[溶瘤重组腺病毒KH901注射后的肿瘤选择性复制、细胞毒性及GM-CSF产生]

Shen Fu-bing, Chang Jian-hua, Yang Chun, Li Jin, Guo Yong, Yi Bo, Li Hong-xia, Ye Xue-fei, Wang Li

National Chengdu Center of Safety Evaluation of Traditional Chinese Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.

Sichuan Da Xue Xue Bao Yi Xue Ban. 2007 Jan;38(1):31-4.

OBJECTIVE

To study the tumor-selective replication, cytotoxicity and GM-CSF production of the recombinant virus in KH901 injection used to infect the cells cultured in vitro.

METHODS

A panel of tumor and normal cells was infected with recombinant adenovirus in KH901 and wild-type adenovirus type 5 at a MOI of 2 PPC, the cells were harvested at 72 hours after infection and made a titer after three cycles of freeze/thaw; A panel of tumor and normal cells was infected with recombinant adenovirus KH901 at MOI of 1 or 10 PPC. For 24 hours after infection the medium was harvested to determine the biological activity of GM-CSF; A panel of tumor and normal cells was infected with KH901 of recombinant adenovirus and wild-type adenovirus type 5 at MOIs of 0, 0.1, 1, 10, 100, and 1000 PPC. At 7 days after infection, cell viability was determined by the MTT assay, and ECso was determined too.

RESULTS

The data showed that wild-type adenovirus type 5 replicated efficiently in and killed both the tumor and normal cells, however, the recombinant adenovirus in KH901 replicated hugely in tumor cells [(2526.4+/-136.8)-(2796.6+/-104.6) TCID50/cell), and produced significant amount of GM-CSF [(1177. 793 +/-6.62)-(3924.497+/-17.79) IU/(10(6) cell x 24h)] and killed the tumor cells [EC50: (0.31+/-0.06)-(0.19+/- 0.01) pfu/cell] while was replicating poorly in non-permissive human normal cells [(56.8+/-9.2)-(90.1+/-14.4) TCID50/ cell], and producing very small amount of GM-CSF [(13.397+/-0.82) IU/(10(6)cell x 24 h)] and attenuating human primary cells killed [EC50: (92.33 +/- 9.12)-(121.20 +/- 19.94) pfu/cell], with which there was statistically a significant difference between wild-type adenovirus type 5 and recombinant adenovirus in KH901 (P<0.05).

CONCLUSION

In vitro studies show that the tumor-selective replication, cytotoxicity, GM-CSF production of recombinant adenovirus lead the injection KH901 containing the recombinant adenovirus, as oncolytic agent, to have a potential utility for the treatment of solid tumors.

目的

研究用于感染体外培养细胞的KH901注射液中重组病毒的肿瘤选择性复制、细胞毒性及GM-CSF产生情况。

方法

用MOI为2个感染复数（PPC）的KH901重组腺病毒和野生型5型腺病毒感染一组肿瘤细胞和正常细胞，感染后72小时收获细胞，经三次冻融循环后进行滴度测定；用MOI为1或10 PPC的重组腺病毒KH901感染一组肿瘤细胞和正常细胞。感染后24小时收集培养基以测定GM-CSF的生物学活性；用MOI为0、0.1、1、10、100和1000 PPC的重组腺病毒KH901和野生型5型腺病毒感染一组肿瘤细胞和正常细胞。感染后7天，通过MTT法测定细胞活力，并测定半数有效浓度（EC50）。

结果

数据显示，野生型5型腺病毒在肿瘤细胞和正常细胞中均能高效复制并杀伤细胞，然而，KH901中的重组腺病毒在肿瘤细胞中大量复制[（2526.4±136.8）-（2796.6±104.6）半数组织培养感染剂量（TCID50）/细胞]，产生大量GM-CSF[（1177.793±6.62）-（3924.497±17.79）国际单位/（10⁶细胞×24小时）]并杀伤肿瘤细胞[EC50：（0.31±0.06）-（0.19±0.01）蚀斑形成单位（pfu）/细胞]，而在非允许性人正常细胞中复制较差[（56.8±9.2）-（90.1±14.4）TCID50/细胞]，产生极少量GM-CSF[（13.397±0.82）国际单位/（10⁶细胞×24小时）]，对人原代细胞杀伤作用减弱[EC50：（92.33±9.12）-（121.20±19.94）pfu/细胞]，野生型5型腺病毒与KH901中的重组腺病毒之间存在统计学显著差异（P<0.05）。