Basic fibroblast growth factor-transfected bone marrow mesenchymal stem cell transplantation for chronic obstructive pulmonary disease

doi:10.3969/j.issn.2095-4344.1750

Abstract

Abstract:

BACKGROUND: The imbalance between pro-inflammatory and anti-inflammatory responses plays a key role in the pathogenesis of chronic obstructive pulmonary disease. The research team attempted to cure the chronic obstructive pulmonary disease by reducing the intrapulmonary inflammatory response, promoting the regeneration and repair of alveolar epithelial or bronchial epithelial cells, and alleviating or reversing the pathological procession of the disease.
OBJECTIVE: To observe the expression of interleukin-10 and interleukin-4 after transplantation of basic fibroblast growth factor (bFGF)-transfected bone marrow mesenchymal stem cells (BMSCs) and cell differentiation in rats with chronic obstructive pulmonary disease.
METHODS: BMSCs were isolated and cultured using the whole bone marrow adhesion method. 150 healthy Sprague-Dawley rats were randomly selected, 30 of which were randomly selected as normal controls and the remaining rats were used to make a chronic obstructive pulmonary disease model by lipopolysaccharide combined with fumigation. Model rats were then randomly divided into chronic obstructive pulmonary disease group (model group), BMSCs group, pcDNA3.1-BMSCs group, and bFGF-pcDNA3.1-BMSCs group (n=30 per group). The rats in each group were sacrificed at 7, 14 and 28 days. The pathological changes of the lung tissues were observed by hematoxylin-eosin staining. The levels of interleukin-10 and interleukin-4 in the peripheral blood were detected by ELISA. The mRNA levels of interleukin-10 and interleukin-4 in the lung tissues were detected by qRT-PCR. The differentiation of BMSCs in the lung tissues was detected by immunofluorescence staining.
RESULTS AND CONCLUSION: On the 28^th day after modeling, the pathological changes of the lung tissues were significantly improved in the three BMSCs groups relative to the model group. The expressions of interleukin-10 and interleukin-4 in peripheral blood and lung tissues at the same observation time point were significantly higher in the three BMSCs groups than the model group (P < 0.05). Although there was no significant difference between the BMSCs and pcDNA3.1-BMSCs group, higher levels of interleukin-10 and interleukin-4 were detected in the bFGF-pcDNA3.1-BMSCs group than the other BMSCs groups (P < 0.05). On the 28^th day after modeling, some cells in the lung tissues in the three BMSCs groups were concurrently positive for CM-Dil and SPC or CC16. Moreover, there were more positive cells in the bFGF-pcDNA3.1-BMSCs group as compared with the other BMSCs groups. In conclusion, transplantation of bFGF-transfected BMSCs for chronic obstructive pulmonary disease in rats can reduce pathological changes of the lung tissue, enhance anti-inflammatory effects, and promote differentiation of BMSCs into alveolar epithelial cells and bronchial epithelial cells.

Key words: chronic obstructive pulmonary disease, bone marrow mesenchymal stem cells, basic fibroblast growth factor, gene transfection, anti-inflammatory cytokines, alveolar epithelial cells, bronchial epithelial cells, National Natural Science Foundation of China

CLC Number:

Wu Huala, Zhou Xiangxiang, Zhong Yulan, Gan Xin. Basic fibroblast growth factor-transfected bone marrow mesenchymal stem cell transplantation for chronic obstructive pulmonary disease[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3378-3385.

Figures/Tables 3

2.1 骨髓间充质干细胞的形态、鉴定和标记、转染结果原代骨髓间充质干细胞为典型的纺锤形、梭形或三角形，局部可见小集落形成。提取、培养的第4代细胞经流式细胞术检测显示CD29、CD44高表达，而基本不表达造血干细胞的标记CD34、CD45，见图1。碱性成纤维细胞生长因子质粒转染骨髓间充质干细胞24-48 h，在荧光显微镜下观察可见部分细胞呈绿色荧光，提示质粒成功转染，见图2。对照组未见绿色荧光。 2.2 肺组织病理学改变各实验组分别在干预第28天留取部分右肺中叶进行病理苏木精-伊红染色，在光学显微镜下观察病理改变，慢性阻塞性肺疾病组病理改变相对于正常对照组：肺泡腔不规则扩大，可见部分肺间隔断裂，数个肺泡融合成一个肺大疱；支气管、肺间质血管周围可见炎症细胞浸润，见图3B，符合典型慢性阻塞性肺疾病的病理改变。BMSCs组、pcDNA3.1-BMSCs组、bFGF-pcDNA3.1-BMSCs组：均有典型的慢性阻塞性肺疾病病理改变。在相同视野下，肺泡腔不规则扩大，可见部分肺泡间隔断裂，肺大疱形成，气管管壁及肺间质血管管壁有少数炎性细胞浸润。BMSCs组、pcDNA3.1-BMSCs组之间无明显差异；与BMSCs组、pcDNA3.1-BMSCs组比较，bFGF-pcDNA3.1-BMSCs组病变较轻，见图3C-E。 2.3 各组大鼠外周血白细胞介素10、白细胞介素4水平各组在干预第7，14，28天留取外周血，通过双抗夹心ELISA法检测外周血中白细胞介素10、白细胞介素4水平，结果显示：在相同时间节点，3个治疗组外周血中白细胞介素10、白细胞介素4水平高于慢性阻塞性肺疾病组(均P < 0.01)，BMSCs组、pcDNA3.1-BMSCs组外周血白细胞介素10、白细胞介素4水平差别不大，但bFGF-pcDNA3.1- BMSCs组较之二者相对较高，差异有显著性意义(均P < 0.05)，见表1。 2.4 各组大鼠肺组织中白细胞介素10、白细胞介素4 mRNA的表达各组分别在干预第7，14，28天留取部分右肺中叶，匀浆研磨后通过qRT-PCR检测肺组织中白细胞介素10、白细胞介素4 mRNA表达。在相同时间节点，3个治疗组肺组织中白细胞介素10、白细胞介素4表达高于慢性阻塞性肺疾病组(均P < 0.01)，BMSCs组、pcDNA3.1-BMSCs组肺组织中的白细胞介素10、白细胞介素4表达差别不大，但bFGF-pcDNA3.1-BMSCs组较之二者相对较高，差异有显著性意义(均P < 0.05)，见图4。 2.5 骨髓间充质干细胞在肺组织中的分化各实验组分别在干预第28天留取部分右肺中叶进行新鲜冰冻切片，在荧光显微镜下观察CM-Dil染色阳性细胞分布情况，选择CM-Dil阳性细胞较多的切片进行免疫荧光染色，观察骨髓间充质干细胞在肺组织中分化情况。荧光显微镜下3个治疗组的肺组织切片中均有少数橙红色荧光的细胞，同时显淡绿色荧光(CM-Dil和SPC/CC16双染色标记)，见图5-10。肺泡壁有少量显橙红色荧光的细胞(CM-Dil标记)，同时显淡绿色荧光(SPC-FIFC标记)，支气管壁有少量显橙红色荧光的细胞(CM-Dil标记)，同时显淡绿色荧光(CC16-FIFC标记)，相对于BMSCs组、pcDNA3.1-BMSCs组，bFGF-pcDNA3.1-BMSCs组可发现双染的阳性细胞较多。"

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