| Malignancy is a group of diseases that are hazard to human's health or even their lives and difficult to cure. With the development of molecular biology, the new treatment modalities, like gene therapy, are actively tested to find alternatives for currently used strategies. Gene therapy, especially suicide gene therapy, has already become a brand-new anti-tumor method following the conventional methods such as surgical excision, irradiation therapy, and chemical therapy.Although suicide gene therapy has already been applied to clinical trials and has gained good results, there have been a lot of evidences which indicated that it would be impossible to cure tumors by suicide gene therapy along. Why? It is because there are still some of tumor cells remained though suicide gene therapy has the ability to eliminate the majority of tumor cells and thus the research works on the field of enhancing the effects of suicide gene therapy on eliminating the tumor residues have become a hot spot. Combination suicide gene therapy with other approaches is one of the most promising way. And thus improving the antitumor inflammatory-immunological microcircumstance of suicide gene therapy is an important strategy to augment its effect. Combination gene therapy involving suicide gene and immuno-gene is a major protocol at present.This kind of combination protocol is to transduce suicide gene and immuno-gene into tumor cells together at the same time. There are lots of problems in this protocol. More virus vector will be needed and the side effect tendency will be greater; The process will be more complicated and will cost much more; The genes transduced into tumor cells will interfere each other so that it is hard to promise genes to express or express in a long time.Clinical and experimental researches have demonstrated that many Chinese medicine or compounds have the effect on augmenting immunity. At the same time, Chinese medicine has its advantages of more convenient administration and low cost and less side effects. The combination protocol which involves suicide gene therapy and Chinese medicine might be a more feasible and more effective one.The system involving herpes simplex virus thymidine kinase and ganciclovir (HSV-tk/GCV) is one of the most frequently utilized forms of gene therapy and it has beentested on many Malignant tumors. So it was selected in our study. The combination protocol of suicide gene therapy and Liuwei Dihuang decoction was put in trial on the therapy of hepatocarcinoma and melanoma in mice to probe into the feasibility of establishing the combination protocol of integrated traditional and western medicine to tumor treatment. 1 Materials and methods1.1 The systematic construction of suicide gene therapy involving herpes simplex virus thymidine kinase gene and GCVThe plasmid (pIC19R/MCl-tk) was originally kindly given by professor Huang L. who was in pharmacological department of University of Pittsburgh, and then was offered by Dr. Yagang Zhao, who is a member of our group and is working in the Guangzhou military area chief hospital. Firstly the plasmid (pIC19R/MCl-tk) was transduced into receptor bacteria DH5ct, and amplified in ampicillin containing medium (lOOmg/L); the plasmid was extracted and then digested by EcoK VBamH I and identified through gel electrophoresis. The HSV-tk cDNA was confirmed in the plasmid. The HSV-tk cDNA was orientationally cloned into the retroviral vector plasmid (pLXSN) by using DNA recombinant technique. Recombinant retroviral vector pLXSN-tk was transduced into receptor bacteria DH5a, and amplified in ampicillin containing medium (lOOmg/L). Then it was extracted and identified by restriction endonuclease digestion and DNA sequencing. By the use of PolyFect transfection technique, recombinant retroviral vector-pLXSN-HSV-tk was tansduced into packing cells (cell line PT67). Then, the stable virus-producing packaging cell line clones were obtained by G418 (400mg/L) screening for 2 weeks. NIH3T3 cells were transfected by the virus in the supernatant of packaging cell culture, screening in 300 mg/L G418 selective culture medium for over 2 weeks and the virus titer was detected. The obtained recombinant retrovirus in the supernatant of packaging cell culture was applied to infect human gastric carcinomatous cells (cellline SGC-7901) and the transfected cell was established by being screened in 350mg/L G418 cell culture medium for 2 weeks 2 days later after viral infection. The transfected cells were detected by spot-blotting to determine whether it contained the HSV-tk gene. Finally, for observing the ganciclovir (GCV) sensibility, cells were treated with GCV (100 mg/L) and the surviving cells were detected.1.2 Hepatocarcinoma as a target disease for experimental treatmentHepatocarcinoma cells (cell line H22) were transfected by the recombinant retrovirus in the culture supernatant of packaging cells, screened in 350mg/L G418 selective culture medium for 2 weeks after transfection and the anti-G418 cell clones were obtained. The killing effect of GCV (lOOmg/L) was observed in vitro. After the killing effect was confirmed in vitro, the transfected cells (H22/tk4) were mixed with untransfected cells (H22/tk") in proportion of 1:4. The Kunming mice were randomly allocated to six groups: the normal control group (for the immunity detection, which was detected by others), the control group of tumor model, the group of suicide gene therapy (treated with GCV), thegroup of Liuwei Dihuang treatment and the combination group (treated with suicide gene therapy and Liuwei Dihuang Bolus together). 0.9%Nacl was injected into the mice of the normal control group and the mixed cells (2xlO6 mixed cells/mouse) were inoculated into the sub-axillary of the mice of the other groups. From the sixth day after tumor cells inoculated on, mice in the group of suicide gene therapy and the combination group had been treated with GCV (lOOmg/L) for 10 days. Mice in the group of Liuwei Dihuang treatment and the combination group had been treated with Liuwei Dihuang bolus for 14 days from the next day after tumor cells inoculated. After all, the anti-tomor effects were observed. 1.3 Melanoma as a target disease for experimental treatmentMelanoma cells (cell line B16) were transfected by the recombinant retrovirus in the culture supernatant of packaging cells, screened in 350mg/L G418 selective culture medium for 2 weeks after transfection and the anti-G418 cell clones were obtained. The killing effect of GCV (lOOmg/L) was observed in vitro. After the killing effect was confirmed, the transfected cells (B16/tk+) were mixed with untransfected cells(B167tk-) in proportion of 1:4. The C57BL/6J mice were randomly allocated to six groups: the normal control group (for the immunity detection, which was detected by others), the control group of tumor model, the group of Liuwei Dihuang treatment, the group of suicide gene therapy (treated with GCV) and the combinant group (treated with suicide gene therapy and Liuwei Dihuang Bolus together). 0.9%Nacl was injected into the mice of the normal control group and the mixed cells (l*106 mixed cells/mouse) were inoculated into the sub-axillary of the mice of the other groups. From the seventh day after tumor cells inoculated on, the mice in the group of suicide gene therapy and the combination group had been treated with GCV(100mg/L) for 20 days, the mice in the group of Liuwei Dihuang treatment and the combination group had been treated with Liuwei Dihuang bolus for 25 days from the next day after tumor cells inoculated, After all, the anti-tomor effects were observed. 2 Results2.1 The systematic construction of suicide gene therapy involving herpes simplex virus thymidine kinase gene and GCVA 1.7kb gene fragment, which was what we wanted, in the plasmid (pIC19R/MCl-tk) was found by gel electrophoresis after the plasmid had been digested by EcoRl/BamR I. The result of gel electrophoresis of pLXSN-tk after being digested by EcoRl IBamW I showed two DNA bands (about 1.7kb and 5.9 kb), which met with its physical map. DNA sequencing showed HSVl-tk was inserted into the recombinant retro viral vector pLXSN-tk successfully. The recombinant retroviral vector-pLXSN-tk was tansduced into packing cells. Then, we obtained the anti-G418 positive cell strain PT67/tk which could excrete retroviral virus. The titer of the virus was l><107cfu/mL. After viral infection of SGC7901, we also obtained the anti-G418 positive cell strain SGC7901-tk. Spot blotting showed the HSV-tk gene was existed in tumor cells. GCV treatment showed an evident toxic effect on the SGC7901/tk but not on SGC7901. We demonstrated that the HSV-tk gene in virus couldexpress and the expressed product showed a biological activity.2.2 Hepatocarcinoma as a target disease for experimental treatmentAfter the transfection of retrovirus to Hepatocarcinoma cells, GCV treatment showed a killing effect on the H22/tk in vitro. It indicated that tk gene was transfected into H22 cells successfully and the tk gene showed a biological activity.The results in vivo include that: 1) tumor growth inhibition was observed in the groups with treatment by comparing tumor volume and tumor growth curve. The tumor volume was reduced in the groups with treatment than that in the control group of tumor model, the difference were statistically significant by comparing tumor volume in the combination group with that in the control group of tumor model at the time when the treatment finished (P<0.05); 2) the average tumor weights(1.5825±1.2380 in control group of tumor model, 0.9904±0.9796 in the group of Liuwei Dihuang treatment, 0.8498±0.7163 in the group of suicide gene therapy along, 0.5859±0.3786 in combination group,unit:g), were much less in the combination group, which was significant by comparing with that in control group of tumor model (PO.05). 3) tumor inhibition rate indicated that the combination group is the best (46.3% in the group of Liuwei Dihuang treatment, 37.4% in the group of suicide gene therapy, 63% in combination group). 4) pathological examination showed less density of tumor cells, more fibro-connective tissue hyperplasy within the tumor or outside the tumor, more leukocyte (lymphocyte and neutrophilic granulocyte) infiltration in the treatment groups, especially in the combination group.2.3 Melanoma as a target disease for experimental treatmentAfter the transfection of retrovirus to B16 cells, GCV showed an evident toxic effect on the B16/tk. It indicated that tk gene was transfected into B16 successfully and the tk gene showed biological activity.The results in vivo include that: 1) tumors appeared in the combination group was the latest (8 days later than control); 2) the tumor take rate in the combination group was the lowest among groups (80% in control group of tumour model, 77.8% in the group of Liuwei Dihuang treatment, 45.5% in the group of suicide gene therapy, 30.0% in combination group). 3) the tumor growth inhibition was observed in the groups with treatment by comparing tumor volume and tumor growth curve. The tumor volume was reduced in the groups with treatment than that in the control group of tumor model, the difference were statistically significant by comparing tumor volume in the combination group with that in the control group of tumor model at the time when the treatment finished (PO.05); 4) the average tumor weight was much less in the combination group than that in the control group of tumor model (0.9583±1.3260 in the control group of tumor model, 0.4369±0.6937 in the group of Liuwei Dihuang treatment, 0.3566±0.8569 in the group of suicide gene therapy, 0.1234±0.2538 in combination group,unit:g), and the difference was statistically significant by comparing with that in control group of tumor model (PO.05). 5) tumor inhibition rate indicated that the combination group is the best (62.8% in the group of suicide gene therapy, 87.1% in the combination group). 6) patho- logical examination showed less density of...
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