發(fā)布時間：2018-11-09 19:05

【摘要】：目的 1、人工體外分離培養(yǎng)兔骨髓間充質(zhì)干細(xì)胞（Bone marrow mesenchymal stem cellsBMSCs）并誘導(dǎo)其分化為骨髓源神經(jīng)樣細(xì)胞。 2、兔頸上交感神經(jīng)節(jié)（superior cervical sympathetic ganglion SCSG）損毀模型的建立。 3、將骨髓源神經(jīng)樣細(xì)胞植入到SCSG損傷模型體內(nèi)，觀察模型兔瞳孔的變化，并通過測定損傷局部及海馬組織中腦源性神經(jīng)因子（brain-derived neurotrophic factor，BDNF）、睫狀神經(jīng)生長因子（Ciliary Neurotrophic Factor，CNTF）的表達(dá)情況，探討骨髓源神經(jīng)樣細(xì)胞移植對兔頸上交感神經(jīng)節(jié)受損修復(fù)作用及可能的作用機制。 方法 利用持針鉗壓榨兔SCSG，制備單側(cè)（右側(cè)）SCSG損傷模型。將體外經(jīng)密度離心聯(lián)合貼壁方法分離培養(yǎng)得到BMSCs，用堿性成纖維細(xì)胞生長因子（Basic FibroblastGrowth Facto，，bFGF）預(yù)誘導(dǎo)，再經(jīng)CNTF誘導(dǎo)分化為骨髓源神經(jīng)樣細(xì)胞。隨機地將模型兔分為三組，每組15只。A組為對照組，B組培養(yǎng)液組，C組為細(xì)胞移植組。模型制作成功第7天時，A組無處理，B組經(jīng)耳緣靜脈注入細(xì)胞培養(yǎng)液1ml，C組注入含骨髓源神經(jīng)樣細(xì)胞1×106個的細(xì)胞培養(yǎng)液1ml。移植后1天、7天、10天、14天、21天觀察兔瞳孔變化，移植后第21天處死兔，分離神經(jīng)節(jié)，進行HE染色，并采用免疫組化分別檢測兔SCSG之BDNF和CNTF的表達(dá)水平。取兔海馬區(qū)標(biāo)本進行HE染色并檢測BDNF的表達(dá)水平。 結(jié)果 1、兔瞳孔直徑(mm)的比較：頸上交感神經(jīng)節(jié)損傷后1、7、10、14、21天時，A組兔瞳孔直徑分別為：5.81+0.42，5.84+0.34，5.68+0.29，5.91+0.31，5.80+0.36；B組兔瞳孔直徑分別為：5.84+0.32，5.74+0.32，5.63+0.37，5.83+0.27，5.58+0.23；C組兔瞳孔直徑分別為：5.64+0.33，5.71+0.35，6.78+0.33，7.97+0.28，8.05+0.21。C組在10天、14天時間點，瞳孔直徑均明顯大于A組（P0.05）。A組、B組不同時間點比較無明顯差異（P0.05）。C組不同時間點比較，差異明顯（P0.05），14天、21天瞳孔直徑明顯大于1天、7天、10天（P0.05），14天與21天比較，瞳孔直徑差異無統(tǒng)計學(xué)意義（P0.05）。 2、BDNF免疫組化染色：A組、B組、C組兔海馬區(qū)BDNF陽性細(xì)胞數(shù)分別為：17.13+2.19，18.53+1.35，20.20+4.29，C組比A組、B組BDNF陽性細(xì)胞數(shù)明顯要多，差異有顯著性（（P0.05）），A組與B組比較無統(tǒng)計學(xué)意義（P0.05）。三組兔損傷側(cè)SCSG之BDNF陽性細(xì)胞數(shù)分別為：6.67+1.11，6.80+1.26，12.18+7.93，C組比A組、B組BDNF陽性細(xì)胞數(shù)明顯要多，差異有顯著性（（P0.05）），A組與B組比較無統(tǒng)計學(xué)意義（P0.05）。 3、CNTF免疫組化染色：A組、B組、C組CNTF陽性細(xì)胞數(shù)分別為：15.6+1.83，15.6+1.54，37.73+7.26，C組明顯高于A組、B組（P0.05），而A組與B組比較無統(tǒng)計學(xué)意義（P0.05）。 結(jié)論 1、骨髓源神經(jīng)樣細(xì)胞移植治療兔SCSG損傷，可促進其受損神經(jīng)功能的修復(fù)。 2、骨髓源神經(jīng)樣細(xì)胞移植修復(fù)兔SCSG損傷的機制可能與上調(diào)BDNF及CNTF有關(guān)。
[Abstract]:Objective 1. Rabbit bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cellsBMSCs) were cultured in vitro and induced to differentiate into bone marrow derived neural cells (BMSCs). 2. The (superior cervical sympathetic ganglion SCSG) lesion model of superior cervical sympathetic ganglion in rabbits was established. 3. Bone marrow-derived neuronal cells were implanted into SCSG injury model to observe the changes of pupillary, and to measure the brain derived neurofactor (brain-derived neurotrophic factor,BDNF) in local and hippocampal tissue. The expression of ciliary nerve growth factor (Ciliary Neurotrophic Factor,CNTF (ciliary nerve growth factor) and its possible mechanism in repairing the injury of superior cervical sympathetic ganglion (SCG) in rabbits were investigated by bone marrow-derived nerve-like cell transplantation. Methods unilateral (right) SCSG injury model was established by pressing rabbit SCSG, with needle holding forceps. BMSCs, was preinduced by basic fibroblast growth factor (Basic FibroblastGrowth Facto,bFGF) by density centrifugation and adherent method in vitro, and then differentiated into bone marrow-derived nerve-like cells induced by CNTF. The model rabbits were randomly divided into three groups: control group (group A), culture medium group (group B) and cell transplantation group (group C). On the 7th day after the model was made, no treatment was found in group A, and 1ml of cell culture medium (1 ml) containing 1 脳 106 myeloid nerve cells was injected into group B via auricular vein. Pupillary changes were observed on day 1, day 7, day 10, day 14 and day 21 after transplantation. The ganglion was isolated and stained with HE on the 21st day after transplantation. The expression of BDNF and CNTF in rabbit SCSG were detected by immunohistochemistry. The hippocampal specimens of rabbits were stained with HE and the expression of BDNF was detected. Results 1 comparison of pupillary diameter (mm) in rabbits: the pupil diameter of group A was 5.81 0.42n 5.84 0.45.68 0.295.91 0.31 鹵5.80 0.36 on day 1421 after injury of superior cervical sympathetic ganglion. The diameter of pupil in group A was 5.81 0.42n 5.84 0.45.68 0.295.91 0.31 and 5.80 0.36 respectively. The pupil diameter of group B was 5.84 0.32 ~ 5.74 0.32 ~ 5.63 0.37 ~ 5.83 0.27 ~ 5.58 and 0.23 respectively. The pupillary diameter of group C was 5.64 0.33 / 5.71 0.35 / 5.71 0.335.78 0.337.97 0.337.97 0.280.280.05 / 0.21.C respectively. The pupil diameter of group C was significantly larger than that of group A at 10 days and 14 days (P0.05). A group). There was no significant difference at different time points in group B (P0.05). The pupil diameter of group B was significantly larger than that of 1 day, 7 days, 10 days (P0.05), 14 days and 21 days, respectively (P0.05). There was no significant difference in pupil diameter (P0.05). 2the number of BDNF positive cells in hippocampus of group A, group B and group C were 17.13 2.19, 18.53 1.35 and 20.20 4.29, respectively, and the number of positive cells of BDNF in group B was significantly higher than that in group A. There was significant difference (P0.05), A group and B group compared with no statistical significance (P0.05). The number of BDNF positive cells in the injured side of SCSG in the three groups was 6.67 1.116.80 1.26 1.26 12.18 7.93 渭 g / L respectively compared with group A, and the number of BDNF positive cells in group B was significantly higher than that in group A (P0.05),). There was no significant difference between group A and group B (P0.05). 3the number of CNTF positive cells in group A, group B and group C were 15.6 1.83n 15.6 15.6 1.54 + 37.73 7.26C, respectively, higher than those in group A (P0.05), group B (P 0.05), group B (P 0.05). There was no significant difference between group A and group B (P0.05). Conclusion 1. Bone marrow-derived neuron-like cell transplantation can promote the repair of injured nerve function in rabbits with SCSG injury. 2. The mechanism of bone marrow derived neuroid cell transplantation in repairing SCSG damage in rabbits may be related to up-regulation of BDNF and CNTF.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

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