论文部分内容阅读
目的:探讨清热解毒、凉血化瘀中药联合大鼠骨髓间充质干细胞(BMSCs)移植对急性肝衰竭大鼠(ALF)肝脏细胞凋亡的影响。方法:40只清洁级雄性SD大鼠,随机分为空白组5只,造模组35只,造模组采用D-氨基半乳糖(D-Gal N,1.2 g·kg~(-1))联合脂多糖(LPS,5μg·kg~(-1))建立急性肝衰竭大鼠模型,造模后12 h取存活大鼠随机分为模型组、复方甘草酸苷组、茵陈四苓颗粒组、干细胞移植组及茵陈四苓颗粒联合干细胞移植组(联合组),联合组给予干细胞门静脉移植及茵陈四苓颗粒灌胃(每次1.5 g·kg~(-1)),干细胞组给予单纯干细胞门静脉移植(1.0×106个/m L),余各组给予等体积生理盐水门静脉注射,茵陈四苓颗粒组给予茵陈四苓颗粒灌胃,复方甘草酸苷组给予复方甘草酸苷灌胃(每次7.5 mg·kg~(-1)),余各组给予等体积生理盐水灌胃,干预后120 h处死大鼠。检测各组大鼠血清丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),总胆红素(TIBL)变化,苏木-伊红(HE)染色观察肝组织病理改变,计算肝脏炎症活动度,原位末端转移酶标记技术(TUNEL)检测肝脏细胞凋亡指数(AI),蛋白免疫印迹法(Western blot)检测大鼠肝组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达,实时荧光定量PCR(Real-time PCR)测定肝组织中B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)mRNA的表达。结果:急性肝衰竭大鼠造模后肝功能异常,联合组与模型组、复方甘草酸苷组、茵陈四苓颗粒组及干细胞移植组比较,大鼠血清AST,ALT,TBIL降低,肝炎活动度及凋亡指数明显降低(P<0.05);联合组大鼠肝组织内Caspase-3蛋白的表达明显降低,Bax mRNA的表达降低,Bcl-2 mRNA表达明显升高,与模型组及各干预组比较差异有统计学意义(P<0.05)。结论:清热解毒凉血化瘀中药联合干细胞移植能够改善肝功能,通过下调Caspase-3蛋白表达,上调Bcl-2 mRNA,下调Bax mRNA的表达来抑制肝脏细胞凋亡,效果优于单纯干预,两者对肝细胞凋亡的抑制具有协同作用。
Objective: To investigate the effects of detoxification, cooling blood and removing blood stasis combined with bone marrow derived mesenchymal stem cells (BMSCs) transplantation on hepatic apoptosis in acute liver failure rats (ALF). Methods: Forty clean male SD rats were randomly divided into blank group (n = 5) and model group (n = 35). D-Gal N Acute hepatic failure rat model was established by lipopolysaccharide (LPS, 5μg · kg -1). The rats were randomly divided into model group, compound glycyrrhizin group, Yinchen Siling Granule group, stem cells Transplantation group and Yinchen Siling granule combined with stem cell transplantation group (combination group). The combination group was given stem cell portal vein transplantation and Yinchen Siling granule gavage (1.5 g · kg -1 each time). Stem cell group was given single stem cell portal vein graft (1.0 × 106 / m L), the other groups were given an equal volume of saline intravenous injection Yinchen Siling Granule group Yinchen Siling granules gavage, compound glycyrrhizin group given compound glycyrrhizin gavage (7.5 mg · kg ~ (-1)), the other groups were given an equal volume of normal saline gavage, rats were killed 120 h after intervention. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TIBL) were detected in each group. The pathological changes of liver were observed by HE staining , The activity of liver inflammation was calculated, the apoptosis index (AI) of hepatocytes was detected by TUNEL, Western blot was used to detect the expression of caspase- (Bcl-2) and Bcl-2-related protein (Bax) mRNA in liver tissue were detected by Real-time PCR. Results: Compared with model group, compound glycyrrhizin group, Yinchen Siling Granule group and stem cell transplantation group, the serum AST, ALT, TBIL, hepatic activity, (P <0.05). Caspase-3 protein expression, Bax mRNA expression and Bcl-2 mRNA expression in hepatic tissue of the combination group were significantly decreased compared with the model group and each intervention group The difference was statistically significant (P <0.05). Conclusion: The combination of heat-clearing and detoxicating, cooling-blood and removing blood stasis with traditional Chinese medicine and stem cell transplantation can improve hepatic function, and inhibit hepatic cell apoptosis by down-regulating Caspase-3 protein expression, up-regulating Bcl-2 mRNA and down-regulating Bax mRNA expression, Inhibition of hepatocyte apoptosis has a synergistic effect.