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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 investigate the curative effect of rat bone marrow mesenchymal stem cells (MSCs) transplanted by spleen transplantation on acute liver failure and to observe the migration of MSCs in vivo. METHODS: Bone marrow of one male SD rat was collected. Density gradient centrifugation and adherent culture were used to isolate, purify and amplify MSCs from male Sprague-Dawley rats. Immunohistochemical staining was used to observe the expression of MSCs Surface marker. Twenty-four female rats with acute hepatic failure model were established by D-galactosamine and tumor necrosis factor-α (TNF-α), and were randomly divided into two groups: experimental group (n = 12) Mice were transplanted spleen within 24 h after transplantation. Blank control group (n = 12) received intraperitoneal injection of 0.5 mL of normal saline. The levels of ALT, TBIL and ALB in the two groups of rats after transplantation were detected by PCR, and the Y sex determination gene (SRY gene) expression, parallel HE staining to observe the pathological changes of liver tissue. Results Generation 4 MSCs expressed CD44 and CD29 but did not express CD34. After 72 h MSCs transplantation, 5 rats (41.7%) survived in the experimental group and 3 rats (25.0%) survived in the blank control group. There was significant difference in the survival rate between the two groups (P <0.05) , Experimental group higher. The MSY of male rat bone marrow MSCs were transplanted into the spleen of female rats, and the expression of SRY gene was detected in the liver of female rats. The HE staining showed that the liver function of the rats in experimental group was obvious at 4 weeks after transplantation improve. Compared with the control group, the levels of ALT and TBIL in the experimental group were significantly lower than those in the blank control group (P <0.05) at 1 and 2 weeks after transplantation ). Conclusion MSCs can migrate and colonize the damaged liver after spleen transplantation, which can replace the function of hepatocytes.