骨髓间充质干细胞经脾移植治疗大鼠急性肝功能衰竭的实验研究

Study on Mesenchymal Stem Cells Transplantation Through The Spleen in Treatment of Acute Liver Failure in Rat

目的探讨大鼠骨髓间充质干细胞(MSCs)经脾移植治疗急性肝功能衰竭的疗效,并观察MSCs在体内的迁徙情况。方法收集1只SD雄性大鼠胫骨及股骨的骨髓,采用密度梯度离心联合贴壁培养法分离、纯化及扩增雄性SD大鼠的骨髓MSCs,再行免疫组化染色以观察第4代骨髓MSCs的表面标志物。联合应用D-氨基半乳糖和肿瘤坏死因子-α(TNF-α)建立24只雌性大鼠急性肝功能衰竭模型,将其随机分为2组:实验组(n=12)大鼠于造模后24 h行骨髓MSCs脾内移植;空白对照组(n=12)仅于脾内注射0.5 mL生理盐水。2组大鼠于移植后均取血检测丙氨酸氨基转移酶(ALT)、总胆红素(TBIL)及白蛋白(ALB)水平,采用PCR法检测大鼠肝脏组织中Y性别决定区基因(SRY基因)的表达,并行HE染色观察肝脏组织的病理学改变。结果第4代MSCs表达CD44和CD29,但不表达CD34。MSCs移植72 h及以后,实验组存活5只大鼠(41.7%),空白对照组存活3只大鼠(25.0%),2组大鼠的存活率比较差异有统计学意义(P<0.05),实验组较高。将雄性大鼠的骨髓MSCs移植于雌性大鼠的脾内后,在雌性大鼠肝脏中能检测到SRY基因的表达;且HE染色结果显示,实验组大鼠的肝功能在移植后4周明显改善。移植后与空白对照组比较,实验组各时点的ALT和TBIL水平均较低(P<0.05);移植后1周和2周,实验组的ALB水平均高于空白对照组(P<0.05)。结论骨髓MSCs经脾内移植后迁徙并定居于受损的肝脏内,可替代肝细胞的功能。

Objective To evaluate the therapeutic effect of transplantation of mesenchymal stem cells （MSCs）through the spleen for acute live failure in rat, and to observe migration of transplanted MSCs in vivo. Methods One male SD rat was sacrificed to collect MSCs, and MSCs were isolated, expanded, and purified by density gradient centrifugation combined with adhere culture method. The surface antigen expressions of MSCs in the fourth generation were detected by immunohistochemistry method. Twenty-four female rats were given D-galactosamine and tumor necrosis factor α （TNF-α） to establish models of acute liver failure, and then divided into experimental group and blank control group, each group enrolled 12 rats. MSCs of male rat were transplanted into the spleen of female acute liver failure rats in experimental group at 24 hours after model establishment, but rats of blank control group were injected saline （0.5 mL）.After the MSCs transplantation, blood samples of rats in 2 groups were got to test levels of serum alanine aminotransferase（ALT）, total bilirubin （TBIL）, and albumin （ALB）. PCR method was used to determine the expression of sex determining region Y gene （SRY gene）, and HE staining was used to observe the pathological change of liver tissues of rats in 2 groups. Results The MSCs of the fourth generation expressed CD44 and CD29, but didn’t express CD34. There were 5 （41.7%） and 3 rats （25.0%） survived at 72 hours, in 1 week and 2 weeks after MSCs transplantation in experimental group and blank control group, respectively, and the survival rate was higher in experimental group （P〈0.05）. The expression of SRY mRNA was detected in rats of experimental group, as well as the damage of liver tissues in rats of experimental group improved. Compared with blank control group, the levels of ALT and TBIL were lower in experimental group at all time points after MSCs transplantation （P〈0.05）, but in 1 week and 2 weeks after MSCs transplantation, the levels of ALB in experimental group were higher （P〈0.05）. Conclusion MSCs can migrate to liver tissue, settle down, and exert the function of replacing hepatocyte after it has been transplanted into the spleen.