兰州大学机构库 >学院待认领
葛根素对同型半胱氨酸诱导的骨髓间充质干细胞损伤的干预研究
Alternative TitleThe effect of Puerarin on the damage of Mesenchymal stem cells induced by Homocysteine
何柏林
Thesis Advisor汤旭磊
2009-05-31
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword葛根素 同型半胱氨酸 骨髓间充质干细胞 增殖 分化 氧化损伤
Abstract目的 观察葛根素对大鼠骨髓间充质干细胞(MSCs)增殖、分化能力的影响,探讨葛根素(Pue)对同型半胱氨酸(Hcy)诱导MSCs的损伤的保护作用及其机制。 方法 ①全骨髓细胞自然贴壁法分离大鼠四肢骨MSCs,以细胞的形态特征及具有体外诱导分化为成骨细胞的能力来鉴定MSCs,采用第三、四代细胞进行实验。②随机将细胞分为空白对照组、葛根素(10-7~1 mmol/L,Pue)组和同型半胱氨酸(0.01~20mmol/L,Hcy)组,分别观察细胞1~4d的增殖及诱导分化,2、4、6、8d的ALP活性。③上述不同浓度葛根素和同型半胱氨酸;预孵育SMCs48h, 测定细胞内的微量丙二醛(mMDA)含量、超氧化物歧化酶(SOD)和总抗氧化能力(T-AOC)。④用上述不同浓度葛根素预孵育MSCs 48h,加入10mmol/L Hcy继续孵育24h(Pue+Hcy10组);用10mmol/L Hcy预孵育24h,换不同浓度葛根素孵48h(Hcy10+Pue组),分别测定各组细胞的MTT与ALP活性。⑤先以10-2mmol/L Pue孵育SMCs 48h,再加(Hcy10+Pue10-2组)或不加(Pue10-2组)10mmol/LHcy 24h后测定细胞内的mMDA含量、SOD和T-AOC活性,并与空白组和单纯Hcy10组对照。 结果 1. Pue对MSCs的影响:①对增殖、分化的影响:与对照组相比,10-5~10-2mmol/L葛根素MTT值和ALP活性均增加(P<0.01),且组间两两比较以10-2mmol/L Pue组最高。1mmol/L Pue组最低且低于对照组。②对氧化指标的影响:与对照组相比,10-3~10-1 mmol/L Pue mMDA含量下降,而SOD和T-AOC升高,均以10-2mmol/L Pue最显著(P<0.01)。2.Hcy对MSCs的影响:①对增殖、分化的影响:在低浓度(0.05~0.5mmol/L)时,MTT值(P<0.05)、ALP活性较正常对照组明显升高(P<0.01)。在高浓度(5~20mmol/L)时MTT值、ALp活性明显降低(P<0.01),均以0.5mmol/L升高最显著(P<0.001);②对氧化指标的影响:各组mMDA含量升高(20mmol/L除外),SOD和T-AOC下降。均以0.5mmol/L Hcy最显著(P<0.01)。3. Pue对Hcy10损伤MSCs增殖和分化的影响:与Hcy10组比较, Pue+Hcy10和Hcy10+Pue组中10-4~10-2 mmol/L Pue使受损MSCs的MTT值升高(P<0.01);10-5~10-2mmol/LPue使ALP活性增加(P<0.01),都以10-2mmol/L影响更为显著(P<0.001),且损伤前加入Pue能起到更好的保护作用(P<0.01)。 4. Pue对Hcy10损伤MSCs氧化指标的影响:与对照组相比,10mmol/LHcy组的mMDA含量显著增加而SOD和T-AOC显著降低(P﹤0.01); Pue使受损MSCs的mMDA含量显著减少(P﹤0.01),而SOD活性和T-AOC则显著增强(P<0.01),但仍不能使mMDA含量(P<0.05)和T-AOC(P﹤0.05)恢复正常。 结论:①Pue与Hcy对MSCs的增殖与分化的作用存在双向性。②Pue在体外对Hcy介导MSCs的氧化损伤具有保护作用。
Other AbstractObjeetive To investigate the Protection of Puerarin on Mesenchymal stem cells(MSCs)by observing the effects of Puerarin and Homocysteine on the proliferation,differentiation function of MSCs damaged by Homocysteine invitro. Methods ①The limb bones of Wistar rats obtained in aseptic condition. After by nature anchor MSCs isolation and purified MSCs were cultured for further study. MSCs were identified by determine activity of alkaline phosphatase(ALP)and the capability of inducing and differentiation. The passage 3、4 osteoblast cells were used in experiment. ② MSCs were randomly divided into three groups: the normal control group,various concentrations of puerarin(10-7~1mmol/LPue)groups and Homocysteine ( 0.01~20mmol/L,)groups. The following study were continued: The OD values on proliferation of MSCs(from the 1st to 4th days) and expressionof ALP(the 2nd 4 th 6 th 8th days )after culture or induction in each group were determined by microplate reader. ③MSCs were treated with various concentrations of puerarin groups and Homocysteine 48h. the content of malondialdehyde(MDA),the activity of superoxide dismutase(SOD) and the total antioxidative cpability(T-AOC) were tested and compared.④Mesenchymal stem cells were treated with Pue 48 hours, and meanwhile treated with 10mmol/L Hcy (Hcy10) 24 hours (Pue+Hcy10)and treated with Hcy10 in 24 hours, then incubated with Pue 48 hours(Hcy10+Pue).Then the proliferation and differentiation in each group were determined by MTT assay and activity of ALP. ⑤The MSCs were treated with 10-2 mmol/L Pue 48 hours, then treated with 10mmol/L(Hcy10+Pue group) or not(Pue10-2 group)24 hours,were determined by the content of MDA, the activity of SOD and T-AOC , comparing with the control group and Hcy10 group. Results 1.The effect of MSCs on Pue: ①Proliferation and differentiation: Comparing with the control group, we found the proliferation of MSCs and the expression of ALP(p<0.01) at the concentrations of(10-5~10-2) mmol/L Pue,have significantly increased in groups, espceially in the concentration of 10-2 mmol/L(P<0.01). However,1 mmol/L puerarin decreased the activity of ALP(P<0.01).②Oxidation indicators: The Pue group showe significantly decrease in the content of mMDA, but markedly increase at the activity of SOD and T-AOC(P<0.01).espceially in the concentration of 10-2mmol/L(P<0.01). 2. The effect of MSCs on Hcy:①Proliferation and differentiation: Comparing with the control group in MTT va...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223417
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
何柏林. 葛根素对同型半胱氨酸诱导的骨髓间充质干细胞损伤的干预研究[D]. 兰州. 兰州大学,2009.
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