DNA双链损伤修复酶Ku70在高糖环境加重布比卡因神经毒性损伤中的作用研究

发布时间：2018-05-24 21:45

本文选题：高糖环境 + 布比卡因　；参考：《南方医科大学》2017年博士论文

【摘要】：第一部分体外细胞实验目的探讨高糖环境对布比卡因作用下SH-SY5Y细胞神经毒性、修复酶Ku70表达的影响。方法用正常和含葡萄糖25、50、100mM的培养基处理SH-SY5Y细胞1、3、7 d后测定蛋白γ-H2AX和Ku70的表达;确定高糖的作用时间和作用浓度。检测布比卡因浓度梯度下细胞活力。建立布比卡因毒性损伤的细胞模型。实验进一步分为对照组、高糖组、布比卡因组、高糖+布比卡因组。检测Ku70 mRNA、修复酶Ku70、凋亡蛋白cleaved caspase-3的表达和细胞内ROS含量、彗星尾距值、凋亡细胞比例。构建Ku70过表达细胞株,验证Ku70在高糖环境加重布比卡因对SH-SY5Y细胞神经毒性中的重要作用统计学处理计量资料以x ±s表示,SPSS 20.0软件分析,高糖或布比卡因单一因素作用后各组之间的比较采用单因素方差分析。高糖与布比卡因两因素作用后各组之间的比较采用双向方差分析,P0.05为差异有统计学意义。结果SH-SY5Y细胞在布比卡因作用后Ku70表达增加。高糖环境抑制Ku70增加布比卡因作用后神经细胞凋亡。过表达Ku70能减轻布比卡因的神经毒性。结论高糖环境抑制Ku70的表达加剧布比卡因的神经毒性,Ku70作为此损伤修复的关键酶。第二部分体内大鼠实验目的观察2型糖尿病大鼠对鞘内注射布比卡因神经毒性损伤及修复酶Ku70表达的影响方法高糖、高脂饮食结合腹腔注射链脲佐菌素建立2型糖尿病大鼠模型。分组:对照组(Con)、糖尿病组(DM)、布比卡因组(Bup)、糖尿病+布比卡因组(DM + Bup)。Bup和DM + Bup组为鞘内注射2.5%布比卡因30ul,其它组注射等量生理盐水。阻滞前和阻滞后6、12、24 h分别测定足底机械痛阈(PWMT)和热痛反应潜伏期(PWTL)的变化。注射24h后取脊髓腰膨大部位测定丙二醛(MDA)、超氧化物歧化酶(SOD)、Ku70与cleavedcaspase-3的表达。HE染色观察脊髓损伤情况,Tunel法检测凋亡细胞。统计学处理计量资料以x± s表示,SPSS 20.0软件分析,PWMT和PWTL值的比较采用重复测量的方差分析。MDA、SOD、Ku70、cleaved caspase-3、凋亡细胞比例的比较采用双向方差分析,组间比较采用两独立样本t检验,P0.05为差异有统计学意义。结果与注射前相比,Bup组注射24 h后PWMT和PWTL无明显变化(P=0.680;P=0.730),DM+Bup 组注射 24 h 后 PWMT 和 PWTL 明显降低(P=0.000;p = 0.000)。高糖环境与布比卡因皆可导致MDA增加和SOD活性抑制,且具有协同作用。与Bup组相比,DM+Bup组抑制修复酶Ku70的表达((= 4.475,P = 0.000);同时增加 cleaved caspase-3的表达和凋亡细胞的比例(t=-4.707,P= 0.000;t =-20.041,P = 0.000)。结论2型糖尿病大鼠加重布比卡因鞘内注射后脊髓神经的毒性损伤,抑制修复酶Ku70的表达。
[Abstract]:The first part was to investigate the effects of high glucose on the neurotoxicity and Ku70 expression of SH-SY5Y cells induced by bupivacaine in vitro. Methods the expression of 纬 -H2AX and Ku70 in SH-SY5Y cells was determined after treated with normal and glucose 2550100mm medium for 1d and 3d, and the action time and concentration of high glucose were determined. Cell viability was measured at bupivacaine concentration gradient. To establish a cell model of bupivacaine toxicity injury. The experiment was further divided into control group, high glucose group, bupivacaine group and high glucose bupivacaine group. Ku70 mRNAs, repair enzyme Ku70, expression of apoptotic protein cleaved caspase-3, ROS content in cells, comet tail distance and the proportion of apoptotic cells were detected. Ku70 overexpression cell lines were constructed to verify the important role of Ku70 in increasing the neurotoxicity of bupivacaine to SH-SY5Y cells in high glucose environment. Statistical analysis was performed with x 卤s to indicate the neurotoxicity of Bupivacaine to SH-SY5Y cells. Single factor ANOVA was used to compare the effects of high glucose or bupivacaine. After two factors of high glucose and bupivacaine, the difference was statistically significant by two-way variance analysis (P0.05). Results the expression of Ku70 in SH-SY5Y cells increased after bupivacaine treatment. High glucose environment inhibited Ku70 to increase neuronal apoptosis after bupivacaine. Overexpression of Ku70 can reduce the neurotoxicity of bupivacaine. Conclusion High glucose environment inhibits the expression of Ku70 and exacerbates the neurotoxicity of bupivacaine. The second part was to observe the effect of type 2 diabetic rats on intrathecal bupivacaine neurotoxicity and the expression of repair enzyme Ku70 in rats with type 2 diabetes mellitus. High fat diet combined with intraperitoneal injection of streptozotocin was used to establish type 2 diabetic rat model. Groups: control group, diabetic group, bupivacaine group, diabetic bupivacaine group, DM Bup).Bup and DM Bup group were injected intrathecally with 2.5% bupivacaine 30ul. the other groups were injected with the same amount of normal saline. The changes of PWMTT and PWTL of plantar mechanical pain threshold (PWMTT) and thermal pain response latency (PWTL) were measured before and after 6 days and 24 hours after block, respectively. 24 hours after injection, malondialdehyde (MDA) MDAA was measured at the lumbar expansion site of spinal cord, and the expression of cleavedcaspase-3 and SODX Ku70 were detected by HE staining. The apoptotic cells were detected by Tunel method after spinal cord injury. The statistical data were analyzed by means of x 卤s software SPSS20.0. The comparison of PWMT and PWTL values was performed by repeated measurement ANOVA .MDA-SODX Ku70cleaved caspase-3, and the proportion of apoptotic cells was compared by bidirectional variance analysis. There was significant difference between the two groups by t test of two independent samples (P0.05). Results there was no significant change in PWMT and PWTL in the Bup group 24 h after injection compared with that before injection. The PWMT and PWTL decreased significantly 24 hours after injection of P0. 680 and 0. 730 in DM Bup group (P < 0. 000p = 0. 000). Both high glucose and bupivacaine could increase MDA and inhibit the activity of SOD. Compared with Bup group, the expression of inhibitory repair enzyme Ku70 in DM Bup group was 4.475 (P = 0.000), and the ratio of cleaved caspase-3 expression and apoptotic cells was increased (P = 0.000 ~ (-0.707) (P = 0.000). Conclusion Type 2 diabetic rats increase the toxic injury of spinal cord after intrathecal injection of bupivacaine and inhibit the expression of repair enzyme Ku70.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R614;R587.1

【相似文献】