本文選題：丙戊酸 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《河北北方學(xué)院》2017年碩士論文

【摘要】：為了探討腹腔注射丙戊酸(VPA)輔佐骨髓間充質(zhì)干細(xì)胞(BMSCs)對(duì)大鼠脊髓損傷(SCI)后神經(jīng)功能恢復(fù)的作用。本實(shí)驗(yàn)選用3-4周齡健康雄性SD幼鼠,體外分離培養(yǎng)BMSCs,對(duì)第3代BMSCs進(jìn)行流式細(xì)胞儀檢測(cè),收集備用。把60只成年雄性SD大鼠隨機(jī)分為5組:假手術(shù)組(A組)、模型組(B組)、丙戊酸組(C組)、移植組(D組)和聯(lián)合組(E組)。動(dòng)物脊髓損傷模型選用改良的Allen’s法制作。丙戊酸組傷后給予腹腔注射VPA(300 mgkg-1),每12 h給一次藥直至取材;移植組在損傷段脊髓注入BMSCs l mL濃度為1×106 mL-1;聯(lián)合組同時(shí)注入VPA和BMSCs,假手術(shù)組和模型組注射等體積的0.9%氯化鈉。大鼠在傷后7 d、14 d先行后肢(BBB)運(yùn)動(dòng)功能評(píng)分,隨后處死取材。用蘇木精-伊紅染色法(HE)染色觀(guān)察脊髓空洞面積,用免疫組化法檢定半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白表達(dá)情況,通過(guò)脫氧核糖核苷酸末端轉(zhuǎn)移酶介導(dǎo)的缺口末端標(biāo)記法(TUNEL)染色觀(guān)察脊髓組織切片神經(jīng)細(xì)胞凋亡情況。實(shí)驗(yàn)結(jié)果顯示,BBB評(píng)分:假手術(shù)組、模型組、丙戊酸組、移植組和聯(lián)合組在SCI后7 d分別為(21),(2.22±0.33),(3.80±0.53),(3.81±0.83),(4.02±0.83);在SCI后14 d分別為(21),(4.51±0.41),(7.25±0.95),(7.32±0.55),(9.76±0.95)。假手術(shù)組運(yùn)動(dòng)功能正常,3個(gè)治療組的BBB評(píng)分明顯比模型組高(P0.05)。在SCI后第14 d聯(lián)合組的評(píng)分明顯高于丙戊酸組和移植組(P0.05)。HE染色結(jié)果:模型組、丙戊酸組、移植組和聯(lián)合組損傷區(qū)域的面積分別是(4.57±0.26)mm2,(3.34±0.21)mm2,(3.51±0.18)mm2,(2.43±0.35)mm2。聯(lián)合組損傷空洞及瘢痕組織面積明顯比其余的3個(gè)組低(P0.05)。免疫組化染色:假手術(shù)組、模型組、丙戊酸組、移植組和聯(lián)合組在SCI后7 d的Caspase-3免疫組化陽(yáng)性目標(biāo)面密度分別為(1.51±0.23)%,(28.51±5.23)%,(24.39±4.65)%,(24.51±3.81)%,(20.73±3.43)%。在SCI后14 d的Caspase-3免疫組化陽(yáng)性目標(biāo)面密度分別為(1.64±0.41)%,(26.22±4.81)%,(21.21±4..04)%,(21.47±3.32)%,(16.15±3.09)%。假手術(shù)組少量caspase-3陽(yáng)性表達(dá),3個(gè)治療組的caspase-3表達(dá)明顯低于模型組(P0.05)。而聯(lián)合組的caspase-3陽(yáng)性表達(dá)明顯低于丙戊酸組和移植組(P0.05)。細(xì)胞凋亡結(jié)果:假手術(shù)組、模型組、丙戊酸組、移植組和聯(lián)合組在SCI后14 d的凋亡指數(shù)分別為(4.18±0.39)%,(40.51±3.81)%,(19.95±2..04)%,(20.87±2.32)%,(12.83±1.09)%。傷后14 d假手術(shù)組存在少量凋亡陽(yáng)性細(xì)胞。與模型組相比,3個(gè)治療組的凋亡指數(shù)較低(P0.05),而3個(gè)治療組之間,聯(lián)合組凋亡指數(shù)明顯比丙戊酸組和移植組低(P0.05)。腹腔注射VPA輔佐BMSCs移植對(duì)SCI后運(yùn)動(dòng)神經(jīng)功能的恢復(fù)效果顯著,其機(jī)制可能和下調(diào)caspase-3的表達(dá),抑制凋亡有關(guān)。
[Abstract]:To investigate the effect of intraperitoneal injection of valproic acid (VPA) combined with bone marrow mesenchymal stem cells (BMSCs) on the recovery of neural function after spinal cord injury (SCI) in rats. BMSCs were isolated and cultured in vitro from 3-4 week old male SD rats. The third generation BMSCs were detected by flow cytometry and collected. Sixty adult male Sprague-Dawley rats were randomly divided into 5 groups: sham operation group (group A), model group (group B), valproic acid group (group C), transplantation group (group D) and combined group (group E). The animal spinal cord injury model was made by modified Allen's method. The valproic acid group was intraperitoneally injected with VPA-300 mg / kg ~ (-1) of BMSCs every 12 hours, the transplantation group was injected with BMSCs (1 脳 10 ~ 6 mL / mL) in the injured spinal cord, and the combined group was injected with VPA and BMSCs, 0.9% sodium chloride was injected into the sham operation group and the model group. The motor function of hind limbs was scored 14 days after injury, and then killed. The area of syringomyelia was observed by hematoxylin-eosin staining and the expression of Caspase-3 protein was detected by immunohistochemistry. Apoptosis of neurons in spinal cord sections was observed by dNT-mediated dUTP Nick end labeling (Tunel) staining. The results showed that the scores of BBB in sham operation group, model group, valproic acid group, transplantation group and combined group were 2.22 鹵0.33, 3.81 鹵0.83C, 4.02 鹵0.83N, 7.32 鹵0.555D, 7.32 鹵0.555L, 4.02 鹵0.83g / d after sci, respectively. The motor function of sham-operation group was normal, and BBB score of three treatment groups was significantly higher than that of model group (P 0.05). On the 14th day after sci, the score of the combined group was significantly higher than that of the valproic acid group and the transplantation group. The result of HE staining: the area of the injured area in the model group, valproic acid group, transplantation group and combined group was 4.57 鹵0.26 mm ~ 2 + 3.34 鹵0.21 鹵0.21 鹵0.18 mm ~ (2) and 2.43 鹵0.35 mm ~ (2) ~ 2 respectively. The area of injury cavity and scar tissue in the combined group was significantly lower than that in the other three groups (P 0.05). Immunohistochemical staining: the positive surface density of Caspase-3 in sham-operation group, model group, valproic acid group, transplantation group and combined group were 1.51 鹵0.23 and 28.51 鹵5.23 respectively on the 7th day after sci. The density of Caspase-3 immunocytochemical positive target was 24.39 鹵4.65 + 24.51 鹵3.81 and 20.73 鹵3.43 respectively in sham operation group, model group, valproic acid group, transplantation group and combined group on the 7th day after sci. On the 14th day after sci, the positive surface density of Caspase-3 was 1.64 鹵0.41 and 21.21 鹵4.81, respectively. The density of Caspase-3 was 21.47 鹵3.32 and 16.15 鹵3.09. A small amount of caspase-3 positive expression was found in the sham-operation group, and the caspase-3 expression in the three treatment groups was significantly lower than that in the model group (P 0.05). The positive expression of caspase-3 in combined group was significantly lower than that in valproic acid group and transplantation group (P 0.05). Results: the apoptotic index of sham operation group, model group, valproic acid group, transplantation group and combined group were 4.18 鹵0.39 on the 14th day after sci. The apoptotic index was 19.95 鹵2.81 and 20.87 鹵1.09 in the sham operation group, model group, valproic acid group, transplantation group and combined group respectively on the 14th day after sci. There were a few apoptotic positive cells in sham-operation group 14 days after injury. Compared with the model group, the apoptotic index of the three treatment groups was lower than that of the valproic acid group and the transplantation group, but the apoptotic index of the combined group was significantly lower than that of the valproic acid group and the transplantation group. The effect of intraperitoneal injection of VPA-assisted BMSCs on the recovery of motor nerve function after sci may be related to the down-regulation of caspase-3 expression and inhibition of apoptosis.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R651.2

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