高效抗逆转录病毒治疗对骨髓间充质干细胞向成骨细胞分化的影响

doi:10.3877/cma.j.issn.1674-1358.2017.04.002

目的

观察高效抗逆转录病毒药物血清干预骨髓间充质干细胞（BMSC）增殖及成骨分化的研究，探讨抗病毒药物对骨代谢的影响。

方法

采用3种HAART药物分别制备药物血清干预BMSC增殖及分化，CCK法检测细胞增殖情况，碱性磷酸酶、钙茜素及油红O染色观察细胞成骨、成脂分化，PCR检测BMSC向成骨分化过程中Runx 2和OPG基因表达水平。

结果

替诺福韦酯药物血清组与大鼠血清组比较，药物血清干预72 h时（A_{72 h}）值差异具有统计学意义（F = 15.42、P = 0.004）。BMSC成骨诱导分化试验中，与对照组比较药物血清组干预组碱性磷酸酶染色阳性细胞减少，茜素红染色钙结节减少，其中替诺福韦酯药物血清组改变最为显著，而且成脂诱导分化中与对照组比较成脂样细胞增多。BMSC成骨诱导分化，PCR检测与对照组比较替诺福韦酯药物血清组Runx 2表达减少，OPG表达增加（F = 11.81、P = 0.026，F = 10.14、P = 0.033）。

结论

HAART药物可能抑制BMSC增殖和成骨分化，其中替诺福韦酯的影响最为显著。

关键词: 高效抗逆转录病毒治疗, 骨髓间充质干细胞, 成骨细胞

Objective

To investigate the intervention of three high active antiretroviral therapy (HAART) drugs serum on proliferation and osteogenic differentiation of bone mesenchymal stem cell (BMSCs), and explore the effects of HAART drugs on bone metabolism.

Methods

Rats’ serums containing three kinds of HAART drug were prepared to intervene the culture and differentiation of BMSCs. Proliferation of BMSCs was measured by CCK assay. Alkaline phosphatase, calcium alizarin and oil red O staining were used to observed the differentiation of cells osteogenesis and adipogenesis. The levels of gene expression of Runx 2 and osteoprotegerin (OPG) in the osteogenetic differentiation of BMSCs were tested by RT-PCR.

Results

Compared with the rat serum group, A_{72 h} value of three kinds of drug serum group were significantly different (F = 15.42, P = 0.004). Compared with the control group, alkaline phosphatase positive cells which reflected osteogenesis differentiation decreased in drug serum group, and calcium nodules decreased in alizarin red staining test, in which tenofovir disoproxil containing group was the most significantly secreased. Fat cells which reflect adipogenic induction increased. In PCR detection, compared with control group, the expression of Runx 2 decreased and OPG expression increased in tenofovir disproxil containing group (F = 11.81, P = 0.026; F = 10.14, P = 0.033).

Conclusions

HAART drugs suppressed the proliferation and osteogenic differentiation of BMSCs which the influence of tenofovir disoproxil was most significant.

Key words: High active antiretroviral therapy, Bone mesenchymal stem cell, Osteoblast

表1 内参基因与目的基因引物序列

基因		序列（3'→5'）	产物大小（bp）
GAPDH			228
	F	GGCAAGTTCAACGGCACAG
	R	CGCCAGTAGACTCCACGACA
OPG			152
	F	CGGATGGGTTCTTCTCAGGT
	R	GCACAGGGTGACATCTATTCCA
Runx 2			130
	F	TGCTTCATTCGCCTCACAAAC
	R	TTGCAGCCTTAAATGACTCGG

表1 内参基因与目的基因引物序列

基因		序列（3'→5'）	产物大小（bp）
GAPDH			228
	F	GGCAAGTTCAACGGCACAG
	R	CGCCAGTAGACTCCACGACA
OPG			152
	F	CGGATGGGTTCTTCTCAGGT
	R	GCACAGGGTGACATCTATTCCA
Runx 2			130
	F	TGCTTCATTCGCCTCACAAAC
	R	TTGCAGCCTTAAATGACTCGG

图1 胎牛血清、大鼠血清、三种（3TC、TDF和LPV/r）药物血清对BMSC增殖的影响

表2 CCK检测BMSC增殖A_{450 nm}值（n = 6）

分组	0 h	24 h	48 h	72 h
胎牛血清	0.169 ± 0.026	0.298 ± 0.057	0.492 ± 0.051	0.787 ± 0.066
大鼠血清	0.170 ± 0.024	0.273 ± 0.030	0.373 ± 0.047	0.516 ± 0.020
3TC药物血清	0.173 ± 0.029	0.289 ± 0.040	0.369 ± 0.022	0.527 ± 0.031
TDF药物血清	0.171 ± 0.024	0.277 ± 0.034	0.343 ± 0.022	0.455 ± 0.022^a
LPV/r药物血清	0.169 ± 0.024	0.285 ± 0.041	0.379 ± 0.018	0.488 ± 0.018
DMEM	0.176 ± 0.026	0.202 ± 0.022	0.225 ± 0.062	0.235 ± 0.029

表2 CCK检测BMSC增殖A_{450 nm}值（n = 6）

分组	0 h	24 h	48 h	72 h
胎牛血清	0.169 ± 0.026	0.298 ± 0.057	0.492 ± 0.051	0.787 ± 0.066
大鼠血清	0.170 ± 0.024	0.273 ± 0.030	0.373 ± 0.047	0.516 ± 0.020
3TC药物血清	0.173 ± 0.029	0.289 ± 0.040	0.369 ± 0.022	0.527 ± 0.031
TDF药物血清	0.171 ± 0.024	0.277 ± 0.034	0.343 ± 0.022	0.455 ± 0.022^a
LPV/r药物血清	0.169 ± 0.024	0.285 ± 0.041	0.379 ± 0.018	0.488 ± 0.018
DMEM	0.176 ± 0.026	0.202 ± 0.022	0.225 ± 0.062	0.235 ± 0.029

图2 观察3种药物血清与大鼠血清干预BMSC向成骨细胞、成脂细胞分化的影响

图3 Runx 2和OPG电泳图

图4 大鼠血清、3种药物血清（3TC、TDF和LPV/r）对BMSC成骨分化中Runx 2和OPG基因相对表达量的影响

表3 PCR检测比较不同药物血清对BMSC成骨分化的影响（?Ct）（n = 6）

组别	Runx 2 mRNA	OPG mRNA
大鼠血清	1.000 ± 0.170	1.000 ± 0.194
3TC药物血清	0.962 ± 0.152	1.085 ± 0.056
TDF药物血清	0.620 ± 0.089	1.414 ± 0.114
LPV/r药物血清	0.920 ± 0.089	1.131 ± 0.055

表3 PCR检测比较不同药物血清对BMSC成骨分化的影响（?Ct）（n = 6）

组别	Runx 2 mRNA	OPG mRNA
大鼠血清	1.000 ± 0.170	1.000 ± 0.194
3TC药物血清	0.962 ± 0.152	1.085 ± 0.056
TDF药物血清	0.620 ± 0.089	1.414 ± 0.114
LPV/r药物血清	0.920 ± 0.089	1.131 ± 0.055

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Effects of High active antiretroviral therapy on the osteogenesis of bone marrow mesenchymal stem cell

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Effects of High active antiretroviral therapy on the osteogenesis of bone marrow mesenchymal stem cell