【摘要】：[研究背景和目的] 急性肺損傷(acute lung injury,ALI)是臨床上常見(jiàn)的危重病,起病急劇,發(fā)展迅速,病情兇險(xiǎn),預(yù)后惡劣,死亡率高,發(fā)病機(jī)制復(fù)雜,目前臨床上缺乏有效的治療措施。因此,尋找新的治療策略是目前臨床上治療ALI亟待解決的重要問(wèn)題。 肺泡上皮細(xì)胞喪失和過(guò)度凋亡是ALI發(fā)展過(guò)程中的重要事件,決定ALI發(fā)生、發(fā)展和預(yù)后,在ALI發(fā)生、發(fā)展過(guò)程中起重要作用。因此,抑制肺泡上皮細(xì)胞凋亡和促進(jìn)肺泡上皮修復(fù)被認(rèn)為是目前ALI治療的重要目標(biāo)和作用的細(xì)胞靶點(diǎn)。 骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMMSC)是一種具有自我更新和多向分化潛能的多功能干細(xì)胞,目前被認(rèn)為是一種較為理想的治療性細(xì)胞,在細(xì)胞替代治療、器官組織的修復(fù)、再生和重構(gòu)以及基因治療等方面具有廣闊臨床應(yīng)用前景。新近研究表明,輸入外源性或遷移的內(nèi)源性干細(xì)胞(stem cells)能定植在損傷的肺組織內(nèi),參與受損的肺組織的修復(fù)和重構(gòu),能夠治療ALI/ARDS.肺纖維化、慢性阻塞性肺疾病(chrocnic obstructive pulmonary disease, COPD),這為急、慢性肺部疾病治療提供了新的策略和新思路。然而,干細(xì)胞治療急、慢性肺部疾病作用和機(jī)制尚不清楚。 綜上所述,我們認(rèn)為BMMSC可以作為ALI的治療性細(xì)胞,推測(cè)其治療作用和機(jī)制可能與抗炎癥、抗肺水腫、抗凋亡及其旁分泌效應(yīng)有關(guān)。為了證明這-假說(shuō),本研究在LPS誘導(dǎo)的小鼠ALI模型上研究BMMSC抗炎癥、抗肺水腫和抗凋亡作用,在肺泡Ⅱ細(xì)胞與BMMSC共培養(yǎng)體系上研究BMMSC抗肺泡Ⅱ上皮細(xì)胞凋亡的旁分泌效應(yīng)機(jī)制,為呼吸性疾病提供新的治療策略和作用的靶點(diǎn)。 [研究方法] 1.BMMSC體外生長(zhǎng)生物學(xué)特性研究方法 (1)采用全骨髓貼壁法體外分離培養(yǎng)BMMSC; (2)光學(xué)顯微鏡下計(jì)數(shù)細(xì)胞生長(zhǎng)數(shù)量、觀測(cè)細(xì)胞培養(yǎng)形態(tài)和定向誘導(dǎo)分化的細(xì)胞形態(tài)； (3)流式細(xì)胞儀分析BMMSC表面標(biāo)志物和細(xì)胞周期。 2.在整體水平上,研究BMMSC的抗炎、抗水腫和抗凋亡作用的評(píng)價(jià)方法： (1)氣管內(nèi)滴注LPS建立急性肺損傷小鼠模型,實(shí)驗(yàn)隨機(jī)分為3組(每組n=18只)：①對(duì)照組：經(jīng)氣管滴注PBS,30min后尾靜脈注射100μl的PBS；②損傷組：經(jīng)氣管滴注LPS,30min后尾靜脈注射100μ1的PBS；③治療組：經(jīng)氣管滴注LPS,30min后尾靜脈注入100μl BMMSC的PBS懸液; (2)Gustavo Matute-Bello病理學(xué)評(píng)分方法光鏡下觀察肺組織損傷程度； (3)血?dú)夥治鰞x檢測(cè)動(dòng)脈血氧分壓(PaO2)； (4)熒光顯微鏡下觀察BMMSC在肺內(nèi)的定植； (5)常規(guī)方法測(cè)定肺濕干(W/D)比值； (6)ELISA測(cè)定肺組織髓過(guò)氧化物酶水平和肺泡灌洗液(BALF)中IL-6、TNF-a、IL-10含量； (7)考馬斯亮藍(lán)(CBB)染料結(jié)合法測(cè)定BALF中蛋白含量; (8)血細(xì)胞計(jì)數(shù)儀檢測(cè)BALF中中性粒細(xì)胞數(shù)量； (9)TUNEL原位檢測(cè)肺組織細(xì)胞凋亡率； (10)免疫組化染色檢測(cè)肺組織肺泡上皮細(xì)胞caspase3、bcl-2和bax表達(dá)水平; (11)透射電鏡觀察肺泡Ⅱ性上皮細(xì)胞凋亡。 3.在細(xì)胞水平上,研究BMMSC抗肺泡Ⅱ型上皮細(xì)胞凋亡作用機(jī)制的評(píng)價(jià)方法 (1)選用人肺泡上皮細(xì)胞株(A549細(xì)胞株)與人骨髓間充質(zhì)干細(xì)胞株(第3代hBMMSC株),建立Transwell非接觸分層共培養(yǎng)體系。實(shí)驗(yàn)分4組(每組6孔,5×106細(xì)胞/孔)：①空白對(duì)照組,PBS+A549;②LPS損傷組,LPS+A549;③BMMSC對(duì)照組,PBS+A549+BMMSC④BMMSC干預(yù)組,LPS+A549+BMMSC。 (2)Annexin V/PI雙染色流式細(xì)胞儀檢測(cè)A549細(xì)胞凋亡率； (3)Western blotting檢測(cè)A549細(xì)胞caspase3、bcl-2和bax蛋白表達(dá)水平; (4)透射電鏡觀察A549細(xì)胞凋亡特征性超微結(jié)構(gòu)； (5)ELISA測(cè)定共培養(yǎng)液中的KGF、HGF含量； (6)熒光定量PCR檢測(cè)BMMSC KGF和HGF mRNA水平。 [研究結(jié)果] 1.BMMSC體外生長(zhǎng)生物學(xué)特性研究結(jié)果 (1)4-6周齡小鼠BMMSC在5%～10%胎牛血清濃度和5～8×107/L接種密度時(shí)生長(zhǎng)活力強(qiáng),生長(zhǎng)速度最快,收獲細(xì)胞數(shù)量最多,被設(shè)定為獲得高濃度、高純度、高活性的BMMSC的最佳條件； (2)在設(shè)定的最佳條件下,體外培養(yǎng)小鼠骨髓BMMSC呈集落性生長(zhǎng),大部分細(xì)胞為G0/G1期未分化細(xì)胞。細(xì)胞形態(tài)主要呈梭形,類(lèi)似成纖維細(xì)胞,其表達(dá)CD14-、CD34-、CD45和CD90+、CD105+、CD106+,具有貼壁生長(zhǎng)特性和多向分化能力,符合間充質(zhì)干細(xì)胞的體外生長(zhǎng)的主要生物學(xué)特性。 2.BMMSC在急性肺損傷中的抗炎、抗水腫和抗凋亡作用研究結(jié)果 (1)BMMS能夠定植于LPS致肺損傷小鼠的肺組織內(nèi),減輕LPS致ALI小鼠的肺組織結(jié)構(gòu)損傷程度,提高動(dòng)脈血Pa02水平； (2)小鼠BMMSC能降低LPS致ALI小鼠的肺組織MPO含量和PMN數(shù)量、IL-6和TNF-α水平,提高IL-10含量; (3)小鼠BMMSC能降低LPS致ALI小鼠肺W/D比值和肺泡內(nèi)蛋白質(zhì)含量； (4)小鼠BMMSC能夠減少LPS致ALI小鼠肺組織肺泡上皮細(xì)胞凋亡及促凋亡蛋白caspase3和bax的表達(dá),增加抗凋亡蛋白bcl-2表達(dá)。 3.BMMSC體外抗肺泡Ⅱ型上皮細(xì)胞凋亡作用機(jī)制研究結(jié)果 (1)LPS能體外誘導(dǎo)體外培養(yǎng)的A549細(xì)胞凋亡增加,最適的濃度和最佳時(shí)間為10μg/ml LPS作用于A549細(xì)胞24h; (2)LPS能促進(jìn)A549細(xì)胞促凋亡蛋白caspase-3、bax表達(dá),降低抗凋亡蛋白bcl-2表達(dá)； (3)BMMSC能促進(jìn)A549細(xì)胞抗凋亡蛋白bcl-2表達(dá),抑制促凋亡蛋白caspase-3、bax蛋白表達(dá)； (4)與LPS誘導(dǎo)的A549細(xì)胞共培養(yǎng),BMMSC能夠旁分泌生長(zhǎng)因子KGF和HGF,增加KGF和HGF mRNA表達(dá)。 [結(jié)論] 1.在設(shè)定的4-6周齡小鼠,5%-10%胎牛血清濃度和5-8×107/L接種密度的最佳條件下,小鼠BMMSC表達(dá)CD14-.CD34-.CD45和CD90+.CD105+.CD106+,具有貼壁生長(zhǎng)特性和多向分化能力,符合間充質(zhì)干細(xì)胞體外生長(zhǎng)的主要生物學(xué)特性。 2.小鼠BMMSC可定植在LPS致ALI小鼠的肺組織內(nèi),能減輕其肺組織結(jié)構(gòu)損傷程度和改善肺功能,具有抗炎癥反應(yīng)和抗肺水腫和抗肺泡上皮細(xì)胞凋亡的作用。 3.與LPS誘導(dǎo)的A549細(xì)胞共培養(yǎng),BMMSC能促進(jìn)A549細(xì)胞表達(dá)抗凋亡蛋白bcl-2和抑制促凋亡蛋白caspase-3和bax的表達(dá),增加旁分泌生長(zhǎng)因子KGF、HGF和KGF.HGF mRNA表達(dá)水平,具有抗細(xì)胞凋亡和旁分泌作用。 4BMMSC旁分泌生長(zhǎng)因子KGF和HGF可能是其抗細(xì)胞凋亡作用的機(jī)制之一
[Abstract]:[background and purpose]
Acute lung injury (ALI) is a common clinical critical disease with a rapid onset, rapid development, dangerous condition, poor prognosis, high mortality and complex pathogenesis. At present, there is no effective treatment in clinic. Therefore, it is an important problem to find a new treatment strategy to treat ALI.
Loss of alveolar epithelial cells and excessive apoptosis are important events in the development of ALI, which determine the occurrence, development and prognosis of ALI and play an important role in the occurrence and development of ALI.
Bone marrow mesenchymal stem cells (BMMSC) are a kind of multifunctional stem cells with self-renewal and multi-differentiation potential. At present, BMMSC is considered to be an ideal therapeutic cell. It has broad clinical applications in cell replacement therapy, organ tissue repair, regeneration and reconstruction, and gene therapy. Recent studies have shown that exogenous or migrating stem cells can be implanted in damaged lung tissue, participate in the repair and reconstruction of damaged lung tissue, and can treat ALI/ARDS. Pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), which is the treatment of acute and chronic lung diseases. Therapy offers new strategies and new ideas. However, the role and mechanism of stem cell therapy for acute and chronic lung diseases are still unclear.
In conclusion, we suggest that BMMSC can be used as a therapeutic cell for ALI, and its therapeutic effect and mechanism may be related to anti-inflammation, anti-pulmonary edema, anti-apoptosis and paracrine effect. To study the paracrine effect of BMMSC on apoptosis of alveolar II epithelial cells in co-culture system with BMMSC, and to provide a new therapeutic strategy and target for respiratory diseases.
[research methods]
Research methods of biological characteristics of 1.BMMSC in vitro growth
(1) BMMSC was isolated and cultured in vitro by whole bone marrow adherent method.
(2) The number of cell growth was counted under optical microscope, and the morphology of cell culture and differentiation were observed.
(3) flow cytometry was used to analyze the surface markers and cell cycle of BMMSC.
2. to evaluate the anti-inflammatory, anti edema and anti apoptotic effects of BMMSC on the whole.
(1) Acute lung injury model was established by intratracheal LPS infusion in mice, and the experiment was randomly divided into three groups (n = 18 mice in each group): control group: PBS was dripped through trachea, 100 ml PBS was injected into tail vein after 30 minutes; injury group: LPS was dripped through trachea, 100 mu 1 PBS was injected into tail vein after 30 minutes; treatment group: LPS was dripped through trachea, and 100 mu 1 PBS was injected into tail vein after 30 minutes. PBS suspension of the L BMMSC.
(2) Gustavo Matute-Bello pathological grading method was used to observe the degree of lung tissue injury under light microscope.
(3) arterial blood oxygen partial pressure (PaO2) was detected by blood gas analyzer.
(4) the colonization of BMMSC in lung was observed under fluorescence microscope.
(5) the ratio of lung wet dry (W/D) was measured by routine method.
(6) ELISA determined the level of myeloperoxidase in lung tissue and IL-6, TNF-a and IL-10 in bronchoalveolar lavage fluid (BALF).
(7) Coomassie brilliant blue (CBB) dye binding assay was used to determine the protein content in BALF.
(8) the number of neutrophils in BALF was detected by blood cell counter.
(9) the apoptosis rate of lung tissue was detected by TUNEL in situ.
(10) immunohistochemical staining was used to detect the expression levels of Caspase3, Bcl-2 and Bax in alveolar epithelial cells of lung tissue.
(11) apoptosis of alveolar epithelial cells was observed by transmission electron microscope.
3. to evaluate the mechanism of BMMSC on apoptosis of alveolar type II epithelial cells at cellular level.
(1) Transwell non-contact stratified co-culture system was established by using human alveolar epithelial cell line (A549 cell line) and human bone marrow mesenchymal stem cell line (hBMMSC line of the third generation). The experiment was divided into four groups (6 holes in each group, 5 *106 cells/hole): blank control group, PBS + A549; LPS + A549; LPS + A549; BMMSC control group, PBS + A549 + MSC intervention group, LPS + A + A549 + MSC; BMMSC intervention group. 549+BMMSC.
(2) Annexin V/PI double staining flow cytometry was used to detect the apoptosis rate of A549 cells.
(3) Western blotting was used to detect the expression level of Caspase3, Bcl-2 and Bax protein in A549 cells.
(4) transmission electron microscopy was used to observe the characteristic ultrastructure of apoptosis in A549 cells.
(5) ELISA was used to determine the content of KGF and HGF in co culture medium.
(6) fluorescence quantitative PCR was used to detect BMMSC KGF and HGF mRNA levels.
[results]
Biological characteristics of 1.BMMSC in vitro growth
(1) BMMSC of 4-6 week-old mice grew vigorously at the concentration of 5%-10% fetal bovine serum and the density of 5-8 *107/L. The growth rate was the fastest and the number of harvested cells was the largest. BMMSC with high concentration, purity and activity was set as the best condition.
(2) Under the optimal conditions, BMMSC in vitro grew in colonies, most of which were undifferentiated cells in G0/G1 phase. The cells were spindle-shaped, similar to fibroblasts, and expressed CD14-, CD34-, CD45 and CD90+, CD105+, CD106+, which had the characteristics of adherent growth and multi-directional differentiation, and accorded with mesenchymal stem cells in vitro. The main biological characteristics of growth.
Anti inflammatory, anti edema and anti apoptotic effects of 2.BMMSC in acute lung injury
(1) BMMS can be implanted into the lung tissue of LPS-induced lung injury mice, reduce the degree of lung tissue damage in LPS-induced ALI mice, and increase the level of Pa02 in arterial blood.
(2) BMMSC could decrease the content of MPO, the quantity of PMN, the levels of IL-6 and TNF-alpha, and increase the content of IL-10 in lung tissue of ALI mice induced by LPS.
(3) mouse BMMSC can reduce the W/D ratio and protein content in alveolar of LPS induced ALI mice.
(4) BMMSC could decrease the apoptosis of alveolar epithelial cells and the expression of pro-apoptotic proteins caspase-3 and bax, and increase the expression of anti-apoptotic protein Bcl-2 in ALI mice induced by LPS.
Effect of 3.BMMSC on apoptosis of alveolar type II epithelial cells in vitro
(1) LPS could induce apoptosis of A549 cells in vitro. The optimal concentration and time of LPS treatment were 10 ug/ml for 24 hours.
(2) LPS can promote the expression of Pro apoptotic protein caspase-3 and Bax in A549 cells, and decrease the expression of anti apoptotic protein bcl-2.
(3) BMMSC could promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein caspase-3 and Bax in A549 cells.
(4) BMMSC can paracrine growth factor KGF and HGF and increase the expression of KGF and HGF mRNA in A549 cells co-cultured with LPS.
[Conclusion]
1. BMMSC expressed CD14-. CD34-. CD45 and CD90+. CD105+. CD106+ under the optimal conditions of 4-6 weeks old mice, 5% - 10% fetal bovine serum concentration and 5-8 *107/L inoculation density. BMMSC had the characteristics of adherent growth and multidirectional differentiation, which accorded with the main biological characteristics of mesenchymal stem cell growth in vitro.
2. BMMSC can be implanted in the lung tissue of ALI mice induced by LPS. It can alleviate the degree of lung tissue damage and improve lung function. It has the effects of anti-inflammatory reaction, anti-pulmonary edema and anti-alveolar epithelial cell apoptosis.
3. BMMSC can promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein caspase-3 and bax, increase the expression of paracrine growth factor KGF, HGF and KGF.
4BMMSC paracrine growth factor KGF and HGF may be one of the mechanisms of its anti apoptotic effect.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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本文編號(hào)：2212552