附子多糖对深低温冻存血管保存效果的影响研究  

Effects of fuzi-polysaccharides on cryopreservation of blood vessels

作　　者：钟郁佳 史业弘 王成 ZHONG Yujia;SHI Yehong;WANG Cheng(The Fifth Affiliated Hospital of Zunyi(Zhuhai)Medical University,Zhuhai 519100,China)

机构地区：[1]遵义医科大学第五附属(珠海)医院,519100

出　　处：《环球中医药》2023年第1期39-45,共7页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(81960824)。

摘　　要：目的观察不同浓度附子多糖(fuzi-polysaccharides,FPS)对深低温冻存大鼠血管组织的保护作用,并探讨其对线粒体凋亡通路相关因子的调控机制。方法将36只SPF级雄性SD大鼠随机分成6组,对照组、冻存模型组、FPS 0.1 mg/mL组、FPS 1 mg/mL组、FPS 10 mg/mL组和FPS 20 mg/mL组,每组6只。取大鼠腹主动脉后,冻存模型组使用灭菌注射用水,FPS浓度组分别用含不同浓度FPS作冷冻保护剂(0.1 mg/mL、1 mg/mL、10 mg/mL、20 mg/mL),均经程序降温后放入-196℃液氮内保存1周。采用苏木精—伊红(hematoxylin-eosin,HE)染色法进行病理切片观察血管组织形态变化;蛋白免疫印迹法(western blot,WB)检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、细胞色素c(cytochrome c,Cyt-c)、半胱氨酸天冬氨酸蛋白酶-9(cysteine-dependent aspartate-specifc proteases-9,Caspase-9)蛋白表达。实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测Bcl-2、Bax mRNA表达。对照组取材后直接进行上述指标的检测。结果(1)HE染色结果显示:与对照组相比,冻存模型组血管组织结构损伤严重;从FPS浓度1 mg/mL起,FPS组血管组织病理变化较冻存模型组改善,内膜连续性开始增强,中膜层纤维组织排列相对紧密,外膜可见;其中,FPS 10 mg/mL组、FPS 20 mg/mL组血管组织结构保存相对完整。(2)WB结果显示:与对照组相比,冻存模型组Bax、Cyt-c、Caspase-9蛋白表达水平均显著增加(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.05);与冻存模型组相比,FPS可以显著下调Bax、Cyt-c、Caspase-9蛋白表达(P<0.05),上调Bcl-2蛋白表达(P<0.05),且FPS 10 mg/mL组、FPS 20 mg/mL组效果更显著,但两者之间差异无统计学意义(P>0.05)。(3)qRT-PCR结果显示:与对照组比较,冻存模型组Bcl-2 mRNA表达水平明显降低(P<0.05),Bax mRNA表达水平明显增加(P<0.05);与冻存模型组比较,FPS 10 mg/mL组可以显著上调Bcl-2 mRNA表达水平、下�Objective To observe the protective effect of different concentrations of Fuzi-polysaccharides(FPS)on cryopreserved vascular tissue of rats.Methods 36 SPF male SD rats were randomly divided into 6 groups:control group,cryopreserved model group,FPS 0.1 mg/mL group,FPS 1 mg/mL group,FPS 10 mg/mL group and FPS 20 mg/mL group,with 6 rats in each group.After the abdominal aorta of rats was removed,sterilized water for injection was used in the cryopreservation model group,in the FPS concentration group,different concentrations of FPS were used as cryoprotectants(0.1 mg/mL,1 mg/mL,10 mg/mL,20 mg/mL),all the samples were stored in liquid nitrogen at-196℃for 1 week.The method of HE-staining was used to observe the morphological changes of vascular tissue.The expressions of Bcl-2,Bax,Cyt-c,Caspase-9 were detected by Western Blot.The expressions of Bcl-2 and Bax mRNA were detected by RT-PCR.The control group was directly tested for the above indicators after sampling.Results(1)HE staining results show that compared with the control group,the vascular tissue structure of the cryopreserved model group was seriously damaged.Starting from the concentration of FPS 1 mg/mL,the pathological changes of vascular tissue in the FPS group were better than that in the model group.The continuity of the intima begins to increase,the fibrous tissue of the media layer was relatively densely arranged,and the adventitia was visible;Among them,the vascular tissue structure of FPS 10 mg/mL group and FPS 20 mg/mL group was relatively intact.(2)The results of Western Blot showed that compared with the control group,the expression of Bax,Cyt-c and Caspase-9 protein in the cryopreserved model group was increased(P<0.05),while the expression level of Bcl-2 protein was decreased(P<0.05).Compared with the cryopreserved model group,FPS could significantly down-regulate the levels of Bax,Cyt-c and Caspase-9 protein and up-regulate the expression of Bcl-2 protein(P<0.05),and the higher concentration of FPS(10 mg/mL,20 mg/mL)had the most significant ef

关 键 词：深低温冻存 附子多糖 冷冻保护剂 血管 线粒体凋亡通路 凋亡 

分 类 号：R285[医药卫生—中药学]