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目的通过观察大鼠烧伤血清对3T3-L1前脂肪细胞增殖活性和成脂分化能力的影响,探索烧伤对脂肪代谢的影响。方法选择成年雄性SD大鼠48只,随机分为假伤组及烧伤1、4、7、14、21 d组,每组8只。各烧伤组大鼠背部制备30%总体表面积的Ⅲ度烫伤模型,分别于对应时间点收集血清;假伤组不作处理,取血清作为正常对照。采用各组血清培养3T3-Ll前脂肪细胞,MTT法检测细胞增殖活性,选择增殖能力最低组血清作为后续刺激血清。取3T3-Ll前脂肪细胞分别采用假伤血清(A组)、烧伤血清(B组)、假伤血清成脂诱导(C组)、烧伤血清成脂诱导(D组)培养。倒置显微镜下观察细胞生长情况,诱导培养7 d后行油红O染色,进行脂肪细胞计数并测量吸光度(A)值,实时定量PCR及Western blot检测过氧化物酶体增殖物激活受体γ(preoxisome proliferator-activated receptorγ,PPAR-γ)和脂蛋白脂酶(lipoprotein lipase,LPL)基因及蛋白表达。结果 MTT检测示,与其余各组比较,烧伤4、7 d组细胞增殖能力显著降低(P<0.05),其中7 d组最低(P<0.05),取烧伤7 d组大鼠血清作为后续刺激血清。倒置显微镜观察示,培养后A、B组细胞形态无明显变化;诱导培养后C、D组细胞呈脂肪细胞样变,其中C组最显著。油红O染色示,C组细胞染色呈阳性,D组为弱阳性,余均为阴性;脂肪细胞计数以及A值比较示,A组高于B组、C组高于D组,差异均有统计学意义(P<0.05)。实时定量PCR及Western blot检测示,B组PPAR-γ基因相对表达量明显低于A组(P<0.05),但LPL基因相对表达量差异无统计学意义(P>0.05);PPAR-γ、LPL蛋白表达量比较差异无统计学意义(P>0.05)。D组PPAR-γ、LPL基因及蛋白表达量均显著优于C组(P<0.05)。结论大鼠烧伤7 d血清可致3T3-Ll前脂肪细胞成脂分化能力下降。
OBJECTIVE: To investigate the effects of burn on the proliferation and adipogenic differentiation of 3T3-L1 preadipocytes and explore the effects of burn on fat metabolism. Methods Forty-eight adult male Sprague-Dawley rats were randomly divided into 4 groups: sham-injury group and burn-injury group 1, 4, 7, 14 and 21 d, with 8 in each group. The 3rd degree scald model with 30% of the total surface area was prepared on the back of rats in each burn group, and the sera were collected at the corresponding time points. The sham group was not treated and the serum was taken as the normal control. The 3T3-L1 preadipocytes were cultured in each group, the cell proliferative activity was detected by MTT assay, and the serum with the lowest proliferative potential was selected as the subsequent stimulated serum. The 3T3-L1 preadipocytes were cultured in sham injury group (group A), burn serum (group B), sham group (group C) and burn group (group D). Cell growth was observed under an inverted microscope. Oil red O staining was performed 7 days after induction, and the number of adipocytes was measured and the absorbance (A) value was measured. Real-time PCR and Western blot were used to detect the expression of peroxisome proliferator-activated receptor γ preoxisome proliferator-activated receptorγ, PPAR-γ) and lipoprotein lipase (LPL) gene and protein expression. Results MTT assay showed that compared with the other groups, the proliferation of cells in 4 and 7 d after burn group was significantly decreased (P <0.05), and the lowest in 7 d group (P <0.05). Serum from 7 d burn group was used as follow-up stimulus serum. Inverted microscopy showed that there was no significant change in the morphology of cells in groups A and B after culturing. Cells in groups C and D showed adipocyte-like changes after induction, of which C was the most significant. Oil red O staining showed that cells in group C were stained positive, group D was weakly positive, and others were negative. Adipocyte count and A value showed that group A was higher than group B and group C was higher than group D Statistical significance (P <0.05). Real-time quantitative PCR and Western blot showed that the relative expression level of PPAR-γ gene in group B was significantly lower than that in group A (P <0.05), but there was no significant difference in the relative expression level of LPL (P> 0.05) LPL protein expression was no significant difference (P> 0.05). The mRNA and protein expressions of PPAR-γ and LPL in group D were significantly better than those in group C (P <0.05). Conclusion Serum of 3T3-L1 preadipocytes can decrease adipogenic differentiation in rats after 7-day burn.