沙利度胺对胶原诱导性关节炎大鼠VEGF、TNF-α表达及红细胞生成的影响
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摘要
类风湿关节炎( rheumatoid arthritis, RA)是一类以慢性、对称性、多关节炎症为主要表现的自身免疫性疾病,其侵犯的靶器官主要是关节滑膜,最终可影响关节功能甚至致残。RA滑膜新生血管的生成是产生和维持血管翳的重要标志,也是造成滑膜炎、血管翳生长、骨破坏的主要原因。因此抑制滑膜组织血管生成、减少其引起的骨破坏将成为治疗RA的重要方法。RA滑膜组织中有多种促血管生成因子高表达,其中血管内皮生长因子(VEGF)在滑膜炎进展及新生血管形成过程中的作用至关重要。VEGF的表达受多种因素影响,肿瘤坏死因子-α(TNF-α)、缺氧、白细胞介素-1(IL-1)、转化生长因子β(TGF-β)及CD40-CD40L等均可以上调其表达。做为RA细胞因子网络瀑布启动的起点,TNF-α在RA病程中发挥着枢纽作用,它与VEGF相互促进,相互影响,加速病变的进程。
贫血是RA最常见的关节外表现,常与RA的活动相一致。目前认为RA患者的贫血多属慢性病贫血(ACD),是活动性RA的一种常见特征,可能与铁吸收异常、巨噬细胞释放铁障碍、促红细胞生成素(EPO)表达缺陷等有关。另外细胞因子如TNF-α、IL-6等已被证实与ACD发病明显相关。它们可以直接抑制红系祖细胞增殖、抑制EPO的产生、迟化骨髓对EPO的反应及干扰铁代谢等多个环节。
沙利度胺因免疫调节、抗炎及抗血管生成作用又重新成为临床研究的热点,证实它对某些肿瘤、风湿性疾病、难治性血液病以及多种原因不明的疾病疗效显著。迄今为止仅有少数关于沙利度胺治疗RA的临床报导,对其疗效褒贬不一,作用机制亦不明确,无远期疗效的临床及相关动物实验资料,也未见其对RA伴贫血患者红系生成方面的临床及动物实验的报道。
为此,本实验在传统的胶原诱导型关节炎基础上加以改进,通过多次皮内注射Ⅱ型胶原与弗氏完全佐剂,成功地建立了关节炎伴贫血的动物模型。从细胞水平、形态学、流式检测、分子生物学等多种角度,有机结合体内和离体试验,以VEGF与TNF-α为切点,以动态观察的方式研究并探讨沙利度胺对关节炎大鼠的炎性活动、血管生成、滑膜细胞表达及凋亡的影响以及对红细胞生成作用及可能机制,进一步评价不同剂量的沙利度胺在实验性关节炎伴贫血大鼠的疗效及临床应用价值,试图为该药在RA新靶点治疗——阻断血管生成方面及选择无明显骨髓抑制的抗风湿药物方面提供一定的实验依据。实验内容主要包括以下四个部分:
第一部分:VEGF、TNF-α在胶原诱导性关节炎大鼠病程中的动态表达及沙利度胺的影响
目的:探讨VEGF、TNF-αmRNA及其蛋白在胶原诱导性关节炎大鼠血清及滑膜中的动态表达及其与关节炎活动程度的关系、滑膜微血管密度的变化及沙利度胺对上述指标的影响。
方法:将128只雄性Wistar大鼠随机分5组,Ⅰ组:正常对照组(n=24),Ⅱ组:造模组(n=26),Ⅲ组:造模组+高剂量沙利度胺治疗组(200mg/kg/d, n=26),Ⅳ组:造模组+沙利度胺低剂量治疗组(100mg/kg/d, n=26),Ⅴ组:造模组+甲氨喋呤(MTX)治疗组(2.7mg/kg/w, n=26)。通过注射Ⅱ型胶原与完全弗氏佐剂(complete freud’s adjuvant, CFA)制作胶原诱导型关节炎(collagen-induced arthritis, CIA)动物模型,各CIA模型组又根据初次免疫后不同时间点(7、14、21、28、35、42及60天)各分为7个亚组。造模后12天开始给药。应用关节炎指数(arthritis index, AI)、足爪厚度、99mTc-MDP核素显像、x线、电镜及病理改变评价模型建立情况;RT-PCR、ELISA及免疫组织化学方法检测VEGF及TNF-αmRNA和其蛋白在不同时期关节炎大鼠血清及滑膜组织中含量的动态变化;采用CD31免疫组织化学方法及微血管计数(microvessel density, MVD)法了解滑膜组织新生血管情况;研究沙利度胺对上述指标的影响并与MTX比较,同时比较不同剂量沙利度胺的作用有无差别。
结果:①CIA大鼠的AI指数、足爪厚度及核素浓聚程度在21天达到高峰,其中关节部位核素的变化敏感性及客观性均优于其它指标,更能够反应关节的早期病变。②CIA大鼠滑膜组织中的VEGF和TNF-αmRNA在造模后21天表达水平分别为0.75±0.06和2.01±0.13,明显高于同期对照组,P<0.01。仅高剂量沙利度胺组可以下调VEGF mRNA的表达,高、低剂量沙利度胺和MTX组都能够下调TNF-αmRNA的表达。③免疫组织化学染色结果显示CIA大鼠滑膜层中VEGF和TNF-α蛋白的表达以造模后21天最强,分别为15683.15±319.23和9037.71±403.03,显著高于同期对照组值,P<0.01。且都与大鼠足爪厚度改变呈正相关,r值分别为0.915和0.664,P<0.01。高、低剂量沙利度胺和MTX都能降低滑膜层中VEGF和TNF-α蛋白的含量,高剂量沙利度胺起效最快。④CIA大鼠血清中VEGF和TNF-α的浓度也在造模后21天达峰值,分别为2147.3±37.56 pg/mL和2685.0±112.09 pg/mL,与同期对照组值比较显著升高,P<0.01。3个治疗组都能降低血清VEGF和TNF-α的浓度,高剂量沙利度胺与MTX作用接近。⑤CIA大鼠滑膜的MVD在造模后28天达峰值为22.45±2.43,与同期对照组比较差异显著,P<0.01。3个治疗组中大鼠滑膜的MVD均明显降低,沙利度胺起效时间早于MTX。⑥CIA大鼠血清与滑膜中VEGF和TNF-α都与大鼠足爪厚度呈正相关,P<0.05;其中VEGF与滑膜MVD的变化也呈显著正相关,P<0.01。
结论:CIA大鼠病程中血清和滑膜组织中VEGF和TNF-α存在高表达,并与关节肿胀程度相一致,关节滑膜组织内的新生血管明显增加,沙利度胺均可有效下调VEGF和TNF-αmRNA及其蛋白的表达,减少滑膜组织的MVD,改善CIA大鼠的关节症状,效果优于或等同于MTX。高剂量沙利度胺对上述指标的影响更显著。
第二部分:沙利度胺对关节炎大鼠滑膜细胞VEGF、TNF-α表达水平和对细胞凋亡的影响
目的:探讨沙利度胺对脂多糖(LPS)刺激的成纤维细胞样的滑膜细胞(FLS)VEGF和TNF-αmRNA及其蛋白表达的作用及对FLS凋亡的影响。
方法:无菌获取关节炎大鼠膝关节的滑膜组织,应用体外FLS的培养技术进行滑膜细胞的分离与培养。实验共分6组:对照组,LPS组,LPS+高剂量沙利度胺组(50 mg/L), LPS+中剂量沙利度胺组(5 mg/L),LPS+低剂量沙利度胺组(0.5 mg/L)组及LPS+MTX组。倒置显微镜及透射电镜观察体外培养的FLS的形态;ELISA方法检测FLS培养上清中VEGF和TNF-α的含量;RT-PCR、Western Blot方法检测FLS中VEGF和TNF-αmRNA及蛋白表达的变化;膜联蛋白(Annexin-V)/碘化丙啶(PI)双标记流式细胞术(flow cytometry, FCM)测定药物对FLS凋亡的影响。
结果:①CIA大鼠的原代滑膜组织分离培养24小时后开始贴壁,10～14天长满培养瓶底并可传代,3代以后主要以梭性或菱形的FLS为主。②ELISA测定LPS刺激后FLS培养上清中的VEGF、TNF-α含量分别为859.00±22.74pg/mL和1315.20±19.38pg/mL,明显高于对照组,P<0.01。沙利度胺能够降低FLS培养上清中的VEGF、TNF-α含量,浓度越高作用越强,其作用强度低于MTX。③RT-PCR、Western Blot结果示沙利度胺可以下调LPS刺激后FLS中VEGF和TNF-αmRNA及蛋白表达水平,抑制程度呈部分剂量依赖性。④沙利度胺有诱导FLS凋亡的作用,在一定浓度范围内呈剂量依赖性。处理48小时后各组凋亡率分别为:8.07%±1.45%、5.72%±1.42%和0.40%±0.04%,高中剂量沙利度胺组的凋亡率与LPS单独处理组的凋亡率(0.24%±0.05%)比较统计学差异显著,P<0.01;高中低剂量沙利度胺组的坏死率分别为:20.34%±4.43%、12.96%±4.32%和1.94%±1.16%,与LPS单独处理组的坏死率(0.13%±0.09%)比较统计学差异显著,P<0.01。高剂量沙利度胺与MTX组之间无显著性差异,P>0.05。⑤透射电镜下可观察到典型的细胞凋亡征象,细胞浆致密,染色质浓缩,沿着核膜排列,形成不同形状和大小的块状,可见凋亡小体。结论:FLS可以通过体外细胞培养及传代法获得并逐渐纯化,沙利度胺可以诱导FLS凋亡,并能够抑制其分泌和表达VEGF、TNF-α,且呈部分剂量依赖关系。
第三部分:沙利度胺对胶原诱导性关节炎伴贫血大鼠血红蛋白的影响
目的:研究并探讨沙利度胺对胶原诱导性关节炎伴贫血大鼠的治疗作用及临床应用价值。
方法:在传统的Ⅱ型胶原诱导性关节炎大鼠模型基础上加以改进,制作关节炎伴贫血的动物模型,具体方法及分组同第一部分,并根据初次免疫后不同时间点(14、21、28、35、42及60天)各分为6组。测定不同时间点血红蛋白浓度,了解贫血出现时间及规律;测定模型组血清铁、血清总铁结合力、血清铁蛋白、促红细胞生成素(EPO),获取骨髓液观察骨髓细胞形态、测定骨髓内外铁变化了解贫血类型;测定并分析不同时间点血清TNF-α浓度、大鼠足爪厚度及与血红蛋白三者之间的相关性;分析EPO与血红蛋白之间的相关性。
结果:①关节炎大鼠在造模后28天开始出现贫血,血红蛋白(121.75g/L±9.18g/L)较正常对照组(147.25 g/L±5.74 g/L)明显下降,P<0.01。②CIA模型鼠的贫血特点:血清铁、总铁结合力明显降低,血清铁蛋白及EPO升高明显,骨髓红系增生活跃,内铁减低,外铁无明显变化,红细胞形态基本正常。③高剂量沙利度胺组在造膜60天与同期模型组比较血红蛋白显著上升,P<0.05。④模型组大鼠血清TNF-α浓度与足爪厚度呈正相关,P<0.01;血红蛋白与TNF-α浓度呈负相关,P<0.05;血红蛋白与足爪厚度无明显相关,P>0.05;⑤模型组大鼠血红蛋白含量与EPO浓度之间无显著相关,P>0.05。
结论:通过改良CIA模型的方法,可成功制作出关节炎伴贫血的大鼠模型。关节炎肿胀程度越大,TNF-α浓度越高;TNF-α浓度越高,贫血也越严重。沙利度胺能够改善贫血及大鼠关节肿胀,降低血清TNF-α浓度,有望为临床RA伴ACD患者的治疗开辟一条新途径。
第四部分:沙利度胺对关节炎伴贫血大鼠红系祖细胞的影响及可能机制
目的:研究并探讨沙利度胺对关节炎伴贫血大鼠红系祖细胞的影响。
方法:雄性Wistar大鼠40只随机分5组,造模方法及处理因素同第三部分。于造模后第3及第8周处死大鼠,剪断股骨两端,获取骨髓液,大鼠淋巴细胞分离液分离获得骨髓单个核细胞(BMMCs),台盼兰染色记数活细胞,在甲基纤维素半固体培养基中进行骨髓红系祖细胞的培养及集落数目的测定;流式细胞学测定骨髓红系祖细胞CD34+及CD71+的表达情况,应用7-氨基放线菌D染色(7AAD)了解CD34+/CD71+细胞凋亡情况。
结果:①关节炎伴贫血大鼠在第3周与第8周BFU-E、CFU-E的数目分别为13.33±4.32BFU-E/2.5×105 BMMCs、16.33±5.44 BFU-E/2.5×10~5 BMMCs和62.27±4.98 CFU-E/2.5×10~5 BMMCs、73.67±7.20 CFU-E/2.5×10~5 BMMCs,明显低于正常组大鼠的值,P<0.05。沙利度胺可以促进BFU-E和CFU-E集落的形成,作用优于MTX。②与正常对照组比较,模型组大鼠CD34+/CD71+的细胞数目明显减少,第3周及第8周分别为0.30%±0.10%和0.47%±0.06%,P<0.05。高剂量沙利度胺可以提高大鼠CD34+/CD71+的细胞数目,第3周及第8周分别为0.52%±0.02%和0.57%±0.11%,P<0.05;低剂量沙利度胺仅在第3周作用明显,为0.44%±0.03%。③模型组大鼠CD34~+/CD71~+细胞的凋亡率与正常对照组比较明显增加,第3周及第8周分别为31.30%±2.64%和21.96%±2.51%,P<0.05。沙利度胺可以降低大鼠CD34~+/CD71~+细胞的凋亡率,高低沙利度胺组凋亡率的值分别为20.57%±2.43%、14.77%±4.52%和23.14%±3.78%、16.04%±2.28%。④模型组大鼠血清中TNF-α浓度与BFU-E数目呈明显负相关,P<0.05,与CD34+/CD71+凋亡细胞的百分率呈明显正相关,P<0.05,而与CD34~+/CD71~+细胞占骨髓有核细胞的比例及CFU-E数目无明显相关,P>0.05。
结论:关节炎伴贫血大鼠骨髓红系形成集落的能力明显下降,CD34~+/CD71~+的细胞数目减少,CD34~+/CD71~+凋亡细胞显著增加。沙利度胺对以上指标有改善作用。TNF-α可能参与关节炎伴贫血的发病机制。
Rheumatoid arthritis (RA) is a kind of autoimmune disease characterized by chronic and symmetrical multijoint inflammation, which result in joint malformation even lead to sever disability. The major damaged-target is synovial membrane. Neovascularization is the major cause of pannus causing synovitis and destroying bone. Therefore, the key point for the treatment of RA is to inhibit synovial neovascularization. Several studies showed that many highly expressed factors contributed to the forming of blood vessels in RA patients, among which vascular endothelial growth factor (VEGF) plays important function in synovitis and neovascularization. The expression of VEGF is regulated by tumor necrosis factor-α(TNF-α), interleukiu-1(IL-1), transforming growth factor-β(TGF-β), CD40-CD40L, hypoxia and other factors. As a key factor in the cytokines net, TNF-αplays vital role and interacts with VEGF, accelerating the progression of RA.
Anemia in RA patients is the most common extra-joint symptom and the degree of anemia is often consistent with the activity of joint inflammation. This kind of anemia is commonly considered as anemia of chronic disease (ACD), and is related with abnormity in iron absorption, obstruction of iron releasing from macrophage and deficient expression of erythropoietin (EPO). Cytokines such as TNF-αand IL-6 are shown to suppress the proliferation of erythroid progenitor cells and the expression of erythropoietin EPO. TNF-αand IL-6 are also shown to interfere the response of bone marrow cells to EPO and iron metabolism.
In the past decade, Thalidomide regains the research hotspot for its immune regulation, anti-inflammation and anti-angiogenesis effects. Studies showed that Thalidomide is effective in the treatment of several diseases such as cancer, rheumatoid disease and refractory anemia. However, there are few reports about the clinical and experimental research on the efficiency and mechanism of thalidomide in the treatment of RA, especially for RA with anemia. We, therefore, established the modified collagen-induced arthritis (CIA ) rats model with anemia by subcutaneous injecting collagen and adjuvant, repetitiously. Through these CIA rats, we made a further study on the effect of thalidomide on joint inflammation, angiogenesis, expression of cytokines, apoptosis and erythropoiesis, in order to select an effective drug in the treatment of RA, without severe side-effects. This experiments consist the following four parts.
Part one: The effect of thalidomide on expression of VEGF and TNF-αin CIA rats.
Objective:To explore the dynamic expression of VEGF and TNF-αmRNA and the protein in the blood and synovial membrane, to analyze the relationship between VEGF, TNF-αand the joint activity, microvessel density in synovial membrane, to study the effect of thalidomide on the expression of VEGF ,TNF-αand the joint activity, microvessel density in CIA rats.
Methods: 128 male Wistar rats were divided into five groups,Ⅰ:normal group (n=24),Ⅱ: Collagen-induced arthritis rats (CIA) model group (n=26),Ⅲ: model group with high dosage of thalidomide group(200mg/kg/d, n=26),Ⅳ: model group with low dosage of thalidomide group(100mg/kg/d, n=26),Ⅴ: model group with Methotrexate ( MTX) group,(2.7mg/kg/w, n=26). CIA rats were made by subcutaneous injecting collagen and complete freud’s adjuvant (CFA). Each group was divided into 7 subgroups according to the time point (7d, 14d, 21d, 28d, 35d, 42d, 60d) after the first injection. Radionuclide imaging,χ-ray, electron microscope, pathological technic were used to evaluate the arthritis index (AI), paw thickness and other parameters of CIA rats. Reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunohis-chemistry were used to detect the expression of VEGF and TNF-αlevel at different time point; microvessel density (MVD) was determined immunohischemistryically by CD31. The effect of Thalidomide and MTX were compared.
Results:①AI, paw thickness, and concentration of radionuclide of CIA rats reached peak value on the 21st day, among which the change of radionuclide in the joints was more sensitive and objective than other index , and had high value in diagnosing early joint disease.②The expression level of VEGF and TNF-αmRNA of CIA rats on the 21st day were 0.75±0.06, 2.01±0.13, which was significantly higher than that of normal group, P<0.01. Only high dose thalidomide could down regulate VEGF mRNA, while both thalidomide and MTX could down regulate TNF-αmRNA.③The results of immunohischemistry test showed that the expression of VEGF and TNF-αin synovial membrane was strongest on the 21st day (15683.15±319.23, 9037.71±403.03), higher than that of normal group, P<0.01. Thalidomide and MTX could reduce the expression of VEGF and TNF-α, and the high dose thalidomide showed the effect the most rapidly.④ELISA results showed the same change of VEGF and TNF-αin blood (2147.3±37.56 and 2685.0±112.09, respectively) as that in synovial membrane of CIA rats, which were higher compared with that in normal group, P<0.01. All the drugs could reduce VEGF and TNF-αlevel, and the effect of high dose thalidomide was almost the same as that of MTX.⑤The MVD of CIA rats was highest on the 28th day (22.45±2.43), higher than that of normal group, P<0.01. All the drugs could decrease MVD of the synovial membrane, and the effect of thalidomide was more rapidly.⑥The level of VEGF and TNF-αin blood and synovial membrane of CIA rats was positively related with the change of paw thickness, P<0.05; the value of VEGF was also positively related with that of MVD, P<0.01.
Conclusion: The expression level of VEGF and TNF-αwas higher in CIA rats, the change of which was correlated with the joint disease. There were increased newly developed microvessels in synovial membrane. Thalidomide could down regulate the expression level of VEGF and TNF-αmRNA and the protein, decrease the MVD, improve the joint disease. The effect of thalidomide was better than or equal to MTX. High dose thalidomide had more effect.
Part two: The effect of thalidomide on the expression of VEGF and TNF-αand the apoptosis of the FLS in CIA rats.
Objective:To explore the effect of thalidomide on the expression of VEGF and TNF-αand the apoptosis on Fibroblast-like synoviocytes (FLS) of CIA rats stimulated by lipopolysaccharide (LPS).
Methods: Axenic synovial membranes from knees of CIA rats were obtained, and synovial cells were separated and cultured in vitro. The cultured cells were divided into six groups as following: normal group, LPS group,LPS+ high dose thalidomide (50mg/L) group, LPS+ middle dose thalidomide (5mg/L) group, LPS+ low dose thalidomide (0.5mg/L) group, LPS+MTX group. The shape of FLS was observed by convert microscope and transmission electron microscope; ELISA was used to determine the expression level of VEGF and TNF-αin the culture medium; RT-PCR and Western Blot method were used to determine VEGF, TNF-αmRNA and protein; Apoptosis of FLS was determine by FCM .
Results:①The original synovial cells were to be confluent after 10～14 day and form a new generation by separated culture. FLS held predominance after 3 generation, which presented shuttle or rhombic shape.②ELISA results showed the expression level of VEGF and TNF-αin the culture medium of LPS group was higher compared with normal group(859.00±22.74pg/mL, 1315.20±19.38pg/mL), P<0.01. Thalidomide could reduce the concentration of VEGF and TNF-α, and the higher the dosage, the better the effect. The effect of MTX was better than thalidomide.③RT-PCR、Western Blot results showed that thalidomide could down regulate the expression level of VEGF and TNF-αmRNA and the protein of FLS, and the effect was partly in dose-dependent manners.④Thalidomide could induce the FLS apoptosis, also in a dose-dependent manner in the definite dosage. The apoptotic rates at 48hr were 8.07%±1.45%, 5.72%±1.42%, and 0.40%±0.04%,among which the former two were higher than that of LPS group (0.24%±0.05%), P<0.01;The nerotic rates at 48hr were 20.34%±4.43%、12.96%±4.32% and 1.94% ±1.16%, higher than that of LPS group(0.13%±0.09%), P<0.01.There was no difference between thalidomide (50mg/L) group and MTX group, P>0.05.⑤Observed with transmission electron microscope, the apoptotic phenomena were obvious: the chromatins condensed and shrunk and aggregated along inside of nuclear membrane to exist on the form of fall, petal and crescent. apoptotic bodies were, sometimes, formed.
Conclusion: FLS cells could be harvested and purified by culturing and passaging. Thalidomide could induce apoptosis of FLS and inhibit the expression of VEGF and TNF-αmRNA and the protein of FLS, partly in dose-dependent manners.
Part three: The effect of thalidomide on the anemia of chronic disease in CIA rats.
Objective:To investigate the effect of thalidomide on the anemia of chronic disease in CIA rats
Methods: The rat model of arthritis accompanied with anemia was established though the modified CIA model, referring to the part one, and each group was divided into 6 subgroups according the time point (14d, 21d, 28d, 35d, 42d, 60d) after the first injection. The hemoglobin concentration and the degree of anemia were determined; Serum iron, total iron binding capacity, ferritin, EPO, morphology of bone marrow, iron inside and outside RBC of BM were determined or observed; Serum TNF-αconcentration and the paw thickness of model rats were measured by the methods mentioned above, and the relationship between these parameters and hemoglobin were analyzed; The relationship between EPO and hemoglobin was analyzed also.
Results:①On the 28th day, anemia appeared in model rats, the hemoglobin was 121.75 g/L±9.18g/L, lower than that of normal group(147.25 g/L±5.74 g/L), P<0.01.②The characteristics of anemia include lower serum iron and total iron binding capacity, higher ferritin and EPO, erythroid cells in BM were in hyperplasia, lower iron inside RBC of BM, unchanged iron outside RBC of BM and the normal shape of RBC.③High dose thalidomide could increase hemoglobin concentration on the 60th day compared with model group on the same time point, P<0.05.④Serum TNF-αconcentration was positively related with the change of paw thickness (P<0.01) , and negatively related with hemoglobin (P<0.05);There was no correlation between hemoglobin and paw thickness (P>0.05).⑤There was no correlation between hemoglobin and EPO concentration (P>0.05).
Conclusion:Model rats with arthritis and anemia were successfully made by the meliorative method. The more serious of arthritis, the higher concentration of TNF-αand the more serious of anemia. Thalidomide could abate joint swelling and anemia, reducing TNF-αconcentration. It is hopeful to apply thalidomide in the treatment of RA with ACD patients.
Part four: The effect of thalidomide on erythroid progenitor cells in CIA rats accompanied with anemia.
Objective : To investigate the effect of thalidomide on erythroid progenitor cells of bone marrow in experimental arthritis and anemia rats. Methods: 40 male Wistar rats were divided into 5 groups. The methods of making model CIA rats grouping were the same as that in part one. Rats were killed on the third and eighth week (the 60th day), and the BM mononuclear cells (BMMNCs) were harvested and cultured in semisolid methylcellulose culture medium. The numbers of BFU-E and CFU-E were counted; The number of CD34+/CD71+cells in BM and the percentage of apoptotic cells were determined with FCM; TNF-αconcentration was determined by ELISA.
Result:①The numbers of BFU-E and CFU-E on the third and eighth week were 13.33±4.32BFU-E/2.5×105 BMMCs,16.33±5.44 BFU-E/2.5×10~5 BMMCs and 62.27±4.98 CFU-E/2.5×10~5 BMMCs,73.67±7.20 CFU-E/2.5×10~5 BMMCs,lower than that of the normal group on the same time point, P<0.05. Thalidomide could increase the formation of BFU-E and CFU-E, which was better than MTX.②The lower number of CD34+/CD71+ cells was in model rats than that of the normal group (P<0.05) and the numbers were 0.30%±0.10% and 0.47%±0.06%, respectively, on the third and eighth week. Thalidomide could also increase the number of CD34~+/CD71~+ cells of model rats. High dosage of thalidomide could increase the number of CD34~+/CD71~+ cells the number is 0.52%±0.02%和0.57%±0.11%,P<0.05;low dosage of thalidomide had the effect just on the 3rd week(0.44%±0.03%).③The percentage of CD34~+/CD71~+ apoptotic cells in model rats (31.30%±2.64%) was higher than that of normal group (21.96%±2.51% ) (P<0.05) and thalidomide could decrease the percentage of CD34+/CD71+ apoptotic cells. Both high and low dosage of thalidomide had the function, the percentage of CD34+/CD71+ was 20.57%±2.43%, 14.77%±4.52% and 23.14%±3.78%, 16.04%±2.28%.④Serum TNF-αconcentration of model rats was significantly negative correlated with the number of BFU-E (P<0.05) , and positive correlated with the percentage of CD34+/CD71+ apoptotic cell (P<0.05), while no correlation was found between the number of CD34+/CD71+ cells and the number of CFU-E (P>0.05).
Conclusion: The ability of forming BFU-E and CFU-E was decreased, the number of CD34~+/CD71~+ cells was lower, and the percentage of CD34~+/CD71~+ apoptotic cell was higher in the BM of rats with arthritis and ACD. Thalidomide could improve these parameters.
贫血是RA最常见的关节外表现,常与RA的活动相一致。目前认为RA患者的贫血多属慢性病贫血(ACD),是活动性RA的一种常见特征,可能与铁吸收异常、巨噬细胞释放铁障碍、促红细胞生成素(EPO)表达缺陷等有关。另外细胞因子如TNF-α、IL-6等已被证实与ACD发病明显相关。它们可以直接抑制红系祖细胞增殖、抑制EPO的产生、迟化骨髓对EPO的反应及干扰铁代谢等多个环节。
沙利度胺因免疫调节、抗炎及抗血管生成作用又重新成为临床研究的热点,证实它对某些肿瘤、风湿性疾病、难治性血液病以及多种原因不明的疾病疗效显著。迄今为止仅有少数关于沙利度胺治疗RA的临床报导,对其疗效褒贬不一,作用机制亦不明确,无远期疗效的临床及相关动物实验资料,也未见其对RA伴贫血患者红系生成方面的临床及动物实验的报道。
为此,本实验在传统的胶原诱导型关节炎基础上加以改进,通过多次皮内注射Ⅱ型胶原与弗氏完全佐剂,成功地建立了关节炎伴贫血的动物模型。从细胞水平、形态学、流式检测、分子生物学等多种角度,有机结合体内和离体试验,以VEGF与TNF-α为切点,以动态观察的方式研究并探讨沙利度胺对关节炎大鼠的炎性活动、血管生成、滑膜细胞表达及凋亡的影响以及对红细胞生成作用及可能机制,进一步评价不同剂量的沙利度胺在实验性关节炎伴贫血大鼠的疗效及临床应用价值,试图为该药在RA新靶点治疗——阻断血管生成方面及选择无明显骨髓抑制的抗风湿药物方面提供一定的实验依据。实验内容主要包括以下四个部分:
第一部分:VEGF、TNF-α在胶原诱导性关节炎大鼠病程中的动态表达及沙利度胺的影响
目的:探讨VEGF、TNF-αmRNA及其蛋白在胶原诱导性关节炎大鼠血清及滑膜中的动态表达及其与关节炎活动程度的关系、滑膜微血管密度的变化及沙利度胺对上述指标的影响。
方法:将128只雄性Wistar大鼠随机分5组,Ⅰ组:正常对照组(n=24),Ⅱ组:造模组(n=26),Ⅲ组:造模组+高剂量沙利度胺治疗组(200mg/kg/d, n=26),Ⅳ组:造模组+沙利度胺低剂量治疗组(100mg/kg/d, n=26),Ⅴ组:造模组+甲氨喋呤(MTX)治疗组(2.7mg/kg/w, n=26)。通过注射Ⅱ型胶原与完全弗氏佐剂(complete freud’s adjuvant, CFA)制作胶原诱导型关节炎(collagen-induced arthritis, CIA)动物模型,各CIA模型组又根据初次免疫后不同时间点(7、14、21、28、35、42及60天)各分为7个亚组。造模后12天开始给药。应用关节炎指数(arthritis index, AI)、足爪厚度、99mTc-MDP核素显像、x线、电镜及病理改变评价模型建立情况;RT-PCR、ELISA及免疫组织化学方法检测VEGF及TNF-αmRNA和其蛋白在不同时期关节炎大鼠血清及滑膜组织中含量的动态变化;采用CD31免疫组织化学方法及微血管计数(microvessel density, MVD)法了解滑膜组织新生血管情况;研究沙利度胺对上述指标的影响并与MTX比较,同时比较不同剂量沙利度胺的作用有无差别。
结果:①CIA大鼠的AI指数、足爪厚度及核素浓聚程度在21天达到高峰,其中关节部位核素的变化敏感性及客观性均优于其它指标,更能够反应关节的早期病变。②CIA大鼠滑膜组织中的VEGF和TNF-αmRNA在造模后21天表达水平分别为0.75±0.06和2.01±0.13,明显高于同期对照组,P<0.01。仅高剂量沙利度胺组可以下调VEGF mRNA的表达,高、低剂量沙利度胺和MTX组都能够下调TNF-αmRNA的表达。③免疫组织化学染色结果显示CIA大鼠滑膜层中VEGF和TNF-α蛋白的表达以造模后21天最强,分别为15683.15±319.23和9037.71±403.03,显著高于同期对照组值,P<0.01。且都与大鼠足爪厚度改变呈正相关,r值分别为0.915和0.664,P<0.01。高、低剂量沙利度胺和MTX都能降低滑膜层中VEGF和TNF-α蛋白的含量,高剂量沙利度胺起效最快。④CIA大鼠血清中VEGF和TNF-α的浓度也在造模后21天达峰值,分别为2147.3±37.56 pg/mL和2685.0±112.09 pg/mL,与同期对照组值比较显著升高,P<0.01。3个治疗组都能降低血清VEGF和TNF-α的浓度,高剂量沙利度胺与MTX作用接近。⑤CIA大鼠滑膜的MVD在造模后28天达峰值为22.45±2.43,与同期对照组比较差异显著,P<0.01。3个治疗组中大鼠滑膜的MVD均明显降低,沙利度胺起效时间早于MTX。⑥CIA大鼠血清与滑膜中VEGF和TNF-α都与大鼠足爪厚度呈正相关,P<0.05;其中VEGF与滑膜MVD的变化也呈显著正相关,P<0.01。
结论:CIA大鼠病程中血清和滑膜组织中VEGF和TNF-α存在高表达,并与关节肿胀程度相一致,关节滑膜组织内的新生血管明显增加,沙利度胺均可有效下调VEGF和TNF-αmRNA及其蛋白的表达,减少滑膜组织的MVD,改善CIA大鼠的关节症状,效果优于或等同于MTX。高剂量沙利度胺对上述指标的影响更显著。
第二部分:沙利度胺对关节炎大鼠滑膜细胞VEGF、TNF-α表达水平和对细胞凋亡的影响
目的:探讨沙利度胺对脂多糖(LPS)刺激的成纤维细胞样的滑膜细胞(FLS)VEGF和TNF-αmRNA及其蛋白表达的作用及对FLS凋亡的影响。
方法:无菌获取关节炎大鼠膝关节的滑膜组织,应用体外FLS的培养技术进行滑膜细胞的分离与培养。实验共分6组:对照组,LPS组,LPS+高剂量沙利度胺组(50 mg/L), LPS+中剂量沙利度胺组(5 mg/L),LPS+低剂量沙利度胺组(0.5 mg/L)组及LPS+MTX组。倒置显微镜及透射电镜观察体外培养的FLS的形态;ELISA方法检测FLS培养上清中VEGF和TNF-α的含量;RT-PCR、Western Blot方法检测FLS中VEGF和TNF-αmRNA及蛋白表达的变化;膜联蛋白(Annexin-V)/碘化丙啶(PI)双标记流式细胞术(flow cytometry, FCM)测定药物对FLS凋亡的影响。
结果:①CIA大鼠的原代滑膜组织分离培养24小时后开始贴壁,10～14天长满培养瓶底并可传代,3代以后主要以梭性或菱形的FLS为主。②ELISA测定LPS刺激后FLS培养上清中的VEGF、TNF-α含量分别为859.00±22.74pg/mL和1315.20±19.38pg/mL,明显高于对照组,P<0.01。沙利度胺能够降低FLS培养上清中的VEGF、TNF-α含量,浓度越高作用越强,其作用强度低于MTX。③RT-PCR、Western Blot结果示沙利度胺可以下调LPS刺激后FLS中VEGF和TNF-αmRNA及蛋白表达水平,抑制程度呈部分剂量依赖性。④沙利度胺有诱导FLS凋亡的作用,在一定浓度范围内呈剂量依赖性。处理48小时后各组凋亡率分别为:8.07%±1.45%、5.72%±1.42%和0.40%±0.04%,高中剂量沙利度胺组的凋亡率与LPS单独处理组的凋亡率(0.24%±0.05%)比较统计学差异显著,P<0.01;高中低剂量沙利度胺组的坏死率分别为:20.34%±4.43%、12.96%±4.32%和1.94%±1.16%,与LPS单独处理组的坏死率(0.13%±0.09%)比较统计学差异显著,P<0.01。高剂量沙利度胺与MTX组之间无显著性差异,P>0.05。⑤透射电镜下可观察到典型的细胞凋亡征象,细胞浆致密,染色质浓缩,沿着核膜排列,形成不同形状和大小的块状,可见凋亡小体。结论:FLS可以通过体外细胞培养及传代法获得并逐渐纯化,沙利度胺可以诱导FLS凋亡,并能够抑制其分泌和表达VEGF、TNF-α,且呈部分剂量依赖关系。
第三部分:沙利度胺对胶原诱导性关节炎伴贫血大鼠血红蛋白的影响
目的:研究并探讨沙利度胺对胶原诱导性关节炎伴贫血大鼠的治疗作用及临床应用价值。
方法:在传统的Ⅱ型胶原诱导性关节炎大鼠模型基础上加以改进,制作关节炎伴贫血的动物模型,具体方法及分组同第一部分,并根据初次免疫后不同时间点(14、21、28、35、42及60天)各分为6组。测定不同时间点血红蛋白浓度,了解贫血出现时间及规律;测定模型组血清铁、血清总铁结合力、血清铁蛋白、促红细胞生成素(EPO),获取骨髓液观察骨髓细胞形态、测定骨髓内外铁变化了解贫血类型;测定并分析不同时间点血清TNF-α浓度、大鼠足爪厚度及与血红蛋白三者之间的相关性;分析EPO与血红蛋白之间的相关性。
结果:①关节炎大鼠在造模后28天开始出现贫血,血红蛋白(121.75g/L±9.18g/L)较正常对照组(147.25 g/L±5.74 g/L)明显下降,P<0.01。②CIA模型鼠的贫血特点:血清铁、总铁结合力明显降低,血清铁蛋白及EPO升高明显,骨髓红系增生活跃,内铁减低,外铁无明显变化,红细胞形态基本正常。③高剂量沙利度胺组在造膜60天与同期模型组比较血红蛋白显著上升,P<0.05。④模型组大鼠血清TNF-α浓度与足爪厚度呈正相关,P<0.01;血红蛋白与TNF-α浓度呈负相关,P<0.05;血红蛋白与足爪厚度无明显相关,P>0.05;⑤模型组大鼠血红蛋白含量与EPO浓度之间无显著相关,P>0.05。
结论:通过改良CIA模型的方法,可成功制作出关节炎伴贫血的大鼠模型。关节炎肿胀程度越大,TNF-α浓度越高;TNF-α浓度越高,贫血也越严重。沙利度胺能够改善贫血及大鼠关节肿胀,降低血清TNF-α浓度,有望为临床RA伴ACD患者的治疗开辟一条新途径。
第四部分:沙利度胺对关节炎伴贫血大鼠红系祖细胞的影响及可能机制
目的:研究并探讨沙利度胺对关节炎伴贫血大鼠红系祖细胞的影响。
方法:雄性Wistar大鼠40只随机分5组,造模方法及处理因素同第三部分。于造模后第3及第8周处死大鼠,剪断股骨两端,获取骨髓液,大鼠淋巴细胞分离液分离获得骨髓单个核细胞(BMMCs),台盼兰染色记数活细胞,在甲基纤维素半固体培养基中进行骨髓红系祖细胞的培养及集落数目的测定;流式细胞学测定骨髓红系祖细胞CD34+及CD71+的表达情况,应用7-氨基放线菌D染色(7AAD)了解CD34+/CD71+细胞凋亡情况。
结果:①关节炎伴贫血大鼠在第3周与第8周BFU-E、CFU-E的数目分别为13.33±4.32BFU-E/2.5×105 BMMCs、16.33±5.44 BFU-E/2.5×10~5 BMMCs和62.27±4.98 CFU-E/2.5×10~5 BMMCs、73.67±7.20 CFU-E/2.5×10~5 BMMCs,明显低于正常组大鼠的值,P<0.05。沙利度胺可以促进BFU-E和CFU-E集落的形成,作用优于MTX。②与正常对照组比较,模型组大鼠CD34+/CD71+的细胞数目明显减少,第3周及第8周分别为0.30%±0.10%和0.47%±0.06%,P<0.05。高剂量沙利度胺可以提高大鼠CD34+/CD71+的细胞数目,第3周及第8周分别为0.52%±0.02%和0.57%±0.11%,P<0.05;低剂量沙利度胺仅在第3周作用明显,为0.44%±0.03%。③模型组大鼠CD34~+/CD71~+细胞的凋亡率与正常对照组比较明显增加,第3周及第8周分别为31.30%±2.64%和21.96%±2.51%,P<0.05。沙利度胺可以降低大鼠CD34~+/CD71~+细胞的凋亡率,高低沙利度胺组凋亡率的值分别为20.57%±2.43%、14.77%±4.52%和23.14%±3.78%、16.04%±2.28%。④模型组大鼠血清中TNF-α浓度与BFU-E数目呈明显负相关,P<0.05,与CD34+/CD71+凋亡细胞的百分率呈明显正相关,P<0.05,而与CD34~+/CD71~+细胞占骨髓有核细胞的比例及CFU-E数目无明显相关,P>0.05。
结论:关节炎伴贫血大鼠骨髓红系形成集落的能力明显下降,CD34~+/CD71~+的细胞数目减少,CD34~+/CD71~+凋亡细胞显著增加。沙利度胺对以上指标有改善作用。TNF-α可能参与关节炎伴贫血的发病机制。
Rheumatoid arthritis (RA) is a kind of autoimmune disease characterized by chronic and symmetrical multijoint inflammation, which result in joint malformation even lead to sever disability. The major damaged-target is synovial membrane. Neovascularization is the major cause of pannus causing synovitis and destroying bone. Therefore, the key point for the treatment of RA is to inhibit synovial neovascularization. Several studies showed that many highly expressed factors contributed to the forming of blood vessels in RA patients, among which vascular endothelial growth factor (VEGF) plays important function in synovitis and neovascularization. The expression of VEGF is regulated by tumor necrosis factor-α(TNF-α), interleukiu-1(IL-1), transforming growth factor-β(TGF-β), CD40-CD40L, hypoxia and other factors. As a key factor in the cytokines net, TNF-αplays vital role and interacts with VEGF, accelerating the progression of RA.
Anemia in RA patients is the most common extra-joint symptom and the degree of anemia is often consistent with the activity of joint inflammation. This kind of anemia is commonly considered as anemia of chronic disease (ACD), and is related with abnormity in iron absorption, obstruction of iron releasing from macrophage and deficient expression of erythropoietin (EPO). Cytokines such as TNF-αand IL-6 are shown to suppress the proliferation of erythroid progenitor cells and the expression of erythropoietin EPO. TNF-αand IL-6 are also shown to interfere the response of bone marrow cells to EPO and iron metabolism.
In the past decade, Thalidomide regains the research hotspot for its immune regulation, anti-inflammation and anti-angiogenesis effects. Studies showed that Thalidomide is effective in the treatment of several diseases such as cancer, rheumatoid disease and refractory anemia. However, there are few reports about the clinical and experimental research on the efficiency and mechanism of thalidomide in the treatment of RA, especially for RA with anemia. We, therefore, established the modified collagen-induced arthritis (CIA ) rats model with anemia by subcutaneous injecting collagen and adjuvant, repetitiously. Through these CIA rats, we made a further study on the effect of thalidomide on joint inflammation, angiogenesis, expression of cytokines, apoptosis and erythropoiesis, in order to select an effective drug in the treatment of RA, without severe side-effects. This experiments consist the following four parts.
Part one: The effect of thalidomide on expression of VEGF and TNF-αin CIA rats.
Objective:To explore the dynamic expression of VEGF and TNF-αmRNA and the protein in the blood and synovial membrane, to analyze the relationship between VEGF, TNF-αand the joint activity, microvessel density in synovial membrane, to study the effect of thalidomide on the expression of VEGF ,TNF-αand the joint activity, microvessel density in CIA rats.
Methods: 128 male Wistar rats were divided into five groups,Ⅰ:normal group (n=24),Ⅱ: Collagen-induced arthritis rats (CIA) model group (n=26),Ⅲ: model group with high dosage of thalidomide group(200mg/kg/d, n=26),Ⅳ: model group with low dosage of thalidomide group(100mg/kg/d, n=26),Ⅴ: model group with Methotrexate ( MTX) group,(2.7mg/kg/w, n=26). CIA rats were made by subcutaneous injecting collagen and complete freud’s adjuvant (CFA). Each group was divided into 7 subgroups according to the time point (7d, 14d, 21d, 28d, 35d, 42d, 60d) after the first injection. Radionuclide imaging,χ-ray, electron microscope, pathological technic were used to evaluate the arthritis index (AI), paw thickness and other parameters of CIA rats. Reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunohis-chemistry were used to detect the expression of VEGF and TNF-αlevel at different time point; microvessel density (MVD) was determined immunohischemistryically by CD31. The effect of Thalidomide and MTX were compared.
Results:①AI, paw thickness, and concentration of radionuclide of CIA rats reached peak value on the 21st day, among which the change of radionuclide in the joints was more sensitive and objective than other index , and had high value in diagnosing early joint disease.②The expression level of VEGF and TNF-αmRNA of CIA rats on the 21st day were 0.75±0.06, 2.01±0.13, which was significantly higher than that of normal group, P<0.01. Only high dose thalidomide could down regulate VEGF mRNA, while both thalidomide and MTX could down regulate TNF-αmRNA.③The results of immunohischemistry test showed that the expression of VEGF and TNF-αin synovial membrane was strongest on the 21st day (15683.15±319.23, 9037.71±403.03), higher than that of normal group, P<0.01. Thalidomide and MTX could reduce the expression of VEGF and TNF-α, and the high dose thalidomide showed the effect the most rapidly.④ELISA results showed the same change of VEGF and TNF-αin blood (2147.3±37.56 and 2685.0±112.09, respectively) as that in synovial membrane of CIA rats, which were higher compared with that in normal group, P<0.01. All the drugs could reduce VEGF and TNF-αlevel, and the effect of high dose thalidomide was almost the same as that of MTX.⑤The MVD of CIA rats was highest on the 28th day (22.45±2.43), higher than that of normal group, P<0.01. All the drugs could decrease MVD of the synovial membrane, and the effect of thalidomide was more rapidly.⑥The level of VEGF and TNF-αin blood and synovial membrane of CIA rats was positively related with the change of paw thickness, P<0.05; the value of VEGF was also positively related with that of MVD, P<0.01.
Conclusion: The expression level of VEGF and TNF-αwas higher in CIA rats, the change of which was correlated with the joint disease. There were increased newly developed microvessels in synovial membrane. Thalidomide could down regulate the expression level of VEGF and TNF-αmRNA and the protein, decrease the MVD, improve the joint disease. The effect of thalidomide was better than or equal to MTX. High dose thalidomide had more effect.
Part two: The effect of thalidomide on the expression of VEGF and TNF-αand the apoptosis of the FLS in CIA rats.
Objective:To explore the effect of thalidomide on the expression of VEGF and TNF-αand the apoptosis on Fibroblast-like synoviocytes (FLS) of CIA rats stimulated by lipopolysaccharide (LPS).
Methods: Axenic synovial membranes from knees of CIA rats were obtained, and synovial cells were separated and cultured in vitro. The cultured cells were divided into six groups as following: normal group, LPS group,LPS+ high dose thalidomide (50mg/L) group, LPS+ middle dose thalidomide (5mg/L) group, LPS+ low dose thalidomide (0.5mg/L) group, LPS+MTX group. The shape of FLS was observed by convert microscope and transmission electron microscope; ELISA was used to determine the expression level of VEGF and TNF-αin the culture medium; RT-PCR and Western Blot method were used to determine VEGF, TNF-αmRNA and protein; Apoptosis of FLS was determine by FCM .
Results:①The original synovial cells were to be confluent after 10～14 day and form a new generation by separated culture. FLS held predominance after 3 generation, which presented shuttle or rhombic shape.②ELISA results showed the expression level of VEGF and TNF-αin the culture medium of LPS group was higher compared with normal group(859.00±22.74pg/mL, 1315.20±19.38pg/mL), P<0.01. Thalidomide could reduce the concentration of VEGF and TNF-α, and the higher the dosage, the better the effect. The effect of MTX was better than thalidomide.③RT-PCR、Western Blot results showed that thalidomide could down regulate the expression level of VEGF and TNF-αmRNA and the protein of FLS, and the effect was partly in dose-dependent manners.④Thalidomide could induce the FLS apoptosis, also in a dose-dependent manner in the definite dosage. The apoptotic rates at 48hr were 8.07%±1.45%, 5.72%±1.42%, and 0.40%±0.04%,among which the former two were higher than that of LPS group (0.24%±0.05%), P<0.01;The nerotic rates at 48hr were 20.34%±4.43%、12.96%±4.32% and 1.94% ±1.16%, higher than that of LPS group(0.13%±0.09%), P<0.01.There was no difference between thalidomide (50mg/L) group and MTX group, P>0.05.⑤Observed with transmission electron microscope, the apoptotic phenomena were obvious: the chromatins condensed and shrunk and aggregated along inside of nuclear membrane to exist on the form of fall, petal and crescent. apoptotic bodies were, sometimes, formed.
Conclusion: FLS cells could be harvested and purified by culturing and passaging. Thalidomide could induce apoptosis of FLS and inhibit the expression of VEGF and TNF-αmRNA and the protein of FLS, partly in dose-dependent manners.
Part three: The effect of thalidomide on the anemia of chronic disease in CIA rats.
Objective:To investigate the effect of thalidomide on the anemia of chronic disease in CIA rats
Methods: The rat model of arthritis accompanied with anemia was established though the modified CIA model, referring to the part one, and each group was divided into 6 subgroups according the time point (14d, 21d, 28d, 35d, 42d, 60d) after the first injection. The hemoglobin concentration and the degree of anemia were determined; Serum iron, total iron binding capacity, ferritin, EPO, morphology of bone marrow, iron inside and outside RBC of BM were determined or observed; Serum TNF-αconcentration and the paw thickness of model rats were measured by the methods mentioned above, and the relationship between these parameters and hemoglobin were analyzed; The relationship between EPO and hemoglobin was analyzed also.
Results:①On the 28th day, anemia appeared in model rats, the hemoglobin was 121.75 g/L±9.18g/L, lower than that of normal group(147.25 g/L±5.74 g/L), P<0.01.②The characteristics of anemia include lower serum iron and total iron binding capacity, higher ferritin and EPO, erythroid cells in BM were in hyperplasia, lower iron inside RBC of BM, unchanged iron outside RBC of BM and the normal shape of RBC.③High dose thalidomide could increase hemoglobin concentration on the 60th day compared with model group on the same time point, P<0.05.④Serum TNF-αconcentration was positively related with the change of paw thickness (P<0.01) , and negatively related with hemoglobin (P<0.05);There was no correlation between hemoglobin and paw thickness (P>0.05).⑤There was no correlation between hemoglobin and EPO concentration (P>0.05).
Conclusion:Model rats with arthritis and anemia were successfully made by the meliorative method. The more serious of arthritis, the higher concentration of TNF-αand the more serious of anemia. Thalidomide could abate joint swelling and anemia, reducing TNF-αconcentration. It is hopeful to apply thalidomide in the treatment of RA with ACD patients.
Part four: The effect of thalidomide on erythroid progenitor cells in CIA rats accompanied with anemia.
Objective : To investigate the effect of thalidomide on erythroid progenitor cells of bone marrow in experimental arthritis and anemia rats. Methods: 40 male Wistar rats were divided into 5 groups. The methods of making model CIA rats grouping were the same as that in part one. Rats were killed on the third and eighth week (the 60th day), and the BM mononuclear cells (BMMNCs) were harvested and cultured in semisolid methylcellulose culture medium. The numbers of BFU-E and CFU-E were counted; The number of CD34+/CD71+cells in BM and the percentage of apoptotic cells were determined with FCM; TNF-αconcentration was determined by ELISA.
Result:①The numbers of BFU-E and CFU-E on the third and eighth week were 13.33±4.32BFU-E/2.5×105 BMMCs,16.33±5.44 BFU-E/2.5×10~5 BMMCs and 62.27±4.98 CFU-E/2.5×10~5 BMMCs,73.67±7.20 CFU-E/2.5×10~5 BMMCs,lower than that of the normal group on the same time point, P<0.05. Thalidomide could increase the formation of BFU-E and CFU-E, which was better than MTX.②The lower number of CD34+/CD71+ cells was in model rats than that of the normal group (P<0.05) and the numbers were 0.30%±0.10% and 0.47%±0.06%, respectively, on the third and eighth week. Thalidomide could also increase the number of CD34~+/CD71~+ cells of model rats. High dosage of thalidomide could increase the number of CD34~+/CD71~+ cells the number is 0.52%±0.02%和0.57%±0.11%,P<0.05;low dosage of thalidomide had the effect just on the 3rd week(0.44%±0.03%).③The percentage of CD34~+/CD71~+ apoptotic cells in model rats (31.30%±2.64%) was higher than that of normal group (21.96%±2.51% ) (P<0.05) and thalidomide could decrease the percentage of CD34+/CD71+ apoptotic cells. Both high and low dosage of thalidomide had the function, the percentage of CD34+/CD71+ was 20.57%±2.43%, 14.77%±4.52% and 23.14%±3.78%, 16.04%±2.28%.④Serum TNF-αconcentration of model rats was significantly negative correlated with the number of BFU-E (P<0.05) , and positive correlated with the percentage of CD34+/CD71+ apoptotic cell (P<0.05), while no correlation was found between the number of CD34+/CD71+ cells and the number of CFU-E (P>0.05).
Conclusion: The ability of forming BFU-E and CFU-E was decreased, the number of CD34~+/CD71~+ cells was lower, and the percentage of CD34~+/CD71~+ apoptotic cell was higher in the BM of rats with arthritis and ACD. Thalidomide could improve these parameters.
引文
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