携带小鼠beta防御素2的间充质干细胞对小鼠恶性腹水的抑制作用  

Inhibitory Effect of Mesenchymal Stem Cells Carrying Murine Beta Defensin 2 on Malignant Ascites in Mice

作　　者：王国庆[1] 徐建容[1] 王瑞[1] 李红霞[1] 徐宁[1] 杜小波[1] 魏于全[1] 

机构地区：[1]生物治疗国家重点实验室四川大学华西医院肿瘤中心,四川成都610041

出　　处：《癌症》2006年第6期657-662,共6页Chinese Journal of Cancer

摘　　要：背景与目的:防御素是天然免疫的重要分子,小鼠beta防御素2(murinebetadefensin2,MBD2)是防御素家族的重要成员,在天然免疫和获得性免疫中具有桥梁作用。本研究观察携带MBD2的间充质干细胞(mesenchymalstemcell,MSC)对小鼠恶性腹水的抑制作用。方法:从BALB/c小鼠肾脏中提取总RNA,通过逆转录聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)得到小鼠MBD2片段。构建、包装表达MBD2的慢病毒Lentivirus系统。用所得病毒感染MSC,经筛选得到稳定表达MBD2的MSC,RT-PCR验证MBD2的表达。取稳定表达MBD2的MSC培养上清用于趋化树突细胞实验。BALB/c小鼠腹腔接种MethA细胞,于接种后第2、4、6、8天腹腔注射筛选所得表达MBD2的MSC或生理盐水、未感染病毒的MSC、感染Null-Lentivirus病毒的MSC,观察恶性腹水产生情况、腹水性状及荷瘤小鼠存活情况。对于长期存活小鼠,再次腹腔注射MethA肿瘤细胞,观察存活情况。结果:在MBD2-Lentivirus感染后的MSC中顺利扩增出MBD2片段。MBD2组细胞培养上清对树突细胞有较强的趋化作用[(43±8)个],明显高于生理盐水(NS)组[(8±1)个]、MSC组[(14±2)个]和Null组[(12±3)个],差异有显著性(P<0.01)。经表达MBD2的MSC处理的荷瘤小鼠腹水血性程度不明显,腹水总量[(3.0±1.0)ml]明显少于NS组[(10.8±1.0)ml]、MSC组[(10.2±1.3)ml]和Null组[(9.8±1.6)ml],差异有显著性(P<0.05)。MBD2组在肿瘤接种后第40天仍有小鼠存活,而各对照组至肿瘤接种后第20天已全部死亡,MBD2组存活时间显著长于对照组(P<0.05)。长期存活的MBD2组小鼠经MethA肿瘤细胞再次腹腔接种,其存活时间仍长于未作任何处理的对照组小鼠。结论:成功构建了以MSC为载体的MBD2基因治疗系统,经腹腔注射可以抑制恶性腹水的生长。BACKGROUND ＆ OBJECTIVE, Murine beta defensin 2 （MBD2） is a small antimicrobial peptide of the innate immune system. It provides a critical link between the innate immune system and the adaptive immune response. This study was to develop a set of MBD2-Ientivirus system, and observe its inhibitory effect on malignant ascites in mice. METHODS： MBD2 RNA was extracted from kidneys of BALB/c mice, and the fragment of MBD2 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. MBD2 plasmid was constructed, and the Ientivirus system expressing MBD2 was produced in 293FT cells. Mesenchymal stem cells （MSCs） were then infected with MBD2-Lentivirus, and stably infected cells （MBD2-MSCs） were selected. The expression of MBD2 was detected by RT-PCR. The biological function of MBD2 was evaluated by dendritic cell （DC） migration experiment. BALB/c mice bearing intraperitoneal MethA tumors were injected intraperitoneally with MBD2-MSCs at the 2nd, 4th, 6th, and 8th day after inoculation. Ascites status and survival status of the mice were observed. Long-term survivors were inoculated intraperitoneally with MethA tumor cells again to observe their survival status, RESULTS：The expression of MBD2 in MBD2-MSCs was verified by RT-PCR, Numbers of migrated DCs were significantly increased in MBD2-MSC group than in normal saline （NS） group, MSC group, and null lentivirus （Null） group （43±8 vs. 8±1, 14±2, and 12±3, P〈0.01）. The volume of tumor ascites was significantly less in MBD2-MSC group than in NS group, MSC group, and Null group [（3.0±1.0） ml vs. （10.8±1.0） ml, （10.2±1.3） ml, and （9.8±1.6） ml, P〈0.05]. Some mice in MBD2 group were still alive 40 days after tumor inoculation, while none in control groups survived up to 20 days after tumor inoculation （P〈 0.05）. After inoculated with MethA tumor cells again, the mice in MBD2-MSC group still survived longer than those in control groups. CONCLUSION. MBD2-Ientivirus gene therapy system is constru

关 键 词：防御素 慢病毒 干细胞 基因治疗 肿瘤 腹水 

分 类 号：R73-36[医药卫生—肿瘤]