间充质干细胞对哮喘小鼠CD4~+CD25~+调节性T细胞和气道炎症的影响
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摘要
支气管哮喘(简称)的本质是变态反应性气道慢性炎症。然而,在哮喘的免疫发病机制中,经典的Th1/Th2失衡理论很难解释哮喘免疫调节失常的全过程,还存在其他免疫学机制参与调节哮喘气道炎症的形成。最近研究发现调节性T细胞(Regulatory T cells, Treg)是CD4+T淋巴细胞的一个亚群,能积极的控制或抑制其他细胞的功能,抑制免疫反应,从而对机体免疫系统产生重要的调节作用。支气管哮喘是气道慢性炎症性疾患,同时也是外周免疫耐受机制发生缺陷的疾病。越来越多的证据表明,Treg与哮喘变态反应性炎症和维持机体自身免疫耐受有着密切的关系。CD4+CD25+Treg由Sakaguchi等首先描述,是天然存在的Treg,又称胸腺源性Treg,是调节性T细胞的主要组成成分,在哮喘的免疫学发病中起着重要的作用。
骨髓间充质干细胞(Mesenchymal stem cells ,MSCs)是骨髓中造血干细胞外的另一类具有高度可塑性的细胞群体,在特定的诱导条件下具有向成骨细胞、软骨细胞、肌腱细胞、肌细胞、神经细胞、脂肪细胞等间质细胞分化的能力,具有广泛的临床应用前景。近年的研究表明,MSCs除具有多向分化潜能外,还具有免疫调节作用。骨髓MSCs可上调CD4+CD25+Treg比例,抑制混合淋巴细胞反应(MLR)中或丝裂原(PHA)刺激引起的T淋巴细胞的增殖。MSCs不表达主要组织相容性复合体II类分子(MHC-II)、Fas配体和T细胞共刺激分子B7-1、B7-2、CD40、CD40L,MSCs不易被宿主免疫细胞识别,并能逃避免疫系统的免疫排斥。异基因MSCs不但可以进入受体的免疫器官,而且可在体内较长期存在。因此,MSCs用于细胞治疗具有独特的优势,但MSCs能否上调哮喘小鼠CD4+CD25+Treg,改善哮喘小鼠气道炎症,具有治疗哮喘的潜在临床应用前景,目前尚未见报道。我们将体外扩增的MSCs植入哮喘小鼠体内,观察MSCs对哮喘小鼠外周血CD4+CD25+Treg及气道炎症的影响,然后采用大鼠抗小鼠CD25+单克隆抗体去除哮喘小鼠外周血CD4+CD25+ Treg,观察哮喘小鼠去除CD4+CD25+Treg后气道炎症的变化以及骨髓间充质干细胞对去除CD4+CD25+ Treg哮喘小鼠气道炎症的影响,探讨MSCs减轻哮喘小鼠气道炎症的可能机制。
本研究包括三个部分:
第一部分小鼠骨髓间充质干细胞的分离、培养及鉴定
第二部分间充质干细胞上调哮喘小鼠外周血CD4+CD25+调节性T细胞减轻气道炎症
第三部分间充质干细胞对去除CD4+CD25+调节性T细胞哮喘小鼠气道炎症的影响
一、小鼠骨髓间充质干细胞的分离、培养及鉴定
目的:建立一种从小鼠骨髓中分离培养间充质干细胞(mMSCs)的高效方法,并在体外进行传代扩增和鉴定。
方法:采取贴壁筛选法分离和纯化小鼠骨髓间充质干细胞(mMSCs),检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。
结果:mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测:CD45,CD11b,CD44及CD29分别为3.34%,2.41%,98.46%,99.36%。第四代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。
结论:贴壁筛选法分离小鼠骨髓MSCs效率和纯度高,稳定性好。分离培养的细胞其形态、流式细胞术检测的表面标记和多向分化潜能符合MSCs的特征。成功分离、培养、扩增小鼠骨髓MSCs为进一步动物实验奠定了基础。
二、间充质干细胞上调哮喘小鼠外周血CD4+CD25+调节性T细胞减轻气道炎症
目的:探讨同种异体骨髓间充质干细胞(MSCs)对哮喘小鼠外周血CD4+CD25+调节性T细胞(Treg)及气道炎症的影响。
方法:将健康6周龄SPF级雌性BALB/c小鼠30只随机分为3组,每组10只,分别为正常对照组(A组)、哮喘模型组(B组)、MSCs处理组(C组),B组和C组以500μg/ml卵白蛋白(OVA)溶液0.2ml腹腔注射致敏,以5%OVA溶液雾化吸入激发建立小鼠哮喘模型,A组以等量生理盐水代替OVA,C组在诱导哮喘的第10天经尾静脉注入小鼠骨髓MSCs,流式细胞术检测小鼠外周血CD4+CD25+Treg占淋巴细胞的比例,并检测小鼠支气管肺泡灌洗液(BALF)中白细胞总数、嗜酸性粒细胞、淋巴细胞、中性粒细胞计数,结合病理切片分析气道炎症情况。
结果:A组、B组、C组小鼠外周血CD4+CD25+Treg占淋巴细胞比例分别为(5.81±0.76)%、(3.21±0.74)%、(5.12±0.34)%,A组和B组比较差异有统计学意义(P<0.01),B组和C组比较差异有统计学意义(P<0.01);A组小鼠BALF中炎症细胞总数、嗜酸性粒细胞计数分别为(5.93±1.86)×104/ml、(0.06±0.04)×104/ml,B组小鼠BALF中炎症细胞总数、嗜酸性粒细胞计数分别为(51.15±7.42)×104/ml、(14.24±2.84)×104/ml,C组小鼠BALF中炎症细胞总数、嗜酸性粒细胞分别为(24.87±3.15)×104/ml、(4.34±0.44)×104/ml,A组和B组比较差异有统计学意义(P<0.01),B组和C组比较差异有统计学意义(P<0.01);病理切片显微镜下观察,A组小鼠气道无明显炎症改变,B组小鼠支气管、血管粘膜下和周围肺组织有明显炎症细胞浸润、气道上皮增生、气道黏液增多、气道上皮部分断裂脱落,C组小鼠气道炎症明显减轻。
结论:静脉输注小鼠骨髓MSCs能上调哮喘小鼠外周血CD4+CD25+Treg比例,同时对哮喘小鼠气道炎症有显著抑制作用。上调CD4+CD25+Treg可能是MSCs抑制哮喘小鼠气道炎症的作用机制。
三、间充质干细胞对去除CD4+CD25+调节性T细胞哮喘小鼠气道炎症的影响
目的:观察小鼠骨髓间充质干细胞对去除CD4+CD25+Treg哮喘小鼠气道炎症的影响,探讨MSCs上调CD4+CD25+调节性T细胞(Treg)在改善哮喘小鼠气道炎症中的作用。
方法:将健康六周龄SPF级雌性BALB/c小鼠40只,随机分为5组,每组8只,分别为正常对照组(A组),哮喘模型组(B组),去除Treg细胞哮喘模型组(C组),MSCs处理组(D组),去除Treg细胞MSCs处理组(E组), B组﹑C组﹑D组和E组以500μg/ml卵白蛋白(OVA)溶液0.2ml腹腔注射致敏,以5%OVA溶液雾化吸入激发建立小鼠哮喘模型,A组以生理盐水代替OVA,C组和E组小鼠在激发前1天尾静脉注射0.25mg大鼠抗小鼠CD25+单克隆抗体,并在激发试验第三天强化注射0.25mg大鼠抗小鼠CD25+单克隆抗体,维持Treg细胞低水平。D组与E组在诱导哮喘的第10天经尾静脉注入0.2ml浓度调整为1×106/ml的MSCs,其余三组经尾静脉注入等量PBS。流式细胞术检测外周血CD4+CD25+Treg占淋巴细胞的比例,检测小鼠支气管肺泡灌洗液(BALF)中白细胞总数、嗜酸性粒细胞、淋巴细胞、中性粒细胞计数,结合病理切片分析气道炎症情况。
结果:A组、B组、C组、D组、E组小鼠小鼠外周血CD4+CD25+Treg占淋巴细胞比例分别为(5.86±0.48)%、(3.60±0.41)%、(0.32±0.11)%、(4.72±0.22)%、(0.33±0.11)%,A组和B组比较差异有统计学意义(P<0.01),B组和C组比较差异有统计学意义(P<0.01);A组BALF炎症细胞总数及Eos分别为(4.93±1.68)×104 /ml、(0.03±0.01)×104 /ml ,B组为(62.84±9.67)×104 /ml、(18.93±3.40)×104 /ml,C组为(80.59±11.34)×104 /ml、(29.26±5.43)×104 /ml,D组为(27.68±4.92)×104 /ml、(4.87±0.55)×104 /ml,E组为(29.59±6.67)×104 /ml、(5.51±1.75)×104 /ml,A组BALF炎症细胞总数及Eos计数与其它四组相比有统计学意义(P<0.01)。B组﹑C组﹑D组和E组BALF炎症细胞总数及Eos计数组间比较有统计学意义(P<0.01);光镜下小鼠气道炎症程度评分正常对照组0 (0~1)分,B组3(2~4)分,C组4 (3~4)分,D组2(1~3)分,E组2 (1~3)分,组间比较有统计学意义(H=32.527,P<0.01),A组小鼠与其它4组比较,炎症改变有显著差别(P<0.01);D组﹑ E组气道炎症改变较B组和C组有所减轻, C组较B组炎症改变有所加重(P<0.05),D组和E组炎症改变无显著差别(P>0.05)。
结论:和未去除外周血CD4+CD25+Treg的哮喘小鼠相比,哮喘小鼠去除外周血CD4+CD25+Treg其气道炎症显著加重,静脉输注MSCs能上调哮喘小鼠外周血CD4+CD25+Treg比例,对哮喘小鼠气道炎症具有显著的抑制作用。去除外周血CD4+CD25+Treg的哮喘小鼠静脉输注MSCs,其气道炎症仍呈现明显抑制作用,与未去除外周血CD4+CD25+Treg哮喘小鼠相比,MSCs对去除外周血CD4+CD25+Treg哮喘小鼠气道炎症的抑制作用有所减弱,但与未去除外周血CD4+CD25+Treg哮喘小鼠相比并无显著性差异。结果进一步表明外周血CD4+CD25+Treg比例下降参与了哮喘小鼠气道炎症的形成,MSCs上调外周血CD4+CD25+Treg比例仅仅是其抑制哮喘小鼠气道炎症的机制之一,MSCs对哮喘小鼠气道炎症的影响还存在其他重要机制。
It’s demonstrated that asthma is characterized of chronic atopic airway inflammation in which the imbalance of Th1/Th2 plays a role. However, the classical theory of the Th1/Th2 imbalance can’t completely explain the immunological pathogenesis of asthma. Some other mechanisms may exist. Recent studies have suggested that Regulatory T cells(Treg),which are a subset of CD4+ T lymphocytes, play an important role in the regulation of the immune system through suppressing the function of other immunocytes and the immunoreaction. Asthma is a chronic atopic inflammatory disease of the airways, and a disease which is insufficient in peripheral immunotolerance. More and more evidences suggested that Treg are closely associated with the atopic inflammation of asthma and the maintenance of peripheral immunotolerance. CD4+CD25+Treg are first described by Sakaguchi, also known as naturally occurring Treg or thymus derived Treg. As a major composition of Treg, they play an important role in the immunological pathogenesis of asthma.
Mesenchymal stem cells (MSCs) are another class of bone marrow stem cells which are different from hematopoietic stem cells. Mesenchymal stem cells can differentiate along multiple lineages such as osteoblasts, chondrocytes, myocytes, tendon cells, neurocytes, adipocytes, etc.In addition, resent studies indicated that MSCs exert an immunoregulatory capacity both in vitro and in vivo. MSCs can upregulate the proportion of CD4+CD25+Treg, thus suppress the proliferative response of the T lymphocytes in the mixed lymphocyte reaction or stimulated by the mitogen PHA. Mesenchymal stem cells don’t express major histocompatibility complexⅡ, Fas ligand and costimulatory molecules of T cells such as B7-1、B7-2、 CD40、CD40L, so that they are not easily recognized by the immunocytes of the host and able to avoid rejection by the host immune system. Heterogenic MSCs can be implanted into the immune organs of the host and survive in a long term. In spite of the advantage in the cytotherapy, it is still unkown whether MSCs can upregulate CD4+CD25+Treg of asthmatic mice and improve its airway inflammation, and furthermore be used in asthmatic therapy. So we design this experiment to observe the influence of MSCs to the peripheral blood CD4+CD25+Treg and the airway inflammation in BALB/c asthmatic mice.
The study includes three parts:
1.Isolation,culture and identification of mouse mesenchymal stem cells from bone marrow
2. Mesenchymal stem cells up-regulating CD4+CD25+ regulatory T cells of peripheral blood and alleviating airway inflammation in asthmatic BALB/c mice
3.The effect of mesenchymal stem cells on airway inflammation in asthmatic BALB/c mice deleted of CD4+CD25+ regulatory T cells
一、Isolation,culture and identification of mouse mesenchymal stem cells from bone marrow
0bjective:To establish an efficient method for isolation and culture of mouse mesenchymal stem cells (mMSCs) from bone marrow, then proliferate and identify the cells in vitro.
Methods:The mononuclear cells were isolated from BALB/c mouse bone marrow and mMSCs were enriched and expanded by using bone marrow adherent culture.Osteogenic and adipogenic induction was performed on the mMSCs. The phenotype was analyzed by Flow Cytometry (FCM) and morphology of the mMSCs was observed under a microscope.
Results : The mMSCs were the adherent cells of similar fibroblastoid morphology. The expressions of CD45,CD11b,CD44 and CD29 were 3.34%,2.41%,98.46%and 99.36%. The mMSCs of the fourth generation could be induced to differentiate into osteoblasts or adipocytes.
Conclusion:The mMSCs can be isolated and expanded by using bone marrow adherent culture which is efficient and stable. The cells had been identified by morphology、biological characteristics and differentiation potentia1 in vitro. We isolated and cultured mouse MSCs from bone marrow in vitro successfully, making it possible for further study.
二、Mesenchymal stem cells up-regulating CD4+CD25+ regulatory T cells of peripheral blood and alleviating airway inflammation in asthmatic BALB/c mice.
0bjective:To observe the effect of MSCs on the CD4+CD25+ regulatory T Cells (Treg) of peripheral blood and the airway inflammation in asthmatic BALB/c mice. Methods:Thirty BALB/c mice were randomly divided into three groups (10 mice in each group): the normal control group (group A), the asthmatic group (group B) and the MSCs group (group C). Group B and group C were sensitized by intraperitoneal injection of 0.2 ml 500μg/ml ovalbumin (OVA) and challenged by inhalation of nebulized 5% OVA solution to establish asthma models. In group A, normal saline of the equal volume was given instead of OVA. Group C was intravenously administered MSCs on the 10th day after sensitization. The proportion of CD4+CD25+ Treg/lymphocytes in peripheral blood was detected by Flow Cytometry. The number of total cells, eosinophils, lymphocytes and neutrophils in BALF was counted to analyze the degree of airway inflammation together with pathological sections.
Results:The proportion of CD4+CD25+ Treg/lymphocytes in peripheral blood was (5.81±0.76)% in group A,(3.21±0.74)% in group B, and (5.12±0.34)% in group C. The difference was significant when group A was compared with group B(P<0.01), and when group B with group C(P<0.01). The number of total cells and eosinophils in BALF was (5.93±1.86)×104 /ml and(0.06±0.04)×104 /ml in group A(,51.15±7.42)×104 /ml and(14.24±2.84)×104/ml in group B(,24.87±3.15)×104/ml and (4.34±0.44)×104 /ml in group C. The numbers were significantly different when group A was compared with group B(P<0.01), and group B with group C(P<0.01). Lung inflammation was examined in HE stained sections. There was no obvious infiltration of inflammatory cells in the airways of group A. However, in group B, remarkable infiltration of inflammatory cells, proliferation and damage of airway epithelia and more mucus in the lumen could be observed in the sections, and the inflammation was attenuated remarkably in group C.
Conclusions:MSCs can up-regulate CD4+CD25+ Treg of peripheral blood and relieve the airway inflammation in asthmatic BALB/c mice. The mechanism that MSCs relieve the airway inflammation in asthmatic BALB/c mice may be up-regulation of CD4+CD25+ Treg.
三、The Effect of Mesenchymal Stem Cells on Airway Inflammation in Asthmatic BALB/c Mice Depleted of CD4+CD25+ Regulatory T Cells
Objective:To study the effect of Mesenchymal Stem Cells on the airway inflammation in asthmatic BALB/c mice depleted of CD4+CD25+ Regulatory T Cells.
Methods:Forty BALB/c mice were randomly divided into five groups(8 mice in each group): the normal control group (group A),the asthmatic group (group B), the asthmatic group depleted of CD4+CD25+Treg (group C),the MSCs group (group D),and the asthmatic group depleted of CD4+CD25+Treg and administrated with MSCs (group E). Except group A, other groups were sensitized by intraperitoneal injection of 0.2 ml 500μg/ml ovalbumin (OVA) and challenged by inhalation of nebulized 5% OVA solution to establish asthma models. In group A, normal saline of the equal volume was given instead of OVA. In group C and group E, each mouse was treated with two doses of 0.25 mg anti-CD25+ mAb– one dose was intravenously injected on the previous day before the OVA challenge, and another was intraperitoneally injected on the third day after the OVA challenge. Group D and group E were intravenously administered with MSCs on the tenth day after sensitization, while other groups were treated with PBS instead. The proportion of CD4+CD25+Treg in peripheral blood was detected by Flow Cytometry. The number of total cells, eosinophils, lymphocytes and neutrophils in BALF was counted to analyze the degree of airway inflammation together with pathological sections.
Results:The proportion of CD4+CD25+Tregs in peripheral blood was (5.86±0.48)% in group A, (3.60±0.41)% in group B, (0.32±0.11)% in group C, (4.72± 0.23)% in group D and(0.33±0.11)% in group E. The proportion was significantly lower in group B than that in group A(P<0.01), as well as in group C than that in group B (P<0.01). The number of total cells and eosinophils in BALF was(4.93±1.68)×104 /ml and(0.03±0.01)×104 /ml in group A(,62.84±9.67)×104 /ml and(18.93±3.40)×104 /ml in group B(,80.59±11.34)×104 /ml and (29.26±5.43)×104 /ml in group C,(27.68±4.92)×104 /ml and (4.87±0.55)×104 /ml in group D, and (29.59±6.67)×104 /ml and (5.51±1.75)×104 /ml in group E. The proportion was significantly different when group A was compared with other groups(P<0.01), and the difference was still significant among group B,C,D and E(P<0.01). The pulmonary pathological score of the group A was 0(0～1), group B was 3(2～4), group C was 4 (3～4),group D was 2(1～3) and group E was 2 (1～3). There was significant difference among the scores of the five groups(H=32.527,P<0.01). The score was significantly lower when group A was compared with other groups(P<0.01),when group D and group E with group B and group C, and when group B with group C (P<0.05). There was no significant difference between the scores of group D and group E(P>0.05).
Conclusions : The asthmatic BALB/c mice have more severe airway inflammation when CD4+CD25+Treg are depleted from peripheral blood. MSCs can up-regulate the CD4+CD25+Treg and thus inhibit the airway inflammation in asthmatic BALB/c mice. This effect is weakened in the mice depleted of CD4+CD25+Treg, compared with those not depleted, even though the difference is not significant. These findings suggest that low level of CD4+CD25+Treg may be an important mechanism of the airway inflammation in asthmatic mice. Increasing the level of CD4+CD25+Treg is just one of the mechanisms of MSCs in alleviating the airway inflammation. Further studies are needed to understand other mechanisms.
骨髓间充质干细胞(Mesenchymal stem cells ,MSCs)是骨髓中造血干细胞外的另一类具有高度可塑性的细胞群体,在特定的诱导条件下具有向成骨细胞、软骨细胞、肌腱细胞、肌细胞、神经细胞、脂肪细胞等间质细胞分化的能力,具有广泛的临床应用前景。近年的研究表明,MSCs除具有多向分化潜能外,还具有免疫调节作用。骨髓MSCs可上调CD4+CD25+Treg比例,抑制混合淋巴细胞反应(MLR)中或丝裂原(PHA)刺激引起的T淋巴细胞的增殖。MSCs不表达主要组织相容性复合体II类分子(MHC-II)、Fas配体和T细胞共刺激分子B7-1、B7-2、CD40、CD40L,MSCs不易被宿主免疫细胞识别,并能逃避免疫系统的免疫排斥。异基因MSCs不但可以进入受体的免疫器官,而且可在体内较长期存在。因此,MSCs用于细胞治疗具有独特的优势,但MSCs能否上调哮喘小鼠CD4+CD25+Treg,改善哮喘小鼠气道炎症,具有治疗哮喘的潜在临床应用前景,目前尚未见报道。我们将体外扩增的MSCs植入哮喘小鼠体内,观察MSCs对哮喘小鼠外周血CD4+CD25+Treg及气道炎症的影响,然后采用大鼠抗小鼠CD25+单克隆抗体去除哮喘小鼠外周血CD4+CD25+ Treg,观察哮喘小鼠去除CD4+CD25+Treg后气道炎症的变化以及骨髓间充质干细胞对去除CD4+CD25+ Treg哮喘小鼠气道炎症的影响,探讨MSCs减轻哮喘小鼠气道炎症的可能机制。
本研究包括三个部分:
第一部分小鼠骨髓间充质干细胞的分离、培养及鉴定
第二部分间充质干细胞上调哮喘小鼠外周血CD4+CD25+调节性T细胞减轻气道炎症
第三部分间充质干细胞对去除CD4+CD25+调节性T细胞哮喘小鼠气道炎症的影响
一、小鼠骨髓间充质干细胞的分离、培养及鉴定
目的:建立一种从小鼠骨髓中分离培养间充质干细胞(mMSCs)的高效方法,并在体外进行传代扩增和鉴定。
方法:采取贴壁筛选法分离和纯化小鼠骨髓间充质干细胞(mMSCs),检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。
结果:mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测:CD45,CD11b,CD44及CD29分别为3.34%,2.41%,98.46%,99.36%。第四代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。
结论:贴壁筛选法分离小鼠骨髓MSCs效率和纯度高,稳定性好。分离培养的细胞其形态、流式细胞术检测的表面标记和多向分化潜能符合MSCs的特征。成功分离、培养、扩增小鼠骨髓MSCs为进一步动物实验奠定了基础。
二、间充质干细胞上调哮喘小鼠外周血CD4+CD25+调节性T细胞减轻气道炎症
目的:探讨同种异体骨髓间充质干细胞(MSCs)对哮喘小鼠外周血CD4+CD25+调节性T细胞(Treg)及气道炎症的影响。
方法:将健康6周龄SPF级雌性BALB/c小鼠30只随机分为3组,每组10只,分别为正常对照组(A组)、哮喘模型组(B组)、MSCs处理组(C组),B组和C组以500μg/ml卵白蛋白(OVA)溶液0.2ml腹腔注射致敏,以5%OVA溶液雾化吸入激发建立小鼠哮喘模型,A组以等量生理盐水代替OVA,C组在诱导哮喘的第10天经尾静脉注入小鼠骨髓MSCs,流式细胞术检测小鼠外周血CD4+CD25+Treg占淋巴细胞的比例,并检测小鼠支气管肺泡灌洗液(BALF)中白细胞总数、嗜酸性粒细胞、淋巴细胞、中性粒细胞计数,结合病理切片分析气道炎症情况。
结果:A组、B组、C组小鼠外周血CD4+CD25+Treg占淋巴细胞比例分别为(5.81±0.76)%、(3.21±0.74)%、(5.12±0.34)%,A组和B组比较差异有统计学意义(P<0.01),B组和C组比较差异有统计学意义(P<0.01);A组小鼠BALF中炎症细胞总数、嗜酸性粒细胞计数分别为(5.93±1.86)×104/ml、(0.06±0.04)×104/ml,B组小鼠BALF中炎症细胞总数、嗜酸性粒细胞计数分别为(51.15±7.42)×104/ml、(14.24±2.84)×104/ml,C组小鼠BALF中炎症细胞总数、嗜酸性粒细胞分别为(24.87±3.15)×104/ml、(4.34±0.44)×104/ml,A组和B组比较差异有统计学意义(P<0.01),B组和C组比较差异有统计学意义(P<0.01);病理切片显微镜下观察,A组小鼠气道无明显炎症改变,B组小鼠支气管、血管粘膜下和周围肺组织有明显炎症细胞浸润、气道上皮增生、气道黏液增多、气道上皮部分断裂脱落,C组小鼠气道炎症明显减轻。
结论:静脉输注小鼠骨髓MSCs能上调哮喘小鼠外周血CD4+CD25+Treg比例,同时对哮喘小鼠气道炎症有显著抑制作用。上调CD4+CD25+Treg可能是MSCs抑制哮喘小鼠气道炎症的作用机制。
三、间充质干细胞对去除CD4+CD25+调节性T细胞哮喘小鼠气道炎症的影响
目的:观察小鼠骨髓间充质干细胞对去除CD4+CD25+Treg哮喘小鼠气道炎症的影响,探讨MSCs上调CD4+CD25+调节性T细胞(Treg)在改善哮喘小鼠气道炎症中的作用。
方法:将健康六周龄SPF级雌性BALB/c小鼠40只,随机分为5组,每组8只,分别为正常对照组(A组),哮喘模型组(B组),去除Treg细胞哮喘模型组(C组),MSCs处理组(D组),去除Treg细胞MSCs处理组(E组), B组﹑C组﹑D组和E组以500μg/ml卵白蛋白(OVA)溶液0.2ml腹腔注射致敏,以5%OVA溶液雾化吸入激发建立小鼠哮喘模型,A组以生理盐水代替OVA,C组和E组小鼠在激发前1天尾静脉注射0.25mg大鼠抗小鼠CD25+单克隆抗体,并在激发试验第三天强化注射0.25mg大鼠抗小鼠CD25+单克隆抗体,维持Treg细胞低水平。D组与E组在诱导哮喘的第10天经尾静脉注入0.2ml浓度调整为1×106/ml的MSCs,其余三组经尾静脉注入等量PBS。流式细胞术检测外周血CD4+CD25+Treg占淋巴细胞的比例,检测小鼠支气管肺泡灌洗液(BALF)中白细胞总数、嗜酸性粒细胞、淋巴细胞、中性粒细胞计数,结合病理切片分析气道炎症情况。
结果:A组、B组、C组、D组、E组小鼠小鼠外周血CD4+CD25+Treg占淋巴细胞比例分别为(5.86±0.48)%、(3.60±0.41)%、(0.32±0.11)%、(4.72±0.22)%、(0.33±0.11)%,A组和B组比较差异有统计学意义(P<0.01),B组和C组比较差异有统计学意义(P<0.01);A组BALF炎症细胞总数及Eos分别为(4.93±1.68)×104 /ml、(0.03±0.01)×104 /ml ,B组为(62.84±9.67)×104 /ml、(18.93±3.40)×104 /ml,C组为(80.59±11.34)×104 /ml、(29.26±5.43)×104 /ml,D组为(27.68±4.92)×104 /ml、(4.87±0.55)×104 /ml,E组为(29.59±6.67)×104 /ml、(5.51±1.75)×104 /ml,A组BALF炎症细胞总数及Eos计数与其它四组相比有统计学意义(P<0.01)。B组﹑C组﹑D组和E组BALF炎症细胞总数及Eos计数组间比较有统计学意义(P<0.01);光镜下小鼠气道炎症程度评分正常对照组0 (0~1)分,B组3(2~4)分,C组4 (3~4)分,D组2(1~3)分,E组2 (1~3)分,组间比较有统计学意义(H=32.527,P<0.01),A组小鼠与其它4组比较,炎症改变有显著差别(P<0.01);D组﹑ E组气道炎症改变较B组和C组有所减轻, C组较B组炎症改变有所加重(P<0.05),D组和E组炎症改变无显著差别(P>0.05)。
结论:和未去除外周血CD4+CD25+Treg的哮喘小鼠相比,哮喘小鼠去除外周血CD4+CD25+Treg其气道炎症显著加重,静脉输注MSCs能上调哮喘小鼠外周血CD4+CD25+Treg比例,对哮喘小鼠气道炎症具有显著的抑制作用。去除外周血CD4+CD25+Treg的哮喘小鼠静脉输注MSCs,其气道炎症仍呈现明显抑制作用,与未去除外周血CD4+CD25+Treg哮喘小鼠相比,MSCs对去除外周血CD4+CD25+Treg哮喘小鼠气道炎症的抑制作用有所减弱,但与未去除外周血CD4+CD25+Treg哮喘小鼠相比并无显著性差异。结果进一步表明外周血CD4+CD25+Treg比例下降参与了哮喘小鼠气道炎症的形成,MSCs上调外周血CD4+CD25+Treg比例仅仅是其抑制哮喘小鼠气道炎症的机制之一,MSCs对哮喘小鼠气道炎症的影响还存在其他重要机制。
It’s demonstrated that asthma is characterized of chronic atopic airway inflammation in which the imbalance of Th1/Th2 plays a role. However, the classical theory of the Th1/Th2 imbalance can’t completely explain the immunological pathogenesis of asthma. Some other mechanisms may exist. Recent studies have suggested that Regulatory T cells(Treg),which are a subset of CD4+ T lymphocytes, play an important role in the regulation of the immune system through suppressing the function of other immunocytes and the immunoreaction. Asthma is a chronic atopic inflammatory disease of the airways, and a disease which is insufficient in peripheral immunotolerance. More and more evidences suggested that Treg are closely associated with the atopic inflammation of asthma and the maintenance of peripheral immunotolerance. CD4+CD25+Treg are first described by Sakaguchi, also known as naturally occurring Treg or thymus derived Treg. As a major composition of Treg, they play an important role in the immunological pathogenesis of asthma.
Mesenchymal stem cells (MSCs) are another class of bone marrow stem cells which are different from hematopoietic stem cells. Mesenchymal stem cells can differentiate along multiple lineages such as osteoblasts, chondrocytes, myocytes, tendon cells, neurocytes, adipocytes, etc.In addition, resent studies indicated that MSCs exert an immunoregulatory capacity both in vitro and in vivo. MSCs can upregulate the proportion of CD4+CD25+Treg, thus suppress the proliferative response of the T lymphocytes in the mixed lymphocyte reaction or stimulated by the mitogen PHA. Mesenchymal stem cells don’t express major histocompatibility complexⅡ, Fas ligand and costimulatory molecules of T cells such as B7-1、B7-2、 CD40、CD40L, so that they are not easily recognized by the immunocytes of the host and able to avoid rejection by the host immune system. Heterogenic MSCs can be implanted into the immune organs of the host and survive in a long term. In spite of the advantage in the cytotherapy, it is still unkown whether MSCs can upregulate CD4+CD25+Treg of asthmatic mice and improve its airway inflammation, and furthermore be used in asthmatic therapy. So we design this experiment to observe the influence of MSCs to the peripheral blood CD4+CD25+Treg and the airway inflammation in BALB/c asthmatic mice.
The study includes three parts:
1.Isolation,culture and identification of mouse mesenchymal stem cells from bone marrow
2. Mesenchymal stem cells up-regulating CD4+CD25+ regulatory T cells of peripheral blood and alleviating airway inflammation in asthmatic BALB/c mice
3.The effect of mesenchymal stem cells on airway inflammation in asthmatic BALB/c mice deleted of CD4+CD25+ regulatory T cells
一、Isolation,culture and identification of mouse mesenchymal stem cells from bone marrow
0bjective:To establish an efficient method for isolation and culture of mouse mesenchymal stem cells (mMSCs) from bone marrow, then proliferate and identify the cells in vitro.
Methods:The mononuclear cells were isolated from BALB/c mouse bone marrow and mMSCs were enriched and expanded by using bone marrow adherent culture.Osteogenic and adipogenic induction was performed on the mMSCs. The phenotype was analyzed by Flow Cytometry (FCM) and morphology of the mMSCs was observed under a microscope.
Results : The mMSCs were the adherent cells of similar fibroblastoid morphology. The expressions of CD45,CD11b,CD44 and CD29 were 3.34%,2.41%,98.46%and 99.36%. The mMSCs of the fourth generation could be induced to differentiate into osteoblasts or adipocytes.
Conclusion:The mMSCs can be isolated and expanded by using bone marrow adherent culture which is efficient and stable. The cells had been identified by morphology、biological characteristics and differentiation potentia1 in vitro. We isolated and cultured mouse MSCs from bone marrow in vitro successfully, making it possible for further study.
二、Mesenchymal stem cells up-regulating CD4+CD25+ regulatory T cells of peripheral blood and alleviating airway inflammation in asthmatic BALB/c mice.
0bjective:To observe the effect of MSCs on the CD4+CD25+ regulatory T Cells (Treg) of peripheral blood and the airway inflammation in asthmatic BALB/c mice. Methods:Thirty BALB/c mice were randomly divided into three groups (10 mice in each group): the normal control group (group A), the asthmatic group (group B) and the MSCs group (group C). Group B and group C were sensitized by intraperitoneal injection of 0.2 ml 500μg/ml ovalbumin (OVA) and challenged by inhalation of nebulized 5% OVA solution to establish asthma models. In group A, normal saline of the equal volume was given instead of OVA. Group C was intravenously administered MSCs on the 10th day after sensitization. The proportion of CD4+CD25+ Treg/lymphocytes in peripheral blood was detected by Flow Cytometry. The number of total cells, eosinophils, lymphocytes and neutrophils in BALF was counted to analyze the degree of airway inflammation together with pathological sections.
Results:The proportion of CD4+CD25+ Treg/lymphocytes in peripheral blood was (5.81±0.76)% in group A,(3.21±0.74)% in group B, and (5.12±0.34)% in group C. The difference was significant when group A was compared with group B(P<0.01), and when group B with group C(P<0.01). The number of total cells and eosinophils in BALF was (5.93±1.86)×104 /ml and(0.06±0.04)×104 /ml in group A(,51.15±7.42)×104 /ml and(14.24±2.84)×104/ml in group B(,24.87±3.15)×104/ml and (4.34±0.44)×104 /ml in group C. The numbers were significantly different when group A was compared with group B(P<0.01), and group B with group C(P<0.01). Lung inflammation was examined in HE stained sections. There was no obvious infiltration of inflammatory cells in the airways of group A. However, in group B, remarkable infiltration of inflammatory cells, proliferation and damage of airway epithelia and more mucus in the lumen could be observed in the sections, and the inflammation was attenuated remarkably in group C.
Conclusions:MSCs can up-regulate CD4+CD25+ Treg of peripheral blood and relieve the airway inflammation in asthmatic BALB/c mice. The mechanism that MSCs relieve the airway inflammation in asthmatic BALB/c mice may be up-regulation of CD4+CD25+ Treg.
三、The Effect of Mesenchymal Stem Cells on Airway Inflammation in Asthmatic BALB/c Mice Depleted of CD4+CD25+ Regulatory T Cells
Objective:To study the effect of Mesenchymal Stem Cells on the airway inflammation in asthmatic BALB/c mice depleted of CD4+CD25+ Regulatory T Cells.
Methods:Forty BALB/c mice were randomly divided into five groups(8 mice in each group): the normal control group (group A),the asthmatic group (group B), the asthmatic group depleted of CD4+CD25+Treg (group C),the MSCs group (group D),and the asthmatic group depleted of CD4+CD25+Treg and administrated with MSCs (group E). Except group A, other groups were sensitized by intraperitoneal injection of 0.2 ml 500μg/ml ovalbumin (OVA) and challenged by inhalation of nebulized 5% OVA solution to establish asthma models. In group A, normal saline of the equal volume was given instead of OVA. In group C and group E, each mouse was treated with two doses of 0.25 mg anti-CD25+ mAb– one dose was intravenously injected on the previous day before the OVA challenge, and another was intraperitoneally injected on the third day after the OVA challenge. Group D and group E were intravenously administered with MSCs on the tenth day after sensitization, while other groups were treated with PBS instead. The proportion of CD4+CD25+Treg in peripheral blood was detected by Flow Cytometry. The number of total cells, eosinophils, lymphocytes and neutrophils in BALF was counted to analyze the degree of airway inflammation together with pathological sections.
Results:The proportion of CD4+CD25+Tregs in peripheral blood was (5.86±0.48)% in group A, (3.60±0.41)% in group B, (0.32±0.11)% in group C, (4.72± 0.23)% in group D and(0.33±0.11)% in group E. The proportion was significantly lower in group B than that in group A(P<0.01), as well as in group C than that in group B (P<0.01). The number of total cells and eosinophils in BALF was(4.93±1.68)×104 /ml and(0.03±0.01)×104 /ml in group A(,62.84±9.67)×104 /ml and(18.93±3.40)×104 /ml in group B(,80.59±11.34)×104 /ml and (29.26±5.43)×104 /ml in group C,(27.68±4.92)×104 /ml and (4.87±0.55)×104 /ml in group D, and (29.59±6.67)×104 /ml and (5.51±1.75)×104 /ml in group E. The proportion was significantly different when group A was compared with other groups(P<0.01), and the difference was still significant among group B,C,D and E(P<0.01). The pulmonary pathological score of the group A was 0(0～1), group B was 3(2～4), group C was 4 (3～4),group D was 2(1～3) and group E was 2 (1～3). There was significant difference among the scores of the five groups(H=32.527,P<0.01). The score was significantly lower when group A was compared with other groups(P<0.01),when group D and group E with group B and group C, and when group B with group C (P<0.05). There was no significant difference between the scores of group D and group E(P>0.05).
Conclusions : The asthmatic BALB/c mice have more severe airway inflammation when CD4+CD25+Treg are depleted from peripheral blood. MSCs can up-regulate the CD4+CD25+Treg and thus inhibit the airway inflammation in asthmatic BALB/c mice. This effect is weakened in the mice depleted of CD4+CD25+Treg, compared with those not depleted, even though the difference is not significant. These findings suggest that low level of CD4+CD25+Treg may be an important mechanism of the airway inflammation in asthmatic mice. Increasing the level of CD4+CD25+Treg is just one of the mechanisms of MSCs in alleviating the airway inflammation. Further studies are needed to understand other mechanisms.
引文
1. HA Awad, DL Butler, GP Boivin, et al. Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng, 1999, 5: 267-277.
2. J Ringe, C Kaps, B Schmitt, et al. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res, 2002, 307: 321-327.
3. JD Mosca, JK Hendricks, D Buyaner, et al. Mesenchymal stem cells as vehicles for gene delivery. Clin Orthop, 2000, 379: S71-S90 (Suppl).
4. HL Jessop, BS Noble, A Cryer. The differentiation of a potential mesenchymal stem cell population within ovine bone marrow. Biochem Soc Trans, 1994, 22 : 248S.
5. Lindolfo da Silva Meirelles, Nance Beyer Nardi. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. British Journal of Haematology, 2003, 123: 702-711.
6. E.Fuchs, J.A. Segre. Stem cells: a new lease on life. Cell 100, 2000, 143-155.
7. Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol, 1999, 27: 1675-1681.
8. Hisha H, Nishino T, Kawamura M et al. Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol, 1995,23:347–352.
9. Fouillard L, Bensidhoum M, Bories D et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia, 2003, 17: 474-476.
10. Short B, Brouard N, Driessen R et al. Prospective isolation of stromal progenitorcells from mouse compact bone. Exp Hematol, 2001, 29:68a.
11. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
12. EJ Schwarz, GM Alexander, DJ Prockop, et al. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther, 1999, 10: 2539-2549.
13. DR Martin, NR Cox, TL Hathcock, et al. Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp Hematol, 2002, 30 : 879–886.
14. S Kadiyala, RG Young, MA Thiede, et al. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant, 1997, 6: 125-134.
15. SM Devine, AM Bartholomew, N Mahmud, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol, 2001, 29: 244-255.
16. S Wakitani, T Goto, SJ Pineda, et al. Mesenchymal cell-based repair of large fullthickness defects of articular cartilage. J Bone Joint Surg, 1994, 76: 579- 592.
17. Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol, 1999, 27: 1675-1681.
18. Hisha H, Nishino T, Kawamura M et al. Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol, 1995,23:347-352.
19. Fouillard L, Bensidhoum M, Bories D et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia, 2003, 17: 474-476.
20. Short B, Brouard N, Driessen R et al. Prospective isolation of stromal progenitor cells from mouse compact bone. Exp Hematol, 2001, 29:68a.
21. Philippe Tropel, Daniele Noel, Nadine Platet, et al. Isolation and characterization of mesenchymal stem cells from adult mouse bone marrow. Experimental Cell Research, 2004, 295: 395-406.
22. Melody Baddoo, Katy Hill, Robin Wilkinson, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. Journal of Cellular Biochemistry, 2003, 89: 1235-1249.
23. Shengkun Sun, Zikuan Guo, Xuren Xiao, et al. Isolation of Mouse Marrow Mesenchymal Progenitors by a Novel and Reliable Method. Stem Cells, 2003, 21:527-535.
24. Alexandra Peister, Jason A. Mellad, Benjamin L. Larson, et al. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood, 2004, 103(5): 1662-1668.
25. Zavifler NJ, Marinova L, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res, 2000, 2(6): 477-488.
26. Pereira RF, Halford KW, O’Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells from bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA, 1995, 92(11): 4851-4861.
27. Jaiswal N, Haynesworth SE, Caplan AI, et al. Osteogenic diffrenciation of purified culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem, 1997, 64(2): 295-312.
1.中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志, 2003, 26: 132-138.
2. Sidney S. Braman MD, FCCP. The Global Burden of Asthma. CHEST, 2006, 130:4S-12S.
3. Global Initiative for Asthma (GINA). Global strategy for asthma management and prevention. Brethesda MD: National Heart, Lung, and Blood Institute; World Health Organization, 2006.
4. Speers JL, Olson SD, Ylostalo J, et al. Differentiation, cell fusion and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci USA, 2003, 100: 2397-2404.
5. Sudeepta Aggarwal, Mark F Pittenger. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005, 105: 1815-1822.
6.宁红梅,金建刚,扈江伟,等.人骨髓间充质干细胞体外对异基因T淋巴细胞表型的影响.中国实验血液学杂志, 2005, 13(1): 43-49.
7. Rita Maccario, Marina Podestà, Antonia Moretta, et al. Interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica, 2005, 90(4): 516-525.
8.田莹,邓宇斌,黄奕俊,等.骨髓间充质干细胞在CD4+ CD25+调节性T细胞减轻大鼠移植物抗宿主反应中的作用.中国实验血液学杂志, 2006, 14 (6) : 1210-1214.
9. Massimo Di Nicola, Carmelo Carlo-Stella, Michele Magni, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 2002, 99(10): 3838-3843.
10. Zhang WeiGe, Wei. Li, Changhong You, et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells & Development, 2004, 13(3): 263-271.
11. Bartholomew A, Sturgeon C, Siatskas M , et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42-8.
12. Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA, 2003, 100: 8407-8411.
13. Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood, 2005, 106: 1755-1761.
14. Zhang J, Li Y, Chen J, et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol, 2005, 195: 16-26.
15. Lazarus HM, Haynesworth SE, Gerson SL, et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557-64.
16. Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant, 2005, 11: 389-398.
17. Koc ON, Day J, Nieder M, et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002;30:215-22.
18. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000, 18: 307-316.
19. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children withosteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA, 2002, 99: 8932-7.
20.李睿,刘恩梅,杨锡强,等.不同饲养环境、种属、佐剂及激发方法对小鼠哮喘炎症的影响.重庆医学, 2006, 35(1): 43-44.
21. Mary Beth Hogan, David N. Weissman, Ann F. Hubbs, et al. Regulation of Eosinophilopoiesis in a Murine Model of Asthma. J Immunol, 2003, 171: 2644-2651.
22. Anne McKay, Bernard P. Leung, Iain B. McInnes, et al. A Novel Anti-Inflammatory Role of Simvastatin in a Murine Model of Allergic Asthma. J Immunol, 2004, 172: 2903-2908.
23. Amy Pastva, Kim Estell, Trenton R. Schoeb, et al. Aerobic Exercise Attenuates Airway Inflammatory Responses in a Mouse Model of Atopic Asthma. J Immunol, 2004, 172: 4520-4526.
24. Wei Duan, Jasmine H. P. Chan, Chui Hong Wong, et al. Anti-Inflammatory Effects of Mitogen-Activated Protein Kinase Inhibitor U0126 in an Asthma Mouse Model. J Immunol, 2004, 172: 7053-7059.
25. Akiko Iwata, Kazumi Nishio, Robert K. Winn, et al. A Broad-Spectrum Caspase Inhibitor Attenuates Allergic Airway Inflammation in Murine Asthma Model. J Immunol, 2003, 170: 3386-3391.
26. Hayashi T, Adachi Y, Hasegawa K, et al. Less sensitivity for late airway inflammation in males than females in BALB/c mice. Scand J Immunol, 2003, 57(6): 562.
27. Blyth DI, Pedrick MS, Savage TJ, et al. Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J Respir Cell Mol Biol, 1996, 45(5): 425-438.
28. Zhang Y, Lamm WJE, Albert RK, et al. Influence of the route of allergen administration and genetic background on the murine allergic pulmonary response. Am J Respir Crit Care Med, 1997, 155(2): 661-669.
29. Shi H, Yokoyama A, Kohno N, et al. Effect of thromboxane A2 inhibitors on allergic pulmonary inflammation in mice. Eur Respir J, 1998, 11(3): 624-629.
30. Sakai K, Yokoyama A, Kohno N, et al. Effect of different sensitizing doses of antigen in a murine model of atopic asthma. Clin Exp Immunol, 1999, 118: 9-15.
31. William R. Henderson, Emil Y. Chi, Charles R. Maliszewski, et al. Soluble IL-4 Receptor Inhibits Airway Inflammation Following Allergen Challenge in a Mouse Model of Asthma. J Immunol, 2000, 164: 1086-1095.
32. Se-Woong Oh, Chong I. Pae, Dong-Keun Lee, et al. Tryptase Inhibition Blocks Airway Inflammation in a Mouse Asthma Model. J Immunol, 2002, 168: 1992-2000.
33. William R. Henderson, Jiangyang Lu, Karen M. Poole, et al. Recombinant Human Platelet-Activating Factor-Acetylhydrolase Inhibits Airway Inflammation and Hyperreactivity in Mouse Asthma Model. J Immunol, 2000, 164: 3360-3367.
34.李建国,胡晓文,檀卫平,等.干扰素-γ质粒基因转染对支气管哮喘小鼠气道炎症的影响.中华结核和呼吸杂志,2005,28(8):530-533.
35. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol, 2000, 28: 875-884.
36.邓为民,韩钦,葛薇等.骨髓源间充质干细胞在异基因小鼠免疫器官内的分布及其免疫调节作用.中国免疫学杂志, 2004, 20: 40-45.
37. Toma C, Pittenger MF, Cahill KS , et al . Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation , 2002 ; 105 : 93 -98.
38. Bartholomew A, Sturgeon C, Siatskas M , et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42-8.
39. Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA, 2003, 100: 8407-8411.
40. Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood, 2005, 106: 1755-1761.
41. Zhang J, Li Y, Chen J, et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol, 2005, 195: 16-26.
42. Lazarus HM, Haynesworth SE, Gerson SL, et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557-64.
43. Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant, 2005, 11: 389-398.
44. Koc ON, Day J, Nieder M, et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002;30:215-22.
45. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000, 18: 307-316.
46. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA, 2002, 99: 8932-7.
47. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004, 363:1439-41.
48.祖莹,李成荣,郑跃杰,等. CD4+CD25+调节性T细胞在儿童哮喘发病机制中的作用初探.中华医学杂志, 2006, 86(1): 35-38.
49.吴奎,孙鲲,毕玉田,等.哮喘小鼠CD4+CD25+调节性T细胞数量及功能的改变.中国呼吸与危重症杂志, 2005, 4 (6): 455-458.
50. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005, 105: 1815-1822.
51. Zhang W, Ge W, Li C, et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells Dev, 2004, 13: 263 -271.
52. Beyth S, Borovsky Z, Mevorach D, et al. Human mesenchymal stem cells alter antigen presenting cellmaturation and induce T-cell unresponsiveness. Blood, 2005, 105: 2214 -2219.
53. Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 2003, 75 (3): 389 -397.
54. Krampera M, Cosmi L, Angeli R. Role for IFN-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells, 2006, 24 (2): 386–389.
1. Massimo Di Nicola,Carmelo Carlo一Stella,Michele Magni,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood,2002, 99(10): 3838-3843.
2. Zhang WeiGe,Wei.Li,Changhong You,et al.Effect of Mesenchymal stem cells on differentiation,maturation,and function of humman monocyte-derived dendritic cells.Stem Cells & Development, 2004, 13(3):263-271.
3.宁红梅,金建刚,扈江伟,等.人骨髓间充质干细胞体外对异基因T淋巴细胞表型的影响.中国实验血液学杂志,2005,13(1):43-49.
4. Sudeepta Aggarwal,Mark F Pittenger.Hunman mesenchymal stem cells modulate allogeneic immune cell responses.Blood,2005,105:1815-1822.
5. Mauro Di Ianni, Beatrice Del Papa, Maria De Ioann,et al. Mesenchymal cells recruit and regulate T regulatory cells. Experimental Hematology 36 (2008) 309-318.
6. Rita Maccario,Marina Podesta,Antonia Moretta,et al. interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica,2005,90(4):516-525.
7.田莹,邓宇斌,黄奕俊,等.骨髓间充质干细胞在CD4+CD25+调节性T细胞减轻大鼠移植物抗宿主反应中的作用.中国实验血液学杂志,2006,14(6):1210-1214.
8. Bartholomew A,Sturgeon C,Siatskas M,et al. Mesenchymal stem cells suppresslymphocyte proliferation in vitro and prolong skin graft survivval in vivo.Exp Hematol 2002;30:42-8.
9. Ortiz LA,Gambelli F,McBride C,et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.Proc Natl Acad Sci USA, 2003, 100: 8407-8411.
10. Zappia E,Casazza S,Pedemonte E,et al.Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood, 2005,106:1755-1761.
11. ZhangJ,Li Y,Chen J,et al.Hunman bone marrow stromal cell treatment improves neurological functional recovery in EAE mice.Exp Neurol, 2005,195:16-26.
12. Lazarus HM,Haynesworth SE, Gerson SL,et aL .Ex vivo expasion and subsequent infusion of human bone marrow-derived stromal progenitor cells(Mesenchymal progenitor cells):implications for therapeutic use,Bone Marrow Transplant 1995;16:557-64.
13. Lazarus HM,Koc ON,Devine SM,et al.Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.Biol Blood Marrow Transplant, 2005, 11:389 -398.
14. Koc ON,Day J, Nieder M, et al.Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy(MLD) and Hurler syndrome (MPS-IH).Bone Marrow Transplant 2002,30:215-22.
15. Koc ON,Gerson SL,Cooper BW,et aL .Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol,2000,18:307-316.
16. Horwitz EM,Gordon PL,Koo WK,et aL .Isolated allogeneic bone marrow-derived mesenchymal stem cells engraft and stimulate growth in children with osteogenesis imperfecta:implications for cell therapy of bone. Proc Natl Acad Sci USA,2002,99:8972-7.
17. Mee-Young Lee, Kyung-Seop Ahn, Ok-Kyung Kwon,et al. Anti-inflammatory and anti-allergic effects of kefir in a mouse asthma model. Immunobiology 212(2007) 647–654.
18. Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, Falorni A, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol 2002; 3:1097 -101.
19. Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, von Boehmer H, et al. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc Natl Acad Sci U S A 2005;102:419-24.
20. Fahlen L, Read S, Gorelik L, Hurst SD, Coffman RL, Flavell RA, et al.T cells that cannot respond to TGF-{beta} escape control by CD4+CD25+regulatory T cells. J Exp Med 2005;201:737-46.
21. Yang JH, Zhang YX, Wang F, et al. Isolation, identification and functional characterization of human CD4+ CD25+ Treg cells from human peripheral blood.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2006;22(2):252-254, 257.
22. Karagiannidis C, Akdis M, Holopainen P, Woolley NJ, Hense G, Ruckert B, et al. Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol 2004;114:1425-33.
23. Doganci A, Eigenbrod T, Krug N, De Sanctis GT, Hausding M,Erpenbeck VJ, et al. The IL-6R alpha chain controls lung CD4+CD25+Treg development and function during allergic airway inflammation in vivo. J Clin Invest 2005; 115:313-25.
24. Tanaka, Hiroshi; Tanaka, Junta;et al. Depletion of CD4+CD25+ Regulatory Cells Augments the Generation of Specific Immune T Cells in Tumor-Draining Lymph Nodes.Jonurnal of Immunotherapy with Emphasis on Tumor Immunology (ISSN:1067-5582).
25. Ton That Ai Long, Shusuke Nakazawa, Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice. International Journal for Parasitology 33 (2003) 175–183.
26. Markus H Frank,Mohamed H Sayegh Immunomodulatory functions of mesenchymal stem cells. The Lancet,2004,363:1411-1413.
27. Farida Djouad,Pascale Plence,Claire Bony,et al.immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.Blood,2003,102(10) :3837-3844.
28. Krampera M,Glennie S,Dyson J,et al.Bone marrow mesenchymal stem cells inhibit the response of na?ve and memory antigen-specific T cells to their cognate peptide.Blood, 2003,101:3722-3729.
29. Keiya Ozawa ,Kazuya Sato , Iekuni Oh ,et al. Cell and gene therapy using mesenchymal stem cells (MSCs), Journal of Autoimmunity 30 (2008) 121-127.
30. Beyth S,Borovsky Z,Mevorach D,et al. Human mesenchymal stem cells alter antigen Presenting cellmaturation and induce T-cell unresponsiveness. Blood, 2005,105:2214-2219.
31. Zhang W,Ge W,Li C,et al. Effects of mesenchymal stem cells on differentiation,maturation,and function of human monocyte-derived dendritic cells.Stem Cells Dev,2004,13:236-271.
32. Krampera M,Glennie S,Dyson J,et al.Bone marrow mesenchymal stem cells inhibit the response of na?ve and memory antigen specific T cell to their cognate peptide.Blood,2003,101(9):3272-3729.
33. Tse WT, Pendleton JD,Beyer WM,et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation. Transplantation, 2003,75(3):389-397.
34. Krampera M , Cosmi L , Angeli R.Role for IFN-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells,2006,24(2):386-389.
2. J Ringe, C Kaps, B Schmitt, et al. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res, 2002, 307: 321-327.
3. JD Mosca, JK Hendricks, D Buyaner, et al. Mesenchymal stem cells as vehicles for gene delivery. Clin Orthop, 2000, 379: S71-S90 (Suppl).
4. HL Jessop, BS Noble, A Cryer. The differentiation of a potential mesenchymal stem cell population within ovine bone marrow. Biochem Soc Trans, 1994, 22 : 248S.
5. Lindolfo da Silva Meirelles, Nance Beyer Nardi. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. British Journal of Haematology, 2003, 123: 702-711.
6. E.Fuchs, J.A. Segre. Stem cells: a new lease on life. Cell 100, 2000, 143-155.
7. Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol, 1999, 27: 1675-1681.
8. Hisha H, Nishino T, Kawamura M et al. Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol, 1995,23:347–352.
9. Fouillard L, Bensidhoum M, Bories D et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia, 2003, 17: 474-476.
10. Short B, Brouard N, Driessen R et al. Prospective isolation of stromal progenitorcells from mouse compact bone. Exp Hematol, 2001, 29:68a.
11. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
12. EJ Schwarz, GM Alexander, DJ Prockop, et al. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther, 1999, 10: 2539-2549.
13. DR Martin, NR Cox, TL Hathcock, et al. Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp Hematol, 2002, 30 : 879–886.
14. S Kadiyala, RG Young, MA Thiede, et al. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant, 1997, 6: 125-134.
15. SM Devine, AM Bartholomew, N Mahmud, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol, 2001, 29: 244-255.
16. S Wakitani, T Goto, SJ Pineda, et al. Mesenchymal cell-based repair of large fullthickness defects of articular cartilage. J Bone Joint Surg, 1994, 76: 579- 592.
17. Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol, 1999, 27: 1675-1681.
18. Hisha H, Nishino T, Kawamura M et al. Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol, 1995,23:347-352.
19. Fouillard L, Bensidhoum M, Bories D et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia, 2003, 17: 474-476.
20. Short B, Brouard N, Driessen R et al. Prospective isolation of stromal progenitor cells from mouse compact bone. Exp Hematol, 2001, 29:68a.
21. Philippe Tropel, Daniele Noel, Nadine Platet, et al. Isolation and characterization of mesenchymal stem cells from adult mouse bone marrow. Experimental Cell Research, 2004, 295: 395-406.
22. Melody Baddoo, Katy Hill, Robin Wilkinson, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. Journal of Cellular Biochemistry, 2003, 89: 1235-1249.
23. Shengkun Sun, Zikuan Guo, Xuren Xiao, et al. Isolation of Mouse Marrow Mesenchymal Progenitors by a Novel and Reliable Method. Stem Cells, 2003, 21:527-535.
24. Alexandra Peister, Jason A. Mellad, Benjamin L. Larson, et al. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood, 2004, 103(5): 1662-1668.
25. Zavifler NJ, Marinova L, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res, 2000, 2(6): 477-488.
26. Pereira RF, Halford KW, O’Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells from bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA, 1995, 92(11): 4851-4861.
27. Jaiswal N, Haynesworth SE, Caplan AI, et al. Osteogenic diffrenciation of purified culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem, 1997, 64(2): 295-312.
1.中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志, 2003, 26: 132-138.
2. Sidney S. Braman MD, FCCP. The Global Burden of Asthma. CHEST, 2006, 130:4S-12S.
3. Global Initiative for Asthma (GINA). Global strategy for asthma management and prevention. Brethesda MD: National Heart, Lung, and Blood Institute; World Health Organization, 2006.
4. Speers JL, Olson SD, Ylostalo J, et al. Differentiation, cell fusion and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci USA, 2003, 100: 2397-2404.
5. Sudeepta Aggarwal, Mark F Pittenger. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005, 105: 1815-1822.
6.宁红梅,金建刚,扈江伟,等.人骨髓间充质干细胞体外对异基因T淋巴细胞表型的影响.中国实验血液学杂志, 2005, 13(1): 43-49.
7. Rita Maccario, Marina Podestà, Antonia Moretta, et al. Interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica, 2005, 90(4): 516-525.
8.田莹,邓宇斌,黄奕俊,等.骨髓间充质干细胞在CD4+ CD25+调节性T细胞减轻大鼠移植物抗宿主反应中的作用.中国实验血液学杂志, 2006, 14 (6) : 1210-1214.
9. Massimo Di Nicola, Carmelo Carlo-Stella, Michele Magni, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 2002, 99(10): 3838-3843.
10. Zhang WeiGe, Wei. Li, Changhong You, et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells & Development, 2004, 13(3): 263-271.
11. Bartholomew A, Sturgeon C, Siatskas M , et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42-8.
12. Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA, 2003, 100: 8407-8411.
13. Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood, 2005, 106: 1755-1761.
14. Zhang J, Li Y, Chen J, et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol, 2005, 195: 16-26.
15. Lazarus HM, Haynesworth SE, Gerson SL, et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557-64.
16. Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant, 2005, 11: 389-398.
17. Koc ON, Day J, Nieder M, et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002;30:215-22.
18. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000, 18: 307-316.
19. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children withosteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA, 2002, 99: 8932-7.
20.李睿,刘恩梅,杨锡强,等.不同饲养环境、种属、佐剂及激发方法对小鼠哮喘炎症的影响.重庆医学, 2006, 35(1): 43-44.
21. Mary Beth Hogan, David N. Weissman, Ann F. Hubbs, et al. Regulation of Eosinophilopoiesis in a Murine Model of Asthma. J Immunol, 2003, 171: 2644-2651.
22. Anne McKay, Bernard P. Leung, Iain B. McInnes, et al. A Novel Anti-Inflammatory Role of Simvastatin in a Murine Model of Allergic Asthma. J Immunol, 2004, 172: 2903-2908.
23. Amy Pastva, Kim Estell, Trenton R. Schoeb, et al. Aerobic Exercise Attenuates Airway Inflammatory Responses in a Mouse Model of Atopic Asthma. J Immunol, 2004, 172: 4520-4526.
24. Wei Duan, Jasmine H. P. Chan, Chui Hong Wong, et al. Anti-Inflammatory Effects of Mitogen-Activated Protein Kinase Inhibitor U0126 in an Asthma Mouse Model. J Immunol, 2004, 172: 7053-7059.
25. Akiko Iwata, Kazumi Nishio, Robert K. Winn, et al. A Broad-Spectrum Caspase Inhibitor Attenuates Allergic Airway Inflammation in Murine Asthma Model. J Immunol, 2003, 170: 3386-3391.
26. Hayashi T, Adachi Y, Hasegawa K, et al. Less sensitivity for late airway inflammation in males than females in BALB/c mice. Scand J Immunol, 2003, 57(6): 562.
27. Blyth DI, Pedrick MS, Savage TJ, et al. Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J Respir Cell Mol Biol, 1996, 45(5): 425-438.
28. Zhang Y, Lamm WJE, Albert RK, et al. Influence of the route of allergen administration and genetic background on the murine allergic pulmonary response. Am J Respir Crit Care Med, 1997, 155(2): 661-669.
29. Shi H, Yokoyama A, Kohno N, et al. Effect of thromboxane A2 inhibitors on allergic pulmonary inflammation in mice. Eur Respir J, 1998, 11(3): 624-629.
30. Sakai K, Yokoyama A, Kohno N, et al. Effect of different sensitizing doses of antigen in a murine model of atopic asthma. Clin Exp Immunol, 1999, 118: 9-15.
31. William R. Henderson, Emil Y. Chi, Charles R. Maliszewski, et al. Soluble IL-4 Receptor Inhibits Airway Inflammation Following Allergen Challenge in a Mouse Model of Asthma. J Immunol, 2000, 164: 1086-1095.
32. Se-Woong Oh, Chong I. Pae, Dong-Keun Lee, et al. Tryptase Inhibition Blocks Airway Inflammation in a Mouse Asthma Model. J Immunol, 2002, 168: 1992-2000.
33. William R. Henderson, Jiangyang Lu, Karen M. Poole, et al. Recombinant Human Platelet-Activating Factor-Acetylhydrolase Inhibits Airway Inflammation and Hyperreactivity in Mouse Asthma Model. J Immunol, 2000, 164: 3360-3367.
34.李建国,胡晓文,檀卫平,等.干扰素-γ质粒基因转染对支气管哮喘小鼠气道炎症的影响.中华结核和呼吸杂志,2005,28(8):530-533.
35. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol, 2000, 28: 875-884.
36.邓为民,韩钦,葛薇等.骨髓源间充质干细胞在异基因小鼠免疫器官内的分布及其免疫调节作用.中国免疫学杂志, 2004, 20: 40-45.
37. Toma C, Pittenger MF, Cahill KS , et al . Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation , 2002 ; 105 : 93 -98.
38. Bartholomew A, Sturgeon C, Siatskas M , et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42-8.
39. Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA, 2003, 100: 8407-8411.
40. Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood, 2005, 106: 1755-1761.
41. Zhang J, Li Y, Chen J, et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol, 2005, 195: 16-26.
42. Lazarus HM, Haynesworth SE, Gerson SL, et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557-64.
43. Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant, 2005, 11: 389-398.
44. Koc ON, Day J, Nieder M, et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002;30:215-22.
45. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000, 18: 307-316.
46. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA, 2002, 99: 8932-7.
47. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004, 363:1439-41.
48.祖莹,李成荣,郑跃杰,等. CD4+CD25+调节性T细胞在儿童哮喘发病机制中的作用初探.中华医学杂志, 2006, 86(1): 35-38.
49.吴奎,孙鲲,毕玉田,等.哮喘小鼠CD4+CD25+调节性T细胞数量及功能的改变.中国呼吸与危重症杂志, 2005, 4 (6): 455-458.
50. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005, 105: 1815-1822.
51. Zhang W, Ge W, Li C, et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells Dev, 2004, 13: 263 -271.
52. Beyth S, Borovsky Z, Mevorach D, et al. Human mesenchymal stem cells alter antigen presenting cellmaturation and induce T-cell unresponsiveness. Blood, 2005, 105: 2214 -2219.
53. Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 2003, 75 (3): 389 -397.
54. Krampera M, Cosmi L, Angeli R. Role for IFN-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells, 2006, 24 (2): 386–389.
1. Massimo Di Nicola,Carmelo Carlo一Stella,Michele Magni,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood,2002, 99(10): 3838-3843.
2. Zhang WeiGe,Wei.Li,Changhong You,et al.Effect of Mesenchymal stem cells on differentiation,maturation,and function of humman monocyte-derived dendritic cells.Stem Cells & Development, 2004, 13(3):263-271.
3.宁红梅,金建刚,扈江伟,等.人骨髓间充质干细胞体外对异基因T淋巴细胞表型的影响.中国实验血液学杂志,2005,13(1):43-49.
4. Sudeepta Aggarwal,Mark F Pittenger.Hunman mesenchymal stem cells modulate allogeneic immune cell responses.Blood,2005,105:1815-1822.
5. Mauro Di Ianni, Beatrice Del Papa, Maria De Ioann,et al. Mesenchymal cells recruit and regulate T regulatory cells. Experimental Hematology 36 (2008) 309-318.
6. Rita Maccario,Marina Podesta,Antonia Moretta,et al. interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica,2005,90(4):516-525.
7.田莹,邓宇斌,黄奕俊,等.骨髓间充质干细胞在CD4+CD25+调节性T细胞减轻大鼠移植物抗宿主反应中的作用.中国实验血液学杂志,2006,14(6):1210-1214.
8. Bartholomew A,Sturgeon C,Siatskas M,et al. Mesenchymal stem cells suppresslymphocyte proliferation in vitro and prolong skin graft survivval in vivo.Exp Hematol 2002;30:42-8.
9. Ortiz LA,Gambelli F,McBride C,et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.Proc Natl Acad Sci USA, 2003, 100: 8407-8411.
10. Zappia E,Casazza S,Pedemonte E,et al.Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood, 2005,106:1755-1761.
11. ZhangJ,Li Y,Chen J,et al.Hunman bone marrow stromal cell treatment improves neurological functional recovery in EAE mice.Exp Neurol, 2005,195:16-26.
12. Lazarus HM,Haynesworth SE, Gerson SL,et aL .Ex vivo expasion and subsequent infusion of human bone marrow-derived stromal progenitor cells(Mesenchymal progenitor cells):implications for therapeutic use,Bone Marrow Transplant 1995;16:557-64.
13. Lazarus HM,Koc ON,Devine SM,et al.Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.Biol Blood Marrow Transplant, 2005, 11:389 -398.
14. Koc ON,Day J, Nieder M, et al.Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy(MLD) and Hurler syndrome (MPS-IH).Bone Marrow Transplant 2002,30:215-22.
15. Koc ON,Gerson SL,Cooper BW,et aL .Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol,2000,18:307-316.
16. Horwitz EM,Gordon PL,Koo WK,et aL .Isolated allogeneic bone marrow-derived mesenchymal stem cells engraft and stimulate growth in children with osteogenesis imperfecta:implications for cell therapy of bone. Proc Natl Acad Sci USA,2002,99:8972-7.
17. Mee-Young Lee, Kyung-Seop Ahn, Ok-Kyung Kwon,et al. Anti-inflammatory and anti-allergic effects of kefir in a mouse asthma model. Immunobiology 212(2007) 647–654.
18. Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, Falorni A, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol 2002; 3:1097 -101.
19. Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, von Boehmer H, et al. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc Natl Acad Sci U S A 2005;102:419-24.
20. Fahlen L, Read S, Gorelik L, Hurst SD, Coffman RL, Flavell RA, et al.T cells that cannot respond to TGF-{beta} escape control by CD4+CD25+regulatory T cells. J Exp Med 2005;201:737-46.
21. Yang JH, Zhang YX, Wang F, et al. Isolation, identification and functional characterization of human CD4+ CD25+ Treg cells from human peripheral blood.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2006;22(2):252-254, 257.
22. Karagiannidis C, Akdis M, Holopainen P, Woolley NJ, Hense G, Ruckert B, et al. Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol 2004;114:1425-33.
23. Doganci A, Eigenbrod T, Krug N, De Sanctis GT, Hausding M,Erpenbeck VJ, et al. The IL-6R alpha chain controls lung CD4+CD25+Treg development and function during allergic airway inflammation in vivo. J Clin Invest 2005; 115:313-25.
24. Tanaka, Hiroshi; Tanaka, Junta;et al. Depletion of CD4+CD25+ Regulatory Cells Augments the Generation of Specific Immune T Cells in Tumor-Draining Lymph Nodes.Jonurnal of Immunotherapy with Emphasis on Tumor Immunology (ISSN:1067-5582).
25. Ton That Ai Long, Shusuke Nakazawa, Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice. International Journal for Parasitology 33 (2003) 175–183.
26. Markus H Frank,Mohamed H Sayegh Immunomodulatory functions of mesenchymal stem cells. The Lancet,2004,363:1411-1413.
27. Farida Djouad,Pascale Plence,Claire Bony,et al.immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.Blood,2003,102(10) :3837-3844.
28. Krampera M,Glennie S,Dyson J,et al.Bone marrow mesenchymal stem cells inhibit the response of na?ve and memory antigen-specific T cells to their cognate peptide.Blood, 2003,101:3722-3729.
29. Keiya Ozawa ,Kazuya Sato , Iekuni Oh ,et al. Cell and gene therapy using mesenchymal stem cells (MSCs), Journal of Autoimmunity 30 (2008) 121-127.
30. Beyth S,Borovsky Z,Mevorach D,et al. Human mesenchymal stem cells alter antigen Presenting cellmaturation and induce T-cell unresponsiveness. Blood, 2005,105:2214-2219.
31. Zhang W,Ge W,Li C,et al. Effects of mesenchymal stem cells on differentiation,maturation,and function of human monocyte-derived dendritic cells.Stem Cells Dev,2004,13:236-271.
32. Krampera M,Glennie S,Dyson J,et al.Bone marrow mesenchymal stem cells inhibit the response of na?ve and memory antigen specific T cell to their cognate peptide.Blood,2003,101(9):3272-3729.
33. Tse WT, Pendleton JD,Beyer WM,et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation. Transplantation, 2003,75(3):389-397.
34. Krampera M , Cosmi L , Angeli R.Role for IFN-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells,2006,24(2):386-389.