霉酚酸衍生物对小鼠T细胞免疫功能的抑制作用

目的研究2种新型霉酚酸衍生物(代号JU95-07B、JU100-07E)在体外对小鼠T细胞免疫功能的影响。方法小鼠脾细胞用抗CD3单克隆抗体(mAb)刺激,同时加入不同浓度(10-4、10-5、10-6、10-7、10-8mol/L)及在不同时间点(刺激细胞后第0、6、12 h)加入同一浓度(10-5mol/L)的霉酚酸衍生物后,用酶联免疫吸附法(ELISA)检测培养上清中IFN-γ和IL-2的水平。同时利用流式细胞术(flow cytometry,FCM)检测霉酚酸衍生物对T细胞IFN-γ和IL-2的分泌、表面分子CD25的表达以及增殖的影响。结果 2种霉酚酸衍生物对抗CD3刺激条件下诱导的小鼠脾细胞产生的IFN-γ和IL-2均具有浓度依赖性抑制作用,并且在抗CD3刺激的不同时间点分别加入2种霉酚酸衍生物均能不同程度地抑制小鼠脾细胞产生IFN-γ和IL-2;同时,与单独使用抗CD3刺激相比,分别加入10-5mol/L浓度的2种霉酚酸衍生物后,均能抑制小鼠T细胞表面分子CD25的表达以及增殖。结论体外实验表明2种霉酚酸衍生物均对小鼠T细胞免疫功能具有明显抑制作用,该研究提示这2种霉酚酸衍生物可以抑制自身免疫性疾病和移植排斥反应。     

BrdU掺入与T细胞的活化和细胞因子表达的关系

目的:探讨利用流式细胞术(FCM)检测细胞增殖(BrdU掺入)与T细胞的活化及细胞因子表达的关系.方法:正常人PBMC经PMA Ionomycin刺激不同时间,在培养结束前1 h加入BrdU,利用抗BrdU以及多种抗细胞表面和抗细胞因子抗体标记,FCM检测.结果:体外刺激PBMC 48 h后,可见T细胞有BrdU掺入,但随着时间的延长,掺入率没有明显增加.对比分析BrdU掺入与活化分子表达的关系表明,CD69在刺激后8 h达高峰,而CD25则需要24 h后达到高峰.另外,BrdU掺入与细胞因子表达没有明显关系,当PBMC刺激后8 h,即有大量IFN-γ的表达,而当培养时间延长到24、48和72 h后,IFN-γ的表达没有明显改变.OKT3 antiCD28刺激T细胞后BrdU的阳性率高于PMA Ionomycin刺激的T细胞,CD8 T细胞掺入率高于CD4 T细胞.结论:利用FCM检测BrdU 细胞可以用于评价T细胞的增殖反应,但在多克隆刺激的条件下,只有少数细胞发生增殖,并且BrdU掺入率受刺激剂种类和时间的影响,BrdU掺入与活化分子和细胞因子的表达均没有明显关系.     

对早期凋亡细胞的吞噬抑制T淋巴细胞活化的实验研究

目的:研究早期凋亡细胞及吞噬早期凋亡细胞的巨噬细胞对T淋巴细胞活化的影响。方法:体外以紫外线照射,诱导出早期凋亡的Jurkat细胞;建立早期凋亡细胞的吞噬模型;ELISA法分析早期凋亡细胞对LPS刺激下巨噬细胞分泌细胞因子的影响及在早期凋亡细胞和吞噬了早期凋亡细胞的巨噬细胞干预下,ConA刺激T淋巴细胞活化后CD69、CD25、CD71表达的变化。结果:巨噬细胞吞噬了早期凋亡细胞后,抑制性细胞因子(TGF_β1)的分泌明显上调,并且在一定程度上抑制了ConA刺激下的T淋巴细胞活化;具体表现为CD69、CD25、CD71等T淋巴细胞活化标志的表达受到明显抑制。当加入TGF_β1中和抗体后,这种抑制作用消失。结论:巨噬细胞吞噬了早期凋亡细胞后抑制ConA刺激下的T淋巴细胞CD69、CD25、CD71的表达,这种抑制作用依赖于的TGF_β1分泌增强。     

乙醇降低人外周血T淋巴细胞活化并增加其凋亡

目的观察乙醇对人外周血T淋巴细胞亚群数量、活化功能及凋亡的影响。方法从健康供血者通过密度离心分离出外周血单个核细胞(PBMC),淋巴细胞与植物血凝素(PHA)和(30、 50)mL/L乙醇共同培养。培养12、 18、 36、 48 h后,采用流式细胞术检测各组淋巴细胞活化功能以及凋亡情况。结果 PBMC与乙醇共培养12 h后,在(30、 50)mL/L的乙醇培养条件下, T淋巴细胞的数量在12 h内无明显变化,但与空白对照相比, PHA组CD8~+ T细胞出现活化,而且50 mL/L乙醇可以抑制CD8~+ T细胞的活化。PBMC与乙醇共培养18 h,与单纯PHA组相比,乙醇和PHA联合组淋巴细胞数量减少,且随着乙醇剂量的增加淋巴细胞数量下降更明显,且PHA刺激T细胞活化减弱。PBMC与乙醇共培养36 h,与单纯PHA组相比,乙醇联合PHA处理组淋巴细胞的数量减少更显著,共培养48 h,淋巴细胞几乎消失。随着PBMC与乙醇共培养时间的延长和乙醇剂量的增加, CD3~+ T淋巴细胞凋亡比例升高。结论较高剂量乙醇处理的外周血T淋巴细胞活化减少,随着乙醇剂量的增加, T淋巴细胞及其亚群的细胞数量减少,可能与促进T淋巴细胞及其亚群凋亡有关。     

儿童癫痫患者T淋巴细胞活化并产生促炎因子

目的检测儿童癫痫患者外周T淋巴细胞活化状态以及细胞因子水平变化。方法选取20名儿童癫痫患者和19名健康对照儿童,采集外周血,分离外周血单个核细胞(PBMC),用细胞膜表面标记抗体流式细胞术检测T淋巴细胞表面共刺激分子CD69、CD25和细胞毒性T淋巴细胞相关蛋白4(CTLA4)的表达,用细胞内因子染色结合流式细胞术检测T细胞细胞因子γ干扰素(IFN-γ)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和IL-17A的表达,利用细胞内因子染色结合流式细胞术检测调节性T淋巴细胞(Treg)表达IL-10的情况。结果与对照组相比,儿童癫痫患者外周血中T淋巴细胞表面高表达共刺激分子CD69、CD25和CTLA4,活化的CD4+T细胞高表达IFN-γ、TNF-α、IL-6和IL-17 A。在儿童癫痫患者高表达IL-10的Treg数目增加。结论儿童癫痫患者外周T淋巴细胞活化并产生细胞因子。     

白藜芦醇对小鼠CD4+T淋巴细胞活化的影响

目的 研究白藜芦醇对小鼠CD4+T细胞活化的影响.方法 将BALB/c鼠(SPF,雌性,9～10周龄)脾脏单个核细胞与不同浓度(0、10、20、40 mmol/L)的白藜芦醇共同孵育30 min后,分别采用刀豆蛋白A(ConA)、抗CD3抗体、抗CD3/抗CD28抗体刺激继续培养;分别共培养6h、36 h后收集细胞采用单克隆抗体抗CD4-PerCP和抗CD69-FITC或抗CD71-FITC染色后,流式细胞术检测CD4+T细胞CD69、CD71的表达.结果 白藜芦醇(≥10 mmol/L)可显著抑制抗CD3抗体引起的CD4+T淋巴细胞CD69表达(P＜0.01),其抑制作用呈剂量依赖关系.白藜芦醇(≥20 mmol/L)可显著抑制由ConA、抗CD3/CD28抗体引起的CD4+T淋巴细胞CD69表达(P＜0.05),其抑制作用呈剂量依赖关系.白藜芦醇(≥10 mmol/L)可显著抑制ConA引起的CD4+T淋巴细胞CD71表达(P＜0.05),其抑制作用呈剂量依赖关系.白藜芦醇(≥20 mmol/L)可显著抑制由抗CD3抗体、抗CD3/抗CD28抗体引起的CD4+T淋巴细胞CD71表达(P＜0.05),其抑制作用呈剂量依赖关系.结论 白藜芦醇具有抑制小鼠CD4+T细胞活化的作用,其抑制作用呈剂量依赖关系.     

雪胆素乙对小鼠淋巴细胞体外活化及增殖的影响

目的分析雪胆素乙(Cucurbitacin IIb,CuIIb)对丝裂原刺激的小鼠淋巴细胞体外活化、增殖及促炎因子表达的影响,探讨其潜在的抗炎效应及作用机制。方法以WST法检测CuIIb对刀豆蛋白A(Con A)刺激的淋巴细胞增殖的作用,利用流式细胞术分析小鼠淋巴细胞活化抗原CD69和CD25以及促炎因子肿瘤坏死因子(TNF-α)和白细胞介素6(IL-6)表达的影响。结果 CuIIb能明显抑制Con A刺激引起的淋巴细胞的增殖作用,并呈现剂量依赖性;经CuIIb处理后,T细胞早期活化标志分子CD69和中期活化分子CD25的表达受到明显抑制;同时,CuIIb以剂量依赖方式抑制Con A诱导的促炎因子TNF-α和IL-6在CD3+T细胞中的表达。结论 CuIIb对小鼠淋巴细胞的活化、增殖均具有明显抑制作用,同时减少炎症相关细胞因子的表达,提示其通过对适应性免疫的调控发挥抗炎效应。     

早期凋亡的T淋巴细胞诱导生成耐受性树突状细胞的实验研究

目的： 探讨早期凋亡T淋巴细胞的抑制性免疫调节性能。方法： 采用定时紫外线照射诱导T淋巴细胞早期凋亡，深低温反复冻融获得坏死T淋巴细胞。体外诱导、纯化并培养骨髓源性不成熟树突状细胞（imDCs）,imDCs分别和早期凋亡或坏死T淋巴细胞共培养。用流式细胞仪、双夹心ELISA、［³H］掺入混合淋巴细胞反应等方法分析imDCs吞噬早期凋亡或坏死T淋巴细胞后，在不同处理条件下，MHC-Ⅱ、CD40、CD80、CD86的表达水平、分泌IL-12 p70以及刺激T淋巴细胞增殖能力的差异。结果： imDCs和坏死细胞碎片共培养后明显趋于成熟，其MHCⅡ和CD40、CD80、CD86的表达水平显著上调；分泌较高水平的IL-12 p70；和同种异体处女T淋巴细胞混合培养后显著刺激处女T淋巴细胞增殖。 imDCs和早期凋亡的T淋巴细胞共培养后,其MHC-Ⅱ、CD40、CD80和CD86的表达维持较低水平；仅分泌较低水平IL-12 p70；和同种异体处女T淋巴细胞混合培养后不能刺激淋巴细胞增殖。此外，早期凋亡的T淋巴细胞孵育上清显著抑制了吞噬坏死细胞碎片后的imDCs表达共刺激分子CD40、CD80、CD86。当TGFβ₁中和抗体和早期凋亡T淋巴细胞同加入imDCs，在表达MHC-Ⅱ、CD40、CD80、CD86，分泌IL-12 p70，刺激处女T淋巴细胞增殖等方面和吞噬坏死T淋巴细的DCs相比无显著差异。结论： 早期凋亡T淋巴细胞通过释放免疫抑制性细胞因子TGFβ₁，诱导imDCs呈现出耐受性DCs（TolDCs）的免疫表型及生物学特征，从而发挥抑制性免疫调节作用。     

脐带间充质干细胞体外对脐带血CD4+ T 淋巴细胞免疫调节作用的研究

目的:研究脐带间充质干细胞(UCMSCs)在体外对异源脐血CD4+ T 淋巴细胞增殖、凋亡及向CD4+ CD25+ 调节性T 细胞(Treg)分化的免疫调控作用。方法:建立不同比例UCMSCs 与植物血凝素(PHA)活化的脐血CD4+ T 淋巴细胞共培养,或与Transwell 共培养体系,检测CD4+ T 淋巴细胞增殖情况、CD4+ CD25+ / CD4+ 比率的变化及调节性T 淋巴细胞标志基因Foxp3 相对表达量的变化。同时,建立UCMSCs 与地塞米松(DXM)刺激的脐血CD4+ T 淋巴细胞共培养,或与Transwell 共培养体系,检测CD4+ T 淋巴细胞凋亡情况。结果:PHA 活化的脐血CD4+ T 淋巴细胞在与UCMSCs 共培养3 d 后,较无UCMSCs对照组相比,细胞数量明显减少(P<0. 05)且CD4+ CD25+ / CD4+ T 淋巴细胞的比率及Foxp3 的相对表达量显著增加(P<0. 01),并随UCMSCs 数量增加,作用增强(P<0. 05)。DXM 诱导的脐血CD4+ T 淋巴细胞与UCMSCs 共培养7 d 后,细胞凋亡比率较无UCMSCs 对照组显著降低(P<0. 01)。上述效应在Transwell 共培养中部分减弱但无法消除。结论:UCMSCs 对脐血CD4+ T细胞介导的免疫反应具有负调节作用,其作用主要表现在对细胞增殖能力和分化的调控而不是促进凋亡。     

抗CD71人-鼠嵌合抗体对活化淋巴细胞的效应

目的　通过体外实验探讨抗CD71人 鼠嵌合抗体对活化淋巴细胞效应的影响 ,并与其鼠源性单克隆抗体进行比较。方法　以丝裂原诱导的人外周血单个核细胞 (PBMC)为靶细胞 ,测定嵌合抗体和鼠源性单抗对其增殖抑制率 ;在新鲜补体存在下 ,测定补体依赖性嵌合抗体介导的细胞毒效应(CDC)。以EBV转化的B细胞为刺激细胞 ,测定两种抗体对其诱导的同种异体PBMC的增殖抑制率 ;以同种异体的PBMC为刺激细胞 ,测定两种抗体在单向、双向混合淋巴细胞培养 (MLC)中的增殖抑制率。结果　嵌合抗体和鼠源性单抗均可明显抑制丝裂原诱导的PBMC的增殖反应 ,且二者抑制率差异无显著性(P >0 .0 5 ) ,其抑制作用随抗体浓度增加而增强 ,PBMC和丝裂原共同孵育 12h后加入抗体的增殖抑制效应最明显 ;在新鲜补体存在下 ,嵌合抗体对丝裂原诱导增殖的PBMC具有CDC作用 ,而鼠源性单抗CDC作用较弱 ;两种抗体对混合淋巴细胞培养反应有明显的抑制作用 ,且嵌合抗体组抑制率明显高于鼠源性单抗组 (P <0 .0 5 )。结论　抗CD71人 鼠嵌合抗体在体外实验中可抑制淋巴细胞的活化及其效应 ,其作用明显强于抗CD71鼠源性单克隆抗体。     

Pain and Effusion and Quadriceps Activation and Strength

Context:

Quadriceps dysfunction is a common consequence of knee joint injury and disease, yet its causes remain elusive.

Objective:

To determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion affect the magnitude of quadriceps dysfunction.

Design:

Crossover study.

Setting:

University research laboratory.

Patients or Other Participants:

Fourteen (8 men, 6 women; age = 23.6 ± 4.8 years, height = 170.3 ± 9.16 cm, mass = 72.9 ± 11.84 kg) healthy volunteers.

Intervention(s):

All participants were tested under 4 randomized conditions: normal knee, effused knee, painful knee, and effused and painful knee.

Main Outcome Measure(s):

Quadriceps strength (Nm/kg) and activation (central activation ratio) were assessed after each condition was induced.

Results:

Quadriceps strength and activation were highest under the normal knee condition and differed from the 3 experimental knee conditions (P < .05). No differences were noted among the 3 experimental knee conditions for either variable (P > .05).

Conclusions:

Both pain and effusion led to quadriceps dysfunction, but the interaction of the 2 stimuli did not increase the magnitude of the strength or activation deficits. Therefore, pain and effusion can be considered equally potent in eliciting quadriceps inhibition. Given that pain and effusion accompany numerous knee conditions, the prevalence of quadriceps dysfunction is likely high.Key Words: arthrogenic muscle inhibition, central activation failure, voluntary activation, muscles

Key Points

• Knee pain and effusion resulted in arthrogenic muscle inhibition and weakness of the quadriceps.
• The simultaneous presence of pain and effusion did not increase the magnitude of quadriceps dysfunction.
• To reduce arthrogenic muscle inhibition and improve muscle strength, clinicians should employ interventions that target removing both pain and effusion.

Quadriceps weakness is a common consequence of traumatic knee joint injury^1,2 and chronic degenerative knee joint conditions.^3,4 Arthrogenic muscle inhibition (AMI), a neurologic decline in muscle activation, results in quadriceps weakness and hinders rehabilitation by preventing gains in strength.⁵ The inability to reverse AMI and restore muscle function can lead to decreased physical abilities,⁶ biomechanical deficits,⁷ and possibly reinjury.⁵ Furthermore, researchers^8,9 have suggested that quadriceps weakness resulting from AMI may place patients at risk for developing osteoarthritis in the knee. In light of the substantial influence of quadriceps AMI on these clinically relevant outcomes, we need to improve our understanding of the factors that contribute to this neurologic decline in muscle activity so efforts to target and reverse it can be implemented and gains in strength can be achieved more easily.Joint injury and disease are accompanied by numerous sequelae (ie, pain, swelling, tissue damage, inflammation), so ascertaining which one ultimately leads to neurologic muscle dysfunction is difficult. Whereas a joint effusion can result in AMI,^10–12 the effects of pain are less understood despite many clinicians attributing AMI to pain. Using techniques that introduce knee pain without accompanying injury may provide insights into the role of pain in eliciting AMI.The degree of knee joint damage may play a role in the quantity of AMI that manifests. Hurley et al^13,14 demonstrated that quadriceps AMI, measured using an interpolated-twitch technique, was greater in patients with extensive traumatic knee injury (eg, fractured tibial plateau, ruptured medial collateral ligament, and medial meniscectomy) than patients with isolated joint trauma (ie, isolated anterior cruciate ligament [ACL] rupture). Similarly, patients with more knee joint symptoms (ie, greater number of symptoms and increased severity of symptoms) may present with greater magnitudes of quadriceps inhibition. Recently, investigators¹⁵ have suggested that patients with more pain display less quadriceps strength, supporting this tenet. Given that effusion and pain often present simultaneously with joint injuries and diseases, such as ACL injury and osteoarthritis, examining both the isolated and cumulative effects of these sequelae appears warranted to determine if they influence the magnitude of muscle inhibition.Experimental joint-effusion and pain models are safe and effective experimental methods that allow for the isolated examination of their effects on muscle function. The effusion model, whereby sterile saline is injected directly into the knee joint capsule,⁷ produces a clinically relevant magnitude of the joint effusion that may be present with traumatic injury. Effusion is thought to activate group II afferents responding to stretch or pressure,^16–18 which in turn may facilitate group Ib interneurons and result in quadriceps AMI.⁵ The pain model involves injecting hypertonic saline into the infrapatellar fat pad to produce anteromedial knee pain similar to that described in patients with patellofemoral pain syndrome.¹⁹ Pain is considered to initiate AMI through activation of group III and IV afferents that act as nocioceptors to signal damage or potential damage to joint structures.^16–18 The firing of these afferents then may lead to facilitation of group Ib interneurons, the flexion reflex, or the gamma loop, ultimately resulting in quadriceps inhibition.²⁰ Thus, these models allow us to create symptoms that are associated with knee injury and have the added benefit of providing a way to examine their effects in isolation.Therefore, the purpose of our study was to determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion would affect the magnitude of quadriceps dysfunction. We hypothesized that pain alone would result in quadriceps inhibition and that the magnitude of inhibition would be greater when effusion and pain were present simultaneously.     

即早基因c-fos与脑血管病及学习记忆

即早基因c-fos是广泛存在于原核细胞和真核细胞的高度保守基因.在正常情况下,c-fos基因参与细胞生长、分化、信息传递、学习和记忆等生理过程,而在病理情况下c-fos基因表达及调控变化与多种疾病的发生和发展有关.C-fos在中枢神经系统的某些部位可有基础水平的表达,但表达很低,当受到如脑缺血、脑出血、痫性发作、应激等刺激后,其在数十分钟内做出反应,在对外界刺激-转录耦联的信忠传递过程中起着核内第三信使的重要作用.     

Opportunities and challenges in the prevention and control of cancer and other chronic diseases: children's diet and nutrition and weight and physical activity

OBJECTIVE: The purpose of this article is to review the role of behavioral research in disease prevention and control, with a particular emphasis on lifestyle- and behavior-related cancer and chronic disease risk factors--specifically, relationships among diet and nutrition and weight and physical activity with adult cancer, and tracking developmental origins of these health-promoting and health-compromising behaviors from childhood into adulthood. METHOD: After reviewing the background of the field of cancer prevention and control and establishing plausibility for the role of child health behavior in adult cancer risk, studies selected from the pediatric published literature are reviewed. Articles were retrieved, selected, and summarized to illustrate that results from separate but related fields of study are combinable to yield insights into the prevention and control of cancer and other chronic diseases in adulthood through the conduct of nonintervention and intervention research with children in clinical, public health, and other contexts. RESULTS: As illustrated by the evidence presented in this review, there are numerous reasons (biological, psychological, and social), opportunities (school and community, health care, and family settings), and approaches (nonintervention and intervention) to understand and impact behavior change in children's diet and nutrition and weight and physical activity. CONCLUSIONS: Further development and evaluation of behavioral science intervention protocols conducted with children are necessary to understand the efficacy of these approaches and their public health impact on proximal and distal cancer, cancer-related, and chronic disease outcomes before diffusion. It is clear that more attention should be paid to early life and early developmental phases in cancer prevention.