鞘内注射人IL-10基因修饰的间质干细胞对慢性骨癌性疼痛大鼠的镇痛作用

The Analgesic Effect of Intrathecal Injection of Human IL-10 Gene-modified Mesenchymal Stem Cells for Chronic Bone Cancer Painful Rats

目的人IL-10基因修饰的间质干细胞(hIL10-rMSCs)经皮鞘内注射入慢性骨癌性疼痛大鼠蛛网膜下腔,观察其在蛛网膜下腔存活情况及其对骨癌性疼痛大鼠的镇痛作用及机制。方法通过将Walker256大鼠腹水癌细胞PBS悬浮液10μL注入大鼠胫骨髓腔内建立骨癌性疼痛大鼠模型,并随机分为hIL10-rMSCs组、空载体-rMSCs组及control组,每组12只,分别经皮鞘内注射hIL10-rMSCs细胞悬液(1×10^6cells·mL^-1 )以及空载体-rMSCs细胞悬液(1×10^ 6cells·mL ^-1 )、生理盐水各30μL;各组大鼠分别于鞘内注射后3、7、14天测定机械性痛觉过敏(PWT)和热痛觉过敏(PWL),同时应用荧光激发、免疫组化及WB技术检测hIL10-rMSCs在蛛网膜下腔存活情况及腰段脊髓小胶质细胞OX-42、IL-1β、IL-6和TNF-α蛋白表达情况。结果与control组和空载体-rMSCs组相比,hIL10-rMSCs组PWT及PWL均明显升高,差异有统计学意义( P <0.05),而空载体-rMSCs组与control组比较差异无统计学意义( P >0.05);hIL10-rMSCs组和空载体-rMSCs组鞘内注射细胞7、14天后在荧光激发下可见绿色荧光细胞,而control组则未见绿色荧光细胞;与control组及空载体-rMSCs组相比,hIL10-rMSCs组脊髓腰段胶质细胞OX-42、IL-1β、IL-6和TNF-α表达明显降低( P <0.05),而这些指标在空载体-rMSCs组与对照组比较则无明显差异( P >0.05)。结论鞘内注射hIL10-rMSCs能通过抑制脊髓小胶质细胞活化,减少促炎细胞因子IL-1β、IL-6、TNF-α的释放在一定程度上减轻大鼠骨癌性疼痛。

OBJECTIVE Human IL-10 gene-modified mesenchymal stem cells (hK-10-rMSCs) were injected into the subarachnoid space of rats with chronic bone cancer pain,in order to observe their survival in the subarachnoid space and its analgesic effect and mechanism in rats with bone cancer pain. METHODS The rat model of bone cancer pain was established by injecting 10μL suspension into the tibial medullary cavity of Walker256 rat ascites cancer cells.The model animals were randomly divided into the hK-10-rMSCs group,the empty vector rMSCs group and the control group,with 12 animals in each group respectively injected with hK-10-rmscs cell suspension (1×10^6cells·mL ^-1 ),the empty vector rMSCs cell suspension (1×10^ 6cells·mL^-1 ),and the normal saline (30μL). Mechanical hyperalgesia (PWT) and thermal hyperalgesia (PWL) were measured at 3,7,and 14 days after intrathecal injection,respectively,and the survival of hK-10-rMSCs in subarachnoid space and the expression of OX-42,IL-1,IL-6,and TNF-α in lumbar spinal glial cells were detected by fluorescence stimulation,immunohistochemical and Western Blotting techniques. RESULTS PWT and PWL were significantly increased in the hK-10-rMSCs group compared with the control group and the empty vector rMSCs group ( P <0.05),while there was no statistically significant difference between the empty vector rMSCs group and the control group ( P >0.05).Green fluorescent cells were observed after 7 or 14 days of intrathecal injection of hil10-rmscs and empty vector rMSCs,while no green fluorescent cells were observed in the control group.Compared with the control group and the empty vector rMSCs group,the expressions of OX-42 and IL-1β,IL-6 and TNF-α in spinal cord and lumbar glial cells in the hIL10-rMSCs group were significantly decreased ( P <0.05).However,there was no significant difference in these indicators between the empty vector rMSCs group and the control group ( P >0.05). CONCLUSION Intrathecal injection of hIL10-rMSCs can alleviate bone cancer pain in rats to some extent by inhibiting the activation of small spinal glial cells and reducing the release of proinflammatory cytokines IL-1β,IL-6 and TNF-α.