Inhibitory Effect Of The C6 Cells Proliferation By Combining Elemene With VEGF Multiclone Antibody

Glioma is a kind of malignant brain tumor, about 35.26-60.96percent of all kinds of nervous system tumor. For malignant glioma,there hasn't a perfect therapy to cure, but we major on theoperation to remove the tumor, and assist with radiotherapy,chemotherapy, immunotherapy and gene therapy i.e. afteroperation. Still, operation is preferred. But operation can'tcompletely remove the whole glioma, and most glioma will recurafter operation, for the invading growing style and non-clearborderline of the tumor. We are still working on a new way toaccomplish this task.As the development of physiology, pathology, biochemistryand molecular biology, we have started making clear the followingthings: the progress of tumor blood vessel, the mechanism oftumor cell and molecule, and the inducing factor and inhibitor. Andcertain studies have showed exiting results about inhibitingangiogenesis therapy by inducing factor and inhibitor. Most of all,we have known the mechanism of vascular endothelial growthfactor (VEGF) and its receptor for signal transmitting, andconfirmed the anti-proliferation effect of malignant tumor usingthe inhibiting angiogenesis therapy policy.Chemotherapy is one of three major strategy of tumor cure,but there are a lot of inconveniences for this method. The effect ofdrugs depends on dose mostly, that is to say, chemotherapy canwork in vivo only when certain blood concentration is reached. Butwhen the blood concentration is beyond a higher line, side effectswill display. So the range of cure dose is limited, and we can onlyincrease the effect by adding drug dose in this range. So we plan ananimal array to combine the elemene and VEGF multicloneantibody.Elemene, isolated from the Chinese medicinal herb RhizomaZedoariae, is mainly composed of beta-elemene, which isdiscovered in China. Elemene has exhibit a confirmed effect in theclinical anti-tumor array, especially in the anti-glioma examination.VEGF derived from the cancinoma is the center factor in theangiogenesis. The production of the VEGF is regulated by growthfactor, cell factor, and other factors, which can not directlycontribute to the angiogenesis, but via the secretion of VEGF.VEGF alternatively directly act on tow kinds of Tyrosine Kinasesreceptor on the surface of vessel endothelial cells, which whenbinding to VEGF can induce self-phosphorylation and the signaltransmitting in vessel endothelial cells, promote the proliferation ofvessel endothelial cell, and eventually increase the blood stream ofthe carcinoma, which leads to the hyperplasia.Our study is major on the research of cooperation betweenelemene and VEGF multiclone antibody, which can provide abetter way to inhibit the proliferation of rat glioma C6 cells.cell culture and suspension prepareGlioma C6 cells, after taken out from the liquid azote wasimmediately putted into the incubator whose temperature wasamong 40-42℃, and was shook completely for 1 minute in orderto melt the cells. Then the tumor cells were transferred to medium,and 5ml of the cell culture was centrifuged at 1000r/min for 5 minto pellet cells. Pellet was resuspended in IMDM containing 3.7g/LNaHCO3, 100U/ml penicillin, 100mg/L streptomycin and 10%decomplemented fetal calf serum (FCS) in 5% carbon dioxide at37℃ in a tissue culture flask. When the flask was full, the culturemedium was removed, and the cells were digested by trypsin. Assoon as the cell started to crimple observed in the microscope, theflask was replenished with medium, and the adherent cells wererepeatedly aspirated for breakaway, which would be centrifuged at1000r/min. After that, the supernatant was removed, and the restwas processed into cell suspension, which was inoculated in theculture flask at a concentration of 10^6 cell/flask. After digested bythe trypsin, supernatants were removed, and the rest was washedby the non-serum culture medium (non-serum DMEM) twice.Then cytometry was completed to regulate the cell concentration at1×10^6/mm3 via the PBS(0.01mol/L, pH 7.4).Animal InoculationThe rats' backs were injected with 1ml the cell suspensiondescribed before after disinfected with iodoform and alky.Observing the situation every two days, we could touch the bumpunder the back skin 4-5 days later, and the diameter of the bumpwould be up to 15mm in about 2 weeks.GroupingWe chose the bumps which had a shape of sphericity orellipse and diameter about 15mm as our tumor model for this array,the others were excluded. We classified all these models randomlyinto 4groups (named Ⅰ,Ⅱ,Ⅲ,Ⅳ), each had 8 rats, which werebred separately (table 1).Group Ⅰ Physiological saline (control)Group Ⅱ elemene (examination)Group Ⅲ VEGF multiclone antibody (examination)Group Ⅳ elemene + VEGF multiclone antibody (examination)AdministrationDrug dose and preparation 20mg/kg elemene, 50ugVEGF multiclone antibody for each rat. We diluted the VEGFmulticlone antibody into a final concentration of 1mg: 1000ml, andthe elemene 100mg: 100ml (1%elemene contained 200mg elemenein 20ml).Administration pathway VEGF multiclone antibody wasinjected in local tumor tissue, while elemene in abdomen. Bothwere administrated every tow days.Administration methodGroup Ⅰ 0.5ml Physiological saline for each ratGroup Ⅱ 0.5ml elemene for each ratGroup Ⅲ 0.5ml VEGF multiclone antibody for each ratGroup Ⅳ 0.5ml elemene + 0.5ml VEGF multiclone antibody foreach ratAfter administration, all rats were observed for making surenone was sick.Detection IndexThe curve of tumor growth:The volume of tumors wasmeasured periodically (V= 1/6πab2), and the curve wascompleted;Measuring the weight of tumor and calculating thesuppression ratio Pathology of tumor;The tumor tissue was fixedby 10% neutral formaldehyde, and then buried by olefin. The masswas sheared into slices of 5um, which were then stained withHematoxylin and Eosin. All of these stained slices were analysedunder the microscope;Flow Cytometry (FCM) of the tumor cellsfor calculating the apoptosis ratio.ResultThrough this array, we can see that in group Ⅳ the growthcurve was obviously lower than in the other groups. Thesuppression ratio of group Ⅱ,Ⅲ,Ⅳ was separately 43.2%,41.3%and 51.9%, which showed that the suppression ratio of group Ⅳwas obviously higher than that of group Ⅱand Ⅲ. Compared withgroup Ⅱand Ⅲ, the tumor tissue observed under microscope wasseriously destroied and contained a few vessels. The apoptosisratio of four group was separately (0.46±0.19)%, (12.9±1.70)%,(11.7±1.70)% and (19.4±1.32)% through the analysis of FCM,which exhibited that the apoptosis ratio of group Ⅳ was higherthan that of group Ⅱand Ⅲ.In a word ,thragh the txamination of tumor's weighe, rdumeand apoptosis ratio,we can see the combination of elemene andVEGF multiclone antibody can obviously inhibit the proliferationof tumor cells, which provides a new way to chemotherapyassisted with antiagiogenesis.ConclusionCompared to control group, Elemene and VEGF multicloneantibody all have the proliferation inhibitory effect of C6 gliomameasured in volume and weight and promote the apoptosis oftumor cells. While combined with VEGF multiclone antibody,elemene can provide a more strength effect in antitumor therapy.