K562来源的外泌体对骨髓间充质干细胞造血和成骨基因表达的影响

doi:10.3877/cma.j.issn.2095-1221.2021.04.003

目的

探讨K562细胞来源的外泌体（EXO）对骨髓间充质干细胞（BMMSCs）支持造血和成骨分化相关因子表达的影响。

方法

利用超速离心法提取K562细胞培养上清中的EXO并进行鉴定。在体外，诱导BMMSCs的成骨分化，根据是否加入EXO分为3组：BMMSCs+PBS、BMMSCs+EXO （25 μg/mL）、BMMSCs+EXO （50 μg/mL）。qRT-PCR和Western blot检测BMMSCs中支持造血和成骨基因的表达，ELISA测定碱性磷酸酶（ALP）活性和细胞基质钙含量。多时间点间比较采用重复测量方差分析，多组间差异采用ANOVA分析，组间两两比较采用Tukey's test检验。

结果

三组各时间点CXC型趋化因子配体12 （CXCL12）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-6 （IL-6）、Runt相关转录因子2 （RUNX2）、碱性磷酸酶（ALP）和Ⅰ型胶原酶（Col Ⅰ） mRNA表达及ALP活性和细胞基质钙含量行重复测量方法分析发现，时间点间、组间、组间×时间点间差异均有统计学意义（P < 0.05）；与BMMSCs+PBS相比，BMMSCs+EXO （25、50 μg/mL）组在第1、3、7天的CXCL12、GM-CSF、RUNX2、ALP和Col ⅠmRNA水平下调，IL-6 mRNA水平上调，差异有统计学意义（P < 0.05）。与BMMSCs+EXO （25 μg/mL）组相比，BMMSCs+EXO （50 μg/mL）组CXCL12 （3 d：0.42±0.11比0.26±0.12，7 d：0.39±0.11比0.24±0.10）、RUNX2 （3 d：0.24±0.10比0.10±0.03）和Col ⅠmRNA水平（1 d：0.74±0.19比0.58±0.20，7 d：0.12±0.16比0.08±0.16）下调，IL-6 mRNA水平（1 d：1.36±0.54比2.14±0.42，3 d：2.46±0.47比3.30±0.42，7 d：3.62±0.49比4.30±0.48）上调，差异有统计学意义（P < 0.05）。Western blot发现，与BMMSCs+PBS相比，BMMSCs+EXO （25、50 μg/mL）组CXCL12 （1.00比0.54±0.10、0.32±0.08）、GM-CSF （1.00比0.46±0.12、0.42±0.03）、RUNX2 （1.00比0.57±0.12、0.45±0.23）、ALP （1.00比0.46±0.06、0.35±0.23）和ColⅠ表达（1.00比0.74±0.15、0.53±0.23）降低，IL-6表达（1.00比5.24±0.25、10.27±0.13）增加，差异有统计学意义（P < 0.05）。与BMMSCs+EXO （25 μg/mL）组相比，BMMSCs+EXO （50 μg/mL）组CXCL12表达（0.54±0.10比0.32±0.08）降低，IL-6表达（5.24±0.25比10.27±0.13）增加，差异有统计学意义（P均< 0.05）。此外，与BMMSCs+PBS组相比，BMMSCs+EXO （25、50 μg/mL）组ALP活性（7 d：1.75±0.58比0.72±0.18、0.58±0.16，14 d：2.78±0.75比1.50±0.32、0.83±0.30）和细胞基质钙含量（14 d：2.73±0.68比1.43±0.42、0.85±0.40）降低，差异有统计学意义（P < 0.05）。

结论

K562细胞来源的EXO影响BMMSCs支持造血和成骨相关因子的表达，抑制了BMMSCs的成骨分化，并且有可能影响其支持造血功能。

关键词: 慢性粒细胞白血病, 骨髓间充质干细胞, 外泌体, 造血, 成骨

Objective

This study was aimed to investigate the effects of K562 cell-derived exosomes on the expression of genes supporting hemopoiesis and osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs) .

Methods

The exosomes in the supernatant of K562 cell culture were isolated by ultracentrifugation and identified. Osteogenic differentiation of BMMSCs was induced in vitro. According to whether exosomes were added, three groups were formed: BMMSCs+PBS, BMMSCs+EXO (25 μg/mL) , and BMMSCs+EXO (50 μg/mL) . The expression of hemopoiesis and osteogenesis-supporting genes in BMMSCs was detected by RT-PCR and Western Blot. ALP activity and cell matrix calcium content were determined by ELISA. Repeated measures ANOVA was used to examine mean differences at different time points, ANOVA analysis to detect differences among multiple groups, and Tukey's test was used for pairwise comparison between groups.

Results

The CXCL12, GM-CSF, IL-6, RUNX2, ALP and ColⅠmRNA expression, alkaline phosphatase activity and cell matrix calcium content in the three groups at each time point were analyzed by repeated measurements and it was found that the differences between time points, groups, and groups×time points were statistically significant (P < 0.05) . Compared with BMMSCs+PBS, on day 1, 3 and 7, CXCL12, GM-CSF, RUNX2, ALP and ColⅠmRNA levels were down-regulated and IL-6 mRNA levels were up-regulated in the BMMSCs+EXO (25 μg/mL and 50 μg/mL) group, and the difference was statistically significant (P < 0.05) . Compared with the BMMSCs+EXO (25 μg/mL) group, the CXCL12 (3 d: 0.42±0.11 vs 0.26±0.12; 7 d: 0.39±0.11 vs 0.24±0.10) , RUNX2 (3 d: 0.24±0.10 vs 0.10±0.03) and ColⅠ (1 d: 0.74±0.19 vs 0.58±0.20; 7 d: 0.12±0.16 vs 0.08±0.16) mRNA levels were down-regulated and IL-6 (1 d:1.36±0.54 vs 2.14±0.42; 3 d:2.46±0.47 vs 3.30±0.42; 7 d:3.62±0.49 vs 4.30±0.48) mRNA levels were up-regulated in the BMMSCs+EXO (50 μg/mL) group, and the difference was statistically significant (P < 0.05) . Compared with BMMSCs+PBS, the expressions of CXCL12 (1.00 vs 0.54±0.10, 0.32±0.08) , GM-CSF (1.00 vs 0.46±0.12, 0.42±0.03) , RUNX2 (1.00 vs 0.57±0.12, 0.45±0.23) , ALP (1.00 vs 0.46±0.06, 0.35±0.23) and ColⅠ (1.00 vs 0.74±0.15,0.53±0.23) were decreased and IL-6 (1.00 vs 5.24±0.25, 10.27±0.13) was increased in the BMMSCs+EXO (25 μg/mL and 50 μg/mL) group, and the difference was statistically significant (P < 0.05) . Compared with the BMMSCs+EXO (25 μg/ml) group, the expression of CXCL12 (0.54±0.10 vs 0.32±0.08) was decreased and IL-6 (5.24±0.25 vs 10.27±0.13) was increased in the BMMSCs+EXO (50 μg/mL) group, and the difference was statistically significant (P < 0.05) . In addition, compared with BMMSCs+PBS, ALP activity (7 d: 1.75±0.58 vs 0.72±0.18, 0.58±0.16; 14 d: 2.78±0.75 vs 1.50±0.32, 0.83±0.30) and cell matrix calcium content (14 d: 2.73±0.68 vs 1.43±0.42, 0.85±0.40) in the BMMSCs+EXO (25 μg/mL and 50 μg/mL) group were decreased, and the difference was statistically significant (P < 0.05) .

Conclusion

K562 cell-derived exosomes affect the expression of hemopoiesis and osteogenesis-supporting genes in BMMSCs, thereby inhibiting osteogenic differentiation of BMMSCs, and may affect the function supporting hemopoiesis.

Key words: Chronic myeloid leukemia, Bone marrow mesenchymal stem cells, Exosome, Hemopoiesis, Osteogenesis

表1 引物序列

基因	序列（5'→3'）	预期扩增产物长度（bp）
CXCL12	上游ACAGATGCCCATGCCGATTCT	159
	下游CCTGAATCCACTTTAGCTTCGGGT
GM-CSF	上游TCTCAGAAATGTTTGACCTAAC	142
	下游GCCCTTGAGCTTGGTGAG
IL-6	上游GGAGACTTGCCTGGTGAAAA	179
	下游CAGGGGTGGTTATTGCATCT
RUNX2	上游TGTCATGGCGGGTAACGAT	147
	下游AAGACGGTTATGGTCAAGGTGAA
ALP	上游ACGTGGCTAAGAATGTCATC	476
	下游CTGGTAGGCGATGTCCTTA
Col Ⅰ	上游GAGGGCCAAGACGAAGACATC	140
	下游CAGATCACGTCATCGCACAAC
GAPDH	上游ATTTGGTCGTATTGGGCG	124
	下游TGGAAGATGGTGATGGGATT

表1 引物序列

基因	序列（5'→3'）	预期扩增产物长度（bp）
CXCL12	上游ACAGATGCCCATGCCGATTCT	159
	下游CCTGAATCCACTTTAGCTTCGGGT
GM-CSF	上游TCTCAGAAATGTTTGACCTAAC	142
	下游GCCCTTGAGCTTGGTGAG
IL-6	上游GGAGACTTGCCTGGTGAAAA	179
	下游CAGGGGTGGTTATTGCATCT
RUNX2	上游TGTCATGGCGGGTAACGAT	147
	下游AAGACGGTTATGGTCAAGGTGAA
ALP	上游ACGTGGCTAAGAATGTCATC	476
	下游CTGGTAGGCGATGTCCTTA
Col Ⅰ	上游GAGGGCCAAGACGAAGACATC	140
	下游CAGATCACGTCATCGCACAAC
GAPDH	上游ATTTGGTCGTATTGGGCG	124
	下游TGGAAGATGGTGATGGGATT

图1 外泌体的特征

图2 K562细胞来源的外泌体对BMMSCs支持造血和成骨分化相关因子蛋白表达的影响

表2 K562细胞来源的外泌体对BMMSCs CXCL12、GM-CSF、IL-6 mRNA表达的影响（ ± s）

分组	实验次数	CXCL12			GM-CSF			IL-6
分组	实验次数	1 d	3 d	7 d	1 d	3 d	7 d	1 d	3 d	7 d
BMMSCs+PBS	3	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
BMMSCs+EXO （25 g/mL）	3	0.83±0.02	0.43±0.04^a	0.41±0.07^a	0.84±0.05^a	0.32±0.06^a	0.25±0.03^a	1.43±0.10^a	2.45±0.45^a	3.62±0.19^a
BMMSCs+EXO （50 g/mL）	3	0.79±0.03^a	0.24±0.05^ab	0.25±0.03^ab	0.74±0.03^ab	0.23±0.04^a	0.18±0.03^ab	2.19±0.20^ab	3.37±0.45^ab	4.34±0.29^ab
时间点间（F/P）		367.423/< 0.001			461.006/< 0.001			234.057/< 0.001
组间×时间点间（F/P）		92.341/< 0.001			115.545/< 0.001			58.901/< 0.001
组间（F/P）		429.316/< 0.001			914.395/< 0.001			81.569/< 0.001

表2 K562细胞来源的外泌体对BMMSCs CXCL12、GM-CSF、IL-6 mRNA表达的影响（ ± s）

分组	实验次数	CXCL12			GM-CSF			IL-6
分组	实验次数	1 d	3 d	7 d	1 d	3 d	7 d	1 d	3 d	7 d
BMMSCs+PBS	3	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
BMMSCs+EXO （25 g/mL）	3	0.83±0.02	0.43±0.04^a	0.41±0.07^a	0.84±0.05^a	0.32±0.06^a	0.25±0.03^a	1.43±0.10^a	2.45±0.45^a	3.62±0.19^a
BMMSCs+EXO （50 g/mL）	3	0.79±0.03^a	0.24±0.05^ab	0.25±0.03^ab	0.74±0.03^ab	0.23±0.04^a	0.18±0.03^ab	2.19±0.20^ab	3.37±0.45^ab	4.34±0.29^ab
时间点间（F/P）		367.423/< 0.001			461.006/< 0.001			234.057/< 0.001
组间×时间点间（F/P）		92.341/< 0.001			115.545/< 0.001			58.901/< 0.001
组间（F/P）		429.316/< 0.001			914.395/< 0.001			81.569/< 0.001

表3 K562细胞来源的外泌体对BMMSCs CXCL12、GM-CSF和IL-6蛋白表达的影响（ ± s）

分组		实验次数	CXCL12	GM-CSF	IL-6
BMMSCs+PBS		3	1.00	1.00	1.00
BMMSCs+EXO （25 μg/mL）		3	0.53±0.06^a	0.46±0.12^a	5.25±0.26^a
BMMSCs+EXO （50 μg/mL）		3	0.35±0.05^ab	0.42±0.04^a	10.27±0.13^ab
	F值		166.180	59.025	2293.417
	P值		< 0.001	< 0.001	< 0.001

表3 K562细胞来源的外泌体对BMMSCs CXCL12、GM-CSF和IL-6蛋白表达的影响（ ± s）

分组		实验次数	CXCL12	GM-CSF	IL-6
BMMSCs+PBS		3	1.00	1.00	1.00
BMMSCs+EXO （25 μg/mL）		3	0.53±0.06^a	0.46±0.12^a	5.25±0.26^a
BMMSCs+EXO （50 μg/mL）		3	0.35±0.05^ab	0.42±0.04^a	10.27±0.13^ab
	F值		166.180	59.025	2293.417
	P值		< 0.001	< 0.001	< 0.001

表4 K562细胞来源的外泌体对BMMSCs RUNX2、ALP和Col Ⅰ mRNA表达的影响（ ± s）

分组		实验次数	RUNX2			ALP			Col Ι
分组		实验次数	1 d	3 d	7 d	1 d	3 d	7 d	1 d	3 d	7 d
BMMSCs+PBS		3	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
BMMSCs+EXO （25 μg/mL）		3	0.54±0.05^a	0.24±0.09^a	0.14±0.09^a	0.74±0.10^a	0.27±0.09^a	0.22±0.02^a	0.77±0.10^a	0.26±0.08^a	0.13±0.02^a
BMMSCs+EXO （50 μg/mL）		3	0.44±0.03^ab	0.10±0.03^ab	0.11±0.07^a	0.64±0.02^a	0.18±0.09^a	0.13±0.07^a	0.56±0.06^ab	0.16±0.07^ab	0.11±0.01^a
	时间点间（F/P）		72.865/< 0.001			83.280/< 0.001			87.076/< 0.001
	组间×时间点间（F/P）		19.161/< 0.001			20.834/< 0.001			23.016/< 0.001
	组间（F/P）		456.905/< 0.001			576.768/< 0.001			11045.320/< 0.001

表4 K562细胞来源的外泌体对BMMSCs RUNX2、ALP和Col Ⅰ mRNA表达的影响（ ± s）

分组		实验次数	RUNX2			ALP			Col Ι
分组		实验次数	1 d	3 d	7 d	1 d	3 d	7 d	1 d	3 d	7 d
BMMSCs+PBS		3	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
BMMSCs+EXO （25 μg/mL）		3	0.54±0.05^a	0.24±0.09^a	0.14±0.09^a	0.74±0.10^a	0.27±0.09^a	0.22±0.02^a	0.77±0.10^a	0.26±0.08^a	0.13±0.02^a
BMMSCs+EXO （50 μg/mL）		3	0.44±0.03^ab	0.10±0.03^ab	0.11±0.07^a	0.64±0.02^a	0.18±0.09^a	0.13±0.07^a	0.56±0.06^ab	0.16±0.07^ab	0.11±0.01^a
	时间点间（F/P）		72.865/< 0.001			83.280/< 0.001			87.076/< 0.001
	组间×时间点间（F/P）		19.161/< 0.001			20.834/< 0.001			23.016/< 0.001
	组间（F/P）		456.905/< 0.001			576.768/< 0.001			11045.320/< 0.001

表5 K562细胞来源的外泌体对BMMSCs RUNX2、ALP和Col Ⅰ蛋白表达的影响（ ± s）

分组	实验次数	RUNX2	ALP	Col Ι
BMMSCs+PBS	3	1.00	1.00	1.00
BMMSCs+EXO （25 μg/mL）	3	0.57±0.10^a	0.47±0.06^a	0.74±0.05^a
BMMSCs+EXO （50 μg/mL）	3	0.45±0.07^a	0.35±0.03^ab	0.54±0.03^ab
F值		50.517	239.267	140.824
P值		< 0.001	< 0.001	< 0.001

表5 K562细胞来源的外泌体对BMMSCs RUNX2、ALP和Col Ⅰ蛋白表达的影响（ ± s）

分组	实验次数	RUNX2	ALP	Col Ι
BMMSCs+PBS	3	1.00	1.00	1.00
BMMSCs+EXO （25 μg/mL）	3	0.57±0.10^a	0.47±0.06^a	0.74±0.05^a
BMMSCs+EXO （50 μg/mL）	3	0.45±0.07^a	0.35±0.03^ab	0.54±0.03^ab
F值		50.517	239.267	140.824
P值		< 0.001	< 0.001	< 0.001

表6 K562细胞来源的外泌体对BMMSCs的ALP活性和细胞基质钙含量的影响

分组		实验次数	ALP活性（mU/mL）			细胞基质钙含量（μmol/dL）
分组		实验次数	1 d	7 d	14 d	1 d	7 d	14 d
BMMSCs+PBS		3	0.28±0.05	1.74±0.39	2.78±0.25	0.25±0.10	1.02±0.20	2.74±0.45
BMMSCs+EXO （25 μg/mL）		3	0.19±0.07	0.74±0.07^a	1.51±0.11^a	0.19±0.05	0.73±0.06^a	1.43±0.12^a
BMMSCs+EXO （50 μg/mL）		3	0.16±0.02	0.59±0.05^a	0.84±0.07^ab	0.16±0.03	0.58±0.06^a	0.78±0.06^ab
	时间点间（F/P）		140.513/< 0.001			103.597/< 0.001
	组间×时间点间（F/P）		18.402/< 0.001			22.332/< 0.001
	组间（F/P）		277.806/< 0.001			175.910/< 0.001

表6 K562细胞来源的外泌体对BMMSCs的ALP活性和细胞基质钙含量的影响

分组		实验次数	ALP活性（mU/mL）			细胞基质钙含量（μmol/dL）
分组		实验次数	1 d	7 d	14 d	1 d	7 d	14 d
BMMSCs+PBS		3	0.28±0.05	1.74±0.39	2.78±0.25	0.25±0.10	1.02±0.20	2.74±0.45
BMMSCs+EXO （25 μg/mL）		3	0.19±0.07	0.74±0.07^a	1.51±0.11^a	0.19±0.05	0.73±0.06^a	1.43±0.12^a
BMMSCs+EXO （50 μg/mL）		3	0.16±0.02	0.59±0.05^a	0.84±0.07^ab	0.16±0.03	0.58±0.06^a	0.78±0.06^ab
	时间点间（F/P）		140.513/< 0.001			103.597/< 0.001
	组间×时间点间（F/P）		18.402/< 0.001			22.332/< 0.001
	组间（F/P）		277.806/< 0.001			175.910/< 0.001

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The effects of K562 cell-derived exosomes on the hematopoietic and osteogenic gene expressions of bone marrow mesenchymal stem cells

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The effects of K562 cell-derived exosomes on the hematopoietic and osteogenic gene expressions of bone marrow mesenchymal stem cells