rBMSCs/Cav1^(F92A)对PAH大鼠肺血管增殖性病变的影响及其机制

Effect of r BMSCs/Cav1^(F92A) on pulmonary vascular proliferation lesions in PAH rats

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 Cav1^F92A modified rat bone marrow mesenchymal stem cells（ r BMSCs/Cav1^F92A） on pulmonary vascular proliferation lesions in rats with pulmonary arterial hypertension（ PAH） and to explore its possible mechanism. Methods Forty Wister rats were randomly divided into the Cav1,Cav1^F92A ,PAH,and normal control groups with 10 in each. PAH was induced by intraperitoneal injection of 1% monocrotaline（ MCT）（ 60 mg/kg） in adult male Wistar rats in the Cav1,Cav1^F92A ,and PAH groups. Meanwhile,rats in the control group were injected with the same volume of normal saline. At week 2 after PAH models were established,1 m L r BMSCs/Cav1 and 1m L r BMSCs/Cav1^F92A（ 1 × 106/m L） were transplantated into the rats of the Cav1 and Cav1^F92A groups by tail vein injection,while rats in the PAH group were not treated. Rats in each group were sacrificed and the lung tissues were dissected after 3weeks of transplantation. The changes of pulmonary vascular lumen and vessel wall thickness were observed under microscope by HE staining. The gene expression of Bbc3,B cell translocation of lung tissue 2（ Btg2）,Dab2,peroxisome proliferator activated receptor（ Ppard）,and Rock1 and leucine rich alpha-2 glycoprotein 1（ Lrg1） in the lung tissues was detected by using real-time quantitative PCR. The protein expression of endothelin 1（ ET-1） and smooth muscle myosin heavy chain（ Myocardin） was investigated by Western blotting. Results Compared with the normal control group,the pulmonary vascular lumen became narrow,nearly closed,and the vascular wall became hyperplastic and hypertrophic significantly in the PAH group. Compared with the PAH group,the thickening degree of pulmonary vascular wall was reduced in the Cav1 and Cav1^F92A groups but more obviously decreased in the Cav1^F92A group,but the pulmonary vascular wall was thicker than that in the normal control group. Compared with the normal control group,the relative mRNA expression of Bbc3 and Btg2 in the lung tissues of the PAH group decreased,but the mRNA expression of Dab2,Ppard,Rock1,and Lrg1 relative,and the protein expression of ET-1 and Myocardin increased（ all P〈0. 01）. Compared with the PAH group,the Bbc3 and Btg2 mRNA expression increased but Dab2,Ppard,Rock1 and Lrg1 mRNA expression decreased in the Cav1 and Cav1^F92A groups,especially in the Cav1^F92Agroup（ P〈0. 05 or P〈0. 01）; the Bbc3 and Btg2 mRNA expression in the lung tissues was higher than that in the PAH and Cav1 groups（ P〈0. 05 or P〈0. 01）. Compared with the PAH and Cav1 groups,the protein expression of ET-1 and Myocardin in the lung tissues decreased in the Cav1^F92Agroup（ both P〈0. 01）.Conclusion r BMSCs/Cav1^F92A can effectively alleviate vascular proliferative lesions in PAH rats by up-regulating the expression of Bbc3 and Btg2,Ppard,Dab2,and Rock1,and down-regulating the expression of ET-1 and Myocardin,and thus inhibiting the vascular smooth muscle cell proliferation,fibrosis,and vascular contraction.