重组人源肿瘤坏死因子受体融合蛋白对大鼠下颌骨缺损的干预效果

doi:10.3969/j.issn.1005-4979.2023.02.004

摘要/Abstract

摘要：

目的：通过大鼠下颌骨缺损再植的实验动物模型，基于Janus激酶-信号转导与转录激活因子（Janus kinase-signal transducers and activators of transcription，JAK-STAT）通路探讨注射重组人源肿瘤坏死因子受体融合蛋白（recombinant human tumor necrosis factor receptor： Fc fusion protein，rhTNFR：Fc）对下颌骨缺损大鼠的干预效果。方法：将70只（雌雄各半）7~8周龄的健康SD大鼠随机分为3组，分别为空白组（10只）、对照组（30只）和实验组（30只）。实验组：于大鼠骨缺损处注入含有RhTNFR：Fc（45 mg/kg）的Bio-Oss骨颗粒，其上覆盖Bio-Gide胶原膜；对照组：于大鼠缺损处注入含25 µL 0.9%氯化钠溶液的Bio-Oss骨颗粒，同样覆盖Bio-Gide胶原膜；空白对照组为健康大鼠。取所有大鼠下颌骨组织，观察其形态学变化，行组织形态计量学测定。实验组和对照组大鼠于术后3、6、9周时被分批处死，取其左侧下颌骨进行炎症因子白细胞介素-6（interleukin-6，IL-6）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）表达水平的检测，同时检测组织中凋亡相关分子Bax、Bcl-2、p-JAK2和p-STAT3蛋白的表达。结果：与对照组比较，实验组的缺损面积显著减少，IL-6和TNF-α的浓度显著下降，而凋亡相关分子Bax、p-JAK2和p-STAT3的表达显著下降，Bcl-2表达显著升高（P<0.05）；与空白组比较，对照组和实验组缺损面积显著增加，缺损模型建立成功。结论：局部注射rhTNFR：Fc与Bio-Oss骨颗粒能够有效地促进下颌骨缺损修复，并且效果显著优于只使用Bio-Oss骨颗粒的组别，其修复作用与抑制细胞凋亡的能力相关，通过实验我们发现JAK2-STAT3细胞信号通路在其中发挥了重要作用。

关键词: 重组人源肿瘤坏死因子受体融合蛋白, 下颌骨缺损, Janus激酶-信号转导与转录激活因子通路

Abstract:

Objective: To investigate the effect of injection of recombinant human tumor necrosis factor receptor: Fc fusion protein (rhTNFR:Fc) on mandibular defects in rats based on Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Methods: On the basis of the random number table method, 70 healthy SD rats aged 7-8 weeks (half male and half female) were divided into 3 groups： blank group (n=10), control group (n=30) and experimental group (n=30). In experimental group, each bone defect location is filled with Bio-Oss bone particles that has been coated in a Bio-Gide collagen membrane and comprises rhTNFR:FC (45 mg/kg). For control group, identical amounts of physiological saline are injected into each bone defect location along with Bio-Oss bone particles that is coated in a Bio-Gide collagen membrane. The blank group was healthy rats. The mandibular tissues of all rats were collected for morphological observation and histomorphometry measurement. The rats in experimental group and control group were sacrificed in batches at 3, 6 and 9 weeks after surgery. The expression levels of interleukin-6 (IL-6) and tumor necrosis factor -α (TNF-α) in the left mandible were measured. The expression levels of Bax, Bcl-2, p-JAK2 and p-STAT3 proteins in tissues were detected. Results: The defect model was established successfully. Compared with the control group, the defect area of the experimental group was significantly reduced, the concentrations of IL-6 and TNF-α were significantly decreased, the protein expression levels of Bax, p-JAK2 and p-STAT3 were also significantly decreased, and Bcl-2 protein expression was considerably increased (P<0.05). Compared with the blank group, the defect area of control group and experimental group increased significantly. Conclusion: The result of this study showed that local injection of rhTNFR:Fc combined with Bio-Oss bone particles can effectively promote the repair of mandibular defects, and its effect was significantly better than that of the group using only Bio-Oss bone particles. Moreover, the repair effect is related to its ability to inhibit apoptosis, which is probably accomplished by inhibiting JAK2-STAT3 signaling pathway.

Key words: recombinant human tumor necrosis factor receptor: Fc fusion protein, mandibular defect, Janus kinase-signal transducers and activators of transcription pathway

中图分类号:

R782

康献刚, 牛军强, 赵冬霞, 霍霁. 重组人源肿瘤坏死因子受体融合蛋白对大鼠下颌骨缺损的干预效果[J]. 《口腔颌面外科杂志》, 2023, 33(2): 83-88.

KANG Xiangang, NIU Junqiang, ZHAO Dongxia, HUO Ji. Intervention effect of recombinant human tumor necrosis factor receptor fusion protein on mandibular defects: An experimental study on the rats[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(2): 83-88.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I2/83

图/表 4

表1 不同组别大鼠的下颌骨缺损面积（mm²，$\bar{x}±s$）

Table 1 The mandibular defect area in different groups of rats（mm²，$\bar{x}±s$）

测量时间	空白组（n=10）	对照组（n=10）	实验组 (n=10)	t值	P值
3周	—	62.25±2.36	55.54±1.83^①	7.154	0.020
6周	—	52.91±3.26	40.43±2.56^①	8.258	0.013
9周	—	46.74±4.02	35.52±3.71^①	12.789	0.009

图1 下颌骨缺损的CT图像 A.缺损形成前的基线图像；B.缺陷产生后6周，对照组的下颌骨恢复情况；C.缺损产生后6周，实验组的下颌骨恢复情况。

Figure 1 CT images of mandibular defects

表2 不同组别大鼠组织中IL-6和TNF-α浓度的比较（$\bar{x}±s$）

Table 2 Comparison of IL-6 and TNF-α levels in different groups of rat tissues（$\bar{x}±s$）

组别	空白组（n=10）	对照组（n=30）	实验组 (n=30)	F值	P值
IL-6 /(pg·mg^-1)	58.46±3.39	153.30±10.17^①	93.78±4.06^②	17.786	0.003
TNF-α/(pg·mg^-1)	52.44±2.78	155.43±10.64^①	112.69±5.37^②	7.594	0.016

图2 各组大鼠组织Bcl-2、Bax、p-JAK2和p-STAT3蛋白表达情况 A.Western blotting检测细胞凋亡相关蛋白及JAK2/STAT3通路蛋白的表达；B—E.利用蛋白半定量法检测各蛋白的表达水平，*表示P<0.05，与空白组比较；#表示P<0.05, ##表示P<0.01,与对照组比较。

Figure 2 The protein expression of Bcl-2, Bax, p-JAK2 and p-STAT3 in three groups

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