p53联合伽玛刀治疗抑制人脑胶质瘤细胞生长的体外研究

目的研究p53基因联合伽玛刀治疗对TJ905胶质瘤细胞系的生长抑制作用。方法野生型p53重组腺病毒载体(Ad-p53)按MOI=100感染人胶质瘤细胞系TJ905,Western蛋白印迹鉴定。感染24小时后,对转染组及对照组细胞分别进行伽玛刀治疗(边缘剂量15Gy),并进行治疗前后生物学特性检测,包括细胞增殖(MTT法、PCNA免疫组化染色)与凋亡(TUNEL法)检测。结果Ad-p53联合伽玛刀治疗TJ905细胞系,较单一治疗具有更高的肿瘤增殖抑制率(P<0.001)和肿瘤细胞凋亡诱导率(P<0.001)。结论p53基因联合伽玛刀治疗TJ905胶质瘤细胞系,能更有效的抑制肿瘤细胞增殖和促进肿瘤细胞凋亡,其效果显著较其中单一治疗为优。     

hTERT反义cDNA对人脑胶质瘤细胞的抑制作用

目的探讨人端粒酶催化亚基(hTERT)反义cDNA(pAdeasy-hTERT)在体外对胶质瘤细胞生长的抑制作用。方法将腺病毒介导的pAdeasy-hTERT作用于人胶质瘤细胞系U251,用MTT法检测细胞存活率、端粒酶重复序列扩增法测定端粒酶活性、Westem杂交鉴定hTERT蛋白的表达、RT-PCR法检测hTERT cDNA水平、PCNA观察肿瘤细胞的凋亡情况以及用流式细胞仪对转染后肿瘤细胞的周期进行分析。结果pAdeasy-hTERT在体外明显抑制肿瘤细胞生长,降低端粒酶活性,抑制hTERT表达。结论pAdeasy-hTERT显著抑制人胶质瘤细胞生长,可成为恶性胶质瘤基因治疗的靶基因。     

反义hTERT抑制恶性胶质瘤生长的体内外研究

目的 探讨腺病毒介导的反义hTERT在体内外对恶性胶质瘤细胞生长的影响.方法 构建含有hTERT反义序列的腺病毒载体在体内外转染恶性胶质瘤细胞系U251,检测肿瘤细胞生长情况、细胞周期变化、端粒酶活性、hTERT蛋白表达等.结果 腺病毒介导的hTERT反义治疗在体外明显抑制肫瘤细胞生长,增殖减慢,凋亡增多,细胞较多聚集在G1/G0期,转染后第6天细胞生存率为46.9%,端粒酶活性和hTERT蛋白表达都明显下降,在体内实验中肿瘤生长明显减慢.结论 腺病毒介导的反义hTERT在体内外能明显抑制恶性胶质瘤细胞的生长.     

p16基因对脑胶质瘤细胞生长及放射敏感性的影响

目的探索p16基因在脑胶质瘤发生发展过程中的作用及p16基因与肿瘤细胞放射敏感性间的关系.方法将外源野生型p16基因导入胶质瘤细胞株U251、C6,筛选阳性克隆.同时以空载体质粒为对照,免疫组化检测p16基因表达,用MTT法测定细胞生长曲线及肿瘤杀伤率.结果转染p16基因的U251、C6细胞有外源p16基因的整合及表达,克隆形成率减少,生长速度明显减慢,对辐射的敏感性增强.结论导入外源野生型p16基因可抑制胶质瘤细胞恶性增殖,提高胶质瘤细胞对辐射的敏感性.     

p16基因抑制脑胶质瘤细胞恶性增殖并诱导细胞凋亡

目的：探索p16基因在脑胶质瘤发生过程中的作用及其与脑胶质瘤细胞凋亡的关系。方法：采用脂质体,磷酸钙＋DMSO休克转染的方法,分别将外源野生型p16基因导入胶质瘤细胞株U251,SWO,观察p16基因短暂转染与长期稳定转染对胶质瘤细胞的作用,并筛选阳性克隆。同时以空载体质粒pCDNA3为对照,免疫组化,Northern杂交检测p16基因表达,对转染后细胞生长的抑制,细胞周期,凋亡及裸鼠致瘤能力的变化进行分析,结果：外源p16基因的高水平表达显著抑制了胶质瘤U251,SWO细胞的生长,克隆形成率减少,肿瘤细胞发生了G1期阻滞,p16基因短暂转染可诱导胶质瘤细胞凋亡,而长期稳定转染无明显的凋亡诱导作用。结论：外源野生型p16基因可抑制胶质瘤细胞恶性增殖并诱导细胞凋亡,肿瘤细胞的异质性特点与p16基因转染后的“自然选择”作用可能是p16基因长期稳定转染无明显凋亡诱导作用的主要机制。     

人脑胶质瘤中p16,p15基因分子的病理学研究

目的:探讨p16、p15基因改变、蛋白表达与脑胶质瘤组织病理的相互关系。方法:应用PCR、银染PCR-SSCP及免疫组化技术检测68例脑胶质瘤中p16、p15基因变化和蛋白表达情况。结果:显示脑胶质瘤中p16、p15基因缺失和P16蛋白丢失主要见于Ⅱ~Ⅳ级肿瘤中,而PCR-SSCP分析未见p16、p15基因点突变。结论:表明脑胶质瘤中p16、p15基因改变是以缺失为主,该基因功能的丧失促进了肿瘤细胞由良性向恶性的演进过程;P16蛋白表达状态可作为判断脑胶质瘤恶性程度及预后的指标。     

重组腺病毒载体介导血管抑素基因治疗大鼠脑胶质瘤的实验研究

目的 研究以重组缺陷型腺病毒为载体的血管抑素基因治疗脑胶质瘤。方法 RTPCR法克隆angiostatin基因,构建携带血管抑素（angiostatin）基因的重组腺病毒载体,体外检测angiostatin的重组腺病毒载体对内皮细胞生长的抑制作用。建立皮下及脑胶质瘤大鼠模型,给予体内基因治疗。结果 克隆得到约1.1kb的angiostatin基因。构建重组腺病毒载体AdhCMV-AGS,体外试验表明,可以强烈抑制内皮细胞的生长。体内试验表明重组腺病毒可以在体内有效表达Angiostatin,并且有效抑制胶质瘤的生长,使荷脑胶质瘤大鼠存活90天以上。结论 以重组腺病毒为载体的血管抑素基因治疗脑胶质瘤效果显著,将是一种基因治疗的新策略。     

脑胶质瘤p16基因CPG岛高甲基化与基因失活的研究

目的探讨脑胶质瘤中p16基因5'端CPG岛高甲基化与该基因失活的相关性.方法应用免疫组化检测50例脑胶质瘤中P16蛋白的表达;应用PCR技术检测脑胶质瘤中p16基因第1、2外显子缺失及第1外显子5'CPG岛高甲基化.结果免疫组化结果显示50例脑胶质瘤组织中27例恶性胶质瘤P16蛋白缺失,23例低级别胶质瘤P16蛋白阳性;9例恶性胶质瘤p16基因纯合性缺失;P16蛋白阴性的恶性胶质瘤中7例显示p16基因CPG岛高甲基化.结论恶性脑胶质瘤中P16蛋白缺失而没有p16基因纯合性缺失,是由于p16基因5'端CPG岛高甲基化后抑制该基因的转录所致.p16基因高甲基化也是该基因失活的重要机制之一.     

p53基因、PTEN基因协同对胶质瘤U251细胞系生长抑制作用的研究

目的探讨p53基因和PTEN基因在脑胶质瘤细胞系U251发生发展过程中的作用机制。方法用不同MOI的p53腺病毒表达载体pAdCMV-p53及空载体pAdCMV-lacZ分别感染表达野生型PTEN基因和突变型PTEN基因的细胞系,RT-PCR及Westernblot方法检测转染效率;并通过MTT检测生长抑制率、流式细胞仪检测细胞周期及TUNEL检测分析细胞凋亡等指标观察p53基因及PTEN基因对U251细胞生长的影响。结果MOI为100时,p53基因可引起U251细胞G0G1期阻滞、诱导细胞凋亡,生长抑制;MOI为50时,U251-p53 PTEN生长抑制率明显高于U251-p53,并能出现细胞凋亡,而U251-p53仅出现少量细胞凋亡。结论p53基因可以通过细胞周期G0G1期阻滞及诱导细胞凋亡抑制胶质瘤细胞系U251的生长;PTEN基因可以促进p53基因对胶质瘤细胞系U251的生长抑制作用,并能增加U251细胞对p53基因诱导凋亡的敏感性。     

重组腺病毒介导HSV-TK基因对胶质瘤细胞的生长抑制

本文构建了带有HSV-TK基因的腺病毒包装质粒pACCMVTK,并获得重组腺病毒AdCMVTK,用其感染胶质母细胞瘤细胞系TJ905,研究了HSV-TK/GCV系统对该细胞系的生长影响。结果发现该系统对胶质母细胞瘤细胞系TJ905生长有明显的抑制作用,以100MOI(重复感染率)的病毒剂量感染细胞,GCV半数抑制浓度IC_(50)仅为5μmol,同时发现HSV-TK/GCV系统对TJ905细胞系有明显的旁观效应。表明该系统有治疗胶质瘤的应用前景。     

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.