本文選題：Nischarin + 脊髓損傷��； 參考：《浙江大學(xué)》2015年碩士論文

【摘要】：目的： 探討Nischarin在脊髓損傷(Spinal cord injury, SCI)后軸突再生中所起的作用,并初步探討其分子機(jī)制。 方法： 1Allen' s撞擊法建立Sprague-Dawley大鼠SCI模型； 2大鼠脊髓內(nèi)微量注射針對Nischarin的shRNA質(zhì)�；騭iRNA小片段與轉(zhuǎn)染試劑,進(jìn)行體內(nèi)轉(zhuǎn)染； 3針對shRNA質(zhì)粒轉(zhuǎn)染,利用GFP熒光蛋白檢測體內(nèi)轉(zhuǎn)染的效率； 4通過RT-PCR方法評價shRNA質(zhì)�；騭iRNA小片段抑制Nischarin的效果； 5連續(xù)6w的曠場試驗對大鼠進(jìn)行BBB行為學(xué)評分,反映大鼠SCI后運動功能恢復(fù)情況； 6連續(xù)6w爬桿試驗對大鼠進(jìn)行后肢腳掌踏地角度FSA、尾根高度指數(shù)RHI和爬桿時間等測試,反映大鼠SCI后運動功能恢復(fù)情況； 7在SCI后6w,脊髓損傷平面以下注射熒光金示蹤劑,逆行示蹤法檢測紅核區(qū)的熒光金分布和強(qiáng)度,反映SCI后軸突再生的情況； 8Western blot和免疫組化法檢測脊髓損傷區(qū)GAP43蛋白的表達(dá),反映軸突再生的情況。 結(jié)果： 1成功建立大鼠SCI模型； 2質(zhì)粒注射區(qū)局部可見GFP熒光,提示shRNA質(zhì)粒成功實現(xiàn)體內(nèi)轉(zhuǎn)染； 3Nis-shRNA質(zhì)粒和Nis-siRNA小片段均能有效抑制Nischarin的轉(zhuǎn)錄水平； 4體重監(jiān)測結(jié)果證明各組間均無顯著差異,提示shRNA和siRNA使用安全； 5曠場試驗中,SCI+Nis-shRNA組的BBB運動功能評分較SCI+Ctl-shRNA組和SCI組高,差異顯著；SCI+Nis-siRNA組的BBB運動功能評分較SCI+Ctl-siRNA組和SCI組高,差異顯著； 6爬桿試驗中,SCI+Nis-shRNA組和SCI+Nis-siRNA組的FSA均較各自的對照組和SCI組低,差異顯著；SCI+Nis-shRNA組和SCI+Nis-siRNA組的RHI均較各自的對照組和SCI組高,差異顯著；但爬桿時間在各組間無顯著差異； 7逆行示蹤實驗中,SCI+Nis-shRNA組和SCI+Nis-siRNA組在紅核的熒光金陽性神經(jīng)元個數(shù)均多于各自的對照組和SCI組； 8SCI后1w和6w, western blot結(jié)果顯示,SCI+Nis-shRNA組和SCI+Nis-siRNA組的GAP43表達(dá)均較各自的對照組和SCI組多,差異顯著；免疫熒光強(qiáng)度顯示,SCI+Nis-shRNA組和SCI+Nis-siRNA組的GAP43表達(dá)較各自的對照組和SCI組多。 結(jié)論： 在損傷脊髓處抑制Nischarin的表達(dá),有利于促進(jìn)SCI大鼠的神經(jīng)元軸突再生和運動功能的康復(fù)。
[Abstract]:Aim: to investigate the role of nischarin in axonal regeneration after spinal cord injury (sci) and its molecular mechanism. Methods: (1) Sprague-Dawley rat sci model was established by Allen's impact method; (2) shRNA plasmids or siRNA fragments targeting Nischarin were microinjected into the spinal cord of rats and transfected with transfection reagent in vivo; (3) shRNA plasmids were transfected with shRNA plasmids. GFP fluorescent protein was used to detect transfection efficiency in vivo; 4 shRNA plasmid or siRNA fragment was used to evaluate the inhibitory effect of nischarin by RT-PCR. The results showed that the recovery of motor function in rats after sci was reflected in 6 consecutive weeks of pole climbing test, which reflected the recovery of motor function of rats after sci by measuring the angle of foot and foot angle, the index of tail root height RHI and the time of climbing pole. (7) at 6 weeks after sci, fluorescent gold tracer was injected below the spinal cord injury plane. The distribution and intensity of fluorescence gold in the red nucleus were detected by retrograde tracing method, which reflected the regeneration of axon after sci. The expression of GAP43 protein in spinal cord injury area was detected by Western blot and immunohistochemistry, which reflected the regeneration of axon. Results: 1 rat sci model was successfully established, 2GFP fluorescence was observed locally in plasmid injection area, indicating that shRNA plasmid was successfully transfected in vivo; 3Nis-shRNA plasmids and Nis-siRNA fragments could effectively inhibit the transcription level of nischarin, and the results of body weight monitoring showed that there was no significant difference among the groups, suggesting that shRNA and siRNA were safe to use. (5) the score of BBB motor function in sci Nis-shRNA group was higher than that in sci Ctl-shRNA group and sci group in open field test, and the BBB motor function score of sci Nis-siRNA group was higher than that of sci Ctl-siRNA group and sci group. 6FSA of sci Nis-shRNA group and sci Nis-siRNA group was lower than that of control group and sci group, and the RHI of sci Nis-shRNA group and sci Nis-siRNA group was higher than that of sci Nis-siRNA group and sci group. 7 in retrograde tracer test, the number of fluorescent gold positive neurons in red nucleus of sci Nis-shRNA group and sci Nis-siRNA group was more than that of control group and sci group. 1 week and 6 weeks after sci, western blot results showed that the expression of GAP43 in sci Nis-shRNA group and sci Nis-siRNA group was significantly higher than that in control group and sci group, and immunofluorescence intensity showed that GAP43 expression in sci Nis-shRNA group and sci Nis-siRNA group was higher than that in sci group and sci group. Conclusion: inhibiting the expression of Nischarin in injured spinal cord is beneficial to promote axonal regeneration and motor function rehabilitation in sci rats.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R651.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 羊明智,吳葉,章亞東,侯樹勛,賈連順;鈣離子及其阻滯劑對脊髓灰質(zhì)c-fos基因表達(dá)的影響[J];中華創(chuàng)傷骨科雜志;2005年02期

2 趙凡;楊有庚;王江;;大鼠完全脊髓橫斷動物模型的建立及完整胚胎脊髓移植后GAP-43 mRNA的表達(dá)[J];中國老年學(xué)雜志;2007年02期

，

本文編號：2113557