羟基喜树碱通过调控NOXA表达预防椎板切除术后硬膜外瘢痕粘连的研究

发布时间：2018-06-25 13:59

本文选题：硬膜外瘢痕粘连 + 羟基喜树碱　；参考：《扬州大学》2017年硕士论文

【摘要】：目的:探讨四种不同浓度梯度的羟基喜树碱(10-Hydroxycamptothecin,HCPT)局部使用对椎板切除术后硬膜外瘢痕粘连的预防作用,并进一步从细胞水平探究其预防硬膜外粘连的具体机制。方法:将72只Sprague-Dawley雄性大鼠平均四组,分别是0.2 mg/ml组、0.1 mg/ml组、0.05 mg/ml和生理盐水对照组。采用1%浓度的戊巴比妥钠对所有大鼠予腹腔注射麻醉后,进行常规术前准备后,在L1-L2附近逐层进入后显露椎体后面,使用微小咬骨钳在L1椎板制造硬脊膜暴露区,面积大小约5mm×2mm,然后将各种不同浓度的HCPT和生理盐水在纱布的保护下局部应用于人工造成的骨缺损区,计时5分钟,然后使用预先准备好的无菌生理盐水冲洗残留药物,在关闭切口之前再次进行创口消毒,手术后连续3天使用抗生素肌肉注射治疗以预防感染。28天后再次进行手术,逐层打开,对硬膜外区域行瘢痕粘连的大体评估,心脏灌注处死后,取下目标椎体还有其周围的组织,对所有标本实行脱钙处理,然后使用石蜡包埋组织,行HE染色法在不同放大率的显微镜下观察不同浓度HCPT对椎板切除术后粘连的预防以及局部成纤维细胞增殖抑制的效果,硬膜外瘢痕中NOXA的表达量的情况由免疫组织化学染色法测定,TUNEL检测用来测定硬膜外瘢痕组织中成纤维细胞的凋亡率变化。体外培养成纤维细胞,予不同浓度的HCPT(0,1,2,4 μg/ml)刺激成纤维细胞,TUNEL染色法观察HCPT刺激成纤维细胞后细胞凋亡率的变化,Western-blot检测凋亡相关蛋白表达情况。予慢病毒载体敲除NOXA基因后,TUNEL染色法检测观察HCPT刺激后的成纤维细胞的凋亡率的变化,Western-blot检测NOXA以及凋亡相关蛋白的表达量变化。结果:本实验中所有的大鼠均未发生瘫痪、感染、死亡等现象。HE染色结果显示:0.2 mg/ml HCPT组、0.1 mg/ml HCPT组以及0.05 mg/ml HCPT组的瘢痕粘连程度明显好于对照组,而高倍镜下观察到HCPT治疗组硬膜外瘢痕组织中成纤维细胞数量明显少于对照组硬膜外瘢痕中的成纤维细胞的数量,差异具有统计学意义(P0.05)。对于硬膜外瘢痕组织中成纤维细胞的TUNEL染色显示:成纤维细胞的凋亡率也升高趋势和HCPT的剂量趋势呈正相关。此外,硬膜外瘢痕组织中免疫组织化学染色结果显示HCPT治疗组中的NOXA表达量明显多于对照组中NOXA的表达量,伴随着HCPT浓度的升高,NOXA的表达量也呈现升高趋势,提示NOXA的表达量的增加可能与HCPT对硬膜外瘢痕粘连的抑制作用有关。体外细胞实验的结果显示HCPT能有效抑制成纤维细胞的细胞活性,TUNEL染色发现所有HCPT处理组的成纤维细胞凋亡率明显多于对照组。Western-blot结果显示应用不同浓度的HCPT刺激成纤维细胞后,NOXA,cleaved-PARP,Bax的表达量随着HCPT浓度的增高而升高,而Bcl-2的表达量则成相反趋势,此外,使用慢病毒技术敲除NOXA之后,我们通过TUNEL染色发现成纤维细胞的凋亡率相应的降低了,而且Western-blot结果也显示凋亡相关蛋白的表达量也随之降低。结论:HCPT可以有效的预防大鼠椎板切除术后硬膜外瘢痕粘连的形成,且其预防作用的潜在机制可能是通过上调NOXA的表达、促进成纤维细胞凋亡。
[Abstract]:Objective: To explore the preventive effect of four different concentration gradient 10-Hydroxycamptothecin (HCPT) on epidural scar adhesion after laminectomy, and to further explore the specific mechanism of preventing epidural adhesion from the level of cell. Methods: the average four groups of 72 Sprague-Dawley male rats were 0.2 mg/ml, respectively. Group, 0.1 mg/ml, 0.05 mg/ml and saline control group. After intraperitoneal injection of pentobarbital, 1% concentrations of sodium pentobarbital were used for routine preoperative preparation, and then the vertebral body was exposed after layer by layer near L1-L2. Small bone nippers were used to make the dural exposed area in the L1 vertebral lamina. The size of the spinal cord was about 5mm * 2mm, and then various kinds of non - The same concentration of HCPT and normal saline were applied to the artificial bone defect area under the protection of gauze for 5 minutes, then the residual drugs were washed with pre prepared aseptic saline, and the wound was sterilized again before closing the incision. After 3 days of operation, antibiotic injection was used to prevent infection after.28 days. After the operation, it was opened by layer by layer to assess the scar adhesion in the epidural area. After the heart perfusion was executed, the target vertebral body and its surrounding tissue were removed, and all the specimens were decalcified. Then the paraffin embedded tissue was used, and the HE staining method was used to observe the different concentration of HCPT under the microscope for laminectomy under the microscope of different magnification. The prevention of posterior adhesion and the effect of local fibroblast proliferation inhibition. The expression of NOXA in epidural scar was measured by immunohistochemical staining. TUNEL detection was used to determine the apoptosis rate of fibroblasts in epidural scar tissue. In vitro culture fibroblasts were cultured with different concentrations of HCPT (0,1,2,4 mu g/ml) spines. The apoptosis rate of HCPT stimulated fibroblasts was observed by TUNEL staining, and the expression of apoptosis related proteins was detected by Western-blot. After the lentivirus vector knockout NOXA gene, the apoptosis rate of fibroblasts stimulated by HCPT was detected by TUNEL staining, Western-blot detection NOXA and apoptosis related Results: all rats in this experiment had no paralysis, infection, and death. The results of.HE staining showed that the degree of scar adhesion in 0.2 mg/ml HCPT group, 0.1 mg/ml HCPT group and 0.05 mg/ml HCPT group was better than that of the control group, and the fibroblasts in the epidural scar tissue of the HCPT treatment group were observed under the high magnification microscope. The number of cells in the epidural scar was significantly less than that in the control group. The difference was statistically significant (P0.05). The TUNEL staining of the fibroblasts in the epidural scar tissue showed that the trend of apoptosis in the fibroblasts was positively correlated with the dose trend of HCPT. In addition, the immune tissues in the epidural scar tissue were immune. The results of chemical staining showed that the expression of NOXA in the HCPT treatment group was more than the NOXA expression in the control group. With the increase of HCPT concentration, the expression of NOXA also showed an upward trend, suggesting that the increase of the expression of NOXA may be related to the inhibitory effect of HCPT on the adhesion of epidural scar. The results of cell experiments in vitro show that HCPT can be found. TUNEL staining showed that the apoptosis rate of fibroblasts in all HCPT treatment groups was significantly more than that of the control group.Western-blot results showed that the expression of NOXA, cleaved-PARP, Bax increased with the increase of HCPT concentration, and the expression of Bcl-2 was opposite to that of the control group. In addition, after using lentivirus technique to knock out NOXA, we found that the apoptosis rate of fibroblasts decreased by TUNEL staining, and the Western-blot results also showed that the expression of apoptosis related proteins also decreased. Conclusion: HCPT can effectively prevent the formation of epidural scar adhesion after laminectomy in rats, and it can be effectively prevented from the formation of the epidural scar adhesion after laminectomy in rats. The potential mechanism of preventive effect may be to promote fibroblast apoptosis by up regulating the expression of NOXA.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687.3

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