益气化瘀补肾方对退变椎间盘细胞蛋白聚糖和Ⅹ型胶原mRNA表达的影响

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Effects of Chinese herbal medicine Yiqi Huayu Bushen Recipe on expressions of aggrecan and type Ⅹ collagen mRNAs in cells from degenerated human intervertebral discs

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doi:10.3736/jcim20111016

Objective: To investigate the effects of serum containing Yiqi Huayu Bushen Recipe, a compound Chinese herbal medicine, on expressions of aggrecan and type Ⅹ collagen mRNAs of the degenerated intervertebral disc cells in patients with cervical spondylotic myelopathy.

Methods: Six inpatients diagnosed as cervical spondylotic myelopathy and undergoing cervical decompression and fusion surgery to remove the degenerated disc tissue were enrolled in this study. Primary intervertebral disc cells were isolated using small tissue mass method. Serum containing Yiqi Huayu Bushen recipe were obtained after SD rats were fed with Yiqi Huayu Bushen recipe for 3 days. MTT method was used to detect proliferation of the first-passage cells isolated from degenerated intervertebral discs and growth curve was drawn. In the following tests, the first-passage cells were cultured with the sera containing drugs and sodium chloride (NaCl), respectively. Proliferation of intervertebral disc cells was detected by MTT method and expressions of aggrecan and type Ⅹ collagen mRNAs were detected by real-time polymerase chain reaction.

Results: The growth curve of the first-passage cells of the degenerated intervertebral discs showed that the time of cell proliferation peak was the sixth day. Compared with the same concentrations of NaCl solution, serum containing Yiqi Huayu Bushen Recipe at 5%, 10% and 20% promoted cell proliferation and expression of aggrecan mRNA and decreased expression of type Ⅹ collagen mRNA (P<0.01). 10% serum containing Yiqi Huayu Bushen recipe was more effective than 5% and 20%.

Conclusion: Yiqi Huayu Bushen Recipe inhibits degeneration of the intervertebral disc cells by regulating collagen and proteoglycan expressions.

Key words: cervical spondylosis, drugs,Chinese herbal, intervertebral disk, aggrecan, collagen type Ⅹ

Ju-bao Li, Hong-yun Zhao, Yong-jian Zhao, Quan Zhou, Ning Li, Peng Xu, Qi Shi, Yong-jun Wang. Effects of Chinese herbal medicine Yiqi Huayu Bushen Recipe on expressions of aggrecan and type Ⅹ collagen mRNAs in cells from degenerated human intervertebral discs. Journal of Chinese Integrative Medicine, 2011, 9(10): 1138-1144.

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URL: http://www.jcimjournal.com/EN/10.3736/jcim20111016

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Gene	Forward primer 5′-3′	Reverse primer 5′-3′	Accession number	Product length (bp)
β-actin	AAGGTGACAGCAGTCGGTT	TGTGTGGACTTGGGAGAGG	NM_177423	195
Aggrecan	GCCAGCACCACCAATGTAAG	TTCAGTAACACCCTCCACGAAC	NM_036445	116
Collagen 10α1	AGTGTTTTACGCTGAACG	TGCTCTCCTCTTACTGCT	NM_000493	123

	Content volume (μL)
Master mix	10
RNase-free water	7
Forward primer	1
Reverse primer	1
cDNA	1
Total volume	20

Group	Optical density value
Group	24 h	48 h
5% saline serum	0.42±0.03	0.67±0.09
10% saline serum	0.71±0.18	0.84±0.21
20% saline serum	1.05±0.34	1.13±0.27
5% Yiqi Huayu Bushen recipe serum	1.68±0.31^**△	1.92±0.36^**△
10% Yiqi Huayu Bushen recipe serum	2.17±0.25^**	2.39±0.44^**
20% Yiqi Huayu Bushen recipe serum	1.53±0.23^**△	1.64±0.49^**△

Group	n	Aggrecan mRNA
Group	n	5 h	48 h	72 h
5% saline serum	6	1.03±0.05	1.06±0.09	1.04±0.03
10% saline serum	6	1.12±0.07	1.27±0.09	1.13±0.05
20% saline serum	6	1.26±0.04	1.23±0.27	1.39±0.12
5% Yiqi Huayu Bushen recipe serum	6	3.08±0.86^**△	5.39±1.23^**△	3.57±1.15^**△
10% Yiqi Huayu Bushen recipe serum	6	6.33±1.12^**	9.50±1.87^**	7.63±1.42^**
20% Yiqi Huayu Bushen recipe serum	6	3.25±0.99^**△	6.28±1.96^**△	4.05±0.34^**△

Group	n	Type Ⅹ collagen mRNA
Group	n	5 h	48 h	72 h
5% saline serum	6	1.02±0.03	1.01±0.02	1.03±0.06
10% saline serum	6	1.01±0.03	1.03±0.02	0.98±0.16
20% saline serum	6	1.03±0.05	1.05±0.04	0.96±0.22
5% Yiqi Huayu Bushen recipe serum	6	0.74±0.14^**△	0.55±0.17^**△	0.73±0.09^**△
10% Yiqi Huayu Bushen recipe serum	6	0.48±0.11^**	0.21±0.03^**	0.35±0.16^**
20% Yiqi Huayu Bushen recipe serum	6	0.63±0.16^**△	0.38±0.05^**△	0.64±0.13^**△

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