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目的观察参附注射液预处理对5-氟胞苷体外兔骨髓间充质干细胞(MSCs)定向分化为心肌样细胞的作用。方法将18只新西兰大耳白兔,随机分为3组,即参附注射液高、低剂量组,对照组,每组6只,先给予参附注射液(5 m L和1 m L)预处理,对照组给予生理盐水3 m L,兔耳缘静脉注射1周后,经兔双侧骼后上棘抽取兔自体骨髓,并在体外对骨髓间充质干细胞进行分离、培养、扩增。各组细胞均分为两个亚组(A组和B组),每组各3例,细胞接种后24 h,进行诱导分化,参附注射液高、低剂量组中,A亚组分别在常规培养液中加入不同浓度的参附注射液(250,50μL·m L-1),同时加用5-氟胞苷(5-aza),B亚组分别在常规培养液中加用5-aza,但不加参附注射液;对照组A亚组加用5-aza,B亚组不加任何药物。诱导24 h后连续培养28d。通过细胞形态学、心肌细胞特异性蛋白(α-肌动蛋白、肌钙蛋白-T)以鉴定诱导分化的效果。结果培养28 d后细胞爬片经HE染色,光学显微镜下观察培养细胞形态与心肌细胞接近程度,由好到差顺序依次为:参附注射液高剂量A组>参附注射液高剂量B组>参附注射液低剂量A组>参附注射液低剂量B组>对照组A组>对照组B组。培养细胞肌钙蛋白T检测显示:同时加入参附注射液及5-aza诱导者骨髓间充质干细胞转化为心肌细胞的阳性强度高于单纯5-aza诱导者,且与剂量有一定的关系,剂量越高,转化为心肌细胞的阳性强度越高;α-肌动蛋白检测显示,同时加入参附注射液诱导者骨髓间充质干细胞转化为心肌细胞的阳性强度高于单纯5-aza诱导,与剂量有一定的关系,剂量越高,转化为心肌细胞的阳性强度越高,但相关性较差,可能与α-肌动蛋白非心肌所特有有关,也可能与样本量较少有关。结论参附注射液与5-氟胞苷联用时对骨髓间充质干细胞向心肌细胞定向分化产生协同促进效应,提高心肌样细胞阳性转化率。
Objective To observe the effect of Shenfu injection preconditioning on the directional differentiation of rabbit bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte in vitro. Methods Eighteen New Zealand white rabbits were randomly divided into three groups, namely Shenfu injection high and low dose group and control group, with 6 rats in each group. Shenfu injection (5 m L and 1 m L) The rats in the control group were treated with normal saline (3 mL) for 1 week, and rabbits’ rabbits were injected intravenously for 1 week. Rabbit autologous bone marrow was extracted from the posterior superior iliac spine of rabbits and the bone marrow mesenchymal stem cells were isolated, cultured and expanded in vitro . The cells in each group were divided into two subgroups (group A and group B), 3 in each group. After 24 h of cell inoculation, the cells were induced to differentiate. In group A and group A, Different concentrations of Shenfu injection (250, 50 μL · m L-1) and 5-aza (5-aza) were added into the routine culture medium. Subgroup B was treated with 5- aza, but without reference compound injection; control group A subgroup plus 5-aza, subgroup B without any drug. After 24 h, the cells were cultured continuously for 28 days. The effect of inducing differentiation was confirmed by cell morphology, cardiomyocyte-specific protein (α-actin, troponin-T). Results After 28 days of culture, the cells were stained with HE, and the morphology of cultured cells was observed under light microscope. The order of appearance of the cells was: Shenfu injection high dose group A> Shenfu injection high dose group B > Shenfu injection low dose group A> Shenfu injection low dose group B> control group A group> control group B group. T cell culture Troponin T showed: simultaneous addition of Shenfu injection and 5-aza induced bone marrow mesenchymal stem cells into cardiomyocytes positive intensity higher than the simple 5-aza inducer, and the dose has a certain relationship, The higher the dose, the higher the positive rate of conversion into cardiomyocytes; α-actin test showed that the positive intensity of simultaneous conversion of MSCs to cardiomyocytes induced by Shenfu injection was higher than that induced by 5-aza alone, And the dose has a certain relationship, the higher the dose, the higher the positive intensity into myocardial cells, but less relevant, may be related to non-specific a-actin myocardium may also be related to the sample size less. Conclusion Shenfu injection combined with 5-fluorocytidine synergizes the directional differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, and improves the positive rate of cardiomyocyte-like cells.