灵芝多糖对斑马鱼存活、发育和衰老的影响

摘要

目的：观察灵芝多糖对斑马鱼存活、发育和衰老的影响。方法取正常受精的斑马鱼卵，在其单细胞发育期注射二胺吗啉代寡核苷酸（mO），制备 p53基因敲除斑马鱼模型。正常斑马鱼卵和 p53基因敲除斑马鱼卵孵育8 h 后，分别加入灵芝多糖1，2和3 g·L-1于28℃继续培养3 d，测定斑马鱼存活率和畸形率，并用衰老相关β-半乳糖苷酶（SA-β-gaI）染色法分析斑马鱼细胞 SA-β-gaI 染色阳性率，用逆转录 PCR 检测衰老相关基因端粒酶逆转录酶基因（ TERT）、抑癌基因 p53、小鼠双微体基因（ mdm2）和 p21基因表达。结果灵芝多糖1和2 g·L-1培养3 d 对野生型斑马鱼的发育无明显影响，3 g·L-1时大部分野生型斑马鱼胚胎细胞表现为畸形，并且存活率仅为48.6%；灵芝多糖1~3 g·L-1对 p53基因敲除斑马鱼的存活和发育无明显影响。灵芝多糖2 g·L-1处理3 d，野生型斑马鱼 SA-β-gaI 染色阳性率与对照组相比明显降低（ P﹤0.01），p53基因敲除斑马鱼 SA-β-gaI 染色阳性率无明显变化。逆转录 PCR 结果表明，灵芝多糖2 g·L-1可降低野生型斑马鱼细胞 p21和 p53基因表达（P﹤0.05，P﹤0.01），对 TERT和 mdm2基因表达无明显影响；对 p53基因敲除斑马鱼 TERT，mdm2，p21和 p53基因表达均无明显影响。结论灵芝多糖2 g·L-1对野生型斑马鱼胚胎细胞复制性衰老具有改善作用，≥3 g·L-1时可导致野生型斑马鱼幼胚发育畸形和死亡。灵芝多糖对斑马鱼衰老的改善作用可能与其降低 p21和 p53基因表达有关。%OBJECTlVE To study the effect of ganoderma poIysaccharide( GLP)on zebrafish ( Danio rerio)survivaI,deveIopment and senescence. METHODS A p53-knockout zebrafish modeI was estabIished by injecting of morphoIino phosphorodiamidate(mO)into normaI zebrafish eggs in the singIe ceII deveIopment stage. Different concentrations of GLP 1,2 and 3 g·L-1 were used to treat both wiId type and p53-knockout zebrafish embryos 8 h post fertiIization(8 hpf)under 28℃ for 3 d. The survivaI and maIformation rates were caIcuIated after 72 hpf,and the effect of GLP on ceII senescence was evaI-uated by senescence associated β-gaIactosidase(SA-β-gaI)staining both in wiId type and p53-knockout zebrafish embryos. In addition,the differentiaI gene expression of cancer inhibitor gene(p53),teIomer-ase reverse transcriptase( TERT),murine doubIe minute2( mdm2)and p21 was examined by RT-PCR both in wiId type and p53-knockout zebrafish embryos after treatment with different concentrations of GLP. RESULTS GLP 1 and 2 g·L-1 had no effect on deveIopment of wiId type zebrafish embryos,but after GLP 3 g·L-1 treatment,most of zebrafish embryos dispIayed maIformation and the survivaI rate was onIy 48.6%. GLP 1,2 and 3 g·L-1 had no effect on survivaI and deveIopment of p53-knockout zebrafish embryos. After GLP 2 g·L-1 treatment for 3 d,the SA-β-gaI staining positive rate of wiId type zebrafish embryos was reduced compared with controI group(P﹤0.01),whiIe there was no significant change in p53-knockout zebrafish embryos. The resuIts of RT-PCR showed that GLP 2 g·L-1 treatment depressed p21 and p53 gene expression(P﹤0.05,P﹤0.01),and had no effect on mdm2 and TERTgene expres-sion of wiId type zebrafish embryos. For p53-knockout zebrafish embryos,the TERT,mdm2,p21 and p53 gene expression dispIayed no significant difference after GLP 2 g·L-1 treatment. CONCLUSlON GLP 2 g·L-1 may improve senescence of wiId type zebrafish embryo ceIIs. GLP ≥3 g·L-1 may Iead to death and abnormaI deveIopment of wiId type zebrafish embryos. The improvement of GLP on senescence of wiId type zebrafish embryo ceIIs might be mediated by its down-reguIation of p21 and p53 gene expression.