TGF-beta1基因修饰的骨髓未成熟树突状细胞来源的exosomes对小鼠炎症性肠病的保护效果及其机制研究
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摘要
自身免疫性疾病是因机体免疫系统对自身成分发生免疫应答而导致的疾病,在人群中有较高的发病率。炎性肠病(inflammatory bowel disease,IBD)包括溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn's disease,CD),主要是由机体对原本处于耐受的正常菌群发生异常的免疫应答所致的非特异性炎性肠疾病。在西方国家发病率为10万分之4-6。在我国,由于近年国人饮食结构的改变,其发病率有逐年上升的趋势,根据国内文献报道,近5年的病例数是上世纪90年代同期的8倍。该病主要症状表现为腹泻、黏液脓血便和腹痛,病久可发生癌变,严重威胁着国人的身体健康。而针对IBD,临床上主要以对症治疗为主,尚没有一种行之有效的治疗手段,因此迫切需要探索一种从根本上治疗的方法。
Transforming Growth Factor-beta1(TGF-beta1)是一种含有112个氨氨基酸,分子量25KD的二聚体蛋白质。在各种属间高度保守,例如人和大鼠、小鼠有完全相同的氨基酸序列。几乎所有细胞都有TGF-beta1的受体,其对细胞的生长、分化的影响是非常广泛的。对T细胞,TGF-beta1可以抑制其增殖,抑制Th1和Th2细胞的分化,抑制CTL的分化,诱导调节性T细胞(Treg)的产生;对B细胞,TGF-beta1可以抑制B细胞的增殖,诱导IgA的类别转换,还可以诱导不成熟和静息B细胞的凋亡。对NK细胞,TGF-beta1可以抑制NK细胞分泌IFN-γ及其溶细胞的功能;对树突状细胞(DC)TGF-beta1抑制DC的成熟及其抗原递呈功能;TGF-beta1还可以抑制活化的巨噬细胞(MΦs),肥大细胞及粒细胞的功能。
目前认为DC是功能最强大的抗原递呈细胞(APC),不同来源及不同成熟状态的DC能够正向或负向调节T细胞的免疫功能。DC有许多亚群,包括髓样DC、浆细胞样DC和朗格罕斯细胞,分别在对免疫反应调节中发挥着不同的作用。除了免疫活化的能力之外,不同亚群的DC在某种生理状态下或某种外来条件的干预下可以发挥免疫抑制功能,诱导中枢及外周免疫耐受。如IL-4或Fas L基因修饰的DC可以抑制小鼠的迟发性超敏反应(delayed-type hypersensitivity,DTH)及胶原诱导的关节炎(collagen-induced arthritis,CIA)。
Exosomes是各种活细胞分泌的,非质膜来源的,直径介于50-100nm的具有脂质双分子层结构的小囊泡。在体内,exosomes对免疫系统发挥着双向调节的作用。肿瘤细胞来源的exosomes携带有肿瘤抗原,能够将肿瘤抗原递呈到DC,活化其抗肿瘤免疫,消除已经建立的小鼠肿瘤。APC来源的exosomes表达MHC-I、MHC-Ⅱ和T细胞共刺激分子,提示其具有免疫调节功能。基因修饰表达vIL-10、Fas L或IL-4的骨髓DC来源的exosomes能够抑制小鼠的迟发型超敏反应及胶原诱导的关节炎,过继实验表明基因修饰的exosmes可以在体内调节APC的功能并诱导Treg的产生,从而发挥系统性的免疫抑制功能。
骨髓来源未成熟DC(imDC)由于其分化不成熟,MHC及各种辅助分子表达较低,因此imDC来源的exosomes可以抑制免疫细胞的活化,诱导机体免疫耐受的产生。基因修饰表达TGF-beta1的imDC能够发挥系统性的免疫抑制功能,促进同种异体小鼠移植心脏的长期存活。Treg在肠道的免疫平衡中发挥着重要的作用,而TGF-beta1可以诱导Treg的产生。当在肠道TGF-beta1信号的缺失的情况下,小鼠表现出更为严重的IBD进展,因此TGF-beta1的信号对肠道的免疫自稳状态的维持将发挥着至关重要的作用。本课题将利用葡聚糖硫酸钠盐(DSS)建立IBD小鼠自身免疫病动物模型,利用TGF-beta1基因修饰的骨髓imDC来源的exosomes(TGF-beta1-exo)腹腔注射模型小鼠,评价TGF-beta1-exo在IBD进展过程中的保护性效果,从而探讨TGF-beta1-exo是否能发挥系统性的免疫抑制功能,恢复肠道的免疫平衡。
第一部分
TGF-beta1基因修饰的骨髓未成熟树突状细胞来源的exosomes的制备鉴定及其体外功能
采用单独的rmGM-CSF骨髓细胞体外培养方法,可以获得具有稳定的不成熟表型的DC,而且纯度也较为理想,平均在60-70%左右。流式细胞术(FACS)鉴定培养7天的细胞,其表面表达小鼠DC的标志物CD11c,低表达MHC-Ⅱ类分子和共刺激分子CD40、CD86。
为了能最高限度的提高AdTGF-beta1对大量DCs的转染效率,我们先用CsCl密度梯度离心将腺病毒进行纯化,然后依据相关文献以重复转染率(MOI)等于50进行病毒转染,在2h的无血清转染过程中,我们将DCs与病毒混合液置于37℃摇床上保持不停震摇,使腺病毒与DCs充分接触,大大提高了转染效率。在高效转染的基础上,我们对经过基因修饰的DCs进行了FACS分析,以明确AdTGF-beta1转基因对DCs成熟过程的影响。结果发现未处理组的细胞表现出未成熟DCs的表型,Ad Lac Z转染并未使这种细胞特征发生很大的变化。Ad TGF-beta1转染后DCs表面的MHC-Ⅱ,CD80,CD86和CD40表达均受到抑制,而对CD80的抑制作用最为明显。
研究证明IL-12是DC分泌的主要细胞因子之一,其主要作用是促使T细胞向T辅助细胞1(Th1)转化并大量分泌IL-2和IFN-γ,从而激发强有力的免疫应答。IL-12的合成分泌能力是DC主要功能指标之一,不仅能够提示DC的成熟状态,而且是DC拥有免疫原性还是耐受原性的指针之一。我们在实验中发现无处理对照组和Ad Lac Z转染组DC在LPS刺激下能够迅速成熟并分泌大量IL-12,而Ad TGF-beta1转染后DC分泌的IL-12较对照为低,在LPS刺激后IL-12浓度的提升幅度较小,反映AdTGF-beta1转染后的DC拥有了对LPS等致成熟因子的抗性。
不同处理组DC与同种小鼠脾T细胞做120h混合淋巴细胞反应,结果显示转对照组DC,Ad TGF-beta1转染的DC刺激同种T细胞的能力明显受到抑制,而Ad LacZ转染的DC刺激同种T细胞的能力较对照组DC有轻微的提升。这与其细胞表型特征及分泌IL-12的能力是一致的。
我们实现了AdTGF-beta1对DC的高效感染,获得了稳定表达未成熟DC表型的Ad TGF-beta1基因修饰DC,并在体外证实了其对T细胞的抑制能力,显示其具有免疫耐受性DC的特征。在此基础上,我们分离纯化了Ad TGF-beta1基因修饰DC上清来源的exosomes,在1*10~6的DC的上清中,前24h可以稳定分离得到1μg的exosomes,此后随着DC的大量凋亡,exosomes的得率相应降低,考虑到随时间的推移,DC会逐渐趋于成熟,我们只收集了前48h的exosomes。对exosomes的电镜分析结果显示,我们分离得到的exosomes主要是直径集中在50-100nm左右的小囊泡,具有典型的脂质双分子层结构,这与文献报道相一致。将exosomes吸附到乳胶颗粒之后,通过FACS对其表型进行了分析,显示AdTGF-beta1-exo低表达MHC-Ⅱ类分子、CD80,几乎不表达CD86与CD40,提示其可能具有免疫调节功能。同时exosomes表面还表达小鼠DC特征性的标志CD11c,证实了其属于DC来源。对exosomes的Westernblot(WB)结果表明,我们分离得到的exosomes表达exosomes特征性的蛋白Tsg101和Hsp70。Tsg101是内体相关的蛋白,参与exosomes的形成,也证实了其内体的来源,而较Ad Lac Z基因修饰DC来源的exosomes(Lac Z-exo)及未处理DC来源的exosomes(Control-exo),TGF-beta1-exo表达较低的Hsp70,而Hsp70在体内可以将免疫耐受状态的T细胞转化为自体反应性T细胞,进一步提示了TGF-beta1-exo具有免疫调节功能。与Control-exo和Lac Z-exo相比,只有TGF-beta1-eXO表达TGF-beta1,说明TGF-beta1基因修饰的DC能够以一种未知的机制将TGF-beta1携带到exosomes上。通过ELISA我们对TGF-beta1-exo上的TGF-beta1进行了定量,经反复冻融与未冻融TGF-beta1-exo上含有的TGF-beta1水平有所差别,分别是15pg和8pg/μg exosomes,说明在exosomes上同时存在着分泌型与膜型的TGF-beta1。在不同处理组exosomes对同种小鼠120h混合淋巴细胞反应影响效应的实验中显示,TGF-betal-exo可以明显抑制小鼠T细胞的增殖,而Control-exo和Lac Z-exo对T细胞亦有微弱的抑制作用TGF-beta1-exo除了对T细胞的抑制作用,我们还发现TGF-beta1-exo还可以抑制Con A引起的淋巴细胞的活化,进一步证实了TGF-beta1-exo的免疫抑制功能。
综上所述,我们成功的分离纯化了Ad TGF-beta1基因修饰DC来源的exosomes,并通过电镜,FACS及WB对其形态,蛋白表达进行了鉴定,并在体外证实了其对T细胞的抑制效果,这为下一步的体内实验奠定了基础。
第二部分
TGF-beta1基因修饰的骨髓未成熟树突状细胞来源的exosomos对DSS诱导的小鼠炎症性肠病的保护作用及其相关机制
为了进一步检验TGF-beta1-exo的免疫抑制功能,明确在体内环境下TGF-beta1-exo是否能同样的发挥免疫抑制的功能,我们将TGF-beta1-exo经腹腔注射小鼠,通过观察小鼠体重的改变情况,大便性状,便血情况,以及肠道组织的病理切片分析,判断TGF-beta1-exo是否能够延缓DSS诱导的小鼠IBD的进展过程,并对其机制进行了探讨。
我们通过让C57BL/6小鼠连续自由饮用浓度为1.5%的DSS 9天,来诱导小鼠IBD的发病。并在小鼠饮用DSS前两天,经腹腔分别注射10μg/只的Control-exo、Lac Z-exo和TGF-beta1-exo,并于饮用DSS后第3天,第5天经腹腔再追加注射10μg/只的各组exosomes。同时设正常饮水的对照组及未经任何处理单纯饮用DSS组。除经TGF-beta1-CXO处理的小鼠,其它各组小鼠在饮用DSS 5天后体重呈进行性下降,至第9天,DSS组、Control-exo组及Lac Z-exo组小鼠的体重分别下降了16%、15%、15%。而TGF-beta1-exo组小鼠的体重在第6天才开始下降,并且下降的程度相对缓慢,至第9天,体重仅下降了5%。除此之外,联合了体重改变,大便性状,便血情况,对小鼠IBD进展情况进行综合评价的每天疾病活跃指数(daily diseaseactivity index,DAI)的评分结果亦显示了TGF-beta1-exo能较大程度的延缓DSS诱导的小鼠的IBD的进展速度。而第7天的各组小鼠便隐血测试结果显示,TGF-beta1-exo组小鼠虽然也出现了较为明显的大便隐血,但与其它各组相比,在便血程度上明显要轻一些。最后,各组小鼠结肠病理切片结果进一步提示了TGF-beta1-exo在小鼠IBD进展过程中的保护作用。在TGF-beta1-exo组小鼠结肠组织的切片中除了可见到粘膜和粘膜下层轻到中等程度的炎症细胞浸润,肠粘膜腺体结构还比较完整。而其它各组小鼠在粘膜层,粘膜下层均可见到重度的炎症细胞浸润,并可见到弥散至肌层的炎症细胞浸润,局部还可见到粘膜结构的破坏,正常腺体结构的消失。
为了探讨TGF-beta1-exo延缓DSS诱导的小鼠IBD进展过程的相关机制,分析TGF-beta1-exo是否能够诱导Treg的产生,从而在一定程度上恢复肠道的免疫平衡,我们分离了各组小鼠脾脏及结肠系膜淋巴结来源的淋巴细胞,分析Foxp3~+ Treg的改变情况。结果我们发现,与其它组小鼠相比,TGF-beta1-exo组小鼠脾脏Foxp3~+Treg并未发生明显的改变,而在炎症局部结肠系膜来源的淋巴细胞中,可以观察到Foxp3~+ Treg的明显上调。为进一步分析这种差别产生的原因,我们用羧基荧光素二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein diacetate succimidyl ester,CFSE)对exosomes进行标记,然后将标记的exosomes与IBD或正常小鼠小鼠脾脏及结肠系膜淋巴结来源的淋巴细胞共培养后,观察不同来源淋巴细胞对exosomes的内化情况。我们发现,与正常小鼠结肠系膜来源的淋巴细胞相比,IBD小鼠结肠系膜来源的淋巴细胞能够内化更多的exosomes,而这种差异在IBD小鼠与正常小鼠的脾脏淋巴细胞中并不能观察到。
综合以上结果可以看出,TGF-beta1-exo能够明显延缓DSS诱导的小鼠IBD的进展速度,而不像它们在体外MLR中观察到的轻微的抑制作用,Contro-exo和LacZ-exo对DSS诱导的小鼠IBD的进展并未显示出任何保护性作用。与其它组exosomes相比,经TGF-beta1-exo处理之后的小鼠,在炎症局部来源的淋巴细胞中,可以观察到Foxp3~+Treg的明显上调,这可能是TGF-beta1-exo能够明显延缓DSS诱导的小鼠IBD的进展速度的机制之一。而能在炎症局部淋巴细胞观察到Foxp3~+Treg的上调而不是脾脏淋巴细胞,原因之一,很有可能是因为炎症局部的淋巴细胞能够富集更多的TGF-beta1-exo而造成局部的高浓度TGF-betal。
Autoimmune disease is a kind of disease that is caused by body immune system responsing to autoantigen.Inflammatory bowel disease(IBD) including ulcerative colitis(UC) and Crohn' s disease(CD) is a kind of non-specific inflammatory bowel disease that is mainly caused by abnormal immune response of immune system to normal intestinal flora.In western countries,incidence of IBD is 4-6 out of one hundred thousand.In our country,because of the change of dietetic structure,incidence of IBD increased every year.Reported by domestic literature,cases in this five years is eight times more than the corresponding period in 90' s of last century.The main symptom of IBD is diarrhea,grume sanguinopurulent stool and bellyache. Furthermore,lasting for a long time,the risk of cancer mutation increases,so IBD threatens the people' s health of our country badly.In clinal,allopathy is the main therapeutic means of IBD and there still has not an effective procedure of treatment,so the demand of exploring a new way to cure IBD thoroughly is urgent.
Transforming Growth Factor-beta 1(TGF-beta1) including 112 amino acid is a dimer protein with molecular weight of 25KD.It is strictly conservative in each species.For example,human,rat and mouse have totally identical sequence.Almost all cells have receptors of TGF-beta1 and its influence of growth,differentiation in cells is very extensive.In T cells,TGF-beta1 can inhibit their proliferation,inhibit the differentiation of Th1 and Th2,inhibit the differentiation of CTL,induce the generation of regulatory T cells(Treg);in B cells,TGF-beta1 can inhibit their proliferation,induce IgA class switch,it also can induce the apoptosis of immature and resting B cells;in NK cells,TGF-beta1 can inhibit the secretion of IFN-γand the cytolytic function of NK cells.In dendritic cells(DC),TGF-beta1 can inhibit their maturation and antigen presenting;furthermore,TGF-beta1 can inhibit the function of active macrophages(MOs) and granulocytes.
DC are the most potent professional antigen presenting cells(APC) that are able to modulate T cell immunity in either a positive or negative manner,depending upon their lineage and state of maturation.There are several subpopulations of DC including myeloid DC,plasmacytoid DC,and Langerhans cells that play different roles in the regulation of the immune responses.In addition to their ability to stimulate immunity, these different DC populations,under certain conditions,are involved in T cell immunosuppression and/or induction of central and peripheral tolerance.For example, IL-4 or Fas L gene modified DC can inhibit delayed-type hypersensitivity(DTH) and collagen-induced arthritis(CIA).
Exosomes are small vesicles released by various live cells,50-100nm,that have structure of lipid bi-layer not derived from cells membrane.In vivo,exosomes play bi-directional regulatory role in immune system.In murine models,exosomes derived from tumor share the tumor antigen can present the antigen to DC,which activate the immunity of anti-tumor of DC and then eradicate established tumor.Exosomes from APC carry MHC-Ⅰ,MHC-Ⅱand T cell costimulatory molecules on their surface, suggesting that they could play important roles in immune regulation.In murine models,exosomes from vIL-10,Fas L or IL-4 gene modified DC can inhibit DTH and CIA.Furthermore,adoptive transfer experiment show that gene modified exosomes can induce the generation of regulatory APC and T cells and then exert the function of systemic immune inhibition.
Bone marrow derived immature DC(MD-imDC) because of their immature stage in differentiation,expressing low level MCHC and costimulatory molecules,so MD-imDC derived exosomes can inhibit the activation of immune cells and induce immunological tolerance.In murine cervical heterotopic heart transplantation model, TGF-beta1 gene modified MD-imDC can exert function of systemic immune inhibition, prolonging the survival of allograft.Treg play a very important role in immune balance in intestine.TGF-beta1 can induce the generation of Treg.Under the condition of losing TGF-beta signal,mice show more severe IBD development,so maintenance of TGF-beta signal is very important in regulating immune homeostasis in the intestine. The aim of this project is to explore if TGF-beta1-exo can play the function of systemic immune inhibition and rebuild immune balance in intestine.In this project dextran sodium sulphate(DSS) was used to induce murine IBD model.Based on this model,TGF-beta1 gene modified DC derived exosomes(TGF-beta1-exo) were pre-injected i.p.and the protective effect of TGF-beta1-exo during IBD development was evaluated.
PARTⅠ.The isolation and identification of exosomes from TGF-beta1 gene modified bone marrow-derived immature DC and their function in vitro.
DCs,the highly effective specialized antigen-presenting cells,play a pivotal role in antigen presentation in the proper context of the required antigenic and costimulatory molecules to induce T cell-mediated immune responses.The functions are highly correlated with their differentiation status.Mature DCs highly express major histocompatibility complex class-Ⅱ(MHC-Ⅱ) molecules and co-stimulatory molecules, such as CD80/86,CD40,providing two signals to activate T lymphocytes to initiate immune responses,while immature DCs which are deficient in these molecules show tolerogenic.So,heterogeneous properties of DCs either tolerogenic or immunogenic determine the outcome of immune responses.The tolerogenic properties of DCs can be amplified by delivery of genes encoding immunosuppressive molecules that are not or seldom produced by DCs.The genes which are the most feasible to induce peripheral tolerance include vIL-10,TGF-beta,CTLA4-Ig,inducible NOS,FasL,and so on. Manipulation of these agents has been shown to render DC tolerogenic characteristics.
Cultured in rmGM-CSF only,we obtained DCs with steady immature phenotype and the purity is ideal averaging between 60-70%.Identified day 7 DCs by FACS,we found DCs expressed CDllc the marker molecule of murine DCs and low level of MHC-Ⅱ,CD40,CD86.
To best improve the infection efficiency of Ad TGF-beta1 to large amount DCs, we purified adenoviruses by CsCl density gradient centrifugation and then infected DCs by MOI50.During the 2h' serum-free infection,we put the mixture of DCs and adenoviruses onto rotatable bed with 37℃and keep the bed rotating which can make the cell and viruses contact completely.By this way,we improved the infection efficiency greatly and based on this,we characterized the immunophenotype of DCs by FACS to define the influence of TGF-beta1 gene modification on DCs maturation.We found the control DCs exhibited features of immature DCs.The infection of Ad Lac Z did not change the features obviously.The expression of MHC-Ⅱ,CD86,CD40 were all inhibited on TGF-beta1 gene modified DCs and the most obvious inhibition was observed in CD80.
IL-12 is one of primary cytokines secreted from DC,which can drive T cells toward help T cell 1(Th1) and their secretion of IL-2 and IFN-γthat result in strong immunological response.The IL-12 level in culture supernatant of mature DC was significantly higher than that of immature DC.After stimulation by LPS,accelerated the maturation of DC.The concentration of IL-12 from the supernatant of TGF-beta1 gene modified DC was lower than the control group,while LPS stimulation widened the differences.
In vitro each groups DC was inactivated and set up in 120 hours primary MLR cultures with naive BALB/c T cells.DC transduced with TGF-beta1 gene,consistent with their surface phenotype and capability of secreting of IL-12,induced a comparatively low level of allogeneic T cell proliferation.DC transduced with Lac Z gene show slightly stronger ability to stimulate allogeneic T cell proliferation than control DC.
We transfected TGF-beta1 gene into DC with ideal efficiency and then obtained TGF-beta1-DC with steady immature phenotype.Furthermore,we validated their inhibitory function to T cells ensuring their immunological tolerant capability.Based on this,we isolated exosomes from supernatant of TGF-beta1 gene modified DC. Supernatant from 1~*10~6DC can produce 1μg exosomes in first 24 hours.After that, alonging with the apoptosis of DC,the production of exosomes also decreased.In view of the maturation of DC with time,we collected the exosomes from first 48h DC.We visualized the exosomes with high purity under the EM,those particles with the sizes at 50-100nm,with clear bi-lipid membrane.After adsorbed onto latex,phenotype of exosomes was detected by FACS.Results showed that TGF-beta1-exo expressed low MHC-Ⅱmolecules,CD80 and undetectable CD86,CD40,which suggested they might bear the function of immune regulation.CD11c the marker of murine DC also could be detected on exosomes which ensured us their genesis of DC.Tsg101 and Hsp70 the characteristic protein of exosomes also could be detected in exosomes by WB.Tsg101 is a marker of endosome and related to the formation of exosomes.Compared with Lac Z-exo and Control-exo,TGF-beta1-exo beared lower Hsp70 which is know to convert T cell tolerance to autoimmunity in vivo.This further suggests the immune regulatory function of exosomes.Compared with Control-exo and Lac Z-exo,only TGF-beta1-exo expressed TGF-beta1.This result showed that TGF-beta1 could be sorted to exosomes through unknown mechanisms.TGF-beta1 was further quantified by ELISA assay and the amount of TGF-beta1 in four cycles of freeze/thaw exosomes and intact exosomes was 15pg/μg and 8pg/μg.This result suggested that there were both secreting and membrane-associated TGF-beta1 in exosomes.In vitro each group exosomes 120 hours primary MLR,TGF-beta1-exo induced a comparatively low level of allogeneic T cell proliferation.While Contro-exo and Lac Z-exo also show slight inhibitory effect.Except for inhibiting T cells proliferation,we also found TGF-beta1-exo could inhibit the activation of lymphocytes induced by Con A.That result made us much more ensure the inhibitory function of TGF-beta1-exo.
In conclusion,we isolated exosomes from TGF-beta1 gene modified DC successfully and characterized them by EM,FACS and WB.Furthermore,we testified their immune inhibitory function in vitro.All those results set up solid foundation for our next therapeutic experiments in vivo.
PARTⅡ.The protective effect of exosomes from TGF-beta1 gene modified bone marrow-derived immature DC in murine inflammatory bowel disease and the underlying mechanisms.
We then examined the in vivo effects of TGF-beta1-exo on the development of IBD using a C57BL/6 murine IBD model induced by DSS.IBD was induced by giving 1.5%DSS for continuous 9 days.Two days before DSS drinking,C57BL/6 were injected i.p.with Control-exo、Lac Z-ex and TGF-beta1-exo at the dose of 10μg/per mouse and on day 3,day 5 after DSS drinking mice were received another injection of each group exosomes i.p.at the same dose.There also had one group with drinking water as normal control and the other drinking DSS only with no treatment as positive control.Compared to control mice,which maintained their weight,the DSS-induced mice(no treatment) suffered extensive weight loss—statistically significant from day 5 after induction,reaching 16%by day 9.Treatment of Contro-exo or Lac Z-exo did not prevent the DSS-induced weight loss and similarly to the group of untreated mice,they all reached 15%by day 9.In contrast,the weight loss of the DSS-induced mice treated with TGF-beta1-exo was significantly lower decreasing by only 5%from their original weight by day 9.The daily monitored disease activity index(DAI,combined score of body weight,bleeding and stool consistency),as well as the intestinal bleeding were also reduced by treatment of TGF-beta1-exo but not by treatment of Contro-exo or Lac Z-exo.Histological assessment of colonic damage in Control-exo,Lac Z-exo or untreated mice,9 days after DSS induction,revealed extensive severe nearly diffuse inflammation involving the mucosa,submucosa and in some cases extending through all intestinal layers(transmural inflammation).This was associated with a pronounced disruption of the normal architecture and crypt loss.In colons of DSS-induced mice treated with TGF-beta1-exo less damage and more conserved glandular structure were revealed,although widespread leukocyte infiltrations were found.
To look into the mechanisms hidden behind the results we had obtained,we isolated lymphocytes from spleens and mesentery lymph nodes(mLNs) of inflammatory site of each group mice and analyzed for relative Foxp3~+ Treg cell nembers.Compared to other group mice,lymphocytes from spleens of TGF-beta1-exo treated mice contained the similar relative Foxp3~+ Treg cell nembers.While among the lymphocytes from mLNs of inflammatory site,the relative Foxp3~+ Treg cell nembers increased statistically.To further analyze the cause of that difference,we labeled exosomes with CFSE and then coincubate CFSE labeled exosomes with lymphocytes from spleens or mLNs of IBD or control mice.We found that lymphocytes from mLNs of IBD mice could internalized more exosomes than control mice but that difference could not be observed between lymphocytes from spleens of IBD and control mice.
In conclusion,the development of IBD induced by DSS of TGF-beta1-exo treated mice was much slower than that of other groups.Unlike the slightly inhibitory effect observed in MLR in vitro,Control-exo and Lac Z-exo could not show any protective effect in the development of IBD induced by DSS.Compared to other groups,the relative Foxp3~+ Treg cell nembers increased statistically among the lymphocytes from mLNs of inflammatory site of TGF-beta1-exo treated mice,which may be one of the mechanisms of TGF-beta1-exo can show the protective effect in the development of IBD induced by DSS.Lymphocytes from inflammatory site could internalize more TGF-beta1-exo that resulted in the high intensity of TGF-beta1 in inflammatory site probably contribute one of the cause of the results that the relative Foxp3~+ Treg cell nembers increased statistically among the lymphocytes from mLNs of inflammatory site but not spleens.
Transforming Growth Factor-beta1(TGF-beta1)是一种含有112个氨氨基酸,分子量25KD的二聚体蛋白质。在各种属间高度保守,例如人和大鼠、小鼠有完全相同的氨基酸序列。几乎所有细胞都有TGF-beta1的受体,其对细胞的生长、分化的影响是非常广泛的。对T细胞,TGF-beta1可以抑制其增殖,抑制Th1和Th2细胞的分化,抑制CTL的分化,诱导调节性T细胞(Treg)的产生;对B细胞,TGF-beta1可以抑制B细胞的增殖,诱导IgA的类别转换,还可以诱导不成熟和静息B细胞的凋亡。对NK细胞,TGF-beta1可以抑制NK细胞分泌IFN-γ及其溶细胞的功能;对树突状细胞(DC)TGF-beta1抑制DC的成熟及其抗原递呈功能;TGF-beta1还可以抑制活化的巨噬细胞(MΦs),肥大细胞及粒细胞的功能。
目前认为DC是功能最强大的抗原递呈细胞(APC),不同来源及不同成熟状态的DC能够正向或负向调节T细胞的免疫功能。DC有许多亚群,包括髓样DC、浆细胞样DC和朗格罕斯细胞,分别在对免疫反应调节中发挥着不同的作用。除了免疫活化的能力之外,不同亚群的DC在某种生理状态下或某种外来条件的干预下可以发挥免疫抑制功能,诱导中枢及外周免疫耐受。如IL-4或Fas L基因修饰的DC可以抑制小鼠的迟发性超敏反应(delayed-type hypersensitivity,DTH)及胶原诱导的关节炎(collagen-induced arthritis,CIA)。
Exosomes是各种活细胞分泌的,非质膜来源的,直径介于50-100nm的具有脂质双分子层结构的小囊泡。在体内,exosomes对免疫系统发挥着双向调节的作用。肿瘤细胞来源的exosomes携带有肿瘤抗原,能够将肿瘤抗原递呈到DC,活化其抗肿瘤免疫,消除已经建立的小鼠肿瘤。APC来源的exosomes表达MHC-I、MHC-Ⅱ和T细胞共刺激分子,提示其具有免疫调节功能。基因修饰表达vIL-10、Fas L或IL-4的骨髓DC来源的exosomes能够抑制小鼠的迟发型超敏反应及胶原诱导的关节炎,过继实验表明基因修饰的exosmes可以在体内调节APC的功能并诱导Treg的产生,从而发挥系统性的免疫抑制功能。
骨髓来源未成熟DC(imDC)由于其分化不成熟,MHC及各种辅助分子表达较低,因此imDC来源的exosomes可以抑制免疫细胞的活化,诱导机体免疫耐受的产生。基因修饰表达TGF-beta1的imDC能够发挥系统性的免疫抑制功能,促进同种异体小鼠移植心脏的长期存活。Treg在肠道的免疫平衡中发挥着重要的作用,而TGF-beta1可以诱导Treg的产生。当在肠道TGF-beta1信号的缺失的情况下,小鼠表现出更为严重的IBD进展,因此TGF-beta1的信号对肠道的免疫自稳状态的维持将发挥着至关重要的作用。本课题将利用葡聚糖硫酸钠盐(DSS)建立IBD小鼠自身免疫病动物模型,利用TGF-beta1基因修饰的骨髓imDC来源的exosomes(TGF-beta1-exo)腹腔注射模型小鼠,评价TGF-beta1-exo在IBD进展过程中的保护性效果,从而探讨TGF-beta1-exo是否能发挥系统性的免疫抑制功能,恢复肠道的免疫平衡。
第一部分
TGF-beta1基因修饰的骨髓未成熟树突状细胞来源的exosomes的制备鉴定及其体外功能
采用单独的rmGM-CSF骨髓细胞体外培养方法,可以获得具有稳定的不成熟表型的DC,而且纯度也较为理想,平均在60-70%左右。流式细胞术(FACS)鉴定培养7天的细胞,其表面表达小鼠DC的标志物CD11c,低表达MHC-Ⅱ类分子和共刺激分子CD40、CD86。
为了能最高限度的提高AdTGF-beta1对大量DCs的转染效率,我们先用CsCl密度梯度离心将腺病毒进行纯化,然后依据相关文献以重复转染率(MOI)等于50进行病毒转染,在2h的无血清转染过程中,我们将DCs与病毒混合液置于37℃摇床上保持不停震摇,使腺病毒与DCs充分接触,大大提高了转染效率。在高效转染的基础上,我们对经过基因修饰的DCs进行了FACS分析,以明确AdTGF-beta1转基因对DCs成熟过程的影响。结果发现未处理组的细胞表现出未成熟DCs的表型,Ad Lac Z转染并未使这种细胞特征发生很大的变化。Ad TGF-beta1转染后DCs表面的MHC-Ⅱ,CD80,CD86和CD40表达均受到抑制,而对CD80的抑制作用最为明显。
研究证明IL-12是DC分泌的主要细胞因子之一,其主要作用是促使T细胞向T辅助细胞1(Th1)转化并大量分泌IL-2和IFN-γ,从而激发强有力的免疫应答。IL-12的合成分泌能力是DC主要功能指标之一,不仅能够提示DC的成熟状态,而且是DC拥有免疫原性还是耐受原性的指针之一。我们在实验中发现无处理对照组和Ad Lac Z转染组DC在LPS刺激下能够迅速成熟并分泌大量IL-12,而Ad TGF-beta1转染后DC分泌的IL-12较对照为低,在LPS刺激后IL-12浓度的提升幅度较小,反映AdTGF-beta1转染后的DC拥有了对LPS等致成熟因子的抗性。
不同处理组DC与同种小鼠脾T细胞做120h混合淋巴细胞反应,结果显示转对照组DC,Ad TGF-beta1转染的DC刺激同种T细胞的能力明显受到抑制,而Ad LacZ转染的DC刺激同种T细胞的能力较对照组DC有轻微的提升。这与其细胞表型特征及分泌IL-12的能力是一致的。
我们实现了AdTGF-beta1对DC的高效感染,获得了稳定表达未成熟DC表型的Ad TGF-beta1基因修饰DC,并在体外证实了其对T细胞的抑制能力,显示其具有免疫耐受性DC的特征。在此基础上,我们分离纯化了Ad TGF-beta1基因修饰DC上清来源的exosomes,在1*10~6的DC的上清中,前24h可以稳定分离得到1μg的exosomes,此后随着DC的大量凋亡,exosomes的得率相应降低,考虑到随时间的推移,DC会逐渐趋于成熟,我们只收集了前48h的exosomes。对exosomes的电镜分析结果显示,我们分离得到的exosomes主要是直径集中在50-100nm左右的小囊泡,具有典型的脂质双分子层结构,这与文献报道相一致。将exosomes吸附到乳胶颗粒之后,通过FACS对其表型进行了分析,显示AdTGF-beta1-exo低表达MHC-Ⅱ类分子、CD80,几乎不表达CD86与CD40,提示其可能具有免疫调节功能。同时exosomes表面还表达小鼠DC特征性的标志CD11c,证实了其属于DC来源。对exosomes的Westernblot(WB)结果表明,我们分离得到的exosomes表达exosomes特征性的蛋白Tsg101和Hsp70。Tsg101是内体相关的蛋白,参与exosomes的形成,也证实了其内体的来源,而较Ad Lac Z基因修饰DC来源的exosomes(Lac Z-exo)及未处理DC来源的exosomes(Control-exo),TGF-beta1-exo表达较低的Hsp70,而Hsp70在体内可以将免疫耐受状态的T细胞转化为自体反应性T细胞,进一步提示了TGF-beta1-exo具有免疫调节功能。与Control-exo和Lac Z-exo相比,只有TGF-beta1-eXO表达TGF-beta1,说明TGF-beta1基因修饰的DC能够以一种未知的机制将TGF-beta1携带到exosomes上。通过ELISA我们对TGF-beta1-exo上的TGF-beta1进行了定量,经反复冻融与未冻融TGF-beta1-exo上含有的TGF-beta1水平有所差别,分别是15pg和8pg/μg exosomes,说明在exosomes上同时存在着分泌型与膜型的TGF-beta1。在不同处理组exosomes对同种小鼠120h混合淋巴细胞反应影响效应的实验中显示,TGF-betal-exo可以明显抑制小鼠T细胞的增殖,而Control-exo和Lac Z-exo对T细胞亦有微弱的抑制作用TGF-beta1-exo除了对T细胞的抑制作用,我们还发现TGF-beta1-exo还可以抑制Con A引起的淋巴细胞的活化,进一步证实了TGF-beta1-exo的免疫抑制功能。
综上所述,我们成功的分离纯化了Ad TGF-beta1基因修饰DC来源的exosomes,并通过电镜,FACS及WB对其形态,蛋白表达进行了鉴定,并在体外证实了其对T细胞的抑制效果,这为下一步的体内实验奠定了基础。
第二部分
TGF-beta1基因修饰的骨髓未成熟树突状细胞来源的exosomos对DSS诱导的小鼠炎症性肠病的保护作用及其相关机制
为了进一步检验TGF-beta1-exo的免疫抑制功能,明确在体内环境下TGF-beta1-exo是否能同样的发挥免疫抑制的功能,我们将TGF-beta1-exo经腹腔注射小鼠,通过观察小鼠体重的改变情况,大便性状,便血情况,以及肠道组织的病理切片分析,判断TGF-beta1-exo是否能够延缓DSS诱导的小鼠IBD的进展过程,并对其机制进行了探讨。
我们通过让C57BL/6小鼠连续自由饮用浓度为1.5%的DSS 9天,来诱导小鼠IBD的发病。并在小鼠饮用DSS前两天,经腹腔分别注射10μg/只的Control-exo、Lac Z-exo和TGF-beta1-exo,并于饮用DSS后第3天,第5天经腹腔再追加注射10μg/只的各组exosomes。同时设正常饮水的对照组及未经任何处理单纯饮用DSS组。除经TGF-beta1-CXO处理的小鼠,其它各组小鼠在饮用DSS 5天后体重呈进行性下降,至第9天,DSS组、Control-exo组及Lac Z-exo组小鼠的体重分别下降了16%、15%、15%。而TGF-beta1-exo组小鼠的体重在第6天才开始下降,并且下降的程度相对缓慢,至第9天,体重仅下降了5%。除此之外,联合了体重改变,大便性状,便血情况,对小鼠IBD进展情况进行综合评价的每天疾病活跃指数(daily diseaseactivity index,DAI)的评分结果亦显示了TGF-beta1-exo能较大程度的延缓DSS诱导的小鼠的IBD的进展速度。而第7天的各组小鼠便隐血测试结果显示,TGF-beta1-exo组小鼠虽然也出现了较为明显的大便隐血,但与其它各组相比,在便血程度上明显要轻一些。最后,各组小鼠结肠病理切片结果进一步提示了TGF-beta1-exo在小鼠IBD进展过程中的保护作用。在TGF-beta1-exo组小鼠结肠组织的切片中除了可见到粘膜和粘膜下层轻到中等程度的炎症细胞浸润,肠粘膜腺体结构还比较完整。而其它各组小鼠在粘膜层,粘膜下层均可见到重度的炎症细胞浸润,并可见到弥散至肌层的炎症细胞浸润,局部还可见到粘膜结构的破坏,正常腺体结构的消失。
为了探讨TGF-beta1-exo延缓DSS诱导的小鼠IBD进展过程的相关机制,分析TGF-beta1-exo是否能够诱导Treg的产生,从而在一定程度上恢复肠道的免疫平衡,我们分离了各组小鼠脾脏及结肠系膜淋巴结来源的淋巴细胞,分析Foxp3~+ Treg的改变情况。结果我们发现,与其它组小鼠相比,TGF-beta1-exo组小鼠脾脏Foxp3~+Treg并未发生明显的改变,而在炎症局部结肠系膜来源的淋巴细胞中,可以观察到Foxp3~+ Treg的明显上调。为进一步分析这种差别产生的原因,我们用羧基荧光素二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein diacetate succimidyl ester,CFSE)对exosomes进行标记,然后将标记的exosomes与IBD或正常小鼠小鼠脾脏及结肠系膜淋巴结来源的淋巴细胞共培养后,观察不同来源淋巴细胞对exosomes的内化情况。我们发现,与正常小鼠结肠系膜来源的淋巴细胞相比,IBD小鼠结肠系膜来源的淋巴细胞能够内化更多的exosomes,而这种差异在IBD小鼠与正常小鼠的脾脏淋巴细胞中并不能观察到。
综合以上结果可以看出,TGF-beta1-exo能够明显延缓DSS诱导的小鼠IBD的进展速度,而不像它们在体外MLR中观察到的轻微的抑制作用,Contro-exo和LacZ-exo对DSS诱导的小鼠IBD的进展并未显示出任何保护性作用。与其它组exosomes相比,经TGF-beta1-exo处理之后的小鼠,在炎症局部来源的淋巴细胞中,可以观察到Foxp3~+Treg的明显上调,这可能是TGF-beta1-exo能够明显延缓DSS诱导的小鼠IBD的进展速度的机制之一。而能在炎症局部淋巴细胞观察到Foxp3~+Treg的上调而不是脾脏淋巴细胞,原因之一,很有可能是因为炎症局部的淋巴细胞能够富集更多的TGF-beta1-exo而造成局部的高浓度TGF-betal。
Autoimmune disease is a kind of disease that is caused by body immune system responsing to autoantigen.Inflammatory bowel disease(IBD) including ulcerative colitis(UC) and Crohn' s disease(CD) is a kind of non-specific inflammatory bowel disease that is mainly caused by abnormal immune response of immune system to normal intestinal flora.In western countries,incidence of IBD is 4-6 out of one hundred thousand.In our country,because of the change of dietetic structure,incidence of IBD increased every year.Reported by domestic literature,cases in this five years is eight times more than the corresponding period in 90' s of last century.The main symptom of IBD is diarrhea,grume sanguinopurulent stool and bellyache. Furthermore,lasting for a long time,the risk of cancer mutation increases,so IBD threatens the people' s health of our country badly.In clinal,allopathy is the main therapeutic means of IBD and there still has not an effective procedure of treatment,so the demand of exploring a new way to cure IBD thoroughly is urgent.
Transforming Growth Factor-beta 1(TGF-beta1) including 112 amino acid is a dimer protein with molecular weight of 25KD.It is strictly conservative in each species.For example,human,rat and mouse have totally identical sequence.Almost all cells have receptors of TGF-beta1 and its influence of growth,differentiation in cells is very extensive.In T cells,TGF-beta1 can inhibit their proliferation,inhibit the differentiation of Th1 and Th2,inhibit the differentiation of CTL,induce the generation of regulatory T cells(Treg);in B cells,TGF-beta1 can inhibit their proliferation,induce IgA class switch,it also can induce the apoptosis of immature and resting B cells;in NK cells,TGF-beta1 can inhibit the secretion of IFN-γand the cytolytic function of NK cells.In dendritic cells(DC),TGF-beta1 can inhibit their maturation and antigen presenting;furthermore,TGF-beta1 can inhibit the function of active macrophages(MOs) and granulocytes.
DC are the most potent professional antigen presenting cells(APC) that are able to modulate T cell immunity in either a positive or negative manner,depending upon their lineage and state of maturation.There are several subpopulations of DC including myeloid DC,plasmacytoid DC,and Langerhans cells that play different roles in the regulation of the immune responses.In addition to their ability to stimulate immunity, these different DC populations,under certain conditions,are involved in T cell immunosuppression and/or induction of central and peripheral tolerance.For example, IL-4 or Fas L gene modified DC can inhibit delayed-type hypersensitivity(DTH) and collagen-induced arthritis(CIA).
Exosomes are small vesicles released by various live cells,50-100nm,that have structure of lipid bi-layer not derived from cells membrane.In vivo,exosomes play bi-directional regulatory role in immune system.In murine models,exosomes derived from tumor share the tumor antigen can present the antigen to DC,which activate the immunity of anti-tumor of DC and then eradicate established tumor.Exosomes from APC carry MHC-Ⅰ,MHC-Ⅱand T cell costimulatory molecules on their surface, suggesting that they could play important roles in immune regulation.In murine models,exosomes from vIL-10,Fas L or IL-4 gene modified DC can inhibit DTH and CIA.Furthermore,adoptive transfer experiment show that gene modified exosomes can induce the generation of regulatory APC and T cells and then exert the function of systemic immune inhibition.
Bone marrow derived immature DC(MD-imDC) because of their immature stage in differentiation,expressing low level MCHC and costimulatory molecules,so MD-imDC derived exosomes can inhibit the activation of immune cells and induce immunological tolerance.In murine cervical heterotopic heart transplantation model, TGF-beta1 gene modified MD-imDC can exert function of systemic immune inhibition, prolonging the survival of allograft.Treg play a very important role in immune balance in intestine.TGF-beta1 can induce the generation of Treg.Under the condition of losing TGF-beta signal,mice show more severe IBD development,so maintenance of TGF-beta signal is very important in regulating immune homeostasis in the intestine. The aim of this project is to explore if TGF-beta1-exo can play the function of systemic immune inhibition and rebuild immune balance in intestine.In this project dextran sodium sulphate(DSS) was used to induce murine IBD model.Based on this model,TGF-beta1 gene modified DC derived exosomes(TGF-beta1-exo) were pre-injected i.p.and the protective effect of TGF-beta1-exo during IBD development was evaluated.
PARTⅠ.The isolation and identification of exosomes from TGF-beta1 gene modified bone marrow-derived immature DC and their function in vitro.
DCs,the highly effective specialized antigen-presenting cells,play a pivotal role in antigen presentation in the proper context of the required antigenic and costimulatory molecules to induce T cell-mediated immune responses.The functions are highly correlated with their differentiation status.Mature DCs highly express major histocompatibility complex class-Ⅱ(MHC-Ⅱ) molecules and co-stimulatory molecules, such as CD80/86,CD40,providing two signals to activate T lymphocytes to initiate immune responses,while immature DCs which are deficient in these molecules show tolerogenic.So,heterogeneous properties of DCs either tolerogenic or immunogenic determine the outcome of immune responses.The tolerogenic properties of DCs can be amplified by delivery of genes encoding immunosuppressive molecules that are not or seldom produced by DCs.The genes which are the most feasible to induce peripheral tolerance include vIL-10,TGF-beta,CTLA4-Ig,inducible NOS,FasL,and so on. Manipulation of these agents has been shown to render DC tolerogenic characteristics.
Cultured in rmGM-CSF only,we obtained DCs with steady immature phenotype and the purity is ideal averaging between 60-70%.Identified day 7 DCs by FACS,we found DCs expressed CDllc the marker molecule of murine DCs and low level of MHC-Ⅱ,CD40,CD86.
To best improve the infection efficiency of Ad TGF-beta1 to large amount DCs, we purified adenoviruses by CsCl density gradient centrifugation and then infected DCs by MOI50.During the 2h' serum-free infection,we put the mixture of DCs and adenoviruses onto rotatable bed with 37℃and keep the bed rotating which can make the cell and viruses contact completely.By this way,we improved the infection efficiency greatly and based on this,we characterized the immunophenotype of DCs by FACS to define the influence of TGF-beta1 gene modification on DCs maturation.We found the control DCs exhibited features of immature DCs.The infection of Ad Lac Z did not change the features obviously.The expression of MHC-Ⅱ,CD86,CD40 were all inhibited on TGF-beta1 gene modified DCs and the most obvious inhibition was observed in CD80.
IL-12 is one of primary cytokines secreted from DC,which can drive T cells toward help T cell 1(Th1) and their secretion of IL-2 and IFN-γthat result in strong immunological response.The IL-12 level in culture supernatant of mature DC was significantly higher than that of immature DC.After stimulation by LPS,accelerated the maturation of DC.The concentration of IL-12 from the supernatant of TGF-beta1 gene modified DC was lower than the control group,while LPS stimulation widened the differences.
In vitro each groups DC was inactivated and set up in 120 hours primary MLR cultures with naive BALB/c T cells.DC transduced with TGF-beta1 gene,consistent with their surface phenotype and capability of secreting of IL-12,induced a comparatively low level of allogeneic T cell proliferation.DC transduced with Lac Z gene show slightly stronger ability to stimulate allogeneic T cell proliferation than control DC.
We transfected TGF-beta1 gene into DC with ideal efficiency and then obtained TGF-beta1-DC with steady immature phenotype.Furthermore,we validated their inhibitory function to T cells ensuring their immunological tolerant capability.Based on this,we isolated exosomes from supernatant of TGF-beta1 gene modified DC. Supernatant from 1~*10~6DC can produce 1μg exosomes in first 24 hours.After that, alonging with the apoptosis of DC,the production of exosomes also decreased.In view of the maturation of DC with time,we collected the exosomes from first 48h DC.We visualized the exosomes with high purity under the EM,those particles with the sizes at 50-100nm,with clear bi-lipid membrane.After adsorbed onto latex,phenotype of exosomes was detected by FACS.Results showed that TGF-beta1-exo expressed low MHC-Ⅱmolecules,CD80 and undetectable CD86,CD40,which suggested they might bear the function of immune regulation.CD11c the marker of murine DC also could be detected on exosomes which ensured us their genesis of DC.Tsg101 and Hsp70 the characteristic protein of exosomes also could be detected in exosomes by WB.Tsg101 is a marker of endosome and related to the formation of exosomes.Compared with Lac Z-exo and Control-exo,TGF-beta1-exo beared lower Hsp70 which is know to convert T cell tolerance to autoimmunity in vivo.This further suggests the immune regulatory function of exosomes.Compared with Control-exo and Lac Z-exo,only TGF-beta1-exo expressed TGF-beta1.This result showed that TGF-beta1 could be sorted to exosomes through unknown mechanisms.TGF-beta1 was further quantified by ELISA assay and the amount of TGF-beta1 in four cycles of freeze/thaw exosomes and intact exosomes was 15pg/μg and 8pg/μg.This result suggested that there were both secreting and membrane-associated TGF-beta1 in exosomes.In vitro each group exosomes 120 hours primary MLR,TGF-beta1-exo induced a comparatively low level of allogeneic T cell proliferation.While Contro-exo and Lac Z-exo also show slight inhibitory effect.Except for inhibiting T cells proliferation,we also found TGF-beta1-exo could inhibit the activation of lymphocytes induced by Con A.That result made us much more ensure the inhibitory function of TGF-beta1-exo.
In conclusion,we isolated exosomes from TGF-beta1 gene modified DC successfully and characterized them by EM,FACS and WB.Furthermore,we testified their immune inhibitory function in vitro.All those results set up solid foundation for our next therapeutic experiments in vivo.
PARTⅡ.The protective effect of exosomes from TGF-beta1 gene modified bone marrow-derived immature DC in murine inflammatory bowel disease and the underlying mechanisms.
We then examined the in vivo effects of TGF-beta1-exo on the development of IBD using a C57BL/6 murine IBD model induced by DSS.IBD was induced by giving 1.5%DSS for continuous 9 days.Two days before DSS drinking,C57BL/6 were injected i.p.with Control-exo、Lac Z-ex and TGF-beta1-exo at the dose of 10μg/per mouse and on day 3,day 5 after DSS drinking mice were received another injection of each group exosomes i.p.at the same dose.There also had one group with drinking water as normal control and the other drinking DSS only with no treatment as positive control.Compared to control mice,which maintained their weight,the DSS-induced mice(no treatment) suffered extensive weight loss—statistically significant from day 5 after induction,reaching 16%by day 9.Treatment of Contro-exo or Lac Z-exo did not prevent the DSS-induced weight loss and similarly to the group of untreated mice,they all reached 15%by day 9.In contrast,the weight loss of the DSS-induced mice treated with TGF-beta1-exo was significantly lower decreasing by only 5%from their original weight by day 9.The daily monitored disease activity index(DAI,combined score of body weight,bleeding and stool consistency),as well as the intestinal bleeding were also reduced by treatment of TGF-beta1-exo but not by treatment of Contro-exo or Lac Z-exo.Histological assessment of colonic damage in Control-exo,Lac Z-exo or untreated mice,9 days after DSS induction,revealed extensive severe nearly diffuse inflammation involving the mucosa,submucosa and in some cases extending through all intestinal layers(transmural inflammation).This was associated with a pronounced disruption of the normal architecture and crypt loss.In colons of DSS-induced mice treated with TGF-beta1-exo less damage and more conserved glandular structure were revealed,although widespread leukocyte infiltrations were found.
To look into the mechanisms hidden behind the results we had obtained,we isolated lymphocytes from spleens and mesentery lymph nodes(mLNs) of inflammatory site of each group mice and analyzed for relative Foxp3~+ Treg cell nembers.Compared to other group mice,lymphocytes from spleens of TGF-beta1-exo treated mice contained the similar relative Foxp3~+ Treg cell nembers.While among the lymphocytes from mLNs of inflammatory site,the relative Foxp3~+ Treg cell nembers increased statistically.To further analyze the cause of that difference,we labeled exosomes with CFSE and then coincubate CFSE labeled exosomes with lymphocytes from spleens or mLNs of IBD or control mice.We found that lymphocytes from mLNs of IBD mice could internalized more exosomes than control mice but that difference could not be observed between lymphocytes from spleens of IBD and control mice.
In conclusion,the development of IBD induced by DSS of TGF-beta1-exo treated mice was much slower than that of other groups.Unlike the slightly inhibitory effect observed in MLR in vitro,Control-exo and Lac Z-exo could not show any protective effect in the development of IBD induced by DSS.Compared to other groups,the relative Foxp3~+ Treg cell nembers increased statistically among the lymphocytes from mLNs of inflammatory site of TGF-beta1-exo treated mice,which may be one of the mechanisms of TGF-beta1-exo can show the protective effect in the development of IBD induced by DSS.Lymphocytes from inflammatory site could internalize more TGF-beta1-exo that resulted in the high intensity of TGF-beta1 in inflammatory site probably contribute one of the cause of the results that the relative Foxp3~+ Treg cell nembers increased statistically among the lymphocytes from mLNs of inflammatory site but not spleens.
引文
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