The Effects Of Her2/neu Gene Silencing On The Proliferation Of Meningioma

Objective：1. This subject is to collect postoperative specimen of meningioma for primaryculture and identified it.2. Human meningioma cells were transfected by Her2siRNA lentiviral，thenobserve the proliferation activity and biological changes of human meningioma cellsbefore and after transfection.Methods:1. The subject collected neurosurgical postoperative of fresh human meningiomatissue, obtained the logarithmic phase meningioma cells through primary culture andsubculture,then used immunohistochemical and FISH method to identify them.2. The meningioma cells was infected by the Her2siRNA lentivirus vectorwhich was chemical synthesis, after1-4days,Observing the lentivirus’ expression todetermine the appropriate multiplicity of infection(MOI) using the invertedmicroscope.3. The experiment was divided into three groups：①C ontrol group：the humanmeningioma cells were cultured with DMEM medium containing10%serum；②Mock group：the meningioma cells were infected with lentivirus of empty vector；③H er2siRNA group：the meningioma cells were infected with lentivirus of Her2siRNA.4.72hours after lentivirus infection, using CCK-8to test the proliferationinhibition rate of meningioma cells from each group；and using RT-PCR to detectmRNA expression of Her2/neu gene was detected by；using the western blot todetermine the expression of Her2、VEGF、Ki-67protein.5. The nude mice has been inoculated with cells from three groups through theirarmpits,then observing the weigh of tumors in the nude mice. Results：1. The fresh specimens collecting from human meningiomas were cultured in10%DMEM culture medium.polygonal cells sticked the wall of the bottle.2. Her2siRNA lentivirus vectors were established by Shanghai HanHeng limitedby share Ltd. The virus titer was1×108TU/ml packaging.3. AS the lentivirus of Her2siRNA infected human meningioma cells, the weakgreen fluorescence was observed after48hours when the MOI value was40,it willenhance after72h.4. The proliferation of meningioma cells were detected by CCK-8method.Compared with control group, the inhibition rate of meningioma cells was highest inHer2siRNA group(P<0.01),the lentivirus began to inhibite the proliferation ofmeningioma cells at48hours after transfection when to72h,it had the strongestinhibitory effect.No significant difference were found in Mock group and highglucose group (P>0.05).5. Expression of RT-PCR for detection of Her2:Compared with Control groupand Mock group, the mRNA level in Her2siRNA group was significantly reduced(P<0.01).There was no significant difference between Control group and Mock group(P>0.05).6. Changes in the expression of Western Blot detection of Her2、VEGF、Ki-67protein:the protein expression of Her2siRNA group were down regulated than thatof control group and Mock group (P<0.01)and no significance were found betweenMock group and high glucose group (P>0.05).7. Nude mice transplant tumor changes:Compared with Control group and Mockgroup,the growth rate of tumor cells inoculated subcutaneously in nude mice wassignificantly slower\reduced in Her2siRNA group(P<0.01)and there was noobvious difference between Mock group and high glucose group (P>0.05).Conclusions: 1. Human meningioma cells were high-efficient cultured by multi-enzymaticdigestion method and transfected successfully.2. The vivo experiments showed that the proliferation ability of meningiomacells would be decreased,the expression of Her2mRNA and Her2、VEGF、Ki-67protein would be reduced after the silencing of Her2/neu gene.3. In vitro,the proliferation activity of transplanted tumor in nude mice wasdecreased after the silencing of Her2/neu gene.