骨癌痛大鼠脊髓背根神经节主要组织相容复合物Ⅱ表达变化的研究

Expression of major histocompatibility class Ⅱ in spinal dorsal root ganglions of rats with cancerous bone pain

摘要:

目的  建立大鼠胫骨的骨癌痛模型，观察骨癌痛发生时脊髓背根神经节内主要组织相容复合物Ⅱ（MHCⅡ）的表达变化情况。方法  雌性SD大鼠33只，随机分为正常组（Naive组，n =9）、假手术组（Sham组，n =12）和骨癌痛组（骨癌痛组，n =12）。骨癌痛组右侧胫骨的骨髓腔内接种Walker 256乳腺癌细胞，Sham组注射相等体积的D-Hanks液。于手术前1天及手术后3、7、10和14 d使用von-Frey丝测定手术侧的机械缩爪阈值；于手术后14 d采集各组（n =9）的L3～L5的右侧背根神经节，每3只大鼠的背根神经节标本作为一组提取总的蛋白质，采用蛋白免疫印迹法（Western blot）检测MHCⅡ的变化情况；于手术后14 d多聚甲醛灌注Sham组（n =3）和骨癌痛组（n =3）大鼠，取L3～L5的右侧背根神经节制作冷冻切片，采用免疫荧光双标染色观察MHCⅡ的变化情况以及与神经元和卫星细胞的关系。结果  手术前各组大鼠的机械缩爪阈值，差异无统计学意义；骨癌痛组在骨癌痛模型建立7 d后较基础值显著降低，差异有统计学意义（P <0.05），且显著低于Naive组，差异有统计学意义（P <0.05）。免疫荧光染色显示MHCⅡ表达于背根神经节的卫星细胞；同时Western blot定量结果发现骨癌痛组的MHCⅡ表达水平显著高于Naive组，而Sham组变化差异无统计学意义。结论  骨癌痛大鼠背根神经节的MHCⅡ表达升高，可能参与骨癌痛的外周敏化。

Abstract:

Objective To investigate the changes in the expression level of major histocompatibility class Ⅱ (MHC Ⅱ) in spinal dorsal root ganglions of rats with cancerous  bone pain (CBP). Methods Thirty-three adult virgin female Sprague-Dawley rats weighing 200-220 g, were randomly divided into three groups: naive group (n = 9), sham group(n = 12), and CBP group (n = 12). Walker 256 rat mammary carcinoma cells were inoculated into the right tibial medullary canal to establish a CBP model, whereas the same volume of D-Hanks solution was injected in the sham group. Mechanical paw withdrawal threshold (MPWT) was examined 1 day before and 3, 7, 10 and 14 days after surgery using calibrated von-Frey filaments applied to the center of ipsilateral hind paw. The L3-L5 ipsilateral dorsal root ganglions (DRGs) were collected 14 days after surgery to test the expression level of MHC Ⅱ by Western blot and immunofluorescent staining. Results The MPWT was not significantly different among the three groups before operation. In the CBP group the MPWT significantly decreased on the 7th day after surgery (P < 0.05), and was significantly lower than that of the naive group (P < 0.05). The immunofluorescent staining identified that MHC Ⅱ was dominantly expressed by satellite cells in the DRGs of the CBP rats. Furthermore, Western blot results showed that expression level of MHC Ⅱ was significantly increased in the CBP group, but not in the sham group, as compared with the naive group. Conclusions The changes in the expression level of MHC Ⅱ in satellite cells of DRGs may be involved in the development of peripheral sensitivity under CBP.