycoprotein in cellular membrance,it mediate absorption of iron throughinterreaction with transferrin,has close relation with grouth, proliferation,differentiation and canceration of cell,it is a ideal report gene inmolecular imaging of MR.In our research,we observe the expression ofTfR in different grades gliomas and normal brain tissue,and analyze therelation of expression with pathology and different pathologicalclassification,discuss the possibility of using TfR as report gene,makeexpression plamid including coding region of TfR, carry out detection andidentification, provide the possibility for the molecular imaging of MR invivo and vitro.Method We collect 38 samples of different grades gliomas and 6samples of normal brain tissue because of cerebral trauma fromDecember 2005 to October 2006 in Neurosurgery of XiangYa hospital ofCentral South University, examine the expression of TfR in glioma andnormal tissue by immunohistochemistry and analyze the relationship ofTfR expression of glioma in the pathological grades and normal tissue.Inaddition,we extract total RNA from cultural U251 cell of humanglioma,make coding gene extron amplification of TfR through ReverseTranscription-Polymerase Chain Reaction(RT-PCR),construct expressionplasmid of pCDNA3.1(+)-TfR after digestion of enzyme andconjunction,do transfection to U251 cell by liposome afteridentification,cause excess expression,detect the efficiency of transientexpression of TfR protein by pEGFPN1-TfR, sieve positive cell ofpCDNA3.1(+)-TfR through G418, choose positive cell clone, detect theexpression of TfR in anteroposterior period through Western blot,FCM,cell immunochemistry and mRNA through RT-PCR, identify the effect of transfection.Result The result show that the expression of TfR in both differentgrades gliomas and normal brain tissue is positive according toimmunohistochemistry of histopathologic slide,the rate of positiveexpression in glioma cell is 100 percent,there are expression in neurn andvascular endothelial cell of normal brain tissue,no evident expression inglial cell,the level of TfR expression in glioma is higher than normaltissue(P＜0.01),and there is positive correlation in TfR expression oftumor and pathologic classification, the expression of TfR in high-gradegliomas is higher than low-grade (P＜0.01), but there are no variabilitiesin the same grade gliomas (P＞0.05). We extract total ribonucleicacid(RNA) of cultural U251 cell,amplify the coding region extron of TfRthrought transcription-polymerase chain reaction(RT-PCR),constructexpression plasmid of pCDNA3.1(+)-TtR successfully, identify thecorrectness through sequencing; the efficiency of transfection to U251cell by liposome is about 25 percent,the expression of TfR protein in thetransient and stable transfection of pCDNA3.1(+)-TfR cell is higher thanno transfection cell (P＜0.01), we got stable positive cell clone of TfRprotein through G418.Conclusion The expression of TfR in glioma is higher than normalbrain tissue,the expression in high-grade glioma is higher thanlow-grade; the higher expression of TfR may play a part in growth,proliferation,differentiation in tumor.We construct pCDNA3.1(+)-TfRexpression plasmid successfully, there are higher expression of TfR aftertransient and stable transfection; we get positive cell clone of containingpCDNA3.1(+)-TfR, establish infarctate foundation to empirical study ofmolecular imaging of MR.