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Liver cancer is one of the most common malignant tumors in China.The efficacy of traditional treatment for liver cancer is unsatisfactory,and the prognosis of the patients is poor.In recent 10 years,with the development of the molecular biological techniques,genetic therapy has become a new and promising approach for liver cancer.Of which,adenovirus mediated herpes simplex virus thymidine kinase (ADV-tk) for the treatment of liver cancer is widely applied.The enzyme secreted by ADV-tk transformed the prodrug gancyclovir (GCV) to the cytotoxic agents and thus to kill the liver cancer cells.The results of multiple animal and clinical experiments showed that ADV-tk/GCV is effective for the treatment of liver cancer.In this article,the recent progress of ADV-tk/GCV in the treatment of liver cancer was reviewed.
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Objective: To study the inhibitory effects of an adenovirus (Ad)-based short hairpin RNA (shRNA) expression system on expression of vascular endothelial growth factor receptor (VEGFR) and on growth of colon carcinoma cells in vitro and in vivo. Methods: RNA interference pAd-Easy/VEGFR vector was used to transfect 293 cells via Lipofectamine 2000. The adenoviral vector carrying VEGFR was used to transfect CW2 cells and the transfection efficiency was determined by fluorescent microscope and flow cytometry. The expression of VEGFR was examined by RT-PCR and Western blotting. The cell growth was observed with MTT method and the growth curve was plotted. Nude mouse was transplanted with CW2 cells to establish colon cancer tumor model and the growth of tumor was observed daily. Micro-vessel density (MVD) was detected by immunohistochemistry. Results: The recombinant pAd-Easy carrying shRNA against VEGFR was verified by sequencing. Flow cytometry showed that the transfection efficiency of CW2 cells was 99.7%. RT-PCR and Western blotting showed that the expression of VEGFR in pAd-Easy/VEGFR group was obviously decreased. The growth curve showed that the cell growth in the pAd-Easy/ VEGFR group was obviously slowed down. We also found that the tumor growth in the nude mouse model was obviously inhibited and the MVD was also decreased. Conclusion: pAd-Easy/VEGFR-mediated VEGFR shRNA can effectively inhibit the expression of VEGFR in CW2 cells and suppress tumor growth in vivo.
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Objective To investigate the effect of exogenous kallikrein on apoptosis of the neurons aroundthe cerebralinfarctareain rats. Methods Thirty rats wjth cerebral infarction induced by middle cerebral artery occlusion(MCAO)were assigned randomly into 3 groups(n=10),namely the blank control group,saline group,and pAdCMV-HTK group.In the pAdCMV-HTK group,kallikrein gene was delivered into the cerebral ischemie lesion via a replication-defective adenovims using stereotaetic injection technique, and the expression of exogenous kallikrein was detected immunohistoehemically.TUNEL staining was performed to evaluate the neuronal apoptosis around the infarct area,and RT-PCR used to detect the mRNA expressions ofbcl-2,bax and caspase-3 in the brain tissues. Results At 24 h aftertreatment there were some HTK expressed cells found in group C and peal(at 72 h after treatment.While compare with group B and group C,there existed significant difference(112±6.1,68±4.2,59±3.9,P<0.05).At 72 h after treatment,the NSS of group C was significantly lower than that ofgruop B and A(6.70±0.16,8.13±0.16,7.93±0.20,P<0.05);7 days after the treatment,the difference was more significant(5.14±0.18,7.82±0.14,7.91±0.10,P<0.01).Apoptotic cells were mostly seen around the infarct area.The ratsinpAdCMV-HTK group showed significantly reduced number of cells positive for TUNEL staining as compared to those in the saline and blank control groups at 3 days(10.1±0.9,16.7±1.1,and 20.4±0.8,respectively)and 7 days after the treatment(15.2±1.2,33.6±1.3,and 28.8±1.7,respectively)(P<0.05).The mRNA levels ofbc1-2.bax and caspasc-3 were elevated in all the groups at 24 h,peaked at 72 h,and decreased gradually till 7 days alter the treatment.Compared with those in the other two groups，bcl-2 mRNA level in the pAdCMV-HTK group increased slightly P>0.05) while bax and caspase-3 mRNA levels decreased markedly(P<0.05) 72 h and 7 days after the treatment.Conclusion Kallikrein can inhibit neuronal apoptosis around the cerebral infarct and improve the neurological fimction of rats following cerebral infarction probably by reducing the expressions of such apoptotic factors as bax and caspase-3.
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Objective To investigate the effects ofkallikrein gene transfer on microvascularproliferation around the cerebral infarct and on the recovery of regional cerebral blood flow (rCBF)following ischemia/reperfusion injury in rats. Methods The rats with cerebral ischemia/reperfusioninjury induced by middle cerebral artery occlusion (MCAO) were randomly assigned into blank controlgroup, saline group, and pAdCMV-HTK treatment group and received corresponding injections into thetissues around the infarct area. Each group was divided into 3 subgroups (n=10) for observation at 12, 24and 72 h after the treatment. The neurological deficits of the rats before and after the treatment wereevaluated using neurological severity scores (NSS), and the expressions of exogenous human tissuekallikrein (HTK) and vascular endothelial growth factor (VEGF) in the brain tissues were detectedimmunohistochemically. TIC staining was performed to measure the changes in the infarct size.14C-iodoantipyrine tracing technique was used to define the rCBF in the rats. Results Compared tothe blank control group, the cerebral infarct size was significantly reduced in pAdCMV-HTK group 24 hafter the treatment, and was further reduced at 72 h (P<0.05). At 24 h after the treatment, the NSS inpAdCMV-HTK group was significantly lower than that in the blank euntrol and saline groups (P<0.05),and was further reduced at 72 h (P<0.01). After MCAO, the VEGF-positive cells were found mostly inthe cortex and the white matter around the infarct area. The expression of VEGF in pAdCMV-HTK groupwas markedly higher than that in the other two groups at 12, 24, and 72 h after the treatment (P<0.05). Inall the 3 groups, the rCBF around the infarct was slightly decreased as compared to that in thecontralateral hemisphere, pAdCMV-HTK slightly increased the rCBF 12 h after the injection (P>0.05),and significant increase in the rCBF occurred 24 h and 72 h after the injection (P<0.05). ConclusionKallikrein gene transfer following cerebral ischemia/reperfusion injury promotes vascular proliferationaround the infarct and increases the rCBF to reduce the infarct volume and attenuate neurological deficitsin rats.
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Objective:To study the inhibitory effects of an adenovirus(Ad)-based short hairpin RNA(shRNA) expression system on expression of vascular endothelial growth factor receptor(VEGFR) and on growth of colon carcinoma cells in vitro and in vivo.Methods: RNA interference pAd-Easy/VEGFR vector was used to transfect 293 cells via Lipofectamine 2000.The adenoviral vector carrying VEGFR was used to transfect CW2 cells and the transfection efficiency was determined by fluorescent microscope and flow cytometry.The expression of VEGFR was examined by RT-PCR and Western blotting.The cell growth was observed with MTT method and the growth curve was plotted.Nude mouse was transplanted with CW2 cells to establish colon cancer tumor model and the growth of tumor was observed daily.Micro-vessel density(MVD) was detected by immunohistochemistry.Results: The recombinant pAd-Easy carrying shRNA against VEGFR was verified by sequencing.Flow cytometry showed that the transfection efficiency of CW2 cells was 99.7%.RT-PCR and Western blotting showed that the expression of VEGFR in pAd-Easy/VEGFR group was obviously decreased.The growth curve showed that the cell growth in the pAd-Easy/VEGFR group was obviously slowed down.We also found that the tumor growth in the nude mouse model was obviously inhibited and the MVD was also decreased.Conclusion: pAd-Easy/VEGFR-mediated VEGFR shRNA can effectively inhibit the expression of VEGFR in CW2 cells and suppress tumor growth in vivo.