本文選題：骨髓間充質干細胞 + 重復移植　； 參考：《華北理工大學》2015年碩士論文

【摘要】：目的本研究通過動物實驗初步探索骨髓間質干細胞重復移植治療腦梗死是否可改善其神經(jīng)功能預后,減少實驗動物腦梗死面積,是更為有效的細胞療法,以及可能作用機制,為臨床應用細胞療法治療腦梗死提供理論依據(jù)。方法1取幼齡SD大鼠長骨骨髓進行培養(yǎng),利用全骨髓貼壁篩選法培養(yǎng)、分離、純化BMSCs。取第3或4代細胞進行移植,移植前24h以終濃度10umol/L的Brd U標記。2用改良的Longa線栓法建立大鼠腦梗死模型,根據(jù)m NSS選出60只大鼠模型,隨機分為對照組,單次組,重復組,每組20只,在建模后24h,對照組注射1ml生理鹽水,單次組和重復組注射3×106個BMSCs,建模后7d,對照組和單次組注射1ml生理鹽水,重復組注射3×106個BMSCs,均通過尾靜脈注射。3建模后7、14d,運用m NSS對大鼠進行神經(jīng)功能評定,然后每組各取5只大鼠進行TTC染色比較各組相對梗死體積。4建模后14d處死大鼠,進行免疫組織化學染色觀察Brdu標記率,單染定性觀察VEGF、Olig-2、Bcl-2、Caspase-3蛋白情況,觀察新生血管內皮、少突膠質細胞增生情況、細胞凋亡。5 Western blot檢測Bcl-2、Caspase-3的蛋白含量。結果1 mNSS結果:建模后3d,單次組及重復組比對照組mNSS低,差異有統(tǒng)計學意義(P0.05),重復組與單次組m NSS低,差異有統(tǒng)計學意義(P0.05);建模后7d,單次組和重復組比對照組m NSS低,差異有統(tǒng)計學意義(P0.01);建模后14d,單次組、重復組m NSS均顯著低于對照組,差異有統(tǒng)計學意義(P0.01),重復組比單次組m NSS低,差異有統(tǒng)計學意義(P0.05)。2建模后7d,單次組及重復組相對梗死體積小于對照組,差異有統(tǒng)計學意義(P0.05),單次組與重復組之間無差異;建模后14d,單次組與對照組相對梗死體積比較,差異有統(tǒng)計學意義(P0.05),重復組與對照組比較,差異有統(tǒng)計學意義(P0.01),重復組與單次組比較,差異有統(tǒng)計學意義(P0.05)。3免疫組織化學染色結果,對照組腦VEGF表達水平低于單次組及重復組,差異有統(tǒng)計學意義(P0.05),單次組與重復組比較有統(tǒng)計學意義(P0.05);對照組腦Olig-2表達低于單次組及重復組,單次組與對照組比較(P0.01),單次組與重復組比較(P0.01),差異有統(tǒng)計學意義;對照組腦Bcl-2表達水平低于單次組及重復組,差異有統(tǒng)計學意義(P0.05),單次組與重復組比較有統(tǒng)計學意義(P0.05);對照組Caspase-3蛋白表達高于單次組及重復組,差異有統(tǒng)計學意義(P0.05)。4 Western blot結果提示:對照組腦組織Bcl-2蛋白表達低于單次組及重復組,差異有統(tǒng)計學意義(P0.01),單次組與重復組比較有統(tǒng)計學意義(P0.05);對照組腦組織Caspase-3蛋白表達高于單次組及重復組,差異有統(tǒng)計學意義(P0.01),單次組與重復組比較有統(tǒng)計學意義(P0.05),提示BMSCs可能通過促進血管內細胞增生、刺激少突膠質細胞再生、減少凋亡治療腦梗死。結論1全骨髓貼壁法培養(yǎng)BMSCs,可迅速增殖、分化、純化。2同種異體BMSCs對大鼠腦梗死有治療作用,且重復移植治療效果優(yōu)于單次移植。3 BMSCs遠期治療效果較好。4線栓法建立大鼠永久性腦梗死模型操作相對簡單,可重復,損傷較小。5 Brd U標記BMSCs,操作簡單,標記率高,對細胞形態(tài)、生長等無影響。6尾靜脈移植BMSCs,可遷移至腦缺血區(qū)域,且未發(fā)現(xiàn)不良反應。7 BMSCs可能通過刺激VEGF蛋白分泌,促進血管再生,改善腦組織血液循環(huán),增加Olig-2蛋白分泌,刺激少突膠質細胞增生,提高軸突傳導能力,并上調Bcl-2蛋白,下降Caspase-3蛋白抑制細胞凋亡,減少梗死體積等機制治療腦梗死。
[Abstract]:Objective to explore the effect of repeated transplantation of bone marrow mesenchymal stem cells in the treatment of cerebral infarction to improve the prognosis of cerebral infarction and reduce the area of cerebral infarction in experimental animals. It is a more effective cell therapy and possible mechanism to provide a theoretical basis for the clinical application of cell therapy in the treatment of cerebral infarction. Method 1 the young age SD was taken. The bone marrow of the long bone of the rat was cultured. The third or 4 generation cells were isolated and purified by the full bone marrow adherence screening method. The BMSCs. Brd U marker of the final concentration of 10umol/L was used to establish the rat model of cerebral infarction with the modified Longa thread thrombus method before transplantation. 60 rat models were selected according to m NSS, and were randomly divided into the control group, the single group and the repeat group. 20 rats in each group were established after modeling 24h, the control group was injected with 1ml physiological saline, the single group and the repeated group were injected with 3 x 106 BMSCs. After modeling 7d, the control group and the single group were injected with 1ml physiological saline, and the repeated group was injected with 3 x 106 BMSCs. The.3 modeling 7,14d was injected through the tail vein, and the rats were evaluated with m NSS, then 5 rats in each group were given 5 rats. The rats were stained with TTC to compare the relative infarct volume in each group, and the rats were killed after the.4 modeling. The Brdu labeling rate was observed by immunohistochemical staining. The VEGF, Olig-2, Bcl-2, Caspase-3 protein were observed by single staining, and the proliferation of oligodendrocytes was observed, and the apoptotic.5 Western blot detected Bcl-2, and the protein contained the protein content. Results 1 mNSS results: after modeling, 3D, single group and repeated group were lower than the control group mNSS, the difference was statistically significant (P0.05). The difference was statistically significant (P0.05) in the repeated group and the single group M NSS; the model 7d, the single group and the repeat group were lower than the control group M NSS, and the difference was statistically significant (P0.01); after modeling, the 14d, single group, and repeated groups were all obvious. The difference was significantly lower than that of the control group (P0.01), the repeat group was lower than the single group M NSS, the difference was statistically significant (P0.05).2 modeling 7d, the relative infarct volume in the single and repeated groups was less than the control group, the difference was statistically significant (P0.05), there was no difference between the single group and the repeated group; after modeling 14d, the single group and the control group were relative infarct bodies. The difference was statistically significant (P0.05). The difference was statistically significant (P0.01) compared with the control group (P0.01). The difference was statistically significant (P0.05) with statistical significance (P0.05), and the level of VEGF expression in the control group was lower than that of the single group and the repeated group, and the difference was statistically significant (P0.05). The comparison group had statistical significance (P0.05); the brain Olig-2 expression in the control group was lower than the single group and the repeated group, the single group was compared with the control group (P0.01), the single group and the repeated group were compared (P0.01), the difference was statistically significant; the level of Bcl-2 expression in the control group was lower than that of the single group and the repeated group (P0.05), the single group and the repeat group ratio were statistically significant (P0.05). The expression of Caspase-3 protein in the control group was higher than that of the single group and the repeated group, the difference was statistically significant (P0.05).4 Western blot results suggested that the expression of Bcl-2 protein in the brain tissue of the control group was lower than that of the single group and the repeat group, the difference was statistically significant (P0.01), and there was a statistical significance (P0.05) in the single group and the repeat group (P0.05); the difference was statistically significant (P0.05) in the control group (P0.05); and the difference was statistically significant (P0.05) in the single group and the repeat group (P0.05); The expression of Caspase-3 protein in the brain tissue was higher than that of the single group and repeated group. The difference was statistically significant (P0.01). There was a significant difference between the single group and the repeated group (P0.05). It suggested that BMSCs could stimulate the proliferation of the cells in the blood vessels, stimulate the regeneration of oligodendrocytes and reduce the apoptosis in the treatment of cerebral infarction. Conclusion 1 full bone marrow adherent method can be used to cultivate BMSCs. Rapid proliferation, differentiation, and purification of.2 allogeneic BMSCs have therapeutic effect on cerebral infarction in rats, and the effect of repeated transplantation is better than that of single transplant.3 BMSCs. It is relatively simple to operate the model of permanent cerebral infarction by.4 line thrombus method, and can be repeated, and the damage is smaller than.5 Brd U, the operation is simple, the labeling rate is high, and the cell mark rate is high, and the cell mark rate is high, and the cell mark rate is high. Morphology, growth and so on without influence of.6 tail vein graft BMSCs, can migrate to cerebral ischemia area, and no adverse reaction.7 BMSCs can stimulate the secretion of VEGF protein, promote vascular regeneration, improve blood circulation of brain tissue, increase the secretion of Olig-2 protein, stimulate oligodendrocyte proliferation, improve the axon conduction ability, and up regulation of Bcl-2 protein and decrease C Aspase-3 protein inhibits apoptosis and reduces infarct volume to treat cerebral infarction.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R743.33

【參考文獻】

相關期刊論文 前1條

1 張洪連;吳曉牧;;骨髓間充質干細胞移植治療腦梗死及存在問題[J];中華腦血管病雜志(電子版);2010年04期

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本文編號：2088487