發(fā)布時(shí)間：2018-11-17 15:29

【摘要】：目的通過(guò)觀察O-IPN模型大鼠一般情況、機(jī)械性刺激敏感程度、背根神經(jīng)節(jié)細(xì)胞形態(tài)學(xué)改變,背根神經(jīng)節(jié)鉑含量及鉑轉(zhuǎn)運(yùn)蛋白OCT2和ATP7A的表達(dá)情況,探討黃芪桂枝五物湯對(duì)奧沙利鉑神經(jīng)毒性的干預(yù)作用及機(jī)制。方法選用42只雌性Wistar大鼠隨機(jī)分為4組,空白組、模型組、西咪替丁(CIM)組和黃芪桂枝五物湯(中藥)組。適應(yīng)性喂養(yǎng)7天后,除空白組(n=6)予以腹腔注射5%葡萄糖注射液2ml/kg外,其余各組(n=12)按照2ml/kg (5mg/kg)的劑量予以腹腔注射奧沙利鉑,每周2次,共4周。同時(shí),空白組、模型組予以0.9%NaCl溶液灌胃1ml/次,每日1次。CIM組予以3ml/kg (30mg/kg)西咪替丁腹腔注射,每日1次,OXA給藥當(dāng)天,西咪替丁于給藥前1小時(shí)腹腔注射。中藥組予以黃芪桂枝五物湯湯劑2.80g/ml,10ml/kg灌胃,每日1次,OXA給藥當(dāng)天,中藥于給藥前1小時(shí)灌胃。持續(xù)性給藥至第28天。每周進(jìn)行2次體重稱量,2次機(jī)械性疼痛閾值檢測(cè),每日觀察大鼠給藥后反應(yīng)、飲食、排泄物等。第29天解剖各組大鼠,取L4-5雙側(cè)背根神經(jīng)節(jié)。組織切片經(jīng)尼氏染色后在油鏡下觀察背根神經(jīng)節(jié)細(xì)胞及核仁形態(tài),運(yùn)用ICP-MS法對(duì)背根神經(jīng)節(jié)內(nèi)的鉑含量進(jìn)行定量檢測(cè),進(jìn)行PCR實(shí)驗(yàn)對(duì)背根神經(jīng)節(jié)中鉑轉(zhuǎn)運(yùn)蛋白OCT2和ATP7A的mRNA表達(dá)情況進(jìn)行檢測(cè)。結(jié)果通過(guò)大鼠行為學(xué)觀察、機(jī)械性刺激實(shí)驗(yàn)以及細(xì)胞形態(tài)學(xué)檢測(cè)證實(shí)O-IPN模型制備成功。結(jié)果表明,使用OXA能夠顯著增加大鼠對(duì)機(jī)械性刺激的敏感度(P0.01),黃芪桂枝五物湯或西咪替丁可在一定程度上緩解大鼠對(duì)機(jī)械性刺激過(guò)度敏感表現(xiàn),但這一效果短期內(nèi)并不顯著,大約在給藥第三周后效果逐漸明顯。細(xì)胞形態(tài)學(xué)觀察結(jié)果顯示,使用奧沙利鉑后背根神經(jīng)元出現(xiàn)細(xì)胞體積縮小,核仁縮小或分裂,尼氏體淡染、紊亂等形態(tài)學(xué)改變,使用黃芪桂枝五物湯和西咪替丁均能夠改善奧沙利鉑導(dǎo)致的大鼠背根神經(jīng)元細(xì)胞及核仁體積縮小、尼氏體結(jié)構(gòu)改變。經(jīng)背根神經(jīng)節(jié)內(nèi)鉑含量檢測(cè)發(fā)現(xiàn),使用OXA后背根神經(jīng)節(jié)內(nèi)可檢測(cè)出較高含量的鉑,而在黃芪桂枝五物湯或西咪替丁干預(yù)下,背根神經(jīng)節(jié)內(nèi)鉑含量均顯著降低(P0.05)。PCR實(shí)驗(yàn)結(jié)果表明：在OXA作用下,大鼠背根神經(jīng)節(jié)Oct2的表達(dá)略升高,Atp7a的表達(dá)稍降低,而在黃芪桂枝五物湯干預(yù)下則出現(xiàn)了相反的結(jié)果,即Oct2的表達(dá)略降低,Atp7a的表達(dá)明顯升高(P0.05),有趣的是,西咪替丁干預(yù)后的結(jié)果卻是Oct2表達(dá)偏高,Atp7a的表達(dá)則基本不變。結(jié)論黃芪桂枝五物湯可以減輕奧沙利鉑慢性神經(jīng)毒性,但對(duì)于急性神經(jīng)毒性效果不顯著,其機(jī)制可能是通過(guò)少量下調(diào)背根神經(jīng)節(jié)細(xì)胞鉑轉(zhuǎn)入蛋白OCT2mRNA的表達(dá),適當(dāng)減少鉑流入背根神經(jīng)元,同時(shí)主要上調(diào)鉑轉(zhuǎn)出蛋白ATP7A mRNA的表達(dá)而促進(jìn)鉑的流出,起到對(duì)抗鉑蓄積于背根神經(jīng)節(jié)的效果,從而緩解O-IPN慢性神經(jīng)毒性表現(xiàn)。
[Abstract]:Objective to observe the general situation, the sensitivity of mechanical stimulation, the morphological changes of dorsal root ganglion cells, the content of platinum in dorsal root ganglion and the expression of platinum transporter OCT2 and ATP7A in O-IPN model rats. To investigate the effect and mechanism of Huangqi Guizhi Wuwu decoction on oxaliplatin neurotoxicity. Methods 42 female Wistar rats were randomly divided into 4 groups: blank group, model group, cimetidine (CIM) group and Huangqi Guizhi Wuwu decoction group. After 7 days of adaptive feeding, oxaliplatin was injected intraperitoneally in the control group (nong6) by intraperitoneal injection of 5% glucose injection (2ml/kg) according to the dose of 2ml/kg (5mg/kg), twice a week for 4 weeks. At the same time, in the blank group, the model group was given 1ml/ with 0.9%NaCl solution once a day, and the CIM group was given 3ml/kg (30mg/kg) cimetidine intraperitoneally, once a day, on the same day as OXA. Cimetidine was injected intraperitoneally 1 hour before administration. The traditional Chinese medicine group was treated with astragalus guizhiwu decoction 2.80 g / ml 10 ml / kg, once a day, one hour before the administration of OXA. The drug was given continuously until the 28 th day. The body weight and mechanical pain threshold were measured twice a week, and the reaction, diet and excretion of rats were observed daily. On the 29 th day, the rats in each group were dissected and L-4-5 bilateral dorsal root ganglion (DRG) was taken. The morphology of dorsal root ganglion cells and nucleoli were observed under oil microscope, and the content of platinum in dorsal root ganglion was quantitatively detected by ICP-MS method. The expression of platinum transporter OCT2 and ATP7A in dorsal root ganglion (DRG) was detected by PCR assay. Results the O-IPN model was successfully prepared by behavioral observation, mechanical stimulation and cell morphology. The results showed that OXA could significantly increase the sensitivity of rats to mechanical stimuli (P0.01). Huangqi Guizhi Wuwu decoction or cimetidine could alleviate the oversensitivity of rats to mechanical stimulation to some extent. However, this effect is not significant in the short term, about the third week after administration of the effect gradually obvious. The morphological changes of dorsal root neurons after oxaliplatin were observed, such as cell volume shrinking, nucleolus shrinking or splitting, Nissl body light staining, disorder and so on. Both Huangqi Guizhi Wuwu decoction and cimetidine could improve the size of dorsal root neurons and nucleoli and change the structure of Nissl body in rats induced by oxaliplatin. A higher level of platinum was detected in dorsal root ganglion (DRG) with OXA, and was treated with astragalus guizhiwuwu decoction or cimetidine. The content of platinum in dorsal root ganglion was significantly decreased (P0.05). PCR experiment showed that the expression of Oct2 in dorsal root ganglion was slightly increased, and the expression of Atp7a was slightly decreased under the action of OXA. However, under the intervention of Huangqi Guizhi Wuwu decoction, the contrary results were found, that is, the expression of Oct2 decreased slightly and the expression of Atp7a increased significantly (P0.05). Interestingly, the result of cimetidine intervention was that the expression of Oct2 was higher than that of control group. The expression of Atp7a was basically unchanged. Conclusion Huangqi Guizhi Wuwu decoction can attenuate the chronic neurotoxicity of oxaliplatin, but it has no significant effect on acute neurotoxicity. The mechanism of Huangqi Guizhi Wuwu decoction may be to down-regulate the expression of platinum transferred protein OCT2mRNA in dorsal root ganglion cells. At the same time, it mainly upregulated the expression of platinum transferrin ATP7A mRNA and promoted the outflow of platinum, which inhibited the accumulation of platinum in dorsal root ganglion and alleviated the chronic neurotoxicity of O-IPN.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R273

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