嗅鞘细胞移植对脊髓损伤大鼠Wnt/β-catenin信号分子影响

摘要/Abstract

摘要： 目的

探讨嗅鞘细胞（olfactory ensheathing cells，OECs）移植后大鼠脊髓组织不同时期Wnt信号分子Wnt-1、β-catenin、GSK-3β、c-myc在损伤部位脊髓的表达变化。

方法

乳鼠嗅球制备OECs，90只成年雄性SD大鼠随机分为3组，以脊髓T₁₀节段为中心，大鼠脊髓损伤模型制备采用Allens'打击法。空白组：T_9-11椎板去除；损伤组：T₁₀脊髓损伤；移植组：T₁₀脊髓损伤，OECs移植。分别于术后1、3、5、7、14 d，每组各取3只大鼠，取以损伤区为中心上下1.5 cm范围内（空白组取椎板去除部位）的脊髓组织，提取总RNA，采用定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）检测脊髓组织中Wnt-1、β-catenin、GSK-3β、c-myc的表达量；术前及术后第7天、第14天观察BBB评分，术后第7天做免疫组化，观察脊髓结构变化。

结果

移植7 d和14 d后，移植组运动功能评分（basso beattie bresnahan，BBB）高于损伤组，差异有统计学意义（P< 0.01）。qPCR结果显示空白组不同时间点各信号分子表达稳定，趋势较为平稳；损伤组Wnt-1、GSK-3β与c-myc总体呈下调趋势，β-catenin有小幅上升，但都呈明显抑制状态；与损伤组比较，移植组除c-myc小幅上调外，Wnt-1、β-catenin与GSK-3β均呈较高表达，明显减轻了损伤后的抑制状态。

结论

脊髓损伤后Wnt/β-catenin通路被抑制，经OECs移植后，大鼠运动功能明显恢复，且Wnt/β-catenin通路抑制状态明显减轻。

关键词:嗅鞘细胞,Wnt/β-catenin,信号分子,脊髓损伤

Abstract: Objective

To investigate the expression of Wnt signaling molecules Wnt-1，β- catenin， GSK-3 β and c-myc in the injured spinal cord of rats after olfactory ensheathing cell transplantation.

Methods

Olfactory ensheathing cells were prepared from the olfactory bulb of suckling rats. Ninety adult male SD rats were randomly divided into three groups. The spinal cord injury model was prepared by Allens' strike method with T₁₀segment of spinal cord as the center. The blank group had T_9-11lamina removal； Injury group had T₁₀spinal cord injury； Transplantation group had T₁₀spinal cord injury and OEC transplantation. Three rats were taken from each group at 1， 3， 5， 7 and 14 days after operation. The spinal cord tissue within 1.5cm above and below the injured area （the vertebral lamina removal site in the blank group） was taken to extract total RNA. The expression of Wnt-1， β-catenin， GSK-3 β and c-myc in spinal cord tissue were detected by semi quantitative RT-PCR； The BBB score was observed preoperative operation and on the 7th and 14th day， and the changes of spinal cord structure were observed by immunohistochemistry on the 7th day after operation.

Results

After 7 and 14 days of transplantation， the BBB score of the cell group was significantly higher than those of injury group （P< 0.01）. QPCR results showed that the expression of signal molecules in the blank group was stable at different time points， and the trend was relatively stable； Injury group Wnt-1， GSK-3 β and c-myc showed a downward trend， β-catenin increased slightly， but all showed obvious inhibition； Compared with the injury group， c-myc in the transplantation group increased slightly， Wnt-1， β-catenin and GSK-3 β were highly expressed， which significantly reduced the inhibitory state after injury.

Conclusion

Wnt/β-catenin pathway is inhibited after spinal cord injury. After olfactory ensheathing cell transplantation， the motor function of rats is significantlyrestored， and Wnt/β-catenin pathway is significantly reduced.

Key words:olfactory ensheathing cell,wnt/β-catenin,signal molecule,spinal cord injury

冯芮, 潘飞, 王晓玉, 陈浩月, 陈悦霞, 郑遵成. 嗅鞘细胞移植对脊髓损伤大鼠Wnt/β-catenin信号分子影响[J]. betway必威登陆网址（betway.com ）学报, 2022, 43(3): 166-171.

Rui FENG, Fei PAN, Xiaoyu WANG, Haoyue CHEN, Yuexia CHEN, Zuncheng ZHENG. Effect of olfactory ensheathing cell transplantation on Wnt/β-catenin signaling molecules in rats with spinal cord injury[J]. Journal of Shandong First Medical Unversity & Shandong Academy of Medical Sciences, 2022, 43(3): 166-171.

图/表12

引物名称	引物序列（5'→3'）
Wnt1-F	CAAGATAGTCAACCGAGGCT
Wnt1-R	GTAGTCGCAGGTGCAGGATT
β-catenin-F	TTTCCCAGTCCTTCACGCAA
β-catenin-R	GCGCTGGACATTAGTGGGAT
GSK-3β-F	AAGCGATTTAAGAACCGAGAGC
GSK-3β-R	GTCCAGCACCAGGTTAAGGT
c-myc-F	CTCGGTGCAGCCCTATTTCA
c-myc-R	TAGCGACCGCAACATAGGAC
GAPDH-F	GCACCGTCAAGGCTGAGAAC
GAPDH-R	TGGTGAAGACGCCAGTGGA

引物名称	引物序列（5'→3'）
Wnt1-F	CAAGATAGTCAACCGAGGCT
Wnt1-R	GTAGTCGCAGGTGCAGGATT
β-catenin-F	TTTCCCAGTCCTTCACGCAA
β-catenin-R	GCGCTGGACATTAGTGGGAT
GSK-3β-F	AAGCGATTTAAGAACCGAGAGC
GSK-3β-R	GTCCAGCACCAGGTTAAGGT
c-myc-F	CTCGGTGCAGCCCTATTTCA
c-myc-R	TAGCGACCGCAACATAGGAC
GAPDH-F	GCACCGTCAAGGCTGAGAAC
GAPDH-R	TGGTGAAGACGCCAGTGGA

时间	空白组	损伤组	移植组	F	P
术前	21.00 ± 0.00	21.00 ± 0.00	21.00 ± 0.00
术后7 d	20.30 ± 0.675^a	3.60 ± 0.699^ch	3.40 ± 1.08^eij	4 581.237	< 0.001
术后14 d	20.90 ± 0.316^b	5.90 ± 0.738^d	13.2 ± 2.09^fgklm	13 140.167	< 0.001
F	21 191.556	136.456	931.918
P	< 0.001	< 0.001	< 0.001

时间	空白组	损伤组	移植组	F	P
术前	21.00 ± 0.00	21.00 ± 0.00	21.00 ± 0.00
术后7 d	20.30 ± 0.675^a	3.60 ± 0.699^ch	3.40 ± 1.08^eij	4 581.237	< 0.001
术后14 d	20.90 ± 0.316^b	5.90 ± 0.738^d	13.2 ± 2.09^fgklm	13 140.167	< 0.001
F	21 191.556	136.456	931.918
P	< 0.001	< 0.001	< 0.001

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.018 ± 0.005	0.542 ± 0.156^※	0.248 ± 0.046^※※	161 388.967	0.002
3	1.004 ± 0.012	0.538 ± 0.026^※	0.249 ± 0.002^※※	423.574	0.034
5	1.002 ± 0.020	0.428 ± 0.020	0.395 ± 0.010^*※	344.620	0.038
7	1.000 ± 0.034	0.275 ± 0.010^***※	0.590 ± 0.025^***※※	939.691	0.023
14	1.008 ± 0.100	0.290 ± 0.549^*	0.159 ± 0.012	188 316.868	0.002
F	269 789.499	54 986.184	47 837.782
P	0.001	0.003	0.003

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.018 ± 0.005	0.542 ± 0.156^※	0.248 ± 0.046^※※	161 388.967	0.002
3	1.004 ± 0.012	0.538 ± 0.026^※	0.249 ± 0.002^※※	423.574	0.034
5	1.002 ± 0.020	0.428 ± 0.020	0.395 ± 0.010^*※	344.620	0.038
7	1.000 ± 0.034	0.275 ± 0.010^***※	0.590 ± 0.025^***※※	939.691	0.023
14	1.008 ± 0.100	0.290 ± 0.549^*	0.159 ± 0.012	188 316.868	0.002
F	269 789.499	54 986.184	47 837.782
P	0.001	0.003	0.003

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.009 ± 0.001	0.829 ± 0.127	2.086 ± 0.100^※※	53 047 036.800	< 0.001
3	1.002 ± 0.086	0.774 ± 0.035^※	2.811 ± 0.030^※※	129 080.212	0.002
5	1.002 ± 0.074	0.933 ± 0.030	2.295 ± 0.062^※※	3 252.773	0.012
7	1.004 ± 0.055	0.953 ± 0.040	3.636 ± 0.165^**※※	3 914.371	0.011
14	1.020 ± 0.014	0.930 ± 0.030	2.735 ± 0.187^※※	17 382.195	0.005
F	1 457 948.810	963.709	14 987.272
P	0.001	0.023	0.006

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.009 ± 0.001	0.829 ± 0.127	2.086 ± 0.100^※※	53 047 036.800	< 0.001
3	1.002 ± 0.086	0.774 ± 0.035^※	2.811 ± 0.030^※※	129 080.212	0.002
5	1.002 ± 0.074	0.933 ± 0.030	2.295 ± 0.062^※※	3 252.773	0.012
7	1.004 ± 0.055	0.953 ± 0.040	3.636 ± 0.165^**※※	3 914.371	0.011
14	1.020 ± 0.014	0.930 ± 0.030	2.735 ± 0.187^※※	17 382.195	0.005
F	1 457 948.810	963.709	14 987.272
P	0.001	0.023	0.006

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.004 ± 0.005	0.959 ± 0.042	1.687 ± 0.206	310 956.261	0.001
3	1.008 ± 0.081^*	0.456 ± 0.030^※	2.853 ± 0.079^※※	218 814.669	0.002
5	1.052 ± 0.075^*	0.471 ± 0.012^*※	2.989 ± 0.100^※※	3 082.888	0.013
7	1.034 ± 0.098^*	0.481 ± 0.010^※	1.281 ± 0.015^**※※	2 895.840	0.013
14	1.007 ± 0.001^*	0.319 ± 0.030^*※	1.823 ± 0.047^***※※	1 313 629.730	0.001
F	394 009.527	163 105.096	7 190.294
P	0.001	0.002	0.008

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.004 ± 0.005	0.959 ± 0.042	1.687 ± 0.206	310 956.261	0.001
3	1.008 ± 0.081^*	0.456 ± 0.030^※	2.853 ± 0.079^※※	218 814.669	0.002
5	1.052 ± 0.075^*	0.471 ± 0.012^*※	2.989 ± 0.100^※※	3 082.888	0.013
7	1.034 ± 0.098^*	0.481 ± 0.010^※	1.281 ± 0.015^**※※	2 895.840	0.013
14	1.007 ± 0.001^*	0.319 ± 0.030^*※	1.823 ± 0.047^***※※	1 313 629.730	0.001
F	394 009.527	163 105.096	7 190.294
P	0.001	0.002	0.008

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.069 ± 0.005	1.604 ± 0.103^※	0.485 ± 0.061^※※	146 153 691.000	< 0.001
3	1.009 ± 0.13	1.971 ± 0.126^※	0.229 ± 0.025^※※	759.476	0.026
5	1.003 ± 0.27^*	0.697 ± 0.015^**※	0.246 ± 0.015^※※	10 054.208	0.007
7	1.192 ± 0.27	0.956 ± 0.020^*※	0.887 ± 0.030^*※	236 729.289	0.001
14	1.029 ± 0.006^*	0.829 ± 0.029^**※	0.262 ± 0.092^※※	19 358 006.800	< 0.001
F	282 809.911	253.439	193.011
P	0.001	0.044	0.051

术后时间（d）	空白组	损伤组	移植组	F	P
1	1.069 ± 0.005	1.604 ± 0.103^※	0.485 ± 0.061^※※	146 153 691.000	< 0.001
3	1.009 ± 0.13	1.971 ± 0.126^※	0.229 ± 0.025^※※	759.476	0.026
5	1.003 ± 0.27^*	0.697 ± 0.015^**※	0.246 ± 0.015^※※	10 054.208	0.007
7	1.192 ± 0.27	0.956 ± 0.020^*※	0.887 ± 0.030^*※	236 729.289	0.001
14	1.029 ± 0.006^*	0.829 ± 0.029^**※	0.262 ± 0.092^※※	19 358 006.800	< 0.001
F	282 809.911	253.439	193.011
P	0.001	0.044	0.051

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