四环素联合桂枝加葛根汤对髓核细胞增殖和相关因子表达的影响

Tetracycline combined with Cinnamon Twig Decoction Plus Kudzuvine Root impacted on nucleus pulposus cells proliferation and correlated factor expressions

中文关键词:  四环素  桂枝加葛根汤  髓核细胞  细胞增殖  大鼠

英文关键词:Tetracycline  Cinnamon Twig Decoction Plus Kudzuvine Root  Nucleus pulposus cells  Cell proliferation  Rat

中文摘要:

  【摘要】 目的：探讨四环素（TET）联合桂枝加葛根汤（CK）对髓核细胞增殖和蛋白聚糖（aggrecan）、Ⅱ型胶原蛋白（type Ⅱ collagen，Col2a）、基质金属蛋白酶13（matrix metalloproteinase-13，MMP-13）和诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）表达的影响。方法：体外分离培养8周龄健康雄性SD大鼠髓核细胞并进行鉴定。然后用药物处理分离成功的髓核细胞：TET组分别应用不同浓度（5，10，15，20，25μg/ml）的TET处理，CK组分别应用低、中、高剂量TET处理，TET+CK组应用20μg/ml TET和中剂量CK处理，对照组不添加药物。采用CCK8方法检测不同药物处理组细胞增殖活力；实时荧光定量PCR（qRT-PCR）检测TET+CK组髓核细胞的aggrecan、Col2a、iNOS和MMP-13的mRNA表达量，免疫印迹（Western blot）检测Aggrecan、Col2a、iNOS和MMP-13的蛋白表达量。利用pcDNA3.1-CMV（+）构建重组质粒pcDNA3.1-iNOS，转染组用pcDNA3.1-iNOS转染髓核细胞，应用20μg/ml TET和中剂量CK处理，空载体组用空载体（pcDNA3.1）转染细胞，应用20μg/ml TET和中剂量CK处理，对照组不添加药物且不转染，应用qRT-PCR检测各组iNOS和MMP-13的mRNA表达量，Western blot检测各组iNOS和MMP-13的蛋白表达量。结果：分离培养的髓核细胞Col2a和Aggrecan免疫组织化学染色阳性细胞比例分别为96%和98%。不同浓度TET或CK处理髓核细胞后细胞活力与对照组比较无显著性差异（P>0.05）。20μg/ml TET和中剂量CK联合作用后髓核细胞的活力、Aggrecan和Col2a的mRNA和蛋白表达量均较对照组显著性升高（P<0.05），iNOS、MMP-13的mRNA和蛋白表达量较对照组显著性下降（P<0.05）。转染pcDNA3.1-iNOS重组质粒后髓核细胞iNOS和MMP-13的mNRA和蛋白表达与对照组和空载体组均显著性升高（P<0.05）。结论：TET联合CK可促进髓核细胞增殖，增加髓核细胞Aggrecan和Col2a mRNA和蛋白的表达，同时可以通过抑制iNOS减少MMP-13 mRNA和蛋白的表达量，为药物预防和治疗椎间盘退变提供了理论依据。

英文摘要:

  【Abstract】 Objectives: To investigate the effects of tetracycline(TET) combined with Cinnamon Twig Decoction Plus Kudzuvine Root(CK) on nucleus pulposus cell proliferation and the expressions of aggrecan, type Ⅱ collagen(Col2a), matrix metalloproteinase-13(MMP-13) and inducible nitric oxide synthase(iNOS). Methods: The nucleus pulposus cells of 8-week-old healthy male SD rats were isolated and identified in vitro. The successfully isolated nucleus pulposus cells were then treated with drug. TET group was treated with different concentrations (5, 10, 15, 20, 25μg/ml) of TET. CK group was treated with low, middle and high doses of CK. TET+CK group was treated with 20μg/ml of TET and middle dose of CK. Control group was treated without drug. Cell viabilities in different drug-treated groups were measured by CCK8 method. The relative mRNA expressions of aggrecan, Col2a, iNOS and MMP-13 in TET+CK group were measured by quantitative real-time polymerase chain reaction(qRT-PCR). The relative protein expressions of aggrecan, Col2a, iNOS, and MMP-13 were measured by Western bolt. The recombinant plasmid pcDNA3.1-iNOS was constructed by pcDNA3.1-CMV(+). The nucleus pulposus cells were transfected with pcDNA3.1-iNOS and treated with 20μg/ml of TET and middle dose of CK in transfer group. Nucleus pulposus cells in blank load transfection group were transfected with pcDNA3.1 and treated with 20μg/ml of TET and middle dose of CK. The control group was treated without drug or transfection. qRT-PCR was used to detect the mRNA expressions of iNOS and MMP-13 in each group. Western blot was used to detect the protein expressions of iNOS and MMP-13 in each group. Results: Immunohistochemical staining of type Ⅱ collagen and aggrecan was obviously positive staining in isolated cultured nucleus pulposus cells, and showed 96% and 98% positive cells respectively. The viability of nucleus pulposus cells with each concentration of TET and CK was not significantly different with that of control group(P＞0.05). The mRNA and protein expression levels of aggrecan and Col2a were significantly up-regulated(P＜0.05), while iNOS and MMP-13 were markedly down-regulated in the cells treated with 20μg/ml of TET and middle dose of CK when compared with those in control group(P＜0.05). The mRNA and protein expression levels of iNOS and MMP-13 were significantly up-regulated after transfection with recombinant plasmid pcDNA3.1-iNOS when compared with those in control group and blank load transfection group. Conclusions: TET combined with CK significantly promotes nucleus pulposus cell proliferation, the mRNA and protein expressions of aggrecan and Col2a, while inhibits the mRNA and protein expression of MMP-13 through down-regulating iNOS expression. It provides theoretical basis for drug prevention and treatment of intervertebral disc degeneration.