氯化锂介导经典Wnt/β-catenin信号通路在大鼠脂肪干细胞增殖和成骨中的作用

Effect of Lithium chloride mediated canonical Wnt/β-catenin signaling on proliferation and osteogenic differentiation of rat adiposed-derived stem cells

目的探讨在体外生长环境下不同浓度氯化锂对脂肪干细胞(ADSCs)增殖和成骨分化的作用及其可能的机制。方法①从3周龄SD大鼠腹股沟脂垫分离出脂肪组织,使用0.1%Ⅰ型胶原酶消化后置于含10%胎牛血清DMEM培养,以0、2.5、5、10、20、40 mmol/L浓度的氯化锂作用ADSCs24、48、72 h,用MTT法测定氯化锂对细胞增殖的作用;②ADSCs加入含0、2.5、5、10、20、40 mmol/L氯化锂的成骨培养液中培养7 d后测定细胞中碱性磷酸酶(AKP)的活性;③用RT-PCR法检测成骨诱导3 d后,不同浓度氯化锂作用7 d时ADSCs中β-catenin、糖原合成激酶3β、AKP的表达。结果在体外环境培养下,低浓度氯化锂(2.5、5、10 mmol/L)促进干细胞增殖,高浓度氯化锂(20、40 mmol/L)抑制细胞增殖。5、10 mmol/L氯化锂促进ADSCs中AKP的活性,但是40 mmol/L具有明显抑制AKP活性的作用。同时,氯化锂能上调β-catenin和AKP的表达。结论氯化锂在体外对大鼠ADSCs增殖有双重影响,并且促进AKP活性和ADSCs成骨分化,具有剂量依赖性。5 mmol/L浓度的氯化锂可作为促进大鼠ADSCs增殖和成骨分化的最适浓度。

Objective To investigate the effect of different concentrations of Lithium chloride on proliferation and osteogenic differentiation of rat adiposed-derived stem cell（ADSCs） in vitro culture and its possible mechanism. Methods ① The ADSCs were harvested from the fat pad in the groin of 3-week-old rats, then maintained in Dul-becco’s modified Eagle’s medium supplemented with 10% fetal bovine serum after digesting with 0 . 1% collagenase type Ⅰ. ADSCs were exposed to Lithium chloride at 0,2. 5,5,10,20,40 mmol/L for 24,48 and 72 hours. The effect of Lithium chloride on cells proliferation was determined by MTT assay.② Alkaline phosphatase（ AKP） ac-tivity in cells was detected after ADSCs cultured for 7 days in osteogenic differentiation medium in 2 . 5 ,5 ,10 ,20 ,40 mmol/L or with no Lithium chloride. ③ The ADSCs were treated with different concentrations of Lithium chloride for 7 days after treating with osteogenic differentiation medium for 3 days, and the expressions ofβ-catenin, glyco-gen synthase kinase-3β, AKP were detected by using RT-PCR method. Results In vitro, low doses of Lithium chloride （2. 5,5 and 10 mmol/L） sitimulated ADSCs proliferation,whereas high doses caused a inhibition of prolif-eration . 5 and 10 mmol/L Lithium chloride induce AKP activity in ADSCs which were induced the differentiation to-wards osteolasts,however 40 mmol/L significantly inhibited AKP activity. Meanwhile,Lithium chloride upregulated the expression ofβ-catenin and AKP. Conclusion In vitro,Lithium chloride has a dual effect on ADSCs prolifera-tion, and it improves AKP activity as well as promoting osteogenic differentiation in a dose-dependent manner. 5 mmol/L Lithium chloride can be used as the optimum concentration for stimulating cell proliferation and promoting osteogenic differentiation in rat ADSCs.