【作者】 孙宏；

【导师】 张荣华；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:本实验旨在通过体外提取、培养并观察3月龄SD大鼠MSCs的生物学特性,研究葛根的有效成分葛根素对体外培养的MSCs增殖及骨向分化的影响,以及通过与成骨分化过程密切相关的Cbfα1 mRNA表达量的观察,探讨中药防治骨代谢疾病的分子生物学机理。方法:1.运用全骨髓贴壁法分离、培养SD大鼠的MSCs,并通过细胞形态学、酶标仪检测吸光值分析生长曲线特点、流式细胞仪分析细胞生长周期和表面标记抗原、多细胞系分化能力等方面鉴定所获得的细胞为MSCs。2.将葛根素溶解于完全培养基中,配制成10^-8mol/L、10^-7 mol/L、10^-6mol/L、10^-5 mol/L和10^-4 mol/L五个浓度的溶液,采用MTT比色法检测不同浓度葛根素对MSCs增殖的影响,筛选出最佳促增殖浓度。3.采用Gomori氏钙钴法对加入葛根素诱导后的细胞进行ALP染色,以确定葛根素是否具有成骨诱导作用,并通过ALP染色阳性细胞表达率,确定葛根素促进骨向分化的最佳诱导浓度。4.采用PNPP法检测,经过最佳诱导浓度的葛根素诱导MSCs向OB分化后的ALP分泌的活性情况,实验分为空白组、最佳诱导浓度的葛根素组和经典诱导组,从而进一步明确其具有促MSCs向OB分化的作用。5.运用RT-PCR的方法,检测成骨诱导体系中与OB分化相关的Cbfα1 mRNA的表达量的变化,分组为空白组、最佳诱导浓度葛根素组、经典诱导组,以明确葛根素治疗OP的分子生物学机制。结果:1.采用全骨髓贴壁法,无菌条件下分离、提取MSCs,24 h后首次全量换液,细胞基本贴壁,13-15 d达到融合,可以进行传代培养。通过对生长曲线的绘制,说明所提取的细胞生长特性分为适应期、快速增殖期和平台期。经过成骨、成脂诱导培养,MSCs分别表现出OB和脂肪细胞表型,经流式细胞仪检测分析第三代的MSCs的生长周期,结果显示大部分细胞处于静止期、少部分细胞处于活跃增殖期,经流式细胞仪分析细胞表面抗原,其中CD29、CD44表达阳性,CD45表达阴性。2.不同浓度的葛根素与MSCs共培养后,经MTT法检测结果显示:1×10^-7mol/L、1×10^-8mol/L葛根素组与空白组比较有统计学意义（p值分别为:p=0.007＜0.01、p=0.004＜0.01）。3.ALP染色阳性细胞表达率显示:1×10^-6mol/L葛根素与空白组相比具有确定的诱导MSCs向OB分化的作用,ALP染色阳性细胞表达率高于空白组但低于经典诱导组（p值分别为:p=0.000＜0.01、p=0.000＜0.01）。4.采用PNPP法对诱导15 d后MSCs向OB分化过程中分泌的ALP进行测定,最佳诱导浓度组(1×10^-6mol/L)和经典诱导组分别与空白组进行比较,有显著差异（p值分别为p=0.000＜0.01、p=0.000＜0.01）,经典诱导组的促分化作用比最佳诱导浓度组强（p=0.000＜0.01）。5.葛根素对诱导体系中Cbfα1 mRNA的表达有上调作用:经典组目的基因的表达强于葛根素组,葛根素组的目的基因表达强于空白组（P＜0.01）。结论:葛根素具有促进体外培养的3月龄SD大鼠第三代MSCs增殖的作用,并能显著诱导其向OB分化,以及在诱导过程中ALP的分泌增多、活性增强。葛根素防治骨代谢疾病的可能机制是,通过促进体外培养的MSCs增殖或直接诱导MSCs向OB分化来实现的。并通过促进ALP的分泌及活性的提高,使分化过程中的OB不断成熟,同时通过上调Cbfα1 mRNA的表达,增强MSCs骨向分化的能力。更多还原

【Abstract】 Objective:This exprement is to explore the mechanism of Puerarin on building up bones on different levels.To identify the optimum conditions of the SD rat MSCs cultured with the active component of Radix Puerariae.To observe the effects on proliferation,differentiation to osteoblast of MSCs cultured by various concentrations of Puerarin and confirms the best concentrations and duration of action.To investigate the expression of Cbfα1 mRNA in induced system with Puerarin on molecular biology level.Methods:1.MSCs of SD rats were isolated and purified by using different attachment method,then identified according to morphology and induced-differentiation potentiality.So we can establish a stable culture system in viro.2.Adding different concentrations of Puerarin to MSCs and observing the proliferation between different groups.3.ALP positive cell counts by the improvement Gomori’s calcium cobalt staining was explored to observe the osteoblastic differentiation potentiality of MSCs induced by different concentration Puerarin.Blank control is the standard medium without Puerarin and positive control is the same one with dexamethasone,sodiumβ-glycerophosphate and vitamine C. Through these we could confirm the most effective concentration of Puerarin which could induce the cells.4.3 groups were divided:Puerarin group（induced by the most effective concentration）,blank control,classic group（classic induce bone formation group,positive control）.The ALP activity and the amount of osteocalcin in cells were measured to indicate the osteoblast function of different groups.5.Semi-quantitative analysis was used to detect the expression of Cbfα1 mRNA during osteodifferentiation through RT-PCR method,grouping was same as above. Results:1.MSCs can culture and ahere to the plastic surface in one day and reached confluence in 15 days.In the conditioned induced,MSCs were successfully differentiated into osteoblast and adipose.2.The OD value of MTT showed that 1×10^-7mol/L and 1×10^-8mol/L were significantly higher than blank groups in the 5 different concentration of Puerarin（P＜0.01）.The results expressed that it can increase the quantity of the MSCs.3.The counts of the ALP positive cell showed that Puerarin addition had a definit induction to osteodifferentiation compared with the blank and significant differentiation（P＜0.01）.4.The ALP activity sequenced as:classic group＞Puerarin group＞blank control（P＜0.01）.5.The expression of the Cbfα1 mRNA followed as:classic group＞Puerarin group＞blank control（P＜0.01）.Conclusion:By the different attachment,we can isolate the MSCs from the marrow of the rats’ bone. The obtained MSCs were purified and had great capacity of proliferation.They can be purified from generation to generation.In different condition of induced,MSCs can differentiate into osteoblast and adipose.Puerarin could accelerate the proliferation and induce the differentiation to osteoblast of MSCs.The mechanism of Pueraialobata（Wild） Ohwi on preventing and curing osteoporosis may be related to accelerating the proliferation as well as inducing the osteodifferentiation by improving the activity of ALP and enhancing the expression of Cbfα1 mRNA.更多还原