2010 30 5 931 Ad-E1A 215123 Ad-E1A BALB /c Ad-E1A RT-PCR E1A H22 BALB /c H22 PBS Ad-E1A 5-Fu 7 10 9 pfu /ml Ad-E1A Ad-E1A 5-Fu 66. 7% 68. 8% P > 0. 05 Ad-E1A PBS 5-Fu P < 0. 01 P < 0. 05 Ad-E1A PBS 5-Fu P < 0. 01 P < 0. 05 Ad-E1A Ad-E1A R739 A 1673-0399 2010 05-0931 - 05 The Oncolytic Adenovirus Ad-E1A in the Gene Therapy of Mouse Ascitic Hepatoma in vivo and Its Effect on Immune Function CAO Jing-yuan YANG Bi-wen QIN Yue-xiang JIAO Yu-juan WANG Xiao-hua WANG Jin-zhi SHENG Wei-hua YANG Ji-cheng MIAO Jing-cheng Dept of Cellular and Molecular Biology Medical College Soochow University Jiangsu Suzhou 215123 China Abstract Objective To research the gene therapy of mouse ascitic hepatoma and its effect on immune function by oncolytic adenovirus Ad-E1A. Methods The E1A recombined adenovirus was proliferated in QBI-293A cells. RT-PCR was used to confirm the transcription of E1A gene in mouse hepatoma H22 cells. The tumor subcutaneous model was established with mouse ascitic hepatoma. The antitumor inhibitive ratio thymus index spleen index and the number of white blood cell lymphocyte neutrophil and monocyte in every group were detected. Results The high titer E1A recombined adenovirus was obtained The titer is 7 10 9 pfu /ml. In tumor subcutaneous model compared with PBS group the inhibitive ratio of Ad-E1A and 5-Fu was 66. 7% and 68. 8% respectively P > 0. 05. Compared with invariable Ad-E1A group and PBS group the thymus index and spleen index of 5-Fu were decreased obviously P < 0. 01& P < 0. 05. The number of white blood cells were equivalence between Ad-E1A group and nontumor group while the group of 5-Fu were drop rapidly P < 0. 01. The number of lymphocyte neutrophil and monocyte were decreased obviously in 5-Fu group. Conclusion The oncolytic adenovirus Ad- 2008 SG315859 08KJB310011 2009-09 - 01 1989 -
932 SUZHOU UVIVERSITY JOURNAL OF MEDICAL SCIENCE 2010 30 5 E1A inhibite H22 ascitic hepatoma obviously and almost has no side effect on immune function. Key words oncolytic adenovirus Ad-E1A mice ascitic hepatoma gene therapy immune function GTCTTGTCATTATC-3 P 2 5 -AAG- CAAGTCCTCGATACATTCCA-3 Sangon DMEM E1A cytopathic effect CPE p53 1. 5 ml 2 000 r /min 1 5 min DMEM -I MHC-I 1 ml- 20 37 3-80 QBI- 293A 10 5 /ml96 100 μl 2 24 h 10-7 10-8 10-9 100 μl 37 5% CO 2 18 h E1A = 10 / H22 - H22 3 100 MOI Ad-GFP 100 MOI 1 1. 1 QBI-293A H22 2 ~ 3 BALB /c H22 Ad- 100 μl 2 10 6 / GFP Ad-E1A 3 12 BALB /c 25 g 10 d E1A P 1 5 -ACGGTTGCAG- 0. 5 cm Ad-E1A PBS 48 h PBS 2 ~ 3 RT-PCR E1A mrna - 12 BALB /c PBS Ad- E1A 5-Fu 4 PBS PBS RPMI1640 Sigma RNA 100 μl Ad-E1A Ad-E1A 100 μl 1 Sangon MMLV 10 8 pfu /ml 5-Fu 5-Fu 20 μg /kg 100 Takara BALB /c μl 1 d 5 14 d 100 4 BALB /c negative 1. 2 - QBI-293A 100 μl 14 d Ad-GFP Ad-E1A 10 ml 5% DMEM 37 5% CO 2 48 h
2010 30 5 933 PBS % = - PBS 100% Ad-E1A H22 E1A β-actin Ad-GFP PBS BALB /c β-actin E1A 1 = g 10 000 g BALB /c 1. 3 1 β-actin of H22 cells 2 β-actin of Ad-GFP 3 β-actin of SAS8. 0 P < Ad-E1A 4 E1A of H22 cell 5 E1A of Ad-GFP 6 E1A 0. 05 of Ad-E1A M DNA marker 2 2. 1 2. 3 Ad-E1A Ad-GFP Ad-E1A 10-8 8 ~ 7 8 2A PBS 2. 053 g 5-Fu 10 9 pfu /ml 7 10 9 pfu /ml Ad-E1A 0. 64g 2. 2 RT-PCR Ad-E1A H22 0. 683g 5-Fu Ad-E1A 68. 8% Ad-GFP Ad-E1A H22 66. 7% PBS 48 h RNA RT-PCR P < 0. 05 2B 1 RT-PCR Ad-E1A 2 Ad-E1A H22 2. 4 Ad-E1A Ad-E1A PBS P > 0. 05 5-Fu PBS P < 0. 05 Ad-E1A PBS 3B Ad-E1A P > 0. 05 5-Fu PBS P < 0. 01 3A
934 SUZHOU UVIVERSITY JOURNAL OF MEDICAL SCIENCE 2010 30 5 3 PBS * P < 0. 05 ** P < 0. 01 Ad-E1A BALB /c A B 2. 5 Ad-E1A P < 0. 01 4B PBS 13. 067 10 9 /L Ad-E1A 13. 875 PBS 10. 53 10 9 /L Ad-E1A 9. 025 10 9 /L 7. 925 10 9 10 9 /L 6. 65 10 9 /L 5-Fu 4. 480 10 9 /L 5-Fu 3. 56 10 9 /L P < 0. 01 4A P < 0. 05 4B PBS Ad-E1A PBS Ad-E1A 1. 2 10 9 /L 2. 35 10 9 /L 1. 33 10 9 /L 2. 5 10 9 /L 0. 3 10 9 /L 5-Fu 0. 95 10 9 /L 5-Fu 0. 48 10 9 /L 0. 44 10 9 /L P > 0. 05 P < 0. 05 4B 5-Fu PBS Ad-E1A 4 negative * P < 0. 05 P > 0. 05 Ad-E1A BALB /c A B
2010 30 5 935 3 Ad-E1A 5 E1A NK CTL E1A 4 E1A E1B E3 5 1 ZHANG Qi WU Mengchao LI Yueming et al. A novel replication-competent adenovirus CNHK500 in the treatment of heptocellular carcinoma in vitro J. The Chinese- German Journal of Clinical Oncology 2004 3 2 70 Ad-E1A 5-Fu 66. 7% - 73. 68. 8% 2 Boyoung Leea Jene Choib Jaesung Kimc et al. Oncolysis of human gastric cancers by an E1B 55kDa-deleted YKL PBS - 1 adenovirus J. Cancer Letters 2002 185 225-233. Ad-E1A PBS 3 YE Zhen Min WANG Xiao Hua ZHONG Jiang et al. The Ad-E1A effect of oncolytic adenovirus on human umbilical vein endothelial cell J. Chinese Journal of Biotechnology 2006 5-Fu Ad-E1A PBS 22 3 397-402. 5-Fu 4 Nikita Avvakumov Joe S Mymryk. New tools for the construction of replication-competent adenoviral vectors with altered E1A regulation J. Journal of Virological Methods 2002 103 41-49. NK 5 Zhang Zi Lai ZOU Wei Guo LUO Chun Xia et al. An armed oncolytic adenovirus system ZD55-gene demonstrating potent anti-tumoral efficacy J. Cell Research 6 2003 13 6 481-489. Ad-E1A 6. M. 2. 2000 808. Ad-E1A 檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷檷 925 8 Kaneto H Kajimoto Y Miyagawa J et al. Beneficial effects of antioxidants in diabetes possible protection of pancreatic beta-cells against glucose toxicity J. Diabetes 1999 48 12 2398-2406. 9 Elsne M Guldbakke B Tiedge M et al. Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin J. Diabetologia 2000 43 12 1528-1533. 10 2003 38 2 85-88.. J.