鼠源性乙酰胆碱受体α亚基97-116肽段(V108A)鼻黏膜耐受对实验性自身免疫性重症肌无力大鼠的影响

目的 研究鼠源性乙酰胆碱受体α亚基97-116肽段丙氨酸替代108缬氨酸[Rα97-116(V108A)]鼻黏膜耐受对实验性自身免疫性重症肌无力(EAMG)大鼠肌无力表现及免疫功能的影响.方法 将采用Rα97-116强化免疫接种方法 成模的22只EAMG大鼠随机分成耐受组和对照组,分别经鼻黏膜给予Rα97-116(V108A)和PBS缓冲液免疫耐受处理10 d后,观察不同时间点两组大鼠体质量、Lennon肌无力评分、血清AChR-ab(ELISA法)及CD28、CTLA4、B7共刺激分子(流式细胞仪)变化.结果 耐受组EAMG大鼠体质量[(228.1±5.8)g]较对照组[(215.0±16.2)g]增加(t=2.395,P＜0.05);肌无力临床评分[耐受组(1.55±0.44)分,对照组(2.10士0.66)分]下降(t=-2.20,P＜0.05);血清AChR-ab(耐受组0.97±0.20,对照组1.27±0.26,t=-2.857,P＜0.05)和外周血CD28、B7-1、B7-2、CTLA4分子的表达(%)耐受组(分别为27.35±7.05、4.73±0.58、2.71±0.35、1.72±0.44)较对照组(分别为40.02±8.81、9.52±1.25、5.88±1.09、2.64±0.47)明显下调(t=3.479、10.861、8.755、4.403,均P＜0.01).结论 Rα97-116(V108A)鼻黏膜耐受能明显减轻EAMG的肌无力症状,并能引起特异性T细胞活化和B细胞免疫功能抑制.     

双类似物鼻粘膜耐受对实验性自身免疫性重症肌无力的影响

目的　在实验性自身免疫性重症肌无力 (EAMG)动物模型采用双类似物进行鼻粘膜免疫耐受 ,观察其临床及免疫功能变化 ,评价疗效并探讨其作用机制。方法　建立Lewis大鼠EAMG动物模型 ,选取经预实验证实有效的最低剂量为治疗量 ,检测致敏同时 (A组 )和缓解期第 1天 (B组 )给予双类似物鼻粘膜免疫耐受治疗后 ,大鼠体重、临床症状、致敏第 35天血清抗AChR抗体IgG含量及其淋巴细胞在不同刺激原作用下的增殖情况。结果　 (1 )治疗后EAMG大鼠体重增加 ,临床症状缓解。 (2 )治疗后血清抗AChR抗体IgG含量 (吸光度 ,A值 ) :A组 (0 98± 0 2 4 )和B组 (0 95± 0 2 6)均少于各自对照组 (分别为 1 1 8± 0 1 0和 1 1 9± 0 1 2 ) ,但A、B组间差异无显著意义。 (3)针对AChR等特异性抗原的淋巴细胞增殖指数 :A组 (1 71± 0 78)和B组 (1 97± 0 56)与对照组 (3 2 4± 1 31和 3 1 9±1 50 )相比均减低 ,增殖反应明显受抑制。结论　双类似物鼻粘膜耐受能明显缓解EAMG的肌无力症状 ,并伴有特异性T、B细胞免疫功能抑制     

TGF—β1基因修饰的树突状细胞移植对EAMG大鼠T淋巴细胞IFN-γ／IFN-γR表达的影响

目的 探讨TGF-β1基因修饰的树突状细胞(DC)移植对实验性自身免疫性重症肌无力(EAMG)大鼠T淋巴细胞IFN-γ分泌及其表面IFN-γ受体(IFN-γR)表达的影响.方法 近交系、8～10周龄、健康雌性Lewis大鼠25只,随机分为健康对照组、EAMG组、DC对照组、pcDNA3-TGF-β1表达质粒转染的DC(pcDNA3-TGF-β1-DC)组与生理盐水对照组5组.除健康对照组外,各组均采用丁氏双鳍电鳐电器官乙酰胆碱受体(AChR)蛋白二次免疫方法复制EAMG大鼠模型.初次免疫后第5天分别皮下注射2×106 DC、pcDNA3-TGF-β1-DC及等体积生理盐水,健康对照组和EAMG组不接受任何治疗.初次免疫后7周,分离脾脏T淋巴细胞,分别应用酶联免疫吸附试验和免疫组化方法检测T淋巴细胞IFN-γ分泌及其表面IFN-γR表达水平.结果 (1)体外培养48 h后,健康对照大鼠T淋巴细胞上清液中IFN-γ水平很低,而EAMG组大鼠T淋巴细胞上清液中IFN-γ水平显著升高(P<0.001).pcDNA3-TGF-β1-DC治疗组IFN-γ水平与EAMG组差异无统计学意义(P>0.05).(2)健康对照大鼠T淋巴细胞经体外培养后可见少量IFN-γR阳性细胞,而EAMG组大鼠T淋巴细胞经体外培养后可见较多IFN-γR阳性细胞,其阳性细胞数及平均吸光度均明显高于健康对照组(P<0.001);pcDNA3-TGF-β1-DC组IFN-γR阳性细胞数较EAMG组明显增多,其阳性细胞平均吸光度亦明显高于EAMG组(均P<0.01).结论 EAMG组大鼠T淋巴细胞IFN-γ分泌及其表面IFN-γR表达水平明显提高;pcDNA3-TGF-β1-DC治疗可进一步诱导IFN-γR表达,调节T淋巴细胞IFN-γ/IFN-γR表达可能是TGF-β1基因修饰的DC治疗EAMG的机制之一.     

共刺激分子CD28：CTLA4/B7在实验性自身免疫性重症肌无力中的作用

目的 研究共刺激分子CD28:CTLA4/B7在实验性自身免疫性重症肌无力(EAMG)发病中的作用.方法 将雌性Lewis鼠随机分为EAMC组和对照组;EAMG组采用人工合成的Ra97-116肽段3次法免疫Lewis鼠,对照组同期注入等量的PBS;3次免疫接种后采用流式细胞术检测CD28、B7-2、B7-1、CTLA4在外周血、淋巴细胞、单核细胞中的表达.结果 EAMG组大鼠成模率75%;与对照组比较,外周血CD28、B7-2B7-1、CTLA4的表达明显增加(P<0.05～0.01);EAMG组大鼠外周血CD28、CTLA4主要在淋巴细胞表达及B7-1、B7-2在淋巴细胞、单核细胞表达显著增加(P<0.05～0.01).结论 EAMG大鼠存在共刺激分子CD28:CTLA4/B7表达异常,共刺激分子CD28:CTLA4/B7可能参与了EAMG的发生、发展.     

青蒿素对实验性自身免疫性重症肌无力大鼠R97-116抗体及细胞因子的影响

目的探讨青蒿素(artemisinin)对实验性自身免疫性重症肌无力(EAMG)大鼠R97-116抗体及干扰素γ(IFN-γ)、白细胞介素17(IL-17)表达水平的影响。方法采用鼠源AChRα亚基97-116肽段免疫方法建立EAMG大鼠模型,将造模成功的大鼠20只随机分为青蒿素小、中、大剂量组和EAMG对照组。青蒿素小、中、大剂量组分别给予15、30、45mg/(kg·d)青蒿素溶液灌胃治疗,1次/d,模型对照组给予等浓度二甲基亚砜水溶液灌胃。评测各组大鼠体质量和临床症状,采用流式细胞术检测淋巴结单个核细胞悬液细胞因子IFN-γ、IL-17水平,ELISA法检测血清抗R97-116IgG/IgG1/IgG2b水平。结果青蒿素中、高剂量组大鼠体质量高于对照组(P0.05);青蒿素各剂量组大鼠临床评分低于对照组(P0.05)。青蒿素各治疗组IFN-γ、IL-17水平均低于对照组(P0.01)。青蒿素15mg/(kg·d)剂量组血清IgG、IgG1、IgG2b水平与对照组比较差异无统计学意义(P0.05);30mg/(kg·d)剂量组血清IgG(P0.05)、IgG1(P0.01)、IgG2b(P0.01)水平低于对照组;45mg/(kg·d)剂量组血清IgG(P0.05)、IgG1(P0.01)水平低于对照组。结论青蒿素能改善EAMG大鼠临床症状,对EAMG大鼠具有免疫调节作用,其机制可能与其通过直接或间接降低血清抗R97-116抗体水平、抑制淋巴结单个核细胞分泌IFN-γ和IL-17促炎性因子有关。     

实验性自身免疫性重症肌无力大鼠T淋巴细胞IFN-γ及其受体表达水平的研究

目的探讨实验性自身免疫性重症肌无力（EAMG）大鼠T淋巴细胞γ-干扰素（IFN-γ）的分泌及其表面IFN-γ受体（IFN-TR）的表达水平。方法健康、雌性Lewis大鼠24只．随机分为正常对照组、完全福（氏）佐剂（CFA）对照组、EAMG组。EAMG组大鼠分别于足垫、腹部及背部皮下多点注射丁（氏）双鳍电鳐的电器官乙酰胆碱受体（AChR）蛋白乳剂1mL，第4周再次注射上述乳剂免疫大鼠。CFA对照组只接受等量的CFA皮下注射。初次免疫后7周．分离各组大鼠脾脏T淋巴细胞并体外培养48h后，应用ELISA和免疫组织化学方法分别检测T淋巴细胞IFN-γ分泌及其表面IFN-γR的表达水平。结果（1）正常大鼠T淋巴细胞体外培养48h后，其上清液中IFN-γ水平很低，而EAMG组大鼠T淋巴细胞培养上清液中IFN-γ水平较CFA对照组与正常组显著增高（P〈0.01）；（2）正常大鼠T淋巴细胞经过体外培养后可见少量IFN-γR阳性细胞．而EAMG组大鼠T淋巴细胞经体外培养后可见较多IFN-γR阳性细胞，其阳性细胞数及其平均吸光度D（λ）值均明显高于CFA对照组与正常组（P〈0.01）。结论EAMG大鼠T淋巴细胞IFN-γ分泌及其表面IFN-γR表达水平明显升高.IFN-γ／IFN-γR的异常表达可能在重症肌无力（MG）的发生过程中发挥重要作用。     

预防性双类似物鼻粘膜耐受对EAMG的影响

目的选择双类似物(Lys262-Ala207)通过不同时间点对实验性自身免疫性重症肌无力(EAMG)模型进行鼻粘膜耐受预防性给药,观察其临床及免疫指标变化,并评价疗效,探讨预防性鼻粘膜耐受在EAMG中的预防作用机制。方法应用乙酰胆碱受体(AChR)加CFA致敏Lewis大鼠建立EAMG模型,并在致敏前10 d(预防耐受A组)及致敏当日(预防耐受B组)给予耐受肽Lvs262-Ala207及相应对照组CA、CB采用相同剂量对照肽MBP-p83-99鼻腔给药。检测给药后A、B组及相应对照组大鼠的体重、临床评分、肌电图、肌肉中AChR含量丢失变化及致敏第42 d血清抗AChR抗体IgG含量。结果急性期和慢性期A、B组体重明显超过相应对照组,临床症状明显轻于相应对照组,慢性期A组体重明显超过B组、病情明显轻于B组;A、B组低频重复电刺激出现衰减反应D5阳性率低于相应对照组;A、B组肌肉AChR含量丢失分别明显低于相应对照组,而A组低于B组;慢性期42 d A、B组IgG含量明显低于相应对照组,同时A组明显低于B组。结论本实验表明,预防耐受的疗效与自身免疫启动时间有关,启动前优于启动时耐受;双类似物鼻粘膜耐受预防不仅可有效地抑制临床症状,且可特异性减低致病性循环抗体含量和减少神经肌接头AChR含量丢失,为采用双类似物鼻粘膜耐受防治人类重症肌无力(MG)提供了依据。     

髓源树突细胞源性外泌体对实验性自身免疫性重症肌无力大鼠NK及NK T细胞的影响

目的探讨他汀类药物诱导髓源树突细胞(BMDCs)源性外泌体(exosomes)对实验性自身免疫性重症肌无力(EAMG)大鼠自然杀伤细胞(NK)及NK T细胞的影响。方法阿托伐他汀和DMSO分别与BMDCs共培养,采用流式细胞术分析其表型。应用梯度离心法提取不同组BMDCs分泌的外泌体(分别记为他汀Dex和对照Dex),将不同组外泌体注射于EAMG大鼠体内,采用双盲法观察EAMG临床症状;通过流式细胞术检测各组大鼠淋巴结NK、NK T和干扰素γ(IFN-γ)阳性细胞、白细胞介素-10(IL-10)阳性细胞的表达水平;采用ELISA方法检测各组血清抗R97-116IgG抗体及其亚型水平。结果他汀类药物能够明显抑制BMDCs表面CD80和CD86的表达水平(CD80:1.10%比22.80%,P0.01;CD86:30.78%比43.93%,P0.01),这些BMDCs可进一步分泌他汀Dex。与对照Dex治疗组比较,他汀Dex治疗组EAMG大鼠淋巴结NK细胞比例增加(2.30%比1.63%,P0.05),淋巴结单个核细胞(MNC)中IFN-γ+细胞比例(1.05%比2.24%,P0.05)、血清抗R97-116IgG抗体及其亚型IgG2a和IgG2b水平均明显降低(IgG:0.44比0.64,IgG2a:0.26比0.35,IgG2b:1.47比1.94;均P0.05)。结论他汀Dex可缓解EAMG大鼠的临床症状,这种作用与上调淋巴结MNC中的NK细胞有关。     

双类似物鼻黏膜耐受治疗实验性自身免疫性重症肌无力的免疫机制

目的　用双类似物鼻黏膜耐受治疗实验性自身免疫性重症肌无力 ( EAMG)大鼠 ,探讨其对机体免疫机制的影响。方法　建立 Lewis大鼠的 EAMG模型 ,在致敏同时 ( A组 )及缓解期第 1天 ( B组 )给予双类似物 3 0 0μg/只。动态评估大鼠临床症状 ,检测外周血 ( PB) ACh R-Ab( Ig G)含量 ,检测急、慢性期 PB单个核细胞 ( MNC)及致敏第 5 0天淋巴结、脾 MNC中的 CD4+ CD2 5 + 细胞数改变。结果　治疗组大鼠临床症状减轻 ;治疗组及对照组大鼠 PB中特异性 ACh R-Ab( Ig G)含量随病程延续而增加 ,但在致敏后第 5及第 7周 ,A、B组特异性 Ig G抗体含量比各自对照组显著减低 ( P <0 .0 5 ) ;双类似物鼻黏膜耐受治疗后 ,EAMG大鼠 PB MNC中的 CD4+ 细胞数减少 ( P<0 .0 5 ) ,CD4+ CD2 5 +细胞数相对增加 ( P<0 .0 5 ) ;A、B组淋巴结和脾 MNC中 CD4+细胞数减少( P<0 .0 5 ) ,CD4+ CD2 5 +细胞数只在 B组相对增加 ( P <0 .0 5 )。结论　双类似物鼻黏膜耐受治疗病情进展中的 EAMG有效 ;伴随临床症状缓解 ,治疗组大鼠 ACh R-Ab( Ig G)含量减少且免疫调节性 CD4+ CD2 5 +细胞数相对增多 ;双类似物治疗 EAMG的可行性为抗原特异性治疗重症肌无力 ( MG)和其他自身免疫性疾病 ( AID)提供了依据。     

设计耐受原并用鼻黏膜耐受治疗EAMG的方法学研究

目的　设计特异性耐受原 ,通过其鼻黏膜耐受治疗对实验性自身免疫性重症肌无力 (EAMG)发病过程的影响 ,以探讨该疗法在模型应用中的可行性。方法　用 Lewis大鼠建立 EAMG模型 ,选取双类似物作为耐受原在致敏同时给予鼻黏膜耐受治疗 ,动态观察大鼠体重及临床评分改变。结果　虽然治疗组大鼠发病率不降低 ,但是在 EAMG急性期和慢性期 ,其临床症状均较对照组减轻 (P <0 .0 5 )。结论　用双类似物鼻黏膜耐受可以达到治疗 EAMG的目的 ,其结果为抗原特异性治疗重症肌无力 (MG)和其他自身免疫性疾病 (AID)提供了依据。     

Mechanisms of the suppression of the nociceptive reflex of the opening of the mouth during stimulation of the structures of the limbic brain

The comparative effectiveness of the inhibitory influence of tetanic stimulation of hypothalamus, amygdala and limbic cortex on EMG-response of m. digastricus evoked by electrical stimulation of tooth pulp nociceptive afferents was studied in cats anesthetized with a mixture of chloralose and nembutal. It was found that inhibition of the EMG-component of the jaw-opening reflex is most pronounced in case of stimulation of medial and lateral region of the hypothalamus, the inhibitory effect of central and medial nuclei of the amygdala is less pronounced and the effect of the limbic cortex is the weakest. It was shown that the mechanism of the antinociceptive effect of tetanic stimulation of the hypothalamus is not related to the concomitant increase of the blood pressure. After stabilization of the blood pressure the suppressive effect of the hypothalamus remains without changes, that points out to a direct, primary, not baro-afferent mechanism of the inhibition of the activity of nociceptive neurons of the trigeminal sensory nuclei. Noradrenaline, injected intravenously, induced a large increase of the blood pressure accompanied by a pronounced inhibition of the pain reflex. Angiotensin causes the same degree of blood pressure elevation without changes in the amplitude of the EMG-response of the pain reflex. Hypothalamic and noradrenergic mechanisms for control of pain sensitivity are discussed.     

药物治疗与合并认知行为治疗对强迫症疗效的比较

目的探讨认知行为心理治疗(CBT)在强迫症(OCD)患者各亚型治疗中的有效性和规律性。方法本研究为临床对照研究。符合入组标准的强迫症患者按患者自愿原则分为两组,治疗观察3、6、12个月。疗效评定分别运用Yale-Brown强迫量表,自拟的自评好转程度量表和临床疗效评定。结果认知行为心理治疗合并药物治疗组31例,临床有效率70.9%,其中治愈率1.8%。单纯药物治疗组24例,临床有效率33.3%。Yale-Brown强迫量表和自评量表得分在6个月和12个月两组有显著差异(P<0.05)。其中强迫症亚型(怕脏型、反复检查型和反复担心型)的疗效比较,怕脏型在治疗3个月末两组间自评量表评分有显著性差异(P<0.05);反复担心型在治疗6个月末两组间Yale-Brown强迫量表总分有显著性差异(P<0.05);反复检查型两组间无统计学差异。结论认知行为心理治疗合并药物治疗强迫症的疗效明显优于单纯药物治疗。强迫症的亚型在治疗中的有效性次序为:反复担心型>怕脏型>反复检查型。     

Characteristics of the sympathetic innervation of the nictitating membrane and of the vasculature of the nose and tongue of the cat

Summary Vasomotor responses from the nasal mucosa and tongue, and contractions of the nictitating membrane, were recorded on stimulation of the cervical sympathetic or internal carotid nerves.Preganglionic sympathetic nerve fibres which elicited a membrane response possessed a lower threshold than those which evoked nasal vasoconstriction, while the latter displayed a lower threshold than fibres which evoked tongue vasoconstriction. The sympathetic vasodilator fibres to the tongue, whose activity was revealed after-receptor blockade, had a similar threshold to the vasoconstrictor fibres.Membrane contraction, nasal vasoconstriction and occasionally tongue vasoconstriction could be evoked by stimulating the internal carotid nerve. The postganglionic fibres innervating the nasal mucosa had a similar threshold to those of the nictitating membrane, which may indicate that there are small myelinated fibres innervating the mucosa.The preganglionic compound nerve action potential had four major components, S₁–S₄. S₁, S₂ and usually S₃ fibres were associated with membrane contraction; S₂, S₃ and sometimes S₁ fibres were associated with nasal vasoconstriction; and S₃, usually S₂ and occasionally S₁ fibres were associated with vasoconstriction in the tongue. It is concluded that each of these three groups of nerve fibres, but not S₄ fibres, may include fibres associated functionally with the three effectors.There was a considerable difference between the relative amplitude of the responses of the three effectors elicited by stimulation of the cervical sympathetic nerve at frequencies between 0.2 and 2 Hz. Vasoconstrictor responses were relatively larger than membrane contractions suggesting differences in the mechanisms of neurotransmission at the neuroeffector junctions.     

Development of the projection from the nucleus of the brachium of the inferior colliculus to the superior colliculus in the ferret

Neurons in the deeper layers of the superior colliculus (SC) have spatially tuned receptive fields that are arranged to form a map of auditory space. The spatial tuning of these neurons emerges gradually in an experience-dependent manner after the onset of hearing, but the relative contributions of peripheral and central factors in this process of maturation are unknown. We have studied the postnatal development of the projection to the ferret SC from the nucleus of the brachium of the inferior colliculus (nBIC), its main source of auditory input, to determine whether the emergence of auditory map topography can be attributed to anatomical rewiring of this projection. The pattern of retrograde labeling produced by injections of fluorescent microspheres in the SC on postnatal day (P) 0 and just after the age of hearing onset (P29), showed that the nBIC-SC projection is topographically organized in the rostrocaudal axis, along which sound azimuth is represented, from birth. Injections of biotinylated dextran amine-fluorescein into the nBIC at different ages (P30, 60, and 90) labeled axons with numerous terminals and en passant boutons throughout the deeper layers of the SC. This labeling covered the entire mediolateral extent of the SC, but, in keeping with the pattern of retrograde labeling following microsphere injections in the SC, was more restricted rostrocaudally. No systematic changes were observed with age. The stability of the nBIC-SC projection over this period suggests that developmental changes in auditory spatial tuning involve other processes, rather than a gross refinement of the projection from the nBIC.     

A contribution to the study of the architecture of the autonomic nervous system of the digestive tract of the rat

Summary The distribution of aminergic and non-aminergic nerve fibres to the different constituents of the wall of the digestive tract in various regions is described. Aminergic fibres synapse with all nervous perikarya. Densely interlacing networks of nerve fibres are found in both layers of the tunica muscularis and in the lamina muscularis mucosae. A finely meshed plexus is observed in relation to the wall of the blood vessels in the wall of the gut. There are many fibres connecting the muscular and the vascular plexus. No nerve fibres have been observed in direct relation to the epithelium.The functional implications of these findings are discussed.