【摘要】：目的:糖尿病痛性神經病變(painful diabetic neuropathy,PDN)是一種發(fā)生在糖尿病患者中以機械刺激性疼痛為特點的疾病。長期糖尿病機械性痛(mechanical allodynia,DMA)對患者的生活造成了諸多不便。本實驗以鏈脲佐菌素(Streptozotocin,STZ)誘導大鼠建立糖尿病痛性神經病變模型,研究經典瞬時受體電位通道6(Transient Receptor Potential Canonical,TRPC6)、下游cAMP反應元件結合蛋白(cAMP response element binding protein,CREB)在模型大鼠背根神經節(jié)(dorsal root ganglia,DRG)中的表達水平及藥物干預后的表達水平變化,及其與大鼠機械性痛覺敏感閾值變化的相互關系。旨在揭示PDN發(fā)病的分子生物學原理,為PDN的臨床治療及預防提供新思路和實驗依據(jù)。方法:(1)將50只清潔級、健康、成年、雄性的斯普拉格-杜勒(Sprague-Dawley,SD)大鼠適應性喂養(yǎng)1周后,隨機將大鼠分為CON組6只、DM組44只,給予DM組大鼠STZ腹腔注射建立DM大鼠模型,CON組則僅予以相同劑量的檸檬酸鹽緩沖液;(2)分別于造模前及造模后第3、7、14、21d測定大鼠血糖、體重與后爪機械性回縮閾值(paw withdrawal threshold,PWT);(3)第3d時,以血糖值≥16.7mmol/L為標準篩選DM模型大鼠;(4)第14d時,用von Frey絲測定大鼠PWT值的變化,按其比造模前基線值下降50%為標準篩選DMA模型大鼠;(5)第15d時,給予CON組及DMA模型組大鼠行鞘內置管術,術后連續(xù)肌內注射青霉素預防性抗感染治療3天,并將DMA模型大鼠隨機分為三組(DMA組、DMA+溶媒組、DMA+SKF96365組),置管后第3天開始鞘內注射SKF96365(65ng/kg),連續(xù)干預5天;(6)分別運用實時定量PCR(quantitative real-time PCR,qRT-PCR)技術和Western blot技術檢測各DMA模型組及CON組大鼠DRGs中TRPC6、pCREB的mRNA及蛋白表達水平。結果:(1)造模前大鼠的血糖、體重、PWT基線值不存在明顯差異(P0.05);(2)STZ注射后第3d,DM組約89%的大鼠血糖顯著升高(血糖16.7 mmol/L),與CON組相比,多飲、多食、多尿、消瘦等糖尿病三多一少癥狀明顯;(3)STZ注射后第14d,DM大鼠約52%出現(xiàn)明顯的雙側后爪機械刺激性痛(即為DMA模型大鼠),PWT值較CON組顯著下降(P0.01),并持續(xù)降低至第21d(P0.01);(4)與CON組相比,DMA組大鼠DRGs中TRPC6與pCREB的mRNA和蛋白表達水平明顯上調(P0.01),DMA組與DMA+溶媒組大鼠DRGs中的TRPC6、pCREB的mRNA和蛋白表達水平無明顯差異(P0.05),DMA+SKF96365組大鼠DRGs中TRPC6、pCREB的mRNA和蛋白表達水平較DMA+溶媒組有所下降(P0.01)。結論:STZ誘導的DMA大鼠DRGs中TRPC6表達上調,與疼痛呈正相關,DMA+SKF96365組TRPC6表達下調,與疼痛的變化相一致,TRPC6可能參與了糖尿病機械痛的發(fā)病過程。TRPC6與pCREB表達水平變化相關,提示TRPC6可能是通過調節(jié)下游核轉錄調節(jié)蛋白pCREB表達,加重疼痛。
[Abstract]:Objective: diabetic painful neuropathy (painful diabetic neuropathy,PDN) is a disease characterized by mechanical irritating pain in diabetic patients. Long-term diabetic mechanical pain (mechanical allodynia,DMA) causes a lot of inconvenience to the life of patients. In this study, streptozotocin (Streptozotocin,STZ) was used to establish diabetic painful neuropathy model in rats. The expression level of classical transient receptor potential channel 6 (Transient Receptor Potential Canonical,TRPC6) and downstream cAMP response element binding protein (cAMP response element binding protein,CREB) in dorsal root ganglion (dorsal root ganglia,DRG) of model rats and the expression level after drug intervention were studied. And its relationship with the changes of mechanical pain sensitivity threshold in rats. The purpose of this study was to reveal the molecular biological principle of PDN and to provide new ideas and experimental basis for clinical treatment and prevention of PDN. Methods: (1) after adaptive feeding of 50 clean, healthy, adult and male Sprague-Dawley,SD rats for one week, the rats were randomly divided into CON group (n = 6) and DM group (n = 44). The DM rat model was established by intraabdominal injection of STZ in DM group, while the CON group was only given the same dose of citrate buffer. (2) on the 3rd, 7th and 14th day after modeling, respectively, the blood glucose, body weight and mechanical retraction threshold (paw withdrawal threshold,PWT); (3 of the posterior claw were measured, the DM model rats were screened with blood sugar value 鈮，

本文編號：2498671