本文選題：高糖環(huán)境 + 布比卡因　； 參考：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：第一部分體外細(xì)胞實(shí)驗(yàn)?zāi)康奶接懜咛黔h(huán)境對(duì)布比卡因作用下SH-SY5Y細(xì)胞神經(jīng)毒性、修復(fù)酶Ku70表達(dá)的影響。方法用正常和含葡萄糖25、50、100mM的培養(yǎng)基處理SH-SY5Y細(xì)胞1、3、7 d后測(cè)定蛋白γ-H2AX和Ku70的表達(dá);確定高糖的作用時(shí)間和作用濃度。檢測(cè)布比卡因濃度梯度下細(xì)胞活力。建立布比卡因毒性損傷的細(xì)胞模型。實(shí)驗(yàn)進(jìn)一步分為對(duì)照組、高糖組、布比卡因組、高糖+布比卡因組。檢測(cè)Ku70 mRNA、修復(fù)酶Ku70、凋亡蛋白cleaved caspase-3的表達(dá)和細(xì)胞內(nèi)ROS含量、彗星尾距值、凋亡細(xì)胞比例。構(gòu)建Ku70過表達(dá)細(xì)胞株,驗(yàn)證Ku70在高糖環(huán)境加重布比卡因?qū)H-SY5Y細(xì)胞神經(jīng)毒性中的重要作用統(tǒng)計(jì)學(xué)處理計(jì)量資料以x ±s表示,SPSS 20.0軟件分析,高糖或布比卡因單一因素作用后各組之間的比較采用單因素方差分析。高糖與布比卡因兩因素作用后各組之間的比較采用雙向方差分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果SH-SY5Y細(xì)胞在布比卡因作用后Ku70表達(dá)增加。高糖環(huán)境抑制Ku70增加布比卡因作用后神經(jīng)細(xì)胞凋亡。過表達(dá)Ku70能減輕布比卡因的神經(jīng)毒性。結(jié)論高糖環(huán)境抑制Ku70的表達(dá)加劇布比卡因的神經(jīng)毒性,Ku70作為此損傷修復(fù)的關(guān)鍵酶。第二部分體內(nèi)大鼠實(shí)驗(yàn)?zāi)康挠^察2型糖尿病大鼠對(duì)鞘內(nèi)注射布比卡因神經(jīng)毒性損傷及修復(fù)酶Ku70表達(dá)的影響方法高糖、高脂飲食結(jié)合腹腔注射鏈脲佐菌素建立2型糖尿病大鼠模型。分組:對(duì)照組(Con)、糖尿病組(DM)、布比卡因組(Bup)、糖尿病+布比卡因組(DM + Bup)。Bup和DM + Bup組為鞘內(nèi)注射2.5%布比卡因30ul,其它組注射等量生理鹽水。阻滯前和阻滯后6、12、24 h分別測(cè)定足底機(jī)械痛閾(PWMT)和熱痛反應(yīng)潛伏期(PWTL)的變化。注射24h后取脊髓腰膨大部位測(cè)定丙二醛(MDA)、超氧化物歧化酶(SOD)、Ku70與cleavedcaspase-3的表達(dá)。HE染色觀察脊髓損傷情況,Tunel法檢測(cè)凋亡細(xì)胞。統(tǒng)計(jì)學(xué)處理計(jì)量資料以x± s表示,SPSS 20.0軟件分析,PWMT和PWTL值的比較采用重復(fù)測(cè)量的方差分析。MDA、SOD、Ku70、cleaved caspase-3、凋亡細(xì)胞比例的比較采用雙向方差分析,組間比較采用兩獨(dú)立樣本t檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果與注射前相比,Bup組注射24 h后PWMT和PWTL無明顯變化(P=0.680;P=0.730),DM+Bup 組注射 24 h 后 PWMT 和 PWTL 明顯降低(P=0.000;p = 0.000)。高糖環(huán)境與布比卡因皆可導(dǎo)致MDA增加和SOD活性抑制,且具有協(xié)同作用。與Bup組相比,DM+Bup組抑制修復(fù)酶Ku70的表達(dá)((= 4.475,P = 0.000);同時(shí)增加 cleaved caspase-3的表達(dá)和凋亡細(xì)胞的比例(t=-4.707,P= 0.000;t =-20.041,P = 0.000)。結(jié)論2型糖尿病大鼠加重布比卡因鞘內(nèi)注射后脊髓神經(jīng)的毒性損傷,抑制修復(fù)酶Ku70的表達(dá)。
[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 緯 -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 鹵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 鹵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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R614;R587.1

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