基因修飾骨髓間充質干細胞腦源性神經營養因子的表達變化

謝茂松，徐國興，黃禮彬

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·實驗論著·

基因修飾骨髓間充質干細胞腦源性神經營養因子的表達變化

謝茂松，徐國興，黃禮彬

Abstract

?AIM: To study the changes of brain-derived neurotrophic factor (BDNF) expression in gene modified bone marrow mesenchymal stem cells (BMSC).

?METHODS: BMSC were divided into blank control group (without transfected BMSC),negative control group (empty vector without BDNF gene transfected BMSC) and experimental group (BDNF gene transfected BMSC).The expression of BDNF mRNA in BMSC was measured by Realtime PCR,and the expression of BDNF in BMSC was measured by ELISA.

?RESULTS: The BDNF mRNA expressions of 3,4,5,6,7 and 8-generation BMSC cells in the experimental group were higher than those in the blank control group and negative control group.The differences were statistically significant (P3: F=491.788,P<0.05; P4: F=380.112,P<0.05; P5: F=1854.929,P<0.05; P6: F=224.540,P<0.05; P7: F=619.155,P<0.05; P8: F=10.092,P<0.05).As the BMSC cells in the experimental group passaging,the BDNF mRNA expressions in the experimental group decreased.The difference of BDNF mRNA expression among different passage cells was statistically significant (F=298.603,P<0.05).The BDNF secretion of 3,4,5,6,7 and 8-generation BMSC cells in the experimental group were higher than those in the blank control group and negative control group.The differences were statistically significant (P3: F=520.609,P<0.05; P4: F=734.520,P<0.05; P5: F=152.847,P<0.05; P6: F=80.372,P<0.05; P7: F=96.083,P<0.05; P8: F=38.532,P<0.05).As the BMSC cells in the experimental group passaging,the BDNF secretion decreased.The difference of BDNF secretion among different passage cells was statistically significant (F=230.084,P<0.05).

?CONCLUSION: Long-term expression of BDNF in BMSC can be enhanced by genetic engineering.

目的：研究基因修飾的骨髓間充質干細胞(bone marrow mesenchymal stem cell，BMSC)分泌腦源性神經營養因子(brain-derived neurotrophic factor，BDNF)的表達變化。

方法：實驗分為空白對照組(未經轉染BMSC細胞)、陰性對照組(采用不含BDNF基因的空載質粒轉染的BMSC細胞)和實驗組(采用含BDNF基因的質粒轉染的BMSC細胞)。Realtime PCR測定基因修飾的BMSC細胞BDNF mRNA表達，ELISA測定基因修飾的BMSC細胞BDNF的分泌表達。

結果：實驗組第3、4、5、6、7 和8代BMSC細胞BDNF mRNA表達高于空白對照組和陰性對照組，組間差異具有統計學意義(P3：F=491.788，P<0.05；P4：F=380.112，P<0.05；P5：F=1854.929，P<0.05；P6：F=224.540，P<0.05；P7：F=619.155，P<0.05；P8：F=10.092，P<0.05)。實驗組BMSC細胞隨著細胞傳代，BDNF mRNA表達逐漸下降，不同代數間差異具有統計學意義(F=298.603，P<0.05)。實驗組3、4、5、6、7和8代BMSC細胞BDNF的分泌表達高于空白對照組和陰性對照組，組間差異具有統計學意義(P3：F=520.609，P<0.05；P4：F=734.520，P<0.05；P5：F=152.847，P<0.05；P6：F=80.372，P<0.05；P7：F=96.083，P<0.05；P8：F=38.532，P<0.05)。實驗組BMSC細胞隨著細胞傳代，BDNF的分泌表達逐漸下降，不同代數間差異具有統計學差異(F=230.084，P<0.05)。

結論：通過基因工程可增強BMSC細胞表達BDNF。

骨髓間充質干細胞；腦源性神經營養因子；基因修飾；視網膜變性疾病；治療

引用：謝茂松，徐國興，黃禮彬.基因修飾骨髓間充質干細胞腦源性神經營養因子的表達變化.國際眼科雜志2016;16(10):1816-1819

0　引言

視網膜變性疾病是嚴重的不可逆性致盲眼病。目前此類疾病尚無有效的治療方法[1-2]。細胞移植治療為視網膜變性疾病的治療點亮了希望。骨髓間充質干細胞(bone marrow mesenchymal stem cell，BMSC)由于多向分化潛能、自體取材方便，無免疫原性等優點，是理想的種子細胞[3-4]。腦源性神經營養因子(brain-derived neurotrophic factor，BDNF)可增加突觸可塑性及神經發生，促進神經細胞生存[5-6]。干細胞治療和基因治療有望為視網膜變性疾病的治療提供新的途徑。本項目研究擬通過基因工程增強BMSC表達BDNF神經營養因子，研究基因修飾的BMSC細胞BDNF的表達變化。

1　材料和方法

1.1材料健康清潔級雄性SD大鼠(福建醫科大學實驗……