阿霉素对原代培养大鼠背根神经节细胞作用机制的研究
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摘要
背根神经节是躯干、四肢痛觉的初级传入神经元,是机体内外环境与脊髓连接的纽带,具有传输和调节机体的各种感觉,接受和传递各种伤害性感受的功能,其在神经病理性疼痛的发生与维持中起着重要作用。
阿霉素(adriamycin,ADM)是一种环蒽类抗生素类广谱抗肿瘤药物,具有自发荧光特性。抗肿瘤剂量具有明显的神经毒性和心脏毒性。近年来的研究证实,静脉内注射阿霉素可以选择性破坏外周感觉神经节,同时它可引起背根神经节细胞坏死而非凋亡。并且阿霉素具有对背根神经节高度亲和性的特点,可以引起周围神经变性,所以在动物实验中也将它作为经典的神经毒物,临床上应用它来损毁背根神经节(dorsal root ganglion,DRG)细胞作疼痛治疗,即选择性感觉神经节化学切除术以治疗难治性疼痛。但对于阿霉素神经细胞毒性机制的研究目前还不明确,存在争议。
目的:
在原代培养大鼠背根神经节细胞的基础上,将不同剂量阿霉素作用于DRG细胞,观察其作用并对其作用机制进行初步探讨。
方法:
用DMEM/F12血清培养基纯化培养新生SD大鼠背根神经节细胞,接种后培养7天,NSE染色进行神经元纯度鉴定后随机分为正常对照组和不同阿霉素浓度组,根据阿霉素剂量不同分为ADM:0.5mg/L;ADM:1.0mg/L;ADM:2.0mg/L;ADM:5.0mg/L;ADM:10mg/L)。各实验组阿霉素作用时间为4小时,对照组换用无血清培养基继续培养4小时。镜下观察细胞的形态学变化,TUNEL法检测细胞凋亡率。在上述实验基础上,再分别测定加入药物前后背根神经节细胞MTT代谢率、乳酸脱氢酶(LDH)活性、超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量的变化。
结果:
(1)原代培养的大鼠背根神经节神经元细胞生长状态正常,有神经突起生长和标记蛋白的表达,可达到90%左右的纯度。
(2)阿霉素实验组的DRG细胞凋亡率与对照组相比有显著差异性(P＜0.05),阿霉素实验组中随着药物浓度的增加凋亡率逐渐升高,但在ADM 10mg/L组中,细胞的凋亡率降低,而且出现了坏死细胞。
(3)阿霉素实验组的MTT代谢率、LDH活性、SOD活性和MDA含量与对照组相比有显著性差异(P＜0.05),阿霉素使背根神经节细胞的LDH活性、MDA含量增加,同时降低了MTT代谢率、SOD活性。阿霉素对MTT代谢率、LDH活性、SOD活性和MDA含量的影响具有剂量依赖性趋势。
结论:
阿霉素对原代培养背根神经节细胞具有细胞毒性作用,低剂量能导致凋亡,凋亡率随着剂量增加而升高,到达一定浓度后凋亡率下降,并且坏死与凋亡共存,其作用机制与氧自由基的产生有关。
Dorsal root ganglion (DRG) cells are the primary afferent neurons of algaesthesis of trunk and extremities. DRG connects the spinal cord with internal and external environment of the organism. It not only transmits and accommodates sensations but also accepts and conveys nociception of the organism, so it plays an important role in the origination and maintenance of neuropathologic pain.
Adriamycin, a broad-spectrum anthracycline antibiotic and anticancer therapeutic agent, has obvious neurotoxic effect and autofluorescence. In the recent years, it was demonstrated that intravenous injection adrinmycin can selective destroy the PNS sensory ganglion, it also cause the DRG cells necrosis but not apoptosis. Also it has highly affinity to the dorsal root ganglion and result in the degeneration of the DRG cells. So it was used in the clinical to treat the neuropathic pain which was named selective chemo-ganglionectomy. However the neurotoxicitive mechanisms of ADM. was not clear, it is controversy.
Objective:
To investigate the effort of Adriamycin(ADM) on in vitro cultured rat dorsal ganglia(DRG) neurons,observe its role and its mechanism of a preliminary study.
Method:
Using DMEM/F12 serum medium purification cultured newborn SD rat dorsal ganglion cells, seven days after innoculation culture. The purification rat cells were evaluated according to cell count and neuronal specific enolase(NSE) immunocytochemistry stain. Then DRG neurons were randomly divided into six groups: normal group and five experimental groups. The experimental groups were pretreated with different concentration of ADM(groupA, 0.5mg/L; group B, lmg/L;group C, 2mg/L; group D,5mg/L; group E, 10mg/L) . All the experimental groups were kept cultivating for 4 hours after ADM pretreated. Then morphological feature of apoptosis were oberverd by termimal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL) was used to detect the apoptosis. While MTT metabolic rate, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA) of the DRG neurons were detected.
Results:
(1) Cultured dorsal root ganglion cells could survive healthily, The purification rate of neurons was 90% or so.
(2) There is statistical difference in the apoptosis rate of DRG nerons between experimental groups and normal group(P<0.05).there was positive correlation between the neurons apoptosis rate and the concentration of ADM.and in the group E, necrosis of the DRG neurons was also observed.
(3) There was statistical difference in MTT metabolic rate; LDH activety;SOD activity; content of MDA between normal group and experimental groups. (P<0.05) . It was found that the LDH activity, content of MDA and the MTT metabolic rate were higher than those in the normal groups,however the activity of SOD decreased. And there was positive correlation between the MTT metabolic rate, LDH activity, SOD activity, content of MDA and the concentration of ADM.
Conclusion:
Our present studies shows that ADM can induce the neurotoxitcity of the DRG neurons, the neurotoxicity include apoptosis an necrosis.also the ADM can increased the MTT metabolic rate, LDH activity, content of MDA and decreased the SOD activity. The oxygen free radical may be one of the neurotoxicitive mechanisms of ADM.
阿霉素(adriamycin,ADM)是一种环蒽类抗生素类广谱抗肿瘤药物,具有自发荧光特性。抗肿瘤剂量具有明显的神经毒性和心脏毒性。近年来的研究证实,静脉内注射阿霉素可以选择性破坏外周感觉神经节,同时它可引起背根神经节细胞坏死而非凋亡。并且阿霉素具有对背根神经节高度亲和性的特点,可以引起周围神经变性,所以在动物实验中也将它作为经典的神经毒物,临床上应用它来损毁背根神经节(dorsal root ganglion,DRG)细胞作疼痛治疗,即选择性感觉神经节化学切除术以治疗难治性疼痛。但对于阿霉素神经细胞毒性机制的研究目前还不明确,存在争议。
目的:
在原代培养大鼠背根神经节细胞的基础上,将不同剂量阿霉素作用于DRG细胞,观察其作用并对其作用机制进行初步探讨。
方法:
用DMEM/F12血清培养基纯化培养新生SD大鼠背根神经节细胞,接种后培养7天,NSE染色进行神经元纯度鉴定后随机分为正常对照组和不同阿霉素浓度组,根据阿霉素剂量不同分为ADM:0.5mg/L;ADM:1.0mg/L;ADM:2.0mg/L;ADM:5.0mg/L;ADM:10mg/L)。各实验组阿霉素作用时间为4小时,对照组换用无血清培养基继续培养4小时。镜下观察细胞的形态学变化,TUNEL法检测细胞凋亡率。在上述实验基础上,再分别测定加入药物前后背根神经节细胞MTT代谢率、乳酸脱氢酶(LDH)活性、超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量的变化。
结果:
(1)原代培养的大鼠背根神经节神经元细胞生长状态正常,有神经突起生长和标记蛋白的表达,可达到90%左右的纯度。
(2)阿霉素实验组的DRG细胞凋亡率与对照组相比有显著差异性(P＜0.05),阿霉素实验组中随着药物浓度的增加凋亡率逐渐升高,但在ADM 10mg/L组中,细胞的凋亡率降低,而且出现了坏死细胞。
(3)阿霉素实验组的MTT代谢率、LDH活性、SOD活性和MDA含量与对照组相比有显著性差异(P＜0.05),阿霉素使背根神经节细胞的LDH活性、MDA含量增加,同时降低了MTT代谢率、SOD活性。阿霉素对MTT代谢率、LDH活性、SOD活性和MDA含量的影响具有剂量依赖性趋势。
结论:
阿霉素对原代培养背根神经节细胞具有细胞毒性作用,低剂量能导致凋亡,凋亡率随着剂量增加而升高,到达一定浓度后凋亡率下降,并且坏死与凋亡共存,其作用机制与氧自由基的产生有关。
Dorsal root ganglion (DRG) cells are the primary afferent neurons of algaesthesis of trunk and extremities. DRG connects the spinal cord with internal and external environment of the organism. It not only transmits and accommodates sensations but also accepts and conveys nociception of the organism, so it plays an important role in the origination and maintenance of neuropathologic pain.
Adriamycin, a broad-spectrum anthracycline antibiotic and anticancer therapeutic agent, has obvious neurotoxic effect and autofluorescence. In the recent years, it was demonstrated that intravenous injection adrinmycin can selective destroy the PNS sensory ganglion, it also cause the DRG cells necrosis but not apoptosis. Also it has highly affinity to the dorsal root ganglion and result in the degeneration of the DRG cells. So it was used in the clinical to treat the neuropathic pain which was named selective chemo-ganglionectomy. However the neurotoxicitive mechanisms of ADM. was not clear, it is controversy.
Objective:
To investigate the effort of Adriamycin(ADM) on in vitro cultured rat dorsal ganglia(DRG) neurons,observe its role and its mechanism of a preliminary study.
Method:
Using DMEM/F12 serum medium purification cultured newborn SD rat dorsal ganglion cells, seven days after innoculation culture. The purification rat cells were evaluated according to cell count and neuronal specific enolase(NSE) immunocytochemistry stain. Then DRG neurons were randomly divided into six groups: normal group and five experimental groups. The experimental groups were pretreated with different concentration of ADM(groupA, 0.5mg/L; group B, lmg/L;group C, 2mg/L; group D,5mg/L; group E, 10mg/L) . All the experimental groups were kept cultivating for 4 hours after ADM pretreated. Then morphological feature of apoptosis were oberverd by termimal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL) was used to detect the apoptosis. While MTT metabolic rate, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA) of the DRG neurons were detected.
Results:
(1) Cultured dorsal root ganglion cells could survive healthily, The purification rate of neurons was 90% or so.
(2) There is statistical difference in the apoptosis rate of DRG nerons between experimental groups and normal group(P<0.05).there was positive correlation between the neurons apoptosis rate and the concentration of ADM.and in the group E, necrosis of the DRG neurons was also observed.
(3) There was statistical difference in MTT metabolic rate; LDH activety;SOD activity; content of MDA between normal group and experimental groups. (P<0.05) . It was found that the LDH activity, content of MDA and the MTT metabolic rate were higher than those in the normal groups,however the activity of SOD decreased. And there was positive correlation between the MTT metabolic rate, LDH activity, SOD activity, content of MDA and the concentration of ADM.
Conclusion:
Our present studies shows that ADM can induce the neurotoxitcity of the DRG neurons, the neurotoxicity include apoptosis an necrosis.also the ADM can increased the MTT metabolic rate, LDH activity, content of MDA and decreased the SOD activity. The oxygen free radical may be one of the neurotoxicitive mechanisms of ADM.
引文
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