本文關(guān)鍵詞：NSCs與PLGA復(fù)合體移植對大鼠脊髓半橫斷損傷的修復(fù)作用　出處：《石河子大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 神經(jīng)干細(xì)胞 脊髓半橫斷損傷 PLGA組織工程支架 運(yùn)動誘發(fā)電位(MEPs) 移植

【摘要】：背景:目前脊髓損傷(spinal cord injury,SCI)的預(yù)防、治療和康復(fù)是目前臨床工作種難以攻克的難題。而生物細(xì)胞學(xué)技術(shù)和組織工程技術(shù)的發(fā)展為治療脊髓損傷帶來新的治療方案。目的:研究乳鼠腦組織源神經(jīng)干細(xì)胞(neural stem cells,NSCs),聯(lián)合聚乳酸-乙醇酸工程支架(Poly-Lactic-co-glycolicacid,PLGA)移植治療SD大鼠脊髓半橫斷損傷的效果。方法:(1)乳鼠NSCs的分離和培養(yǎng)的優(yōu)化:取出生3天的SD乳鼠大腦顳葉皮質(zhì),分別用機(jī)械吹打法、胰蛋白酶消化法分離培養(yǎng),通過免疫熒光法對NSCs進(jìn)行鑒定和誘導(dǎo)分化;分別于培養(yǎng)的第2、5、7天計(jì)數(shù)細(xì)胞球直徑及數(shù)目;通過MTT法比較原代及傳代后第3亞代NSCs克隆增值速率。(2)NSC與PLGA復(fù)合體移植治療SD大鼠脊髓半橫斷損傷修復(fù)作用:將72只雌性SD大鼠分成6組:正常組、假手術(shù)組、手術(shù)組、NSCs移植組、PLGA移植組、NSCs+PLGA復(fù)合體共移植組。制作脊髓半橫斷模型,正常組不做處理,假手術(shù)組僅摘除椎板;其他各組行脊髓右側(cè)半橫斷術(shù);術(shù)后第7天,再次打開脊髓,手術(shù)組于損傷處移入20ul培養(yǎng)液,NSCs移植組移入含NSCs培養(yǎng)液;PLGA移植組將PLGA組織工程支架植入脊髓損傷處;NSCs+PLGA復(fù)合體共移植組將NSCs+PLGA復(fù)合體植入脊髓損傷處。每組大鼠于術(shù)前1d、術(shù)后7d、移植后第1、2、4、8周行BBB評分、IP評分等行為學(xué),于移植后第2、4、8周,行電生理MEPs檢查、病理HE染色。結(jié)果:(1)NSCs的分離和培養(yǎng)的優(yōu)化:機(jī)械吹打組與胰酶消化組獲得的NSCs,行免疫熒光發(fā)現(xiàn),Nestin、NF-200、GFAP染色均陽性,培養(yǎng)第5、7天時,機(jī)械吹打組獲得的細(xì)胞球數(shù)量和直徑均高于相同培養(yǎng)時間點(diǎn)的胰酶消化組,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。機(jī)械吹打組原代和第3亞代細(xì)胞第2、5天細(xì)胞克隆增殖能力均優(yōu)于同時間節(jié)點(diǎn)的胰酶消化組,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)行為學(xué)評價:移植后第4、8周時NSCs移植組、PLGA移植組、NSCs+PLGA復(fù)合體移植組BBB評分、IP評分高于手術(shù)組,差異有統(tǒng)計(jì)學(xué)意義(p0.05);NSCs移植組、PLGA移植兩組比較,差異無統(tǒng)計(jì)學(xué)意義(p0.05)。移植后第4、8周,NSCs+PLGA復(fù)合體移植組BBB評分、IP評分高于NSCs移植組、PLGA移植組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。(3)電生理:運(yùn)動誘發(fā)電位(MEPs)潛伏期比較顯示:移植后第2、4、8周,NSCs移植組、PLGA移植組、NSCs+PLGA復(fù)合體移植組優(yōu)于手術(shù)組(p0.05);移植后第2、4、8周NSCs+PLGA復(fù)合體移植組優(yōu)于NSCs移植組、PLGA移植組(p0.05)。同時間點(diǎn)各組健側(cè)MEPs的潛伏期輕度優(yōu)于手術(shù)側(cè),但是在統(tǒng)計(jì)學(xué)上差異無意義(p0.05)。(4)病理HE染色:各實(shí)驗(yàn)組脊髓組織行HE染色后,發(fā)現(xiàn)移植后第8周時,NSCs移植組、PLGA移植組、NSCs+PLGA移植組損傷處脊髓組織結(jié)構(gòu)較前有所恢復(fù),空洞、瘢痕組織少于手術(shù)組。NSCs+PLGA復(fù)合體移植組周圍組織結(jié)構(gòu)恢復(fù)更好,空洞、瘢痕組織少于NSCs移植組、PLGA移植組。結(jié)論:1.機(jī)械吹打法、胰酶消化法均可成功分離培養(yǎng)、誘導(dǎo)分化NSCs,但是機(jī)械吹打法分離培養(yǎng)的NSCs的克隆增殖能力優(yōu)于胰酶消化法。2.通過行為學(xué)、電生理及病理學(xué)評估發(fā)現(xiàn)NSCs+PLGA復(fù)合體移植對SD大鼠脊髓半橫斷損傷有一定的修復(fù)作用,尤其對后肢運(yùn)動能力的恢復(fù)比較明顯。但是對脊髓損傷的解剖結(jié)構(gòu)的重塑和功能的恢復(fù)作用有限。3.NSCs、PLGA單獨(dú)移植時行為學(xué)、電生理、病理學(xué)評價較手術(shù)組有一定的改善,但改善程度弱于兩者復(fù)合體移植治療。
[Abstract]:Background: the prevention, treatment and rehabilitation of spinal cord injury (SCI) is a difficult problem to be tackled in the current clinical work. The development of biological cytology and tissue engineering will bring new treatment for the treatment of spinal cord injury. Objective: To study the effect of neural stem cells (NSCs) combined with poly (lactic acid glycolic acid) engineering scaffold (Poly-Lactic-co-glycolicacid, PLGA) transplantation on the treatment of spinal cord hemisection injury in SD rats. Methods: (1) NSCs rats were isolated and cultured for optimization: 3 day old SD rat cerebral cortex respectively by mechanical trituration and trypsin digestion method for isolation and cultivation, identification and differentiation of NSCs by immunofluorescence; on the second, fifth, seventh day count and number of training ball diameter the MTT method; through comparison of primary and passage third sub NSCs clone VAT rate. (2) transplantation of NSC and PLGA complex to treat spinal cord hemisection injury in SD rats: 72 female SD rats were divided into 6 groups: normal group, sham operation group, operation group, NSCs transplantation group, PLGA transplantation group and NSCs+PLGA complex transplantation group. Production of spinal cord hemisection model, normal group was not treated, the sham operation group only with each other on the right side of the lamina; spinal cord hemisection; seventh days after surgery, open surgery group in spinal cord injury. Again, in 20ul medium, NSCs medium containing NSCs in the transplantation group; PLGA transplantation group PLGA scaffolds for tissue engineering implantation of spinal cord injury; NSCs+PLGA complex were transplanted NSCs+PLGA complex was implanted into spinal cord injury. Rats in each group were subjected to 1D, 7d, first, second, fourth and 8 weeks after operation, BBB score, IP score and other behaviors. Second, fourth, eighth weeks after transplantation, electrophysiological MEPs examination and pathological HE staining were performed. Results: (1) NSCs isolate and culture: the optimization of the mechanical percussion group and group NSCs obtained by trypsin digestion, immunofluorescence, Nestin, NF-200 and GFAP staining were positive when cultured for fifth, seventh days, cell number and diameter of mechanical percussion group were higher than those obtained by trypsin digestion group culture of the same point in time, the difference was statistically significant (P0.05). Mechanical percussion group primary and third sub cells second, fifth days of cell clone proliferation were better than trypsin digestion group at the same time node, the difference was statistically significant (P0.05). (2) behavioral evaluation: at fourth, eighth weeks after transplantation, BBB score and IP score in NSCs transplantation group, PLGA transplantation group and NSCs+PLGA complex transplantation group were higher than those in operation group, the difference was statistically significant (P0.05), NSCs group and PLGA transplantation group had no significant difference between two groups (P0.05). Fourth, eighth weeks after transplantation, the BBB score and IP score of the NSCs+PLGA complex group were higher than those in the NSCs transplantation group and the PLGA transplantation group, and the difference was statistically significant (P0.05). (3) electrophysiological: motor evoked potentials (MEPs) were compared: second, fourth, eighth weeks after transplantation, NSCs transplantation group, PLGA transplantation group, NSCs+PLGA transplantation group than the complex operation group (P0.05); second, fourth, eighth weeks after transplantation NSCs+PLGA complex transplantation group was better than NSCs transplantation group, PLGA transplantation group (P0.05). At the same time, the latency of MEPs in each group was slightly better than that in the operation side, but there was no significant difference in Statistics (P0.05). (4) pathological HE staining: after the HE staining of spinal cord tissue in each experimental group, it was found that at eighth weeks after transplantation, the spinal cord structure of NSCs transplantation group, PLGA transplantation group and NSCs+PLGA transplantation group recovered earlier than that of the operation group, and the cavity and scar tissue were less than those of the operation group. The surrounding tissue structure of the NSCs+PLGA complex transplanted group was better, and the cavity and scar tissue were less than that of the NSCs transplantation group and the PLGA transplantation group. Conclusion: 1., mechanical blow and trypsin digestion methods can successfully isolate, culture and induce NSCs differentiation, but the NSCs cloning ability of mechanical blow up culture is better than that of trypsin digestion. 2. through behavioral, electrophysiological and pathological evaluation, we found that NSCs+PLGA complex transplantation has certain repair effect on spinal cord hemisection in SD rats, especially for the recovery of hind limb movement ability. However, the reshaping and functional recovery of the anatomical structure of spinal cord injury are limited. The evaluation of behavior, electrophysiology and pathology of 3.NSCs and PLGA was better than that of the operation group, but the degree of improvement was weaker than that of the two groups.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R651.2
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本文編號：1345246