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目的探讨突变型窑蛋白1(Cav1~(F92A))修饰的大鼠骨髓间充质干细胞(r BMSCs/Cav1~(F92A))对肺动脉高压(PAH)大鼠肺血管增殖性病变的影响及其可能的机制。方法将40只Wistar大鼠随机分为窑蛋白1(Cav1)组、Cav1~(F92A)组、PAH组及正常对照组,每组10只。Cav1组、Cav1~(F92A)组、PAH组给予1%野百合碱60 mg/kg腹腔注射建立PAH模型,正常对照组腹腔注射等体积生理盐水。建模后2周,Cav1组、Cav1~(F92A)组分别于尾静脉移植r BMSCs/Cav1、r BMSCs/Cav1~(F92A)各1 m L(1×10~6个/m L),PAH组不予处理。各组移植后3周处死,分离肺组织。HE染色后显微镜下观察肺血管管腔及血管壁厚度改变,采用实时荧光定量PCR法检测肺组织Bbc3、B细胞易位2(Btg2)、Dab2、过氧化物酶体增生物激活受体(Ppard)、Rock1、富亮氨酸alpha-2糖蛋白1(Lrg1)基因表达,采用Western blotting法检测肺组织内皮素1(ET-1)、平滑肌肌球蛋白重链(Myocardin)蛋白表达。结果与正常对照组比较,PAH组肺血管管腔显著狭窄,几乎闭锁,血管壁明显增生肥厚。Cav1组、Cav1~(F92A)组较PAH组肺血管壁增厚程度减轻,以Cav1~(F92A)组减轻更明显,但肺血管壁仍较正常对照组增厚。与正常对照组比较,PAH组肺组织Bbc3、Btg2 mRNA相对表达量均降低,Dab2、Ppard、Rock1、Lrg1 mRNA相对表达量及ET-1、Myocardin蛋白相对表达量均升高(P均<0.01)。与PAH组比较,Cav1组与Cav1~(F92A)组肺组织Btg2 mRNA相对表达量均升高,Dab2、Ppard、Rock1及Lrg1mRNA相对表达量均降低,且Cav1~(F92A)组Ppard、Rock1及Lrg1 mRNA相对表达量降低更明显(P<0.05或<0.01)。Cav1~(F92A)组肺组织Bbc3、Btg2 mRNA相对表达量均高于PAH组、Cav1组(P<0.05或<0.01)。与PAH组、Cav1组比较,Cav1~(F92A)组肺组织ET-1、Myocardin蛋白相对表达量均降低(P均<0.01)。结论 r BMSCs/Cav1~(F92A)可减轻PAH大鼠的肺血管增殖性病变;通过上调Bbc3、Btg2基因表达,下调Ppard、Dab2、Rock1基因及ET-1、Myocardin蛋白表达而抑制血管平滑肌细胞增殖、纤维化及血管收缩可能是其作用机制。
Objective To investigate the effect of rBMSCs / Cav1 ~ (F92A) -modified rat bone marrow-derived mesenchymal stem cells (Cav1 ~ (F92A)) on pulmonary vascular proliferative lesions in rats with pulmonary hypertension (PAH) Possible mechanism. Methods Forty Wistar rats were randomly divided into three groups: Cav1 group, Cav1 ~ (F92A) group, PAH group and normal control group, with 10 rats in each group. The PAH model was established by intraperitoneal injection of 1% monocrotaline 60 mg / kg in Cav1 group, Cav1 ~ (F92A) group and PAH group. The normal control group was injected with the same volume of normal saline by intraperitoneal injection. At 2 weeks after modeling, 1 ml (1 × 10 6 / ml) of rBMSCs / Cav1 and rBMSCs / Cav1 ~ (F92A) were implanted into the tail vein of Cav1 ~ Group will not be processed. Each group was sacrificed 3 weeks after transplantation, lung tissue was isolated. The thickness of pulmonary vascular lumen and vessel wall was observed under microscope with HE staining. The expression of Bbc3, B cell translocation 2 (Btg2), Dab2, Ppard ), Rock1 and leucine-rich alpha-2 glycoprotein 1 (Lrg1) were detected by Western blotting. The expression of endothelin 1 (ET-1) and myocardin in myocardium were detected by Western blotting. Results Compared with the normal control group, the pulmonary vascular lumen of PAH group was significantly narrowed, almost closed, and the vascular wall was hyperplasia hypertrophy. Cav1 ~ (F92A) group of Cav1 ~ (F92A) group than PAH group reduce the degree of pulmonary vascular wall thickening, but the pulmonary vascular wall is still thicker than the normal control group. Compared with the normal control group, the relative expression of Bbc3 and Btg2 mRNA in the lung tissue of PAH group decreased, while the relative expression of Dab2, Ppard, Rock1 and Lrg1 mRNA and the relative expression of ET-1 and Myocardin protein increased (all P <0.01) . Compared with PAH group, the relative expression of Btg2 mRNA in Cav1 ~ (F92A) group was increased, and the relative expression of Dab2, Ppard, Rock1 and Lrg1 mRNA decreased in Cav1 ~ (F92A) group, and Ppard, Rock1 and Lrg1 mRNA relative expression decreased more significantly (P <0.05 or <0.01). The relative expression of Bbc3 and Btg2 mRNA in Cav1 ~ (F92A) group was higher than that in PAH group and Cav1 group (P <0.05 or <0.01). Compared with PAH group and Cav1 group, the relative expression of ET-1 and Myocardin protein in Cav1 ~ (F92A) group decreased (P <0.01). CONCLUSION: r BMSCs / Cav1 ~ (F92A) can reduce pulmonary vascular proliferative lesions in PAH rats and inhibit the proliferation of vascular smooth muscle cells by up-regulating the expression of Bbc3 and Btg2 genes and down-regulating the expressions of Ppard, Dab2, Rock1, ET- , Fibrosis and vasoconstriction may be its mechanism of action.